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A10. Hydrothermal Vents or Primoridal Soup? - Biology

A10.  Hydrothermal Vents or Primoridal Soup? - Biology


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The case for the origin of life in deep sea hydrothermal vents and not in a primordial "Campbell's" soup has been argued convincingly by Lane et al (2010). What's needed for life are reasonably complex molecules and an energy source to drive unfavorable reactions. It's the latter on which that Lane et al focus. This can be surmised from the low energy yield (compared to aerobic respiration) achieved in present day glycolytic (fermentative) pathways from all major domains of life, archaea, bacteria, and eukaryotes.

Background: Based on rRNA sequences, a primordial cell evolved into two different types of cells, one that became bacteria, and another that split further into archaea (single cells, similar to bacteria) and eukaryotic cells (complex cells with internal organelles that eventually formed multicellular organisms). Bacterial and archaea are collected called prokaroytic cells.

In addition, these pathways required the evolution of up to dozen different enzymes to produce their relatively meager energy yield which ultimately depends on the oxidation of an organic molecule by another organic molecule instead of by a powerful oxidant like dioxygen. An anisotropic arrangement of molecules in a concentrated soup could lead to transient chemical potential fluctuations but these would be inefficient and impermanent sources of energy. Effectively the primordial soup would be at equilibrium and hardly expected to provide the energy for synthesis of RNA enzymes and replicators. UV light leads to photo-damage and photolysis not replication of complex molecules. What is needed is a way to drive the synthesis of molecule with high chemical potential energy (like sulfur esters and phosphoanhydrides) compared to their lytic products. These could then provide an energy sources to drive ATP synthesis, for example.

A detailed look a the bioworld shows that the earliest organisms used energy from the collapse of the proton gradient (chemisomotic principle elucidated by Peter Mitchell). All present autotrophs (organisms that can fix CO2 and form complex organic molecules) and many heterotrophs (use complex organic molecules of other organisms for fuel) use redox complexes in membranes coupled to membrane gradients. These complexes would take reduced molecules and pass electrons from them to oxidizing agents (electron acceptors), including O2, CO2, and Fe3+ to form H2O, CH4, and Fe2+. Fermentors also use ATPase membrane enzymes to transport nutrients. Yet genomic analysis of bacteria and archaea show that enzymes involved in fermentation differ significantly, suggesting that they evolved separately towards a convergent function. Structure in common include DNA, RNA, ribosomes and membrane ATPases, which Lane et al suggest were in a the Last Universal Common Cell (LUCA).

All autotrophs produce their energy source by fixing CO2 using either H2 directly or indirectly using H2O and H2S. All of the are available in nonhydrothermal deep sea vents. Volcanic vents, however, are extremely hot (not optimal for organic molecule synthesis), very acidic, and lack hydrogen gas. A different type of nonvolcanic vent, an alkaline hydrothermal one, might produce more conducive as a site of the origin of life. In these vents, water chemically reacts with minerals in the crust (such as olivine) leading to their hydroxylation and subsequent fracture, with promotes more water entry into the crust. It has been reported that there is more water found as hydroxylated minerals in the crust, that there is liquid water in the oceans. These processes result in temperatures up to 200 degrees Celsius and release of hydrogen gas into a moderately alkaline vents into the sea water at temperatures more conducive (70 degrees C) to the origin of life.

Figure: Alkaline Vent

Contributors

  • Prof. Henry Jakubowski (College of St. Benedict/St. John's University)

A10. Hydrothermal Vents or Primoridal Soup? - Biology

Sajini, K K Karun, A Amamath, C H Engelmann, F

The present study investigates the effect of preculture conditions, vitrification and unloading solutions on survival and regeneration of coconut zygotic embryos after cryopreservation. Among the seven plant vitrification solutions tested, PVS3 was found to be the most effective for regeneration of cryopreserved embryos. The optimal protocol involved preculture of embryos for 3 days on medium with 0.6 M sucrose, PVS3 treatment for 16 h, rapid cooling and rewarming and unloading in 1.2 M sucrose liquid medium for 1.5 h. Under these conditions, 70-80 survival (corresponding to size enlargement and weight gain) was observed with cryopreserved embryos and 20-25 percent of the plants regenerated (showing normal shoot and root growth) from cryopreserved embryos were established in pots.

Wakisaka, Keiko Tsuji Ichiyanagi, Kenji Ohno, Seiko Itoh, Masanobu

P -element transposition in the genome causes P-M hybrid dysgenesis in Drosophila melanogaster . Maternally deposited piRNAs suppress P -element transposition in the progeny, linking them to P-M phenotypes however, the role of zygotic piRNAs derived from paternal P elements is poorly understood. To elucidate the molecular basis of P -element suppression by zygotic factors, we investigated the genomic constitution and P -element piRNA production derived from fathers. As a result, we characterized males of naturally derived Q, M' and P strains, which show different capacities for the P -element mobilizations introduced after hybridizations with M-strain females. The amounts of piRNAs produced in ovaries of F1 hybrids varied among the strains and were influenced by the characteristics of the piRNA clusters that harbored the P elements. Importantly, while both the Q- and M'-strain fathers restrict the P -element mobilization in ovaries of their daughters, the Q-strain fathers supported the production of the highest piRNA expression in the ovaries of their daughters, and the M' strain carries KP elements in transcriptionally active regions directing the highest expression of KP elements in their daughters. Interestingly, the zygotic P -element piRNAs, but not the KP element mRNA, contributed to the variations in P transposition immunity in the granddaughters. The piRNA-cluster-embedded P elements and the transcriptionally active KP elements from the paternal genome are both important suppressors of P element activities that are co-inherited by the progeny. Expression levels of the P -element piRNA and KP -element mRNA vary among F1 progeny due to the constitution of the paternal genome, and are involved in phenotypic variation in the subsequent generation.

Blachon, Stephanie Khire, Atul Avidor-Reiss, Tomer

Centrosomes are composed of two centrioles surrounded by pericentriolar material (PCM). However, the sperm and the oocyte modify or lose their centrosomes. Consequently, how the zygote establishes its first centrosome, and in particular, the origin of the second zygotic centriole, is uncertain. Drosophila melanogaster spermatids contain a single centriole called the Giant Centriole (GC) and a Proximal centriole-like (PCL) structure whose function is unknown. We found that, like the centriole, the PCL loses its protein markers at the end of spermiogenesis. After fertilization, the first two centrioles are observed via the recruitment of the zygotic PCM proteins and are seen in asterless mutant embryos that cannot form centrioles. The zygote’s centriolar proteins label only the daughter centrioles of the first two centrioles. These observations demonstrate that the PCL is the origin for the second centriole in the Drosophila zygote and that a paternal centriole precursor, without centriolar proteins, is transmitted to the egg during fertilization. PMID:24532732

Elbl, Paula De Souza, Amanda P. Jardim, Vinicius de Oliveira, Leandro F. Macedo, Amanda F. dos Santos, André L. W. Buckeridge, Marcos S. Floh, Eny I. S.

Three zygotic developmental stages and two somatic Araucaria angustifolia cell lines with contrasting embryogenic potential were analyzed to identify the carbohydrate-mediated responses associated with embryo formation. Using a comparison between zygotic and somatic embryogenesis systems, the non-structural carbohydrate content, cell wall sugar composition and expression of genes involved in sugar sensing were analyzed, and a network analysis was used to identify coordinated features during embryogenesis. We observed that carbohydrate-mediated responses occur mainly during the early stages of zygotic embryo formation, and that during seed development there are coordinated changes that affect the development of the different structures (embryo and megagametophyte). Furthermore, sucrose and starch accumulation were associated with the responsiveness of the cell lines. This study sheds light on how carbohydrate metabolism is influenced during zygotic and somatic embryogenesis in the endangered conifer species, A. angustifolia. PMID:28678868

That the soul of a human person is infused at conception is a metaphysical claim. But given its traditional articulation, it has the empirical consequence that the zygote must have a substantial continuity with the adult person, a continuity which is already determined at conception. This empirical consequence is contradicted by the fact that the zygote may become a hydatidiform mole, or several persons. The metaphysical claim is falsified by the facts.

Larcombe, Matthew J Costa E Silva, João Tilyard, Paul Gore, Peter Potts, Brad M

Many previous studies conclude that pre- zygotic barriers such as mechanical isolation account for most reproductive isolation between pairs of taxa. However, the inheritance and persistence of barriers such as these after the first generation of hybridization is rarely quantified, even though it is a vital consideration in understanding gene flow potential. There is an asymmetrical pre- zygotic mechanical barrier to hybridization between Eucalyptus nitens and Eucalyptus globulus, which completely prevents small-flowered E. nitens pollen from mating with large E. globulus flowers, while the reverse cross is possible. We aimed to determine the relative importance of pre- and post- zygotic barriers in preventing gene flow following secondary contact between E. nitens and E. globulus, including the inheritance of barriers in advanced-generation hybrids. Experimental crossing was used to produce outcrossed E. nitens, E. globulus and their F1, F2, BCg and BCn hybrids. The strength and inheritance of a suite of pre- and post- zygotic barriers were assessed, including 20-year survival, growth and reproductive capacity. The mechanical barrier to hybridization was lost or greatly reduced in the F1 hybrid. In contrast, intrinsic post- zygotic barriers were strong and persistent. Line-cross analysis indicated that the outbreeding depression in the hybrids was best explained by epistatic loss. The removal of strong mechanical barriers between E. nitens and E. globulus allows F1 hybrids to act as a bridge for bi-directional gene flow between these species. However, strong and persistent post- zygotic barriers exist, meaning that wherever F1 hybridization does occur, intrinsic post- zygotic barriers will be responsible for most reproductive isolation in this system. This potential transient nature of mechanical barriers to zygote formation due to additive inheritance in hybrids appears under-appreciated, and highlights the often important role that intrinsic post-mating barriers play

Ma, Xue-Shan Chao, Shi-Bin Huang, Xian-Ju Lin, Fei Qin, Ling Wang, Xu-Guang Meng, Tie-Gang Zhu, Cheng-Cheng Schatten, Heide Liu, Hong-Lin Sun, Qing-Yuan

H3K9 methylation is an important histone modification that is correlated with gene transcription repression. The asymmetric H3K9 dimethylation (H3K9me2) pattern between paternal and maternal genomes is generated soon after fertilization. In the present study, we carefully determined the dynamics of H3K9me2 changes in mouse zygotes , and investigated the regulatory mechanisms. The results indicated that histone methyltransferase G9a, but not GLP, was involved in the regulation of asymmetric H3K9me2, and G9a was the methyltransferase that induced the appearance of H3K9me2 in the male pronucleus of the zygote treated with cycloheximide. We found that there were two distinct mechanisms that regulate H3K9me2 in the male pronucleus. Before 8 h of in vitro fertilization (IVF), a mechanism exists that inhibits the association of G9a with the H3K9 sites. After 10 h of IVF the inhibition of G9a activity depends on yet unknown novel protein(s) synthesis. The two mechanisms of transfer take place between 8–10 h of IVF, and the novel protein failed to inhibit G9a activity in time, resulting in the appearance of a low level de novo H3K9me2 in the male pronucleus. PMID:26639638

Moriyama, Yohsuke Yamazaki, Tomokazu Nomura, Hideo Sasaki, Narie Kawano, Shigeyuki

The active , selective digestion of mtDNA from one parent is a possible molecular mechanism for the uniparental inheritance of mtDNA. In Physarum polycephalum, mtDNA is packed by DNA-binding protein Glom, which packs mtDNA into rod-shaped mt-nucleoids. After the mating, mtDNA from one parent is selectively digested, and the Glom began to disperse. Dispersed Glom was retained for at least 6 h after mtDNA digestion, but disappeared completely by about 12 h after mixing two strains. We identified two novel nucleases using DNA zymography with native-PAGE and SDS-PAGE. One is a Ca2+-dependent, high-molecular-weight nuclease complex (about 670 kDa), and the other is a Mn2+-dependent, high-molecular-weight nuclease complex (440-670 kDa) the activity of the latter was detected as a Mn2+-dependent, 13-kDa DNase band on SDS-PAGE. All mitochondria isolated from myxamoebae had mt-nucleoids, whereas half of the mitochondria isolated from the zygotes at 12 h after mixing had lost the mt-nucleoids. The activity of the Mn2+-dependent nuclease in the isolated mitochondria was detected at least 8 h after mixing of two strains. The timing and localization of the Mn2+-dependent DNase activity matched the selective digestion of mtDNA.

Winkelmann, Traud Ratjens, Svenja Bartsch, Melanie Rode, Christina Niehaus, Karsten Bednarz, Hanna

Somatic embryogenesis has been shown to be an efficient in vitro plant regeneration system for many crops such as the important ornamental plant Cyclamen persicum, for which this regeneration pathway of somatic embryogenesis is of interest for the vegetative propagation of parental lines as well as elite plants. However, somatic embryogenesis is not commercially used in many crops due to several unsolved problems, such as malformations, asynchronous development, deficiencies in maturation and germination of somatic embryos. In contrast, zygotic embryos in seeds develop and germinate without abnormalities in most cases. Instead of time-consuming and labor-intensive experiments involving tests of different in vitro culture conditions and plant growth regulator supplements, we follow a more directed approach. Zygotic embryos served as a reference and were compared to somatic embryos in metabolomic analyses allowing the future optimization of the in vitro system. The aims of this study were to detect differences in the metabolite profiles of torpedo stage somatic and zygotic embryos of C. persicum. Moreover, major metabolites in endosperm and testa were identified and quantified. Two sets of extracts of two to four biological replicates each were analyzed. In total 52 metabolites were identified and quantified in the different tissues. One of the most significant differences between somatic and zygotic embryos was that the proline concentration in the zygotic embryos was about 40 times higher than that found in somatic embryos. Epicatechin, a scavenger for reactive oxygen species, was found in highest abundance in the testa. Sucrose, the most abundant metabolite was detected in significantly higher concentrations in zygotic embryos. Also, a yet unknown trisaccharide, was significantly enriched in zygotic embryos. PMID:26300898

Small, Lawrence E. Dawes, Adriana T.

Establishment of anterior–posterior polarity in the Caenorhabditis elegans zygote requires two different processes: mechanical activity of the actin–myosin cortex and biochemical activity of partitioning-defective (PAR) proteins. Here we analyze how PARs regulate the behavior of the cortical motor protein nonmuscle myosin (NMY-2) to complement recent efforts that investigate how PARs regulate the Rho GTPase CDC-42, which in turn regulates the actin-myosin cortex. We find that PAR-3 and PAR-6 concentrate CDC-42–dependent NMY-2 in the anterior cortex, whereas PAR-2 inhibits CDC-42–dependent NMY-2 in the posterior domain by inhibiting PAR-3 and PAR-6. In addition, we find that PAR-1 and PAR-3 are necessary for inhibiting movement of NMY-2 across the cortex. PAR-1 protects NMY-2 from being moved across the cortex by forces likely originating in the cytoplasm. Meanwhile, PAR-3 stabilizes NMY-2 against PAR-2 and PAR-6 dynamics on the cortex. We find that PAR signaling fulfills two roles: localizing NMY-2 to the anterior cortex and preventing displacement of the polarized cortical actin–myosin network. PMID:28615321

Aten, Quentin T. Jensen, Brian D. Howell, Larry L.

This paper presents a surface-micromachined microelectromechanical system nanoinjector designed to inject DNA into mouse zygotes which are ≈90 μm in diameter. The proposed injection method requires that an electrically charged, DNA coated lance be inserted into the mouse zygote . The nanoinjector's principal design requirements are (1) it must penetrate the lance into the mouse zygote without tearing the cell membranes and (2) maintain electrical connectivity between the lance and a stationary bond pad. These requirements are satisfied through a two-phase, self-reconfiguring metamorphic mechanism. In the first motion subphase a change-point six-bar mechanism elevates the lance to ≈45 μm above the substrate. Inmore » the second motion subphase, a compliant folded-beam suspension allows the lance to translate in-plane at a constant height as it penetrates the cell membranes. The viability of embryos following nanoinjection is presented as a metric for quantifying how well the nanoinjector mechanism fulfills its design requirements of penetrating the zygote without causing membrane damage. Viability studies of nearly 3000 nanoinjections resulted in 71.9% of nanoinjected zygotes progressing to the two-cell stage compared to 79.6% of untreated embryos.« less

In brown algal cells, the centrosome, consisting of a pair of centrioles and the pericentriolar material, is primarily involved in the organization of microtubules (MTs) throughout the cell cycle. In motile cells, the centrioles participate in the formation of flagellar axoneme as flagellar basal bodies, and in somatic cells they play a crucial role in many cellular activities as a part of the centrosome. With respect to the role of the centrosome as a microtubule organizing center (MTOC), brown algal cells resemble animal cells. In most animal fertilization processes, the sperm cell introduces centrioles, the core of the centrosome, into the egg cytoplasm. In this study, the behavior of centrioles from gametogenesis and fertilization to the first cell division of the zygote was examined in the three sexual reproduction patterns occurring in brown algae, i.e., oogamy, anisogamy and isogamy, by electron- and immunofluorescence-microscopy. The pair of centrioles contained in somatic cells was shown to be derived from the male gamete, irrespective of the sexual reproductive pattern. The paternally derived centrioles were duplicated before mitosis and were involved in spindle pole formation. Moreover, MTs from the centrosome play a crucial role in the process of cytokinesis, as the position of centrosomes accompanying daughter nuclei seems to determine the cytokinetic plane. A new approach to clarifying the mode of cytokinesis in brown algae is presented in this study.

Despite extensive work on the mechanisms that generate plasma membrane furrows, understanding how cells are able to dynamically regulate furrow dimensions is an unresolved question. Here, we present an in-depth characterization of furrow behaviors and their regulation in vivo during early Drosophila morphogenesis. We show that the deepening in furrow dimensions with successive nuclear cycles is largely due to the introduction of a new, rapid ingression phase (Ingression II). Blocking the midblastula transition (MBT) by suppressing zygotic transcription through pharmacological or genetic means causes the absence of Ingression II, and consequently reduces furrow dimensions. The analysis of compound chromosomes that produce chromosomal aneuploidies suggests that multiple loci on the X, II, and III chromosomes contribute to the production of differentially-dimensioned furrows, and we track the X-chromosomal contribution to furrow lengthening to the nullo gene product. We further show that checkpoint proteins are required for furrow lengthening however, mitotic phases of the cell cycle are not strictly deterministic for furrow dimensions, as a decoupling of mitotic phases with periods of active ingression occurs as syncytial furrow cycles progress. Finally, we examined the turnover of maternal gene products and find that this is a minor contributor to the developmental regulation of furrow morphologies. Our results suggest that cellularization dynamics during cycle 14 are a continuation of dynamics established during the syncytial cycles and provide a more nuanced view of developmental- and MBT-driven morphogenesis. PMID:29337989

Tang, Lichun Zeng, Yanting Du, Hongzi Gong, Mengmeng Peng, Jin Zhang, Buxi Lei, Ming Zhao, Fang Wang, Weihua Li, Xiaowei Liu, Jianqiao

Previous works using human tripronuclear zygotes suggested that the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system could be a tool in correcting disease-causing mutations. However, whether this system was applicable in normal human (dual pronuclear, 2PN) zygotes was unclear. Here we demonstrate that CRISPR/Cas9 is also effective as a gene-editing tool in human 2PN zygotes . By injection of Cas9 protein complexed with the appropriate sgRNAs and homology donors into one-cell human embryos, we demonstrated efficient homologous recombination-mediated correction of point mutations in HBB and G6PD. However, our results also reveal limitations of this correction procedure and highlight the need for further research.

Ciruna, Brian Weidinger, Gilbert Knaut, Holger Thisse, Bernard Thisse, Christine Raz, Erez Schier, Alexander F

We report a generally applicable strategy for transferring zygotic lethal mutations through the zebrafish germ line. By using a morpholino oligonucleotide that blocks primordial germ cell (PGC) development, we generate embryos devoid of endogenous PGCs to serve as hosts for the transplantation of germ cells derived from homozygous mutant donors. Successful transfers are identified by the localization of specifically labeled donor PGCs to the region of the developing gonad in chimeric embryos. This strategy, which results in the complete replacement of the host germ line with donor PGCs, was validated by the generation of maternal and maternal- zygotic mutants for the miles apart locus. This germ-line replacement technique provides a powerful tool for studying the maternal effects of zygotic lethal mutations. Furthermore, the ability to generate large clutches of purely mutant embryos will greatly facilitate embryological, genetic, genomic, and biochemical studies.

Ciruna, Brian Weidinger, Gilbert Knaut, Holger Thisse, Bernard Thisse, Christine Raz, Erez Schier, Alexander F.

We report a generally applicable strategy for transferring zygotic lethal mutations through the zebrafish germ line. By using a morpholino oligonucleotide that blocks primordial germ cell (PGC) development, we generate embryos devoid of endogenous PGCs to serve as hosts for the transplantation of germ cells derived from homozygous mutant donors. Successful transfers are identified by the localization of specifically labeled donor PGCs to the region of the developing gonad in chimeric embryos. This strategy, which results in the complete replacement of the host germ line with donor PGCs, was validated by the generation of maternal and maternal- zygotic mutants for the miles apart locus. This germ-line replacement technique provides a powerful tool for studying the maternal effects of zygotic lethal mutations. Furthermore, the ability to generate large clutches of purely mutant embryos will greatly facilitate embryological, genetic, genomic, and biochemical studies. PMID:12397179

HOSHINO, YOICHIRO MURATA, NAHO SHINODA, KOICHI

• Aims To develop a procedure for isolating living egg cells and zygotes from Alstroemeria ovules. • Scope An attempt was made to isolate egg cells and zygotes from the ovules of Alstroemeria aurea. The ovules were histologically observed using a clearing procedure which revealed the localization and sizes of the embryo sacs and egg apparatus within the ovules. For the isolation of egg cells, ovules were cut into sections with a surgical blade and treated with an enzyme solution. Subsequently, these ovule sections were dissected using a glass needle under an inverted microscope. Egg cells successfully isolated by this procedure were collected using microcapillaries connected to a micropump. For zygote isolation, ovules were excised from ovaries 24 h after self-pollination. By treating excised ovules with an enzyme solution and subsequently dissecting them using a glass needle, zygotes were successfully isolated from the ovules and collected with a microcapillary. The isolated zygotes were associated with pollen tubes and one of the synergids. Egg cells and zygotes were viable for up to 2 h following isolation, as determined by fluorescein diacetate staining. • Conclusions The procedures for isolating egg cells and zygotes in Alstroemeria were established, and each egg cell and zygote was captured with a microcapillary. PMID:16621859

Fertilization marks the turnover from the gametophyte to sporophyte generation in higher plants. After fertilization, sporophytic development undergoes genetic turnover from maternal to zygotic control: the maternal-to- zygotic transition (MZT). The MZT is thought to be critical for early embryogenesis however, little is known about the time course or developmental impact of the MZT in higher plants. Here, we discuss what is known in the field and focus on techniques used in relevant studies and their limitations. Some significant questions and technical requirements for further investigations are also discussed. © 2015 Elsevier Inc. All rights reserved.

Noah, Alexandre Mboene Niemenak, Nicolas Sunderhaus, Stephanie Haase, Christin Omokolo, Denis Ndoumou Winkelmann, Traud Braun, Hans-Peter

Somatic embryogenesis can efficiently foster the propagation of Theobroma cacao, but the poor quality of resulted plantlet hinders the use of this technique in the commercial scale. The current study has been initiated to systematically compare the physiological mechanisms underlying somatic and zygotic embryogenesis in T. cacao on the proteome level. About 1000 protein spots per fraction could be separated by two-dimensional isoelectric focusing/SDS PAGE. More than 50 of the protein spots clearly differed in abundance between zygotic and somatic embryos: 33 proteins spots were at least 3-fold higher in abundance in zygotic embryos and 20 in somatic embryos. Analyses of these protein spots differing in volume by mass spectrometry resulted in the identification of 68 distinct proteins. Many of the identified proteins are involved in genetic information processing (21 proteins), carbohydrate metabolism (11 proteins) and stress response (7 proteins). Somatic embryos especially displayed many stress related proteins, few enzymes involved in storage compound synthesis and an exceptional high abundance of endopeptidase inhibitors. Phosphoenolpyruvate carboxylase, which was accumulated more than 3-fold higher in zygotic embryos, represents a prominent enzyme in the storage compound metabolism in cacao seeds. Implications on the improvement of somatic embryogenesis in cacao are discussed. Copyright © 2012 Elsevier B.V. All rights reserved.

Rover, Ticiane Simioni, Carmen Hable, Whitney Bouzon, Zenilda L

This study investigates the pattern and performance of cellular structures during the early development of zygotes and embryos of Sargassum cymosum. The early development S. cymosum germlings has already been characterized and compared with the pattern of development established for all fucoid algae, in which the zygote remains attached to the receptacle by mucilage during the establishment of polarity and early cell division. As in the algae Fucus and Silvetia, the first division is transverse across the longer axis of the zygote of S. cymosum. However, the cell that will give rise to the rhizoids is not determined in the first division rather, the formation of this cell occurs with the second division, forming a small cell in the embryo shaded site. Stabilizing polarity during the process of forming a multicellular embryo occurs rapidly. During development, significant cytoplasmic alterations take place. Initially, the cytoplasm shows large clusters of phenolic compounds located in specific parts, but later, in the course of development, these compounds are dispersed in the cytoplasm, although a significant amount remains confined to the nucleus. Moreover, to produce more zygotes and higher growth rates for the germlings, the best conditions found for the species S. cymosum were 22 and 26 °C, respectively.

Shimada, Atsuko Takeda, Hiroyuki

Taking advantage of the characteristics that make hybrids between Japanese and Chinese medaka grow well, albeit sterile, we have developed a method of germ-line replacement in which these hybrids are used as hosts for the production of a maternal- zygotic mutant. The protocol is described herein.

Tereso, Susana Zoglauer, Kurt Milhinhos, Ana Miguel, Célia Oliveira, M Margarida

We compared morphogenesis and accumulation of storage proteins and starch in Pinus pinaster Ait. zygotic embryos with those in somatic embryos grown with different carbohydrate sources. The maturation medium for somatic embryos included 80 microM abscisic acid (ABA), 9 g l(-1) gellam gum and either glucose, sucrose or maltose at 44, 88, 175 or 263 mM in the presence or absence of 6% (w/v) polyethylene glycol (PEG) 4000 MW. Maturation medium containing 44 or 88 mM of a carbohydrate source produced only one or no cotyledonary somatic embryos per 0.6 g fresh mass of culture. The addition of PEG to the basal maturation medium resulted in a low yield of cotyledonary somatic embryos that generally showed incomplete development and anatomical abnormalities such as large intercellular spaces and large vacuoles. High concentrations of maltose also induced large intercellular spaces in the somatic embryonic cells, and 263 mM sucrose produced fewer and less developed cotyledonary somatic embryos compared with 175 mM sucrose, indicating that the effect of carbohydrate source is partially osmotic. Zygotic embryos had a lower dry mass than somatic embryos at the same stage of development. Starch granules followed a similar accumulation pattern in zygotic and somatic embryos. A low starch content was found in cotyledonary zygotic embryos and in somatic embryos developed in the presence of 175 mM maltose or 263 mM glucose. In zygotic embryos and in PEG-treated somatic embryos, protein bodies appeared later and were smaller and fewer than in well-developed somatic embryos grown without PEG. We propose that storage protein concentration might be a marker of embryo quality.

Fan, Yong Li, Rong Huang, Jin Yu, Yang Qiao, Jie

Human embryonic stem cells have shown tremendous potential in regenerative medicine, and the recent progress in haploid embryonic stem cells provides new insights for future applications of embryonic stem cells. Disruption of normal fertilized embryos remains controversial thus, the development of a new source for human embryonic stem cells is important for their usefulness. Here, we investigated the feasibility of haploid and diploid embryo reconstruction and embryonic stem cell derivation using microsurgically repaired tripronuclear human zygotes . Diploid and haploid zygotes were successfully reconstructed, but a large proportion of them still had a tripolar spindle assembly. The reconstructed embryos developed to the blastocyst stage, although the loss of chromosomes was observed in these zygotes . Finally, triploid and diploid human embryonic stem cells were derived from tripronuclear and reconstructed zygotes (from which only one pronucleus was removed), but haploid human embryonic stem cells were not successfully derived from the reconstructed zygotes when two pronuclei were removed. Both triploid and diploid human embryonic stem cells showed the general characteristics of human embryonic stem cells. These results indicate that the lower embryo quality resulting from abnormal spindle assembly contributed to the failure of the haploid embryonic stem cell derivation. However, the successful derivation of diploid embryonic stem cells demonstrated that microsurgical tripronuclear zygotes are an alternative source of human embryonic stem cells. In the future, improving spindle assembly will facilitate the application of triploid zygotes to the field of haploid embryonic stem cells. PMID:23255130

Amarnath, Dasari Wakayama, Sayaka Zhu, Jie Moawad, Adel R Wakayama, Teruhiko Campbell, Keith H S

A high potassium concentration in culture media is considered detrimental to in vitro culture of mouse embryos. Here we show that pig zygotic medium (PZM) containing a higher concentration of potassium, and modified to contain 0.2 mM glucose and 0.01 mM EDTA, supported efficient pre- and post-implantation development of mouse zygotes to blastocysts and live pups, respectively. At first, modified PZM (mPZM) was compared with other culture media such as M16, CZB and KSOM-AA for its ability to support development of in vivo mouse zygotes to the blastocyst stage. The proportions of zygotes reaching 2-cell (94-99%) and blastocyst (90-96%) stages in mPZM and other media were not different. However, hatching rates of blastocysts were different (P zygotes developed to blastocysts in mPZM and KSOM-AA. The proportion of blastocysts developing into live pups was not different between mPZM (49%) and KSOM-AA (44%). Finally, we evaluated whether mPZM could be also used as a fertilization medium. Modified PZM containing 5.56 mM of glucose and 0.4% BSA efficiently supported IVF of mouse gametes. The percent of zygotes cleaving to 2-cell (94-98%) and blastocysts (91-93%) stage was not different from zygotes fertilized in human tubal fluid medium. We concluded that modified pig zygotic medium containing a higher potassium concentration than any other commonly used mouse media supported not only culture of mouse embryos, but also efficient IVF of mouse gametes. Copyright © 2011 Elsevier Inc. All rights reserved.

Yu, Xing-Jiang Yi, Zhaohong Gao, Zheng Qin, Dandan Zhai, Yanhua Chen, Xue Ou-Yang, Yingchun Wang, Zhen-Bo Zheng, Ping Zhu, Min-Sheng Wang, Haibin Sun, Qing-Yuan Dean, Jurrien Li, Lei

Maternal effect genes play critical roles in early embryogenesis of model organisms where they have been intensively investigated. However, their molecular function in mammals remains largely unknown. Recently, we identified a subcortical maternal complex (SCMC) that contains four proteins encoded by maternal effect genes (Mater, Filia, Floped and Tle6). Here we report that TLE6, similar to FLOPED and MATER, stabilizes the SCMC and is necessary for cleavage beyond the two-cell stage of development. We document that the SCMC is required for formation of the cytoplasmic F-actin meshwork that controls the central position of the spindle and ensures symmetric division of mouse zygotes . We further demonstrate that the SCMC controls formation of the actin cytoskeleton specifically via Cofilin, a key regulator of F-actin assembly. Our results provide molecular insight into the physiological function of TLE6, its interaction with the SCMC and their roles in the symmetric division of the zygote in early mouse development.

Charron, Guillaume Landry, Christian R

Although microorganisms account for the largest fraction of Earth's biodiversity, we know little about how their reproductive barriers evolve. Sexual microorganisms such as Saccharomyces yeasts rapidly develop strong intrinsic post- zygotic isolation, but the role of extrinsic isolation in the early speciation process remains to be investigated. We measured the growth of F 1 hybrids between two incipient species of Saccharomyces paradoxus to assess the presence of extrinsic post- zygotic isolation across 32 environments. More than 80% of hybrids showed either partial dominance of the best parent or over-dominance for growth, revealing no fitness defects in F 1 hybrids. Extrinsic reproductive isolation therefore likely plays little role in limiting gene flow between incipient yeast species and is not a requirement for speciation. © 2017 The Author(s).

Kurihara, Daisuke Kimata, Yusuke Higashiyama, Tetsuya Ueda, Minako

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

Remy, Séverine Tesson, Laurent Menoret, Séverine Usal, Claire De Cian, Anne Thepenier, Virginie Thinard, Reynald Baron, Daniel Charpentier, Marine Renaud, Jean-Baptiste Buelow, Roland Cost, Gregory J Giovannangeli, Carine Fraichard, Alexandre Concordet, Jean-Paul Anegon, Ignacio

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes ). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner. © 2014 Remy et al. Published by Cold Spring Harbor Laboratory Press.

Remy, Séverine Tesson, Laurent Menoret, Séverine Usal, Claire De Cian, Anne Thepenier, Virginie Thinard, Reynald Baron, Daniel Charpentier, Marine Renaud, Jean-Baptiste Buelow, Roland Cost, Gregory J. Giovannangeli, Carine Fraichard, Alexandre Concordet, Jean-Paul Anegon, Ignacio

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%–5.13% of microinjected zygotes ). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner. PMID:24989021

Zonies, Seth Motegi, Fumio Hao, Yingsong Seydoux, Geraldine

Polarization of the C. elegans zygote is initiated by ECT-2-dependent cortical flows, which mobilize the anterior PAR proteins (PAR-3, PAR-6 and PKC-3) away from the future posterior end of the embryo marked by the sperm centrosome. Here, we demonstrate the existence of a second, parallel and redundant pathway that can polarize the zygote in the absence of ECT-2-dependent cortical flows. This second pathway depends on the polarity protein PAR-2. We show that PAR-2 localizes to the cortex nearest the sperm centrosome even in the absence of cortical flows. Once on the cortex, PAR-2 antagonizes PAR-3-dependent recruitment of myosin, creating myosin flows that transport the anterior PAR complex away from PAR-2 in a positive-feedback loop. We propose that polarity in the C. elegans zygote is initiated by redundant ECT-2- and PAR-2-dependent mechanisms that lower PAR-3 levels locally, triggering a positive-feedback loop that polarizes the entire cortex.

Niemenak, Nicolas Kaiser, Edward Maximova, Siela N Laremore, Tatiana Guiltinan, Mark J

Two dimensional electrophoresis and nano-LC-MS were performed in order to identify alterations in protein abundance that correlate with maturation of cacao zygotic and somatic embryos. The cacao pod proteome was also characterized during development. The recently published cacao genome sequence was used to create a predicted proteolytic fragment database. Several hundred protein spots were resolved on each tissue analysis, of which 72 variable spots were subjected to MS analysis, resulting in 49 identifications. The identified proteins represent an array of functional categories, including seed storage, stress response, photosynthesis and translation factors. The seed storage protein was strongly accumulated in cacao zygotic embryos compared to their somatic counterpart. However, sucrose treatment (60 g L(-1)) allows up-regulation of storage protein in SE. A high similarity in the profiles of acidic proteins was observed in mature zygotic and somatic embryos. Differential expression in both tissues was observed in proteins having high pI. Several proteins were detected exclusively in fruit tissues, including a chitinase and a 14-3-3 protein. We also identified a novel cacao protein related to known mabinlin type sweet storage proteins. Moreover, the specific presence of thaumatin-like protein, another sweet protein, was also detected in fruit tissue. We discuss our observed correlations between protein expression profiles, developmental stage and stress responses. Copyright © 2015 Elsevier GmbH. All rights reserved.

Liang, Puping Xu, Yanwen Zhang, Xiya Ding, Chenhui Huang, Rui Zhang, Zhen Lv, Jie Xie, Xiaowei Chen, Yuxi Li, Yujing Sun, Ying Bai, Yaofu Songyang, Zhou Ma, Wenbin Zhou, Canquan Huang, Junjiu

Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.

Lete, Marta G Byrne, Richard D Alonso, Alicia Poccia, Dominic Larijani, Banafshé

Regulation of nuclear envelope dynamics is an important example of the universal phenomena of membrane fusion. The signalling molecules involved in nuclear membrane fusion might also be conserved during the formation of both pronuclear and zygote nuclear envelopes in the fertilised egg. Here, we determine that class-I phosphoinositide 3-kinases (PI3Ks) are needed for in vitro nuclear envelope formation. We show that, in vivo, PtdIns(3,4,5)P 3 is transiently located in vesicles around the male pronucleus at the time of nuclear envelope formation, and around male and female pronuclei before membrane fusion. We illustrate that class-I PI3K activity is also necessary for fusion of the female and male pronuclear membranes. We demonstrate, using coincidence amplified Förster resonance energy transfer (FRET) monitored using fluorescence lifetime imaging microscopy (FLIM), a protein-lipid interaction of Rab7 GTPase and PtdIns(3,4,5)P 3 that occurs during pronuclear membrane fusion to create the zygote nuclear envelope. We present a working model, which includes several molecular steps in the pathways controlling fusion of nuclear envelope membranes. © 2017. Published by The Company of Biologists Ltd.

Ansari, Salim Troelenberg, Nicole Dao, Van Anh Richter, Tobias Klingler, Martin

The distinction of anterior versus posterior is a crucial first step in animal embryogenesis. In the fly Drosophila, this axis is established by morphogenetic gradients contributed by the mother that regulate zygotic target genes. This principle has been considered to hold true for insects in general but is fundamentally different from vertebrates, where zygotic genes and Wnt signaling are required. We investigated symmetry breaking in the beetle Tribolium castaneum, which among insects represents the more ancestral short-germ embryogenesis. We found that maternal Tc-germ cell-less is required for anterior localization of maternal Tc-axin, which represses Wnt signaling and promotes expression of anterior zygotic genes. Both RNAi targeting Tc-germ cell-less or double RNAi knocking down the zygotic genes Tc-homeobrain and Tc-zen1 led to the formation of a second growth zone at the anterior, which resulted in double-abdomen phenotypes. Conversely, interfering with two posterior factors, Tc-caudal and Wnt, caused double-anterior phenotypes. These findings reveal that maternal and zygotic mechanisms, including Wnt signaling, are required for establishing embryo polarity and induce the segmentation clock in a short-germ insect. PMID:29432152

Sucar, Sofia Moore, Ginger L. Ard, Melissa E. Ring, Brian C.

The mangrove killifish, Kryptolebias marmoratus, is unique among vertebrates due to its self-fertilizing mode of reproduction involving an ovotestis. As a result, it constitutes a simplistic and desirable vertebrate model for developmental genetics as it is easily maintained, reaches sexual maturity in about 100 days, and provides a manageable number of relatively clear embryos. After the establishment and characterization of an initial mutagenesis pilot screen using N-ethyl-N-nitrosourea, a three-generation genetic screen was performed to confirm zygotic mutant allele heritability and simultaneously score for homozygous recessive mutant sterile F2 fish. From a total of 307 F2 fish screened, 10 were found to be 1° males, 16 were sterile, 92 wild-type, and the remaining 189, carriers of zygotic recessive alleles. These carriers produced 25% progeny exhibiting several zygotic phenotypes similar to those previously described in zebrafish and in the aforementioned pilot screen, as expected. Interestingly, new phenotypes such as golden yolk, no trunk, and short tail were observed. The siblings of sterile F2 mutants were used to produce an F3 generation in order to confirm familial sterility. Out of the 284 F3 fish belonging to 10 previously identified sterile families, 12 were found to be 1° males, 69 were wild-type, 83 sterile, and 120 were classified as */+ (either wild-type or carriers) with undefined genotypes. This screen provides proof of principle that K. marmoratus is a powerful vertebrate model for developmental genetics and can be used to identify mutations affecting fertility. PMID:26801648

Asmarinah Syauqy, Ahmad Umar, Liya Agustin Lestari, Silvia Werdhy Mansyur, Eliza Hestiantoro, Andon Paradowszka-Dogan, Agnieszka

This study aimed to evaluate sperm chromatin maturity and integrity of that injected into good-quality oocytes in an in vitro fertilization-intra cytoplasmic sperm injection (IVF-ICSI) program. A cut-off value of sperm chromatin maturity and integrity was developed as a function of their correlation to the zygote development, i.e., embryo formation and cleavage rate. The study assessed sperm chromatin maturity using aniline blue (AB) staining, whereas toluidine blue (TB) staining was used to assess sperm chromatin integrity. Ejaculates from 59 patients undergoing ICSI and 46 fertile normozoospermic donors for determination of normal values of sperm chromatin status were used in this study. Embryo formation and cleavage rates were observed for the period of 3 days after ICSI. There was a significant difference in the percentage of sperm with mature chromatin between ejaculate from ICSI patients and fertile donor (p=0.020) while there was no significant difference in sperm chromatin integrity of both samples (p=0.120). There was no significant correlation between sperm chromatin maturity and either embryo formation or cleavage rate as well as sperm chromatin integrity to both parameters of zygote development (p>0.05). Furthermore, we found that the cut-off value of sperm chromatin maturity and integrity of the fertile normozoospermic ejaculates were 87.2% and 80.2%, respectively. Using the cut-offs, we found that low sperm chromatin maturity at the level of zygote (p=0.022 r=0.371) whereas poor sperm chromatin integrity at the level of zygote development following ICSI. AB: aniline blue CMA3: chromomycin A3 ICSI: intra cytoplasmic sperm injection IVF: in vitro fertilization PBS: phosphate buffer saline SPSS

Aghaz, Faranak Hajarian, Hadi KaramiShabankareh, Hamed

This study was carried out to investigate the effects of supplementation of potassium simplex optimized medium (KSOM-aa) with various sericin concentrations (0, 0.1, 0.5, 1 and 2.5%) on ovine zygotes . The results indicate that the supplementation of oocyte in vitro culture medium with optimal concentration of sericin (0.1 and 0.5%) may have beneficial effects on developmental competence of in vitro-derived ovine embryos. Copyright © 2015 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Fertilization triggers the assembly of a cell wall around the egg cell of three brown algae, Fucus vesiculosus, F. distichus, and F. inflatus. New polysaccharide polymers are continually being added to the cell wall during the first 24 hours of synchronous embryo development. This wall assembly involves the extracellular deposition of fibrillar material by cytoplasmic vesicles fusing with the plasma membrane. One hour after fertilization a fragmented wall can be isolated free of cytoplasm and contains equal amounts of cellulose and alginic acid with no fucose-containing polymers (fucans) present. Birefringence of the wall caused by oriented cellulose microfibrils is not detected in all zygotes until 4 hours, at which time intact cell walls can be isolated that retain the shape of the zygote . These walls have a relatively low ratio of fucose to xylose and little sulfate when compared to walls from older embryos. When extracts of walls from 4-hour zygotes are subjected to cellulose acetate electrophoresis at pH 7, a single fucan (F(1)) can be detected. By 12 hours, purified cell walls are composed of fucans containing a relatively high ratio of fucose to xylose and high levels of sulfate, and contain a second fucan (F(2)) which is electrophoretically distinct from F(1). F(2) appears to be deposited in only a localized region of the wall, that which elongates to form the rhizoid cell. Throughout wall assembly, the polyuronide block co-polymer alginic acid did not significantly vary its mannuronic (M) to guluronic (G) acid ratio (0.33-0.55) or its block distribution (MG, 54% GG, 30% MM, 16%). From 6 to 24 hours of embryo development, the proportion of the major polysaccharide components found in purified walls is stable. Alginic acid is the major polymer and comprises about 60% of the total wall, while cellulose and the fucans each make-up about 20% of the remainder. During the extracellular assembly of this wall, the intracellular levels of the storage glucan

We demonstrate here the possibility of regenerating phenotypically normal, fertile maize plants via in vitro fertilization of isolated, single sperm and egg cells mediated by electrofusion. The technique leads to the highly efficient formation of polar zygotes , globular structures, proembryos, and transition-phase embryos and to the formation of plants from individually cultured fusion products. Regeneration of plants occurs via embryogenesis and occasionally by polyembryony and organogenesis. Flowering plants can be obtained within 100 days of gamete fusion. Regenerated plants were studied by karyological and morphological analyses, and the segregation of kernel color was determined. The hybrid nature of the plants was confirmed. PMID:12271084

Dilkes, Brian P Dante, Ricardo A Coelho, Cintia Larkins, Brian A

Flow cytometry was used to assess the variability of endoreduplication in endosperms of maize inbred lines. Little variation was found between midwestern dent types, and high levels of endoreduplication were observed in popcorns. Endoreduplication is different between inbred lines by 13-18 days after pollination, and flow cytometric analysis of ploidy level was feasible until 20 DAP. To study the genetic regulation of endoreduplication, four inbreds were crossed to B73 and developing endosperms from both parental, reciprocal F(1), and backcross generations were subjected to flow cytometric analysis. Three measurements of endoreduplication were calculated from these data and analyzed as quantitative genetic traits. Multiple models of trait inheritance were considered including triploid, diploid, sporophytic maternal, and maternal and paternal zygotic nuclear inheritance. Maternal zygotic effects, often considered a form of parental imprinting, and maternal sporophytic effects were detected. To test the feasibility of introgressing a high endoreduplication phenotype into a midwestern dent inbred line, a backcross population was generated from B73 x Sg18. Parental and progeny endoreduplication levels were compared and heritabilities assessed. The heritabilities calculated from these data generally agree with the values calculated in the larger crossing experiments.

Pultz, M A Zimmerman, K K Alto, N M Kaeberlein, M Lange, S K Pitt, J N Reeves, N L Zehrung, D L

We have screened for zygotic embryonic lethal mutations affecting cuticular morphology in Nasonia vitripennis (Hymenoptera Chalcidoidea). Our broad goal was to investigate the use of Nasonia for genetically surveying conservation and change in regulatory gene systems, as a means to understand the diversity of developmental strategies that have arisen during the course of evolution. Specifically, we aim to compare anteroposterior patterning gene functions in two long germ band insects, Nasonia and Drosophila. In Nasonia, unfertilized eggs develop as haploid males while fertilized eggs develop as diploid females, so the entire genome can be screened for recessive zygotic mutations by examining the progeny of F1 females. We describe 74 of >100 lines with embryonic cuticular mutant phenotypes, including representatives of coordinate, gap, pair-rule, segment polarity, homeotic, and Polycomb group functions, as well as mutants with novel phenotypes not directly comparable to those of known Drosophila genes. We conclude that Nasonia is a tractable experimental organism for comparative developmental genetic study. The mutants isolated here have begun to outline the extent of conservation and change in the genetic programs controlling embryonic patterning in Nasonia and Drosophila. PMID:10866651

Redel, Bethany K Beaton, Benjamin P Spate, Lee D Benne, Joshua A Murphy, Stephanie L O'Gorman, Chad W Spate, Anna M Prather, Randall S Wells, Kevin D

Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. A sequence from the mMalat1 gene was cloned downstream of the IRES/Cas9 cassette described above. An mRNA concentration curve was constructed with either commercially available Cas9 mRNA or the IRES/ Cas9/triplex, by injection into porcine zygotes . Blastocysts were genotyped to determine if differences existed in the percent of embryos modified. The concentration curve identified differences due to concentration and RNA type injected. Single step production of Cas9 mRNA provides an alternative source of Cas9 for use in zygote injections.

de Vega-Bartol, José J Simões, Marta Lorenz, W Walter Rodrigues, Andreia S Alba, Rob Dean, Jeffrey F D Miguel, Célia M

It is during embryogenesis that the plant body plan is established and the meristems responsible for all post-embryonic growth are specified. The molecular mechanisms governing conifer embryogenesis are still largely unknown. Their elucidation may contribute valuable information to clarify if the distinct features of embryo development in angiosperms and gymnosperms result from differential gene regulation. To address this issue, we have performed the first transcriptomic analysis of zygotic embryo development in a conifer species (Pinus pinaster) focusing our study in particular on regulatory genes playing important roles during plant embryo development, namely epigenetic regulators and transcription factors. Microarray analysis of P. pinaster zygotic embryogenesis was performed at five periods of embryo development from early developing to mature embryos. Our results show that most changes in transcript levels occurred in the first and the last embryo stage-to-stage transitions, namely early to pre-cotyledonary embryo and cotyledonary to mature embryo. An analysis of functional categories for genes that were differentially expressed through embryogenesis highlighted several epigenetic regulation mechanisms. While putative orthologs of transcripts associated with mechanisms that target transposable elements and repetitive sequences were strongly expressed in early embryogenesis, PRC2-mediated repression of genes seemed more relevant during late embryogenesis. On the other hand, functions related to sRNA pathways appeared differentially regulated across all stages of embryo development with a prevalence of miRNA functions in mid to late embryogenesis. Identification of putative transcription factor genes differentially regulated between consecutive embryo stages was strongly suggestive of the relevance of auxin responses and regulation of auxin carriers during early embryogenesis. Such responses could be involved in establishing embryo patterning. Later in

Background It is during embryogenesis that the plant body plan is established and the meristems responsible for all post-embryonic growth are specified. The molecular mechanisms governing conifer embryogenesis are still largely unknown. Their elucidation may contribute valuable information to clarify if the distinct features of embryo development in angiosperms and gymnosperms result from differential gene regulation. To address this issue, we have performed the first transcriptomic analysis of zygotic embryo development in a conifer species (Pinus pinaster) focusing our study in particular on regulatory genes playing important roles during plant embryo development, namely epigenetic regulators and transcription factors. Results Microarray analysis of P. pinaster zygotic embryogenesis was performed at five periods of embryo development from early developing to mature embryos. Our results show that most changes in transcript levels occurred in the first and the last embryo stage-to-stage transitions, namely early to pre-cotyledonary embryo and cotyledonary to mature embryo. An analysis of functional categories for genes that were differentially expressed through embryogenesis highlighted several epigenetic regulation mechanisms. While putative orthologs of transcripts associated with mechanisms that target transposable elements and repetitive sequences were strongly expressed in early embryogenesis, PRC2-mediated repression of genes seemed more relevant during late embryogenesis. On the other hand, functions related to sRNA pathways appeared differentially regulated across all stages of embryo development with a prevalence of miRNA functions in mid to late embryogenesis. Identification of putative transcription factor genes differentially regulated between consecutive embryo stages was strongly suggestive of the relevance of auxin responses and regulation of auxin carriers during early embryogenesis. Such responses could be involved in establishing embryo patterning

Jin, Bo Seki, Shinsuke Paredes, Estefania Qiu, Juan Shi, Yanbin Zhang, Zhenqiang Ma, Chao Jiang, Shuyan Li, Jiaqi Yuan, Feng Wang, Shu Shao, Xiaoguang Mazur, Peter

In this study, mature female mice of the ICR strain were induced to superovultate, mated, and collected at either zygote or early morula stages. Embryos suspended in 1 M ethylene glycol in PBS containing 10 mg/L Snomax for 15 min, then transferred in sample holder to Linkam cryostage, cooled to and seeded at 7 °C, and then observed and photographed while being cooled to -70 °C at 0.5-20 °C/min. Intracellular ice formation (IIF) was observed as abrupt ''flashing''. Two types of flashing or IIF were observed in this study. Extracellular freezing occurred at a mean of -7.7 °C. In morulae, about 25% turned dark within ±1 °C of extracellular ice formation (EIF). These we refer to as "high temperature'' flashers. In zygotes , there were no high temperature flashers. All the zygotes flashed at temperatures well below the temperature for EIF. Presumably high temperature flashers were a consequence of membrane damage prior to EIF or damage from EIF. We shall not discuss them further. In the majority of cases, IIF occurred well below -7.7 °C these we call ''low temperature'' flashers. None flashed with cooling rate (CR) of 0.5 °C/min in either zygotes or morulae. Nearly all flashed with CR of 4 °C/min or higher, but the distribution of temperatures is much broader with morulae than with zygotes . Also, the mean flashing temperature is much higher with morulae (-20.9 °C) than with zygotes (-40.3 °C). We computed the kinetics of water loss with respect to CR and temperature in both mouse zygotes and in morulae based on published estimates of Lp and it is Ea. The resulting dehydration curves combined with knowledge of the embryo nucleation temperature permits an estimate of the likelihood of IIF as a function of CR and subzero temperature. The agreement between these computed probabilities and the observed values are good. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Sershen Varghese, B Naidoo, C Pammenter, N W

Zygotic embryos from recalcitrant seeds are sensitive to desiccation. In spite of their sensitivity, rapid partial dehydration is necessary for their successful cryopreservation. However, dehydration to water contents (WCs) that preclude lethal ice crystal formation during cooling and rewarming generally leads to desiccation damage. This study investigated the effects of rapid dehydration on selected stress biomarkers (electrolyte leakage, respiratory competence, rate of protein synthesis, superoxide production, lipid peroxidation, antioxidant activity and degree of cellular vacuolation) in zygotic embryos of four recalcitrant-seeded species. Most biomarkers indicated differences in the levels of stress/damage incurred by embryos dried to WCs 0.4 g·g(-1) , within species however, these changes were often unrelated to viability and percentage water loss when data for the four species were pooled for regression analyses. Dehydration-induced electrolyte leakage was, however, positively related with percentage water loss, while biomarkers of cellular vacuolation were positively related with both percentage water loss and viability. This suggests that electrolyte leakage and degree of cellular vacuolation can be used to quantify dehydration-induced stress/damage. Biomarkers such as superoxide production, whilst useful in establishing the nature of the dehydration stress incurred may not be able to distinguish the effects of different WCs/drying times. Irrespective of which biomarker is used, the data suggest that understanding differences in desiccation sensitivity across recalcitrant-seeded species will remain a challenge unless these biomarkers are related to a generic desiccation stress index that integrates the effects of percentage water loss and drying time. © 2016 German Botanical Society and The Royal Botanical Society of the Netherlands.

Chaigne, Agathe Campillo, Clément Voituriez, Raphaël Gov, Nir S. Sykes, Cécile Verlhac, Marie-Hélène Terret, Marie-Emilie

Mitotic spindle position relies on interactions between astral microtubules nucleated by centrosomes and a rigid cortex. Some cells, such as mouse oocytes, do not possess centrosomes and astral microtubules. These cells rely only on actin and on a soft cortex to position their spindle off-centre and undergo asymmetric divisions. While the first mouse embryonic division also occurs in the absence of centrosomes, it is symmetric and not much is known on how the spindle is positioned at the exact cell centre. Using interdisciplinary approaches, we demonstrate that zygotic spindle positioning follows a three-step process: (1) coarse centring of pronuclei relying on the dynamics of an F-actin/Myosin-Vb meshwork (2) fine centring of the metaphase plate depending on a high cortical tension (3) passive maintenance at the cell centre. Altogether, we show that F-actin-dependent mechanics operate the switch between asymmetric to symmetric division required at the oocyte to embryo transition.

Friberg, Urban Rice, William R.

Sisters and brothers are completely unrelated with respect to the sex chromosomes they inherit from their heterogametic parent. This has the potential to result in a previously unappreciated form of genetic conflict between the sex chromosomes, called sexually antagonistic zygotic drive (SA-ZD). SA-ZD can arise whenever brothers and sisters compete over limited resources or there is brother–sister mating coupled with inbreeding depression. Although theory predicts that SA-ZD should be common and influence important evolutionary processes, there is little empirical evidence for its existence. Here we discuss the current understanding of SA-ZD, why it would be expected to elude empirical detection when present, and how it relates to other forms of genetic conflict. PMID:25573714

Aida, Tomomi Nakade, Shota Sakuma, Tetsushi Izu, Yayoi Oishi, Ayu Mochida, Keiji Ishikubo, Harumi Usami, Takako Aizawa, Hidenori Yamamoto, Takashi Tanaka, Kohichi

Although CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by microhomology-mediated end-joining. Here, we identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells. By combining the Exo1 and PITCh-directed donor vectors, we achieved convenient one-step knock-in of gene cassettes and floxed allele both in human cells and mouse zygotes . Our results provide a technical platform for high-throughput knock-in.

Ballesteros, Daniel Sershen Varghese, Boby Berjak, Patricia Pammenter, Norman W

Cryopreservation is the most promising option for the long-term germplasm conservation of recalcitrant-seeded species. However, the variable post-cryo success achieved with the excised zygotic explants traditionally used for cryopreservation has been a concern for some time. Differential drying rates amongst explants of different species, uneven drying amongst explants within a batch of seeds and uneven drying across tissues within individual embryos could be contributory factors to this variable success and these phenomena form the foci of the present study. Using zygotic explants from a range of recalcitrant-seeded species, which included sub-tropical dicotyledonous trees and sub-tropical monocotyledonous geophytes, the study showed that embryo morphology and anatomy are critical determinants of the drying characteristics of the different tissues composing the explant and hence, post-cryo survival. The results suggest that the rates of drying of explants to water contents (WCs) in the theoretically optimal range for successful cryopreservation are species-specific, and that more rapid drying rates may promote post-cryo survival. However, the large variation in WC amongst individual explants in bulk samples challenges the selection of the theoretically optimum WC for cryopreservation. As a consequence of differential drying rates across the different tissues composing explants, either lethal ice crystal damage or desiccation damage may sometimes be likely in tissues responsible for the onwards development of the embryo. Drying times for cryopreservation of such explants should, therefore, be selected on the basis of WC of segments containing root or shoot meristem, rather than embryo bulk WC. Drying intensity and duration also interact with explant morphology and embryo/axis size and anatomy to bring about - or preclude - post-cryo survival. Copyright © 2014 Elsevier Inc. All rights reserved.

Grenier, Lisanne Robaire, Bernard Hales, Barbara F.

Paternal exposures to cancer chemotherapeutics or environmental chemicals may have adverse effects on progeny outcome that are manifested in the preimplantation embryo. The objectives of this study were to determine the impact of paternal exposure to cyclophosphamide, an anticancer alkylating agent, on the formation, chromatin origin and function of micronuclei in cleavage stage rat embryos. Male Sprague-Dawley rats were gavaged with saline or cyclophosphamide (6 mg/kg/day) for 4 weeks and mated to naturally cycling females to collect pronuclear zygotes and 2 to 8 cell embryos. Micronuclear chromatin structure was characterized using confocal microscopy to detect immunoreactivities for H3K9me3, a marker for maternal chromatin, and lamin B, a nuclear membrane marker. DNA synthesis was monitored using EdU (5-ethynyl-2′-deoxyuridine) incorporation. Fertilization by cyclophosphamide-exposed spermatozoa led to a dramatic elevation in micronuclei in cleavage stage embryos (control embryos: 1% to 5% embryos sired by treated males: 70%). The formation of micronuclei occurred during the first zygotic division and was associated with a subsequent developmental delay. The absence of H3K9me3 indicated that these micronuclei were of paternal origin. The micronuclei had incomplete peri-nuclear and peri-nucleolar lamin B1 membrane formation but incorporated EdU into DNA to the same extent as the main nucleus. The formation of micronuclei in response to the presence of a damaged paternal genome may play a role in increasing the rate of embryo loss that is associated with the use of assisted reproductive technologies, parenthood among cancer survivors, and paternal aging. PMID:22110683

Wong, Ming-Kin Guan, Daogang Ng, Kaoru Hon Chun Ho, Vincy Wing Sze An, Xiaomeng Li, Runsheng Ren, Xiaoliang

Metazoan development demands not only precise cell fate differentiation but also accurate timing of cell division to ensure proper development. How cell divisions are temporally coordinated during development is poorly understood. Caenorhabditis elegans embryogenesis provides an excellent opportunity to study this coordination due to its invariant development and widespread division asynchronies. One of the most pronounced asynchronies is a significant delay of cell division in two endoderm progenitor cells, Ea and Ep, hereafter referred to as E2, relative to its cousins that mainly develop into mesoderm organs and tissues. To unravel the genetic control over the endoderm-specific E2 division timing, a total of 822 essential and conserved genes were knocked down using RNAi followed by quantification of cell cycle lengths using in toto imaging of C. elegans embryogenesis and automated lineage. Intriguingly, knockdown of numerous genes encoding the components of general transcription pathway or its regulatory factors leads to a significant reduction in the E2 cell cycle length but an increase in cell cycle length of the remaining cells, indicating a differential requirement of transcription for division timing between the two. Analysis of lineage-specific RNA-seq data demonstrates an earlier onset of transcription in endoderm than in other germ layers, the timing of which coincides with the birth of E2, supporting the notion that the endoderm-specific delay in E2 division timing demands robust zygotic transcription. The reduction in E2 cell cycle length is frequently associated with cell migration defect and gastrulation failure. The results suggest that a tissue-specific transcriptional activation is required to coordinate fate differentiation, division timing, and cell migration to ensure proper development. PMID:27056332

Monteiro, Joana Aires, Rita Becker, Jörg D Jacinto, António Certal, Ana C Rodríguez-León, Joaquín

The activity of ion channels and transporters generates ion-specific fluxes that encode electrical and/or chemical signals with biological significance. Even though it is long known that some of those signals are crucial for regeneration, only in recent years the corresponding molecular sources started to be identified using mainly invertebrate or larval vertebrate models. We used adult zebrafish caudal fin as a model to investigate which and how ion transporters affect regeneration in an adult vertebrate model. Through the combined use of biophysical and molecular approaches, we show that V-ATPase activity contributes to a regeneration-specific H+ ef`flux. The onset and intensity of both V-ATPase expression and H+ efflux correlate with the different regeneration rate along the proximal-distal axis. Moreover, we show that V-ATPase inhibition impairs regeneration in adult vertebrate. Notably, the activity of this H+ pump is necessary for aldh 1 a 2 and mkp3 expression, blastema cell proliferation and fin innervation. To the best of our knowledge, this is the first report on the role of V-ATPase during adult vertebrate regeneration.

Monteiro, Joana Aires, Rita Becker, Jörg D. Jacinto, António Certal, Ana C. Rodríguez-León, Joaquín

The activity of ion channels and transporters generates ion-specific fluxes that encode electrical and/or chemical signals with biological significance. Even though it is long known that some of those signals are crucial for regeneration, only in recent years the corresponding molecular sources started to be identified using mainly invertebrate or larval vertebrate models. We used adult zebrafish caudal fin as a model to investigate which and how ion transporters affect regeneration in an adult vertebrate model. Through the combined use of biophysical and molecular approaches, we show that V-ATPase activity contributes to a regeneration-specific H+ ef`flux. The onset and intensity of both V-ATPase expression and H+ efflux correlate with the different regeneration rate along the proximal-distal axis. Moreover, we show that V-ATPase inhibition impairs regeneration in adult vertebrate. Notably, the activity of this H+ pump is necessary for aldh 1 a 2 and mkp3 expression, blastema cell proliferation and fin innervation. To the best of our knowledge, this is the first report on the role of V-ATPase during adult vertebrate regeneration. PMID:24671205

The development of the rhizoid cells of the green alga Ulva mutabilis was investigated at the ultrastructural level paying special attention to the mechanism of attachment of the plant. Cytochemical data concerning the initial settling of the early zygote are also given. On the basis of histochemical staining and enzyme treatment it is concluded that the adhesive material secreted by the rhizoid cells is chemically different from that secreted by the zygote during the initial settling of the alga.

Katoh, M Cacheiro, N L Cornett, C V Cain, K T Rutledge, J C Generoso, W M

Earlier studies in this laboratory revealed that ethylene oxide (EtO) or ethyl methanesulfonate (EMS) induced high frequencies of midgestation and late fetal deaths, and of malformations among some of the surviving fetuses, when female mice were exposed at the time of fertilization of their eggs or during the early pronuclear stage of the zygote . Effects of the two mutagens are virtually identical. Thus, in investigating the mechanisms responsible for the dramatic effects in the early pronuclear zygotes , the two compounds were used interchangeably in the experiments. First, a reciprocal zygote -transfer study was conducted in order to determine whether the effect is directly on the zygotes or indirectly through maternal toxicity. And second, cytogenetic analyses of pronuclear metaphases, early cleavage embryos, and midgestation fetuses were carried out. The zygote transplantation experiment rules out maternal toxicity as a factor in the fetal maldevelopment. Together with the strict stage specificity observed in the earlier studies, this result points to a genetic cause for the abnormalities. However, the cytogenetic studies failed to show structural or numerical chromosome aberrations. Since intragenic base changes and deletions may also be ruled out, it appears that the lesions in question induced in zygotes by the two mutagens are different from conventional ones and, therefore, could be a novel one in experimental mammalian mutagenesis. Alternatively, the mechanism could involve a non-mutational 'imprinting' process that caused changes in gene expression.

Bevacqua, R. J. Fernandez-Martin, R. Canel, N. G. Gibbons, A. Texeira, D. Lange, F. Vans Landschoot, G. Savy, V. Briski, O. Hiriart, M. I. Grueso, E. Ivics, Z. Taboga, O. Kues, W. A. Ferraris, S.

Transgenic domestic animals represent an alternative to bioreactors for large-scale production of biopharmaceuticals and could also provide more accurate biomedical models than rodents. However, their generation remains inefficient. Recently, DNA transposons allowed improved transgenesis efficiencies in mice and pigs. In this work, Tn5 and Sleeping Beauty (SB) transposon systems were evaluated for transgenesis by simple cytoplasmic injection in livestock zygotes . In the case of Tn5, the transposome complex of transposon nucleic acid and Tn5 protein was injected. In the case of SB, the supercoiled plasmids encoding a transposon and the SB transposase were co-injected. In vitro produced bovine zygotes were used to establish the cytoplasmic injection conditions. The in vitro cultured blastocysts were evaluated for reporter gene expression and genotyped. Subsequently, both transposon systems were injected in seasonally available ovine zygotes , employing transposons carrying the recombinant human factor IX driven by the beta-lactoglobulin promoter. The Tn5 approach did not result in transgenic lambs. In contrast, the Sleeping Beauty injection resulted in 2 lambs (29%) carrying the transgene. Both animals exhibited cellular mosaicism of the transgene. The extraembryonic tissues (placenta or umbilical cord) of three additional animals were also transgenic. These results show that transpositional transgenesis by cytoplasmic injection of SB transposon components can be applied for the production of transgenic lambs of pharmaceutical interest. PMID:28301581

Chu, Van Trung Weber, Timm Graf, Robin Sommermann, Thomas Petsch, Kerstin Sack, Ulrike Volchkov, Pavel Rajewsky, Klaus Kühn, Ralf

The CRISPR/Cas9 system is increasingly used for gene inactivation in mouse zygotes , but homology-directed mutagenesis and use of inbred embryos are less established. In particular, Rosa26 knock-in alleles for the insertion of transgenes in a genomic 'safe harbor' site, have not been produced. Here we applied CRISPR/Cas9 for the knock-in of 8-11 kb inserts into Rosa26 of C57BL/6 zygotes . We found that 10-20 % of live pups derived from microinjected zygotes were founder mutants, without apparent off-target effects, and up to 50 % knock-in embryos were recovered upon coinjection of Cas9 mRNA and protein. Using this approach, we established a new mouse line for the Cre/loxP-dependent expression of Cas9. Altogether, our protocols and resources support the fast and direct generation of new Rosa26 knock-in alleles and of Cas9-mediated in vivo gene editing in the widely used C57BL/6 inbred strain.

Del Carmen Rodríguez-Gacio, María Nicolás, Carlos Matilla, Angel Jesús

In a previous report from the present authors, it was shown that the 1-aminocyclopropane-1-carboxylate (ACC) oxidation may play a crucial role during zygotic embryogenesis of turnip tops seeds. The present study was performed to elucidate the contribution of the silique-wall and seeds in ethylene production during this developmental process. ACC content in the silique wall is only higher than in seeds during the middle phases of zygotic embryogenesis. The ACC-oxidase (ACO) activity peaks in the silique-wall and seeds during the onset of embryogenesis, declining gradually afterwards, being undetectable during desiccation period. Using reverse transcriptase-polymerase chain reaction, one cDNA clone coding for an ACO and called BrACO1, was isolated. The deduced protein for BrACO1 has a molecular weight of 36.8 kDa and a high homology with other crucifer ACOs. The heterologous expression of this cDNA confirmed that BrACO1 is an ACO. The expression of this gene was high during the first phases of silique-wall development, low during the middle phases and undetectable during desiccation. By contrast, BrACO1 transcript was accumulated only in the earliest phases of seed embryogenesis and may participate in the highest ACO activity and ethylene production by seeds at the beginning of embryogenesis. Finally, in this work a correlation between the heterogeneity of Brassica rapa L. cv. Rapa seeds and the ability to oxidize the ACC to ethylene has been demonstrated.

Mateo, Silvia Vidal, Francesca Coll, Lluc Veiga, Anna Boada, Montserrat

This study aims to increase the knowledge about monopronucleated ICSI-derived blastocysts, analyzing trophectoderm biopsies by aCGH and FISH to evaluate their chromosome constitution. Fifteen monopronucleated ICSI-derived blastocysts were studied. Double trophectoderm biopsy was performed and analyzed by FISH and aCGH. The blastocysts were classified according to chromosome constitution. Disagreements between the two techniques were assessed. Results obtained after FISH and aCGH analyses showed the following: 20% (3/15) and 60% (9/15) diploid females, respectively 26.7% (4/15) and 26.7% (4/15) diploid males, respectively and 53.3% (8/15) and 13.3% (2/15) mosaics, respectively. No mosaic male embryos were found using FISH or aCGH. There were disagreements in 40% (6/15) of the cases due to the higher detection of mosaicism by FISH compared to aCGH. The combination of FISH and aCGH has been shown to be a suitable approach to increase the knowledge about monopronucleated ICSI-derived embryos. FISH analysis of blastocysts derived from monopronucleated ICSI zygotes enabled us to conclude that aCGH underestimates haploidy. Some diploid embryos diagnosed by aCGH are in fact mosaic. In cases where these embryos would be used for reproductive purposes, extra analysis of parental genome origin is recommended.

Zhang, John Zhuang, Guanglun Zeng, Yong Grifo, Jamie Acosta, Carlo Shu, Yimin Liu, Hui

Nuclear transfer of an oocyte into the cytoplasm of another enucleated oocyte has shown that embryogenesis and implantation are influenced by cytoplasmic factors. We report a case of a 30-year-old nulligravida woman who had two failed IVF cycles characterized by all her embryos arresting at the two-cell stage and ultimately had pronuclear transfer using donor oocytes. After her third IVF cycle, eight out of 12 patient oocytes and 12 out of 15 donor oocytes were fertilized. The patient's pronuclei were transferred subzonally into an enucleated donor cytoplasm resulting in seven reconstructed zygotes . Five viable reconstructed embryos were transferred into the patient's uterus resulting in a triplet pregnancy with fetal heartbeats, normal karyotypes and nuclear genetic fingerprinting matching the mother's genetic fingerprinting. Fetal mitochondrial DNA profiles were identical to those from donor cytoplasm with no detection of patient's mitochondrial DNA. This report suggests that a potentially viable pregnancy with normal karyotype can be achieved through pronuclear transfer. Ongoing work to establish the efficacy and safety of pronuclear transfer will result in its use as an aid for human reproduction. Copyright © 2016 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Motegi, Fumio Zonies, Seth Hao, Yingsong Cuenca, Adrian A. Griffin, Erik Seydoux, Geraldine

A hallmark of polarized cells is the segregation of the PAR polarity regulators into asymmetric domains at the cell cortex1, 2. Antagonistic interactions involving two conserved kinases, atypical protein kinase C (aPKC) and PAR-1, have been implicated in polarity maintenance1, 2, but the mechanisms that initiate the formation of asymmetric PAR domains are not understood. Here, we describe one pathway used by the sperm-donated centrosome to polarize the PAR proteins in Caenorhabditis elegans zygotes . Before polarization, cortical aPKC excludes PAR-1 kinase and its binding partner PAR-2 by phosphorylation. During symmetry breaking, microtubules nucleated by the centrosome locally protect PAR-2 from phosphorylation by aPKC, allowing PAR-2 and PAR-1 to access the cortex nearest the centrosome. Cortical PAR-1 phosphorylates PAR-3, causing the PAR-3/aPKC complex to leave the cortex. Our findings illustrate how microtubules, independent of actin dynamics, stimulate the self-organization of PAR proteins by providing local protection against a global barrier imposed by aPKC. PMID:21983565

Cook, Jonathan M Charlesworth, Amanda

Developmentally important proteins that are crucial for fertilization and embryogenesis are synthesized through highly regulated translation of maternal mRNA. The Zygote arrest proteins, Zar1 and Zar2, are crucial for embryogenesis and have been implicated in binding mRNA and repressing mRNA translation. To investigate Zar1 and Zar2, the full-length proteins had been fused to glutathione-S-transferase (GST) or MS2 protein tags with minimal inter-domain linkers derived from multiple cloning sites however, these fusion proteins expressed poorly and/or lacked robust function. Here, we tested the effect of inserting additional linkers between the fusion domains. Three linkers were tested, each 17 amino acids long with different physical and chemical properties: flexible hydrophilic, rigid extended or rigid helical. In the presence of any of the three linkers, GST-Zar1 and GST-Zar2 had fewer breakdown products. Moreover, in the presence of any of the linkers, MS2-Zar1 was expressed to higher levels, and in dual luciferase tethered assays, both MS2-Zar1 and MS2-Zar2 repressed luciferase translation to a greater extent. These data suggest that for Zar fusion proteins, increasing the length of linkers, regardless of their physical or chemical properties, improves stability, expression and bioactivity. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]

Gassler, Johanna Flyamer, Ilya M Tachibana, Kikuë

The 3D folding of the genome is linked to essential nuclear processes including gene expression, DNA repair, and replication. Chromatin conformation capture assays such as Hi-C are providing unprecedented insights into higher-order chromatin structure. Bulk Hi-C of millions of cells enables detection of average chromatin features at high resolution but is challenging to apply to rare cell types. This chapter describes our recently developed single-nucleus Hi-C (snHi-C) approach for detection of chromatin contacts in single nuclei of murine oocytes and one-cell embryos ( zygotes ). The step-by-step protocol includes isolation of these cells, extraction of nuclei, fixation, restriction digestion, ligation, and whole genome amplification. Contacts obtained by snHi-C allow detection of chromatin features including loops, topologically associating domains, and compartments when averaged over the genome. The combination of snHi-C with other single-cell techniques in these and other rare cell types will likely provide a comprehensive picture of how chromatin architecture shapes cell identity. © 2018 Elsevier Inc. All rights reserved.

Nomali, Z Ngobese Sershen Berjak, P Pammenter, N W

Cryopreservation, the most promising method for the long-term conservation of recalcitrant (desiccation-sensitive) seed germplasm, is often associated with high viability losses. Cryo-procedures involve a sequence of steps which must be optimised to reduce the impact of the stresses. This study reports on the effects of some of the steps of cryopreservation on the recalcitrant zygotic embryos of the amaryllid, Ammocharis coranica. Embryos were subjected to cryoprotection with glycerol and/or DMSO, rapid (flash) drying, and rapid (>100 degree C s(-1)) or slow (1 degree C s(-1)) cooling. Rapid dehydration (from c. 2.7 to 0.9 g g(-1) over 60 min) and cooling had a detrimental effect on the viability of the embryos, which was exacerbated when these steps were applied sequentially. After cooling, seedling production (30%) was obtained only from embryos that had been cryoprotected with glycerol prior to drying and rapid cooling, while 30% of non-treated embryos and 70% of those that had undergone cathodic protection during flash drying produced callus. Noting that no post-cryo survival of A. coranica embryos had previously been obtained, this study identified cryoprotection with glycerol and the incorporation of cathodic protection during flash drying as promising intervention points for future studies.

Rutledge, J C Generoso, W M

Exposure of female mice to ethylene oxide by inhalation 1 or 6 h after mating produced not only multitemporal death of conceptuses but also high rates of abnormalities among surviving fetuses. In contrast, only marginal effects were observed when females were exposed 9 or 25 h after mating. The abnormalities found among 17 day gestation live fetuses were predominated by hydrops and eye defects, which, together, constitute 54% of all anomalies. Most of the remaining anomalies were distributed among 5 other types: small size, cleft palate, and cardiac, abdominal wall, or extremity and/or tail defects. In a follow-up study, the fetuses of females treated 6 h postmating were examined at 11-15 days gestation and the progression of fetal death and of malformations was studied. Results indicate that the expression of most fetal anomalies does not become apparent until late in gestation. Several of these induced anomalies are similar to common human sporadic birth defects. This new class of experimentally induced fetal anomalies provides a new avenue for investigating zygotic biology and a system for studying the progression of aberrant development.

Wang, Bangmei Li, Kunyu Wang, Amy Reiser, Michelle Saunders, Thom Lockey, Richard F Wang, Jia-Wang

The clustered regularly interspaced short palindromic repeat (CRISPR) gene editing technique, based on the non-homologous end-joining (NHEJ) repair pathway, has been used to generate gene knock-outs with variable sizes of small insertion/deletions with high efficiency. More precise genome editing, either the insertion or deletion of a desired fragment, can be done by combining the homology-directed-repair (HDR) pathway with CRISPR cleavage. However, HDR-mediated gene knock-in experiments are typically inefficient, and there have been no reports of successful gene knock-in with DNA fragments larger than 4 kb. Here, we describe the targeted insertion of large DNA fragments (7.4 and 5.8 kb) into the genomes of mouse embryonic stem (ES) cells and zygotes , respectively, using the CRISPR/HDR technique without NHEJ inhibitors. Our data show that CRISPR/HDR without NHEJ inhibitors can result in highly efficient gene knock-in, equivalent to CRISPR/HDR with NHEJ inhibitors. Although NHEJ is the dominant repair pathway associated with CRISPR-mediated double-strand breaks (DSBs), and biallelic gene knock-ins are common, NHEJ and biallelic gene knock-ins were not detected. Our results demonstrate that efficient targeted insertion of large DNA fragments without NHEJ inhibitors is possible, a result that should stimulate interest in understanding the mechanisms of high efficiency CRISPR targeting in general.

Carroll, Michael Levasseur, Mark Wood, Chris Whitaker, Michael Jones, Keith T McDougall, Alex

In ascidians, as in mammals, sperm trigger repetitive Ca2+-waves that originate from cortical pacemakers situated in the vegetal hemisphere of the zygotes . In ascidians, a vegetal protrusion termed the contraction pole (CP) acts as the Ca2+-wave pacemaker, but the mechanism that underlies the generation of a Ca2+-wave pacemaker is not known. Here, we tested four hypotheses to determine which factors at the CP are involved in setting the pace of the ascidian Ca2+-wave pacemaker: (1) localized Ca2+ influx (2) accumulation of phosphatidylinositol (4,5)bisphosphate [PtdIns(4,5)P2] (3) accumulation of cortical endoplasmic reticulum (cER) and (4) enrichment of the sperm activating factor. We developed a method of dynamically monitoring the location of the CP during fertilization using a plekstrin homology (PH) domain from phospholipase Cdelta1 coupled to green fluorescent protein (GFP) that binds PtdIns(4,5)P2. We found that eggs in Ca2+-free sea water displayed Ca2+ waves that originated from the CP, showing that enhanced CP Ca2+ influx does not determine the origin of the pacemaker. Also, disruption of the PH::GFP-labelled CP once it had formed did not dislodge the Ca2+-wave pacemaker from that site. Next, when we prevented the accumulation of cER at the CP, all of the Ca2+ waves came from the site of sperm-egg fusion and the frequency of Ca2+ oscillations was unaltered. These data show that local Ca2+ influx, the accumulation of PtdIns(4,5)P2 and cER at the CP are not required for Ca2+-wave pacemaker function and instead suggest that a factor associated with the sperm determines the site of the Ca2+-wave pacemaker. Finally, when we injected ascidian sperm extract into the centre of unfertilized ascidian eggs that had been treated with microfilament- and microtubule-disrupting drugs, all the Ca2+ waves still originated from near the plasma membrane, showing that the sperm factor does not require an intact cortex if it is enriched near the plasma membrane (PM). We

Bonetti, Keila A P Quoirin, Marguerite Quisen, Regina C Lima, Suelen C S

The interspecific oil palm hybrid BRS Manicoré (E. guineensis x E. oleifera) has superior agronomic characteristics. However, the germination rate is low (30%) and the process is slow when the seeds are sown in a conventional form. The purpose of this study was to optimize the in vitro germination of zygotic embryos of this hybrid comparing seed lots. The viability of zygotic embryos was evaluated by the tetrazolium test (0.075%) for 4 h. The embryos were cultured on MS and Y3 culture media, with and without the addition of NaH2PO4, as well as on MS, MS1/2 and N6 medium. In MS medium containing NaH2PO4, the germination rate was increased from 40 to 70% in comparison with the medium without sodium phosphate. The comparison between the culture media MS, MS 1/2, N6 and Y3 showed that 75% of zygotic embryos cultured in the Y3 medium formed whole plants (with roots and shoots defined), a higher percentage than embryos cultured on MS, MS 1/2 and N6 media (46, 35 and 17% respectively). In the same Y3 culture medium, the embryos were larger (36% ≥ 2 cm and 30% ≥ 5 cm) than in the other media. Results obtained by the tetrazolium test were similar to those of germination, showing the effect of the genotype of each seed lot. For the germination and development of plantlets it is essential to add NaH2PO4 to a culture medium containing no phosphate or with a low phosphate concentration.

Tenenhaus, Christina Subramaniam, Kuppuswamy Dunn, Melanie A. Seydoux, Geraldine

The CCCH zinc finger protein PIE-1 is an essential regulator of germ cell fate that segregates with the germ lineage during the first cleavages of the Caenorhabditis elegans embryo. We have shown previously that one function of PIE-1 is to inhibit mRNA transcription. Here we show that PIE-1 has a second function in germ cells it is required for efficient expression of the maternally encoded Nanos homolog NOS-2. This second function is genetically separable from PIE-1's inhibitory effect on transcription. A mutation in PIE-1's second CCCH finger reduces NOS-2 expression without affecting transcriptional repression and causes primordial germ cells to stray away from the somatic gonad, occasionally exiting the embryo entirely. Our results indicate that PIE-1 promotes germ cell fate by two independent mechanisms as follows: (1) inhibition of transcription, which blocks zygotic programs that drive somatic development, and (2) activation of protein expression from nos-2 and possibly other maternal RNAs, which promotes primordial germ cell development. PMID:11316796

Liu, Wei Wu, Haiyi Liu, Mengxia Duan, Delin

Artificial seedling production of Sargassum thunbergii is an effective way to relieve pressure on natural resources. In order to improve the utilization of zygotes and reduce the loss of seedlings, studies on the characteristic of the zygotes release, the development of rhizoids, the attachment of germlings, and the influence of jet washing were conducted. Results show that the percent of zygotes released was increased with time in the first 60 h. The capacity of germlings attached to the substratum was significantly increased, especially coincident with the time of the new rhizoids emerged and elongated. The detachment rate of germlings significantly decreased with the delay of starting time of jet washing or the reduction of jet washing velocity. However, the jet washing at any level applied in the experiment could cause considerable loss of germlings within the 20 days after the attachment. Our study provided some parameters to optimize the operation in the early stage of seedling production.

Superina, Simone Borovina, Antonia Ciruna, Brian

Growth factors and morphogens regulate embryonic patterning, cell fate specification, cell migration, and morphogenesis. The activity and behavior of these signaling molecules are regulated in the extracellular space through interactions with proteoglycans (Bernfield et al., 1999 Perrimon and Bernfield 2000 Lander and Selleck 2000 Selleck 2000). Proteoglycans are high molecular-weight proteins consisting of a core protein with covalently linked glycosaminoglycan (GAG) side chains, which are thought to mediate ligand interaction. Drosophila mutant embryos deficient for UDP-glucose dehydrogenase activity (Ugdh, required for GAG synthesis) exhibit abnormal Fgf, Wnt and TGFß signaling and die during gastrulation, indicating a broad and critical role for proteoglycans during early embryonic development (Lin et al., 1999 Lin and Perrimon 2000) (Hacker et al., 1997). Mouse Ugdh mutants also die at gastrulation, however, only Fgf signaling appears disrupted (Garcia-Garcia and Anderson, 2003). These findings suggested a possible divergence in the requirement for proteoglycans during Drosophila and mouse embryogenesis, and that mammals may have evolved alternative means of regulating Wnt and TGFß activity . To further examine the function of proteoglycans in vertebrate development, we have characterized zebrafish mutants devoid of both maternal and zygotic Ugdh/Jekyll activity (MZjekyll). We demonstrate that MZjekyll mutant embryos display abnormal Fgf, Shh, and Wnt signaling activities , with concomitant defects in central nervous system patterning, cardiac ventricular fate specification and axial morphogenesis. Furthermore, we uncover a novel role for proteoglycans in left-right pattern formation. Our findings resolve longstanding questions into the evolutionary conservation of Ugdh function and provide new mechanistic insights into the initiation of left-right asymmetry. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Wu, Xuemei Wang, Pei Brown, Christopher A Zilinski, Carolyn A Matzuk, Martin M

Zygote arrest 1 (ZAR1) is an ovary-specific maternal factor that plays essential roles during the oocyte-to-embryo transition. In mice, the Zar1 mRNA is detected as a 1.4-kilobase (kb) transcript that is synthesized exclusively in growing oocytes. To further understand the functions of ZAR1, we have cloned the orthologous Zar1 cDNA and/or genes for mouse, rat, human, frog, zebrafish, and pufferfish. The entire mouse Zar1 gene and a related pseudogene span approximately 4.0 kb, contain four exons, and map to adjacent loci on mouse chromosome 5. The human ZAR1 orthologous gene similarly consists of four exons and resides on human chromosome 4p12, which is syntenic with the mouse Zar1 chromosomal locus. Rat (Rattus norvegicus) and pufferfish (Fugu rubripes) Zar1 genes were recognized by database mining and deduced protein alignment analysis. The rat Zar1 gene also maps to a region that is syntenic with the mouse Zar1 gene locus on rat chromosome 14. Frog (Xenopus laevis) and zebrafish (Danio rerio) Zar1 orthologs were cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends analysis of ovarian mRNA. Unlike mouse and human, the frog Zar1 is detected in multiple tissues, including lung, muscle, and ovary. The Zar1 mRNA appears in the cytoplasm of oocytes and persists until the tailbud stage during frog embryogenesis. Mouse, rat, human, frog, zebrafish, and pufferfish Zar1 genes encode proteins of 361, 361, 424, 295, 329, and 320 amino acids, respectively, and share 50.8%-88.1% amino acid identity. Regions of the N-termini of these ZAR1 orthologs show high sequence identity among these various proteins. However, the C-terminal 103 amino acids of these proteins, encoded by exons 2-4, contain an atypical eight-cysteine Plant Homeo Domain motif and are highly conserved, sharing 80.6%-98.1% identity among these species. These findings suggest that the carboxyl-termini of these ZAR1 proteins contain an important functional domain that is

Excised zygotic embryos, mericarps ("seeds") and hypocotyls of seedlings of cultivated carrot Daucus carota cv. Scarlet Nantes were evaluated for their ability to generate somatic embryos on a semisolid hormone-free nutrient medium. Neither intact zygotic embryos nor hypocotyls ever produced somatic embryos. However, mericarps and broken zygotic embryos were excellent sources for somatic embryo production (response levels as high as 86%). Somatic embryo formation was highest from cotyledons, but was also observed on isolated hypocotyls and root tips of mature zygotic embryos. On media containing unreduced nitrogen, somatic embryo formation led to the generation of vigorous cultures comprised entirely of somatic embryos at various stages of development which in turn proliferated still other somatic embryos. However, a medium was devised which when 1-5 mM NH4+ was the sole nitrogen source, led only to a proliferation of globular proembryos. Sustained subculturing of these proembryos at 2-3 week intervals enabled establishment of highly uniform cultures in which no further development into more mature stages of embryonic development occurred. These have been maintained, without decline, as morphogenetically competent proembryonic globules for over ten months. A basal medium containing from 1-5 mM NH4+ as the sole nitrogen source appears not to be inductive to somatic proembryo formation. Instead, such a medium is best thought of as permissive to the expression of embryogenically determined cells within zygotic embryos. By excising and breaking or wounding zygotic embryos, constituent cells are probably released from positional or chemical restraints and thus are able to express their innate embryogenic potential. Once a proembryonic culture is established, this medium containing 1-5 mM NH4+ as the sole nitrogen source provides a nonpermissive environment to the development and growth of later embryonic stages, but it does allow the continued formation and

Excised zygotic embryos, mericarps ("seeds") and hypocotyls of seedlings of cultivated carrot Daucus carota cv. Scarlet Nantes were evaluated for their ability to generate somatic embryos on a semisolid hormone-free nutrient medium. Neither intact zygotic embryos nor hypocotyls ever produced somatic embryos. However, mericarps and broken zygotic embryos were excellent sources for somatic embryo production (response levels as high as 86%). Somatic embryo formation was highest from cotyledons, but was also observed on isolated hypocotyls and root tips of mature zygotic embryos. On media containing unreduced nitrogen, somatic embryo formation led to the generation of vigorous cultures comprised entirely of somatic embryos at various stages of development which in turn proliferated still other somatic embryos. However, a medium was devised which when 1-5 mM NH4+ was the sole nitrogen source, led only to a proliferation of globular proembryos. Sustained subculturing of these proembryos at 2-3 week intervals enabled establishment of highly uniform cultures in which no further development into more mature stages of embryonic development occurred. These have been maintained, without decline, as morphogenetically competent proembryonic globules for over ten months. A basal medium containing from 1-5 mM NH4+ as the sole nitrogen source appears not to be inductive to somatic proembryo formation. Instead, such a medium is best thought of as permissive to the expression of embryogenically determined cells within zygotic embryos. By excising and breaking or wounding zygotic embryos, constituent cells are probably released from positional or chemical restraints and thus are able to express their innate embryogenic potential. Once a proembryonic culture is established, this medium containing 1-5 mM NH4+ as the sole nitrogen source provides a nonpermissive environment to the development and growth of later embryonic stages, but it does allow the continued formation and

Meyer, E Butler, A Dubrana, K Duharcourt, S Caron, F

In ciliates, the germ line genome is extensively rearranged during the development of the somatic macronucleus from a mitotic product of the zygotic nucleus. Germ line chromosomes are fragmented in specific regions, and a large number of internal sequence elements are eliminated. It was previously shown that transformation of the vegetative macronucleus of Paramecium primaurelia with a plasmid containing a subtelomeric surface antigen gene can affect the processing of the homologous germ line genomic region during development of a new macronucleus in sexual progeny of transformed clones. The gene and telomere-proximal flanking sequences are deleted from the new macronuclear genome, although the germ line genome remains wild type. Here we show that plasmids containing nonoverlapping segments of the same genomic region are able to induce similar terminal deletions the locations of deletion end points depend on the particular sequence used. Transformation of the maternal macronucleus with a sequence internal to a macronuclear chromosome also causes the occurrence of internal deletions between short direct repeats composed of alternating thymines and adenines. The epigenetic influence of maternal macronuclear sequences on developmental rearrangements of the zygotic genome thus appears to be both sequence specific and general, suggesting that this trans-nucleus effect is mediated by pairing of homologous sequences. PMID:9199294

Reddy, B A Kloc, M Etkin, L D

We have cloned a cDNA (xlan4) from a Xenopus laevis oocyte cDNA library whose cognate mRNA is localized in the animal pole region of full grown oocytes. The cDNA can be translated in vitro to produce a predicted size protein of 35 kDa and, is also expressed in E. coli as a fusion protein. The conceptual protein encoded by the xlan4 cDNA is 17.5% proline rich and possesses several PEST sequences found in proteins with short half-lives. The xlan4 mRNA is 2.6 kb and during early development its titer decreases until the neurula stage after which it begins to reaccumulate. Northern blots on dissected embryos and in situ hybridization revealed that the zygotic expression is limited to the dorsal axial structures consisting primarily of the CNS. UV irradiation of the vegetal pole region immediately following fertilization that produces ventralized embryos results in a loss of zygotic xlan4 expression. In the adult, xlan4 mRNA is limited primarily to the brain. The presence of this mRNA in animal pole region which contributes to the future neural cell lineages suggests that this gene product may function either in the specification of neural cell types or in a neural specific function.

Abdelnour-Esquivel, Ana Engelmann, Florent

This paper presents the development of cryopreservation protocols for zygotic embryos and apices of chayote (Sechium edule Jacq. Sw.), a tropical plant species with recalcitrant seeds. Zygotic embryos of two cultivars, Ccocro negro (CN) and Claudio (Cl) could withstand cryopreservation, with survival percentages of 10 and 30 %, after desiccation to 23 and 19 % moisture content (fresh weight basis), respectively. Apices sampled on in vitro plantlets of cultivars Cl, 13 and JM were successfully cryopreserved using a vitrification technique. Optimal conditions included the culture of mother-plants for 22 days on medium containing 0.3 M sucrose, culture of excised apices on the same medium for 1 day, loading of apices for 20 min with 2M glycerol + 0.4M glycerol, treatment with a series of diluted PVS2 solution (60 % PVS2 followed by 80 % PVS2 solution for 15 min (cultivar Cocoro Blanco [CB]) or 30 min (cultivars CN and Cl) at each concentration), rapid freezing and thawing, washing of shoot-tips with a 1.2 M sucrose solution, followed by recovery on media with progressively decreasing sucrose concentrations until the standard concentration of 0.1 M was reached. The highest survival percentages achieved ranged between 17 and 38 %, depending on the cultivar.

Cebrian-Serrano, Alberto Zha, Shijun Hanssen, Lars Biggs, Daniel Preece, Christopher

Genome manipulation in the mouse via microinjection of CRISPR/Cas9 site-specific nucleases has allowed the production time for genetically modified mouse models to be significantly reduced. Successful genome manipulation in the mouse has already been reported using Cas9 supplied by microinjection of a DNA construct, in vitro transcribed mRNA and recombinant protein. Recently the use of transgenic strains of mice overexpressing Cas9 has been shown to facilitate site-specific mutagenesis via maternal supply to zygotes and this route may provide an alternative to exogenous supply. We have investigated the feasibility of supplying Cas9 genetically in more detail and for this purpose we report the generation of a transgenic mice which overexpress Cas9 ubiquitously, via a CAG-Cas9 transgene targeted to the Gt(ROSA26)Sor locus. We show that zygotes prepared from female mice harbouring this transgene are sufficiently loaded with maternally contributed Cas9 for efficient production of embryos and mice harbouring indel, genomic deletion and knock-in alleles by microinjection of guide RNAs and templates alone. We compare the mutagenesis rates and efficacy of mutagenesis using this genetic supply with exogenous Cas9 supply by either mRNA or protein microinjection. In general, we report increased generation rates of knock-in alleles and show that the levels of mutagenesis at certain genome target sites are significantly higher and more consistent when Cas9 is supplied genetically relative to exogenous supply. PMID:28081254

Almoguera, C. Coca, M. A. Jordano, J.

We have isolated and sequenced Ha UbiS, a cDNA for a dry-seed-stored mRNA that encodes tetraubiquitin. We have observed differential accumulation of tetraubiquitin mRNAs during sunflower (Helianthus annuus L.) zygotic embryogenesis. These mRNAs were up-regulated during late embryogenesis and reached higher prevalence in the dry seed, where they were found to be associated mainly with provascular tissue. UbiS mRNA, as confirmed by Rnase A protection experiments, accumulated also in response to heat shock, but only in leaves and later during postgerminative development. These novel observations demonstrate expression during seed maturation of specific plant polyubiquitin transcripts and developmental regulation of their heat-shock response. Using ubiquitin antibodies we also detected discrete, seed-specific proteins with distinct temporal expression patterns during zygotic embryogenesis. Some of these patterns were concurrent with UbiS mRNA accumulation in seeds. The most abundant ubiquitin-reacting proteins found in mature seeds were small (16-22 kD) and acidic (isoelectric points of 6.1-7.4). Possible functional implications for UbiS expression elicited from these observations are discussed. PMID:12228401

A full factorial crossing experiment with five females and five males of each of two coregonid species from upper Lake Constance was used to test for intrinsic post- zygotic incompatibilities during early ontogeny. Up until shortly before hatching, there was no difference in embryo mortality between homo and heterologous crosses. A maternal effect on mortality was found in both species, but paternal effects and female-male interactions were absent. Thus, genetic incompatibility during early ontogeny does not appear to prevent introgressive hybridization, suggesting that genetic divergence between these species is maintained primarily by pre- zygotic barriers. The recent genetic homogenizations of coregonid species flocks in European alpine lakes may have been caused by a flattening of adaptive landscapes through eutrophication, but intensive stocking with larvae obtained in hatcheries from artificially fertilized eggs is also likely to be a contributing factor. To safeguard diversity among sympatric coregonids, it is important to re-establish ecological conditions conducive to species divergence and to revise traditional management strategies. © 2015 The Fisheries Society of the British Isles.

Crispo, M Mulet, A P Tesson, L Barrera, N Cuadro, F dos Santos-Neto, P C Nguyen, T H Crénéguy, A Brusselle, L Anegón, I Menchaca, A

While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes . When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and

Crispo, M. Mulet, A. P. Tesson, L. Barrera, N. Cuadro, F. dos Santos-Neto, P. C. Nguyen, T. H. Crénéguy, A. Brusselle, L. Anegón, I. Menchaca, A.

While CRISPR/Cas9 technology has proven to be a valuable system to generate gene-targeted modified animals in several species, this tool has been scarcely reported in farm animals. Myostatin is encoded by MSTN gene involved in the inhibition of muscle differentiation and growth. We determined the efficiency of the CRISPR/Cas9 system to edit MSTN in sheep and generate knock-out (KO) animals with the aim to promote muscle development and body growth. We generated CRISPR/Cas9 mRNAs specific for ovine MSTN and microinjected them into the cytoplasm of ovine zygotes . When embryo development of CRISPR/Cas9 microinjected zygotes (n = 216) was compared with buffer injected embryos (n = 183) and non microinjected embryos (n = 173), cleavage rate was lower for both microinjected groups (P zygote CRISPR/Cas9 microinjection were transferred to 29 recipient females resulting in 65.5% (19/29) of pregnant ewes and 41.5% (22/53) of newborns. From 22 born lambs analyzed by T7EI and Sanger sequencing, ten showed indel mutations at MSTN gene. Eight showed mutations in both alleles and five of them were homozygous for indels generating out-of frame mutations that resulted in premature stop codons. Western blot analysis of homozygous KO founders confirmed the absence of myostatin, showing heavier body weight than wild type counterparts. In conclusion, our results demonstrate that CRISPR/Cas9 system was a very efficient tool to generate gene KO sheep. This technology is quick and easy to perform and less expensive than previous techniques, and can be applied to obtain genetically modified animal models of interest for biomedicine and

Jusof, Wan-Hafizah Wan Khan, Nor-Ashikin Mohamed Noor Rajikin, Mohd Hamim Satar, Nuraliza Abdul Mustafa, Mohd-Fazirul Jusoh, Norhazlin Dasiman, Razif

The evolutionary significance of the interaction between paternal and maternal genomes in fertilized zygotes is a very interesting and challenging question. Wang et al. developed the concept of epigenetic game theory, and they try to use this concept to explain the interaction between paternal and maternal genomes in fertilized zygotes [1]. They emphasize that the embryogenesis can be considered as an ecological system in which two highly distinct and specialized gametes coordinate through either cooperation or competition, or both, to maximize the fitness of embryos under Darwinian selection. More specifically, they integrate game theory to model the pattern of coordination of paternal genome and maternal genomes mediated by DNA methylation dynamics, and they called this epigenetic game theory.

Inoue, Daigo Wittbrodt, Joachim Gruss, Oliver J

Centrosomes are the main microtubule organizing centers in animal cells. In particular during embryogenesis, they ensure faithful spindle formation and proper cell divisions. As metazoan centrosomes are eliminated during oogenesis, they have to be reassembled upon fertilization. Most metazoans use the sperm centrioles as templates for new centrosome biogenesis while the egg's cytoplasm re-prepares all components for on-going centrosome duplication in rapidly dividing embryonic cells. We discuss our knowledge and the experimental challenges to analyze zygotic centrosome reformation, which requires genetic experiments to enable scrutinizing respective male and female contributions. Male and female knockout animals and mRNA injection to mimic maternal expression of centrosomal proteins could point a way to the systematic molecular dissection of the process. The most recent data suggest that timely expression of centrosome components in oocytes is the key to zygotic centrosome reformation that uses male sperm as coordinators for de novo centrosome production. © 2018 WILEY Periodicals, Inc.

Rawe, V Y Olmedo, S Brugo Nodar, F N Ponzio, R Sutovsky, P

The assembly of nuclear pore complexes (NPC) and their cytoplasmic stacks, annulate lamellae (AL), promote normal nucleocytoplasmic trafficking and accompany pronuclear development within the mammalian zygote . Previous studies showed that a percentage of human oocytes fertilized in vitro failed to develop normal pronuclei and cleave within 40-48 h post insemination. We hypothesized that an aberrant recruitment of NPC proteins, nucleoporins and/or NPC preassembled into AL, might accompany human fertilization arrest. We explored NPC and AL assembly in unfertilized human oocytes, and fertilized and arrested zygotes by immunofluorescence with an NPC- and AL-specific antibody, mAb 414, and by transmission electron microscopy. Major NPC or AL assembly was not observed in the unfertilized human oocytes. Once fertilization took place, the formation of AL was observed throughout the cytoplasm and near the developing pronuclei with NPC. On the contrary, NPC assembly was disrupted in the arrested zygotes , whereas AL were clustered into large sheaths. This was accompanied by the lack of NPC incorporation into the nuclear envelopes. We conclude that the aberrant assembly of NPC and AL coincides with early developmental failure in humans.

Morel, Alexandre Trontin, Jean-François Corbineau, Françoise Lomenech, Anne-Marie Beaufour, Martine Reymond, Isabelle Le Metté, Claire Ader, Kevin Harvengt, Luc Cadene, Martine Label, Philippe Teyssier, Caroline Lelu-Walter, Marie-Anne

Cotyledonary somatic embryos (SEs) of maritime pine are routinely matured for 12 weeks before being germinated and converted to plantlets. Although regeneration success is highly dependent on SEs quality, the date of harvesting is currently determined mainly on the basis of morphological features. This empirical method does not provide any accurate information about embryo quality with respect to storage compounds (proteins, carbohydrates). We first analyzed SEs matured for 10, 12 and 14 weeks by carrying out biological (dry weight, water content) and biochemical measurements (total protein and carbohydrate contents). No difference could be found between collection dates, suggesting that harvesting SEs after 12 weeks is appropriate. Cotyledonary SEs were then compared to various stages, from fresh to fully desiccated, in the development of cotyledonary zygotic embryos (ZEs). We identified profiles that were similar using hierarchical ascendant cluster analysis (HCA). Fresh and dehydrated ZEs could be distinguished, and SEs clustered with fresh ZEs. Both types of embryo exhibited similar carbohydrate and protein contents and signatures. This high level of similarity (94.5 %) was further supported by proteome profiling. Highly expressed proteins included storage, stress-related, late embryogenesis abundant and energy metabolism proteins. By comparing overexpressed proteins in developing and cotyledonary SEs or ZEs, some (23 proteins) could be identified as candidate biomarkers for the late, cotyledonary stage. This is the first report of useful generic protein markers for monitoring embryo development in maritime pine. Our results also suggest that improvements of SEs quality may be achieved if the current maturation conditions are refined.

Yu, Hong-Hao Zhao, Heng Qing, Yu-Bo Pan, Wei-Rong Jia, Bao-Yu Zhao, Hong-Ye Huang, Xing-Xu Wei, Hong-Jiang

Dystrophinopathy, including Duchenne muscle dystrophy (DMD) and Becker muscle dystrophy (BMD) is an incurable X-linked hereditary muscle dystrophy caused by a mutation in the DMD gene in coding dystrophin. Advances in further understanding DMD/BMD for therapy are expected. Studies on mdx mice and dogs with muscle dystrophy provide limited insight into DMD disease mechanisms and therapeutic testing because of the different pathological manifestations. Miniature pigs share similar physiology and anatomy with humans and are thus an excellent animal model of human disease. Here, we successfully achieved precise DMD targeting in Chinese Diannan miniature pigs by co-injecting zygotes with Cas9 mRNA and sgRNA targeting DMD . Two piglets were obtained after embryo transfer, one of piglets was identified as DMD -modified individual via traditional cloning, sequencing and T7EN1 cleavage assay. An examination of targeting rates in the DMD -modified piglet revealed that sgRNA:Cas9-mediated on-target mosaic mutations were 70% and 60% of dystrophin alleles in skeletal and smooth muscle, respectively. Meanwhile, no detectable off-target mutations were found, highlighting the high specificity of genetic modification using CRISPR/Cas9. The DMD -modified piglet exhibited degenerative and disordered phenotypes in skeletal and cardiac muscle, and declining thickness of smooth muscle in the stomach and intestine. In conclusion, we successfully generated myopathy animal model by modifying the DMD via CRISPR/Cas9 system in a miniature pig.

Garrels, Wiebke Mátés, Lajos Holler, Stephanie Dalda, Anna Taylor, Ulrike Petersen, Björn Niemann, Heiner Izsvák, Zsuzsanna Ivics, Zoltán Kues, Wilfried A.

Genetic engineering can expand the utility of pigs for modeling human diseases, and for developing advanced therapeutic approaches. However, the inefficient production of transgenic pigs represents a technological bottleneck. Here, we assessed the hyperactive Sleeping Beauty (SB100X) transposon system for enzyme-catalyzed transgene integration into the embryonic porcine genome. The components of the transposon vector system were microinjected as circular plasmids into the cytoplasm of porcine zygotes , resulting in high frequencies of transgenic fetuses and piglets. The transgenic animals showed normal development and persistent reporter gene expression for >12 months. Molecular hallmarks of transposition were confirmed by analysis of 25 genomic insertion sites. We demonstrate germ-line transmission, segregation of individual transposons, and continued, copy number-dependent transgene expression in F1-offspring. In addition, we demonstrate target-selected gene insertion into transposon-tagged genomic loci by Cre-loxP-based cassette exchange in somatic cells followed by nuclear transfer. Transposase-catalyzed transgenesis in a large mammalian species expands the arsenal of transgenic technologies for use in domestic animals and will facilitate the development of large animal models for human diseases. PMID:21897845

Jacobi, Ashley M Rettig, Garrett R Turk, Rolf Collingwood, Michael A Zeiner, Sarah A Quadros, Rolen M Harms, Donald W Bonthuis, Paul J Gregg, Christopher Ohtsuka, Masato Gurumurthy, Channabasavaiah B Behlke, Mark A

Genome editing using the CRISPR/Cas9 system requires the presence of guide RNAs bound to the Cas9 endonuclease as a ribonucleoprotein (RNP) complex in cells, which cleaves the host cell genome at sites specified by the guide RNAs. New genetic material may be introduced during repair of the double-stranded break via homology dependent repair (HDR) if suitable DNA templates are delivered with the CRISPR components. Early methods used plasmid or viral vectors to make these components in the host cell, however newer approaches using recombinant Cas9 protein with synthetic guide RNAs introduced directly as an RNP complex into cells shows faster onset of action with fewer off-target effects. This approach also enables use of chemically modified synthetic guide RNAs that have improved nuclease stability and reduces the risk of triggering an innate immune response in the host cell. This article provides detailed methods for genome editing using the RNP approach with synthetic guide RNAs using lipofection or electroporation in mammalian cells or using microinjection in murine zygotes , with or without addition of a single-stranded HDR template DNA. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Sisunandar Rival, Alain Turquay, Patricia Samosir, Yohannes Adkins, Steve W

The present study aimed at exploring the fidelity of coconut (Cocos nucifera L.) plants recovered from cryopreservation. Zygotic embryos from various different cultivars were cryopreserved following four successive steps, namely: rapid dehydration, rapid freezing, rapid thawing and in vitro recovery followed by acclimatization. At the end of the acclimatization period, the seedlings were compared to counterparts of the same age, which were produced from non-cryopreserved embryos. Both series were submitted to morphological, cytological and molecular comparisons. No significant differences in terms of growth rates could be measured. In addition, no morphological variation could be detected through the measurement of shoot elongation rates, production of opened leaves, and the number and total length of primary roots. Karyotype analysis revealed the same chromosome number (2n = 32) in all studied cultivars independently of cryopreservation. No significant differences could be observed between control and cryopreserved material concerning the type of chromosomes, the length of the long and short arms, the arm length ratio and the centromeric index. However, idiogram analysis did show a greater number of black banding on chromosomes isolated from cryopreserved material. Genetic and epigenetic fidelity was assessed through microsatellite (SSR) analysis and global DNA methylation rates no significant differences would be observed between genomic DNAs isolated from seedlings originating from cryopreserved embryos and respective controls. In conclusion, our results suggest that the method of cryopreservation under study did not induce gross morphological, genetic or epigenetic changes, thus suggesting that it is an appropriate method to efficiently preserve coconut germplasm.

Raveux, Aurélien Vandormael-Pournin, Sandrine Cohen-Tannoudji, Michel

Microinjection of the CRISPR/Cas9 system in zygotes is an efficient and comparatively fast method to generate genetically modified mice. So far, only few knock-in mice have been generated using this approach, and because no systematic study has been performed, parameters controlling the efficacy of CRISPR/Cas9-mediated targeted insertion are not fully established. Here, we evaluated the effect of several parameters on knock-in efficiency changing only one variable at a time. We found that knock-in efficiency was dependent on injected Cas9 mRNA and single-guide RNA concentrations and that cytoplasmic injection resulted in more genotypic complexity compared to pronuclear injection. Our results also indicated that injection into the pronucleus compared to the cytoplasm is preferable to generate knock-in alleles with an oligonucleotide or a circular plasmid. Finally, we showed that Cas9D10A nickase variant was less efficient than wild-type Cas9 for generating knock-in alleles and caused a higher rate of mosaicism. Thus, our study provides valuable information that will help to improve the future production of precise genetic modifications in mice.

Piacentino, Michael L Chung, Oliver Ramachandran, Janani Zuch, Daniel T Yu, Jia Conaway, Evan A Reyna, Arlene E Bradham, Cynthia A

Skeletal patterning in the sea urchin embryo requires coordinated signaling between the pattern-dictating ectoderm and the skeletogenic primary mesenchyme cells (PMCs) recent studies have begun to uncover the molecular basis for this process. Using an unbiased RNA-Seq-based screen, we have previously identified the TGF-ß superfamily ligand, LvBMP5-8, as a skeletal patterning gene in Lytechinus variegatus embryos. This result is surprising, since both BMP5-8 and BMP2/4 ligands have been implicated in sea urchin dorsal-ventral (DV) and left-right (LR) axis specification. Here, we demonstrate that zygotic LvBMP5-8 is required for normal skeletal patterning on the left side, as well as for normal PMC positioning during gastrulation. Zygotic LvBMP5-8 is required for expression of the left-side marker soxE, suggesting that LvBMP5-8 is required for left-side specification. Interestingly, we also find that LvBMP5-8 knockdown suppresses serotonergic neurogenesis on the left side. While LvBMP5-8 overexpression is sufficient to dorsalize embryos, we find that zygotic LvBMP5-8 is not required for normal DV specification or development. In addition, ectopic LvBMP5-8 does not dorsalize LvBMP2/4 morphant embryos, indicating that, in the absence of BMP2/4, BMP5-8 is insufficient to specify dorsal. Taken together, our data demonstrate that zygotic LvBMP5-8 signaling is essential for left-side specification, and for normal left-side skeletal and neural patterning, but not for DV specification. Thus, while both BMP2/4 and BMP5-8 regulate LR axis specification, BMP2/4 but not zygotic BMP5-8 regulates DV axis specification in sea urchin embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

Orsi, Guillermo A Joyce, Eric F Couble, Pierre McKim, Kim S Loppin, Benjamin

The Drosophila I-R type of hybrid dysgenesis is a sterility syndrome (SF sterility) associated with the mobilization of the I retrotransposon in female germ cells. SF sterility results from a maternal-effect embryonic lethality whose origin has remained unclear since its discovery about 40 years ago. Here, we show that meiotic divisions in SF oocytes are catastrophic and systematically fail to produce a functional female pronucleus at fertilization. As a consequence, most embryos from SF females rapidly arrest their development with aneuploid or damaged nuclei, whereas others develop as non-viable, androgenetic haploid embryos. Finally, we show that, in contrast to mutants affecting the biogenesis of piRNAs, SF egg chambers do not accumulate persistent DNA double-strand breaks, suggesting that I-element activity might perturb the functional organization of meiotic chromosomes without triggering an early DNA damage response.

Uzbekova, Svetlana Roy-Sabau, Monica Dalbiès-Tran, Rozenn Perreau, Christine Papillier, Pascal Mompart, Florence Thelie, Aurore Pennetier, Sophie Cognie, Juliette Cadoret, Veronique Royere, Dominique Monget, Philippe Mermillod, Pascal

Background Zygote arrest 1 (ZAR1) is one of the few known oocyte-specific maternal-effect genes essential for the beginning of embryo development discovered in mice. This gene is evolutionary conserved in vertebrates and ZAR1 protein is characterized by the presence of atypical plant homeobox zing finger domain, suggesting its role in transcription regulation. This work was aimed at the study of this gene, which could be one of the key regulators of successful preimplantation development of domestic animals, in pig and cattle, as compared with human. Methods Screenings of somatic cell hybrid panels and in silico research were performed to characterize ZAR1 chromosome localization and sequences. Rapid amplification of cDNA ends was used to obtain full-length cDNAs. Spatio-temporal mRNA expression patterns were studied using Northern blot, reverse transcription coupled to polymerase chain reaction and in situ hybridization. Results We demonstrated that ZAR1 is a single copy gene, positioned on chromosome 8 in pig and 6 in cattle, and several variants of correspondent cDNA were cloned from oocytes. Sequence analysis of ZAR1 cDNAs evidenced numerous short inverted repeats within the coding sequences and putative Pumilio-binding and embryo-deadenylation elements within the 3'-untranslated regions, indicating the potential regulation ways. We showed that ZAR1 expressed exclusively in oocytes in pig ovary, persisted during first cleavages in embryos developed in vivo and declined sharply in morulae and blastocysts. ZAR1 mRNA was also detected in testis, and, at lower level, in hypothalamus and pituitary in both species. For the first time, ZAR1 was localized in testicular germ cells, notably in round spermatids. In addition, in pig, cattle and human only shorter ZAR1 transcript variants resulting from alternative splicing were found in testis as compared to oocyte. Conclusion Our data suggest that in addition to its role in early embryo development highlighted by

Hariton, Eduardo Kim, Keewan Mumford, Sunni L Palmor, Marissa Bortoletto, Pietro Cardozo, Eden R Karmon, Anatte E Sabatini, Mary E Styer, Aaron K

To evaluate the association of oocyte donor-recipient characteristics, oocyte donor response, and live birth pregnancy rate following fresh donor oocyte IVF-ET. Retrospective cohort study. Academic reproductive medicine practice. Two hundred thirty-seven consecutive fresh donor oocyte IVF-ET cycles from January 1, 2007 to December 31, 2013 at the Massachusetts General Hospital Fertility Center. None. Live birth rate per cycle initiated. The mean (±SD) age of oocyte donors and recipients was 27.0 ± 3.7 and 41.4 ± 4.6 years, respectively. Oocyte donor demographic/reproductive characteristics, ovarian reserve testing, and peak serum E 2 during ovarian stimulation were similar among cycles which did and did not result in live birth, respectively. Overall implantation, clinical pregnancy, and live birth pregnancy rates per cycle initiated were 40.5%, 60.8%, and 54.9%, respectively. The greatest probability of live birth was observed in cycles with >10 oocytes retrieved, mature oocytes, oocytes with normal fertilization ( zygote -two pronuclear stage), and cleaved embryos. The number of oocytes (total and mature), zygotes , and cleaved embryos are associated with live birth following donor oocyte IVF cycles. These findings suggest that specific peri-fertilization factors may be predictive of pregnancy outcomes following donor oocyte IVF cycles. Copyright © 2017 American Society for Reproductive Medicine. All rights reserved.

Peng, Jin Wang, Yong Jiang, Junyi Zhou, Xiaoyang Song, Lei Wang, Lulu Ding, Chen Qin, Jun Liu, Liping Wang, Weihua Liu, Jianqiao Huang, Xingxu Wei, Hong Zhang, Pumin

Precise genome modification in large domesticated animals is desirable under many circumstances. In the past it is only possible through lengthy and burdensome cloning procedures. Here we attempted to achieve that goal through the use of the newest genome-modifying tool CRISPR/Cas9. We set out to knockin human albumin cDNA into pig Alb locus for the production of recombinant human serum albumin (rHSA). HSA is a widely used human blood product and is in high demand. We show that homologous recombination can occur highly efficiently in swine zygotes . All 16 piglets born from the manipulated zygotes carry the expected knockin allele and we demonstrated the presence of human albumin in the blood of these piglets. Furthermore, the knockin allele was successfully transmitted through germline. This success in precision genomic engineering is expected to spur exploration of pigs and other large domesticated animals to be used as bioreactors for the production of biomedical products or creation of livestock strains with more desirable traits.

Nakagawa, Yoshiko Sakuma, Tetsushi Nishimichi, Norihisa Yokosaki, Yasuyuki Takeo, Toru Nakagata, Naomi Yamamoto, Takashi

Robust reproductive engineering techniques are required for the efficient and rapid production of genetically modified mice. We have reported the efficient production of genome-edited mice using reproductive engineering techniques, such as ultra-superovulation, in vitro fertilization (IVF) and vitrification/warming of zygotes . We usually use vitrified/warmed fertilized oocytes created by IVF for microinjection because of work efficiency and flexible scheduling. Here, we investigated whether the culture time of zygotes before microinjection influences the efficiency of producing knock-in mice. Knock-in mice were generated using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system and single-stranded oligodeoxynucleotide (ssODN) or PITCh (Precise Integration into Target Chromosome) system, a method of integrating a donor vector assisted by microhomology-mediated end-joining. The cryopreserved fertilized oocytes were warmed, cultured for several hours and microinjected at different timings. Microinjection was performed with Cas9 protein, guide RNA(s), and an ssODN or PITCh donor plasmid for the ssODN knock-in and the PITCh knock-in, respectively. Different production efficiencies of knock-in mice were observed by changing the timing of microinjection. Our study provides useful information for the CRISPR-Cas9-based generation of knock-in mice. © 2017. Published by The Company of Biologists Ltd.

Maximova, Siela N Florez, Sergio Shen, Xiangling Niemenak, Nicolas Zhang, Yufan Curtis, Wayne Guiltinan, Mark J

Theobroma cacao L. is a tropical fruit tree, the seeds of which are used to create chocolate. In vitro somatic embryogenesis (SE) of cacao is a propagation system useful for rapid mass-multiplication to accelerate breeding programs and to provide plants directly to farmers. Two major limitations of cacao SE remain: the efficiency of embryo production is highly genotype dependent and the lack of full cotyledon development results in low embryo to plant conversion rates. With the goal to better understand SE development and to improve the efficiency of SE conversion we examined gene expression differences between zygotic and somatic embryos using a whole genome microarray. The expression of 28,752 genes was determined at 4 developmental time points during zygotic embryogenesis (ZE) and 2 time points during cacao somatic embryogenesis (SE). Within the ZE time course, 10,288 differentially expressed genes were enriched for functions related to responses to abiotic and biotic stimulus, metabolic and cellular processes. A comparison ZE and SE expression profiles identified 10,175 differentially expressed genes. Many TF genes, putatively involved in ethylene metabolism and response, were more strongly expressed in SEs as compared to ZEs. Expression levels of genes involved in fatty acid metabolism, flavonoid biosynthesis and seed storage protein genes were also differentially expressed in the two types of embryos. Large numbers of genes were differentially regulated during various stages of both ZE and SE development in cacao. The relatively higher expression of ethylene and flavonoid related genes during SE suggests that the developing tissues may be experiencing high levels of stress during SE maturation caused by the in vitro environment. The expression of genes involved in the synthesis of auxin, polyunsaturated fatty acids and secondary metabolites was higher in SEs relative to ZEs despite lack of lipid and metabolite accumulation. These differences in gene

Background Theobroma cacao L. is a tropical fruit tree, the seeds of which are used to create chocolate. In vitro somatic embryogenesis (SE) of cacao is a propagation system useful for rapid mass-multiplication to accelerate breeding programs and to provide plants directly to farmers. Two major limitations of cacao SE remain: the efficiency of embryo production is highly genotype dependent and the lack of full cotyledon development results in low embryo to plant conversion rates. With the goal to better understand SE development and to improve the efficiency of SE conversion we examined gene expression differences between zygotic and somatic embryos using a whole genome microarray. Results The expression of 28,752 genes was determined at 4 developmental time points during zygotic embryogenesis (ZE) and 2 time points during cacao somatic embryogenesis (SE). Within the ZE time course, 10,288 differentially expressed genes were enriched for functions related to responses to abiotic and biotic stimulus, metabolic and cellular processes. A comparison ZE and SE expression profiles identified 10,175 differentially expressed genes. Many TF genes, putatively involved in ethylene metabolism and response, were more strongly expressed in SEs as compared to ZEs. Expression levels of genes involved in fatty acid metabolism, flavonoid biosynthesis and seed storage protein genes were also differentially expressed in the two types of embryos. Conclusions Large numbers of genes were differentially regulated during various stages of both ZE and SE development in cacao. The relatively higher expression of ethylene and flavonoid related genes during SE suggests that the developing tissues may be experiencing high levels of stress during SE maturation caused by the in vitro environment. The expression of genes involved in the synthesis of auxin, polyunsaturated fatty acids and secondary metabolites was higher in SEs relative to ZEs despite lack of lipid and metabolite accumulation

Wang, Miranda Ly, Michael Lugowski, Andrew Laver, John D Lipshitz, Howard D Smibert, Craig A Rissland, Olivia S

In animal embryos, control of development is passed from exclusively maternal gene products to those encoded by the embryonic genome in a process referred to as the maternal-to- zygotic transition (MZT). We show that the RNA-binding protein, ME31B, binds to and represses the expression of thousands of maternal mRNAs during the Drosophila MZT. However, ME31B carries out repression in different ways during different phases of the MZT. Early, it represses translation while, later, its binding leads to mRNA destruction, most likely as a consequence of translational repression in the context of robust mRNA decay. In a process dependent on the PNG kinase, levels of ME31B and its partners, Cup and Trailer Hitch (TRAL), decrease by over 10-fold during the MZT, leading to a change in the composition of mRNA-protein complexes. We propose that ME31B is a global repressor whose regulatory impact changes based on its biological context.

Von Neumann and Morgenstern published the Theory of Games and Economic Behavior in 1944, describing game theory as a model in which intelligent rational decision-makers manage to find their best strategies in conflict, cooperative or other mutualistic relationships to acquire the greatest benefit [1]. This model was subsequently incorporated in ecology to simulate the fitness of a species during natural selection, designated evolutionary game theory (EGT) [2]. Wang et al. proposed epiGame, taking paternal and maternal genomes as intelligent players that compete, cooperate or both during embryogenesis to maximize the fitness of the embryo [3]. They further extended game theory to an individual or single cell environment. During early zygote development, DNA methylation is reprogrammed such that the paternal genome is demethylated before the maternal genome. After the reset, the blastocyst is re-methylated during embryogenesis. At that time, the paternal and maternal genomes have a conflict of interest related to the expression of their own genes. The proposed epiGame models such interactive regulation between the parental genomes to reach a balance for embryo development (equation (2)).

Laver, John D Li, Xiao Ray, Debashish Cook, Kate B Hahn, Noah A Nabeel-Shah, Syed Kekis, Mariana Luo, Hua Marsolais, Alexander J Fung, Karen Yy Hughes, Timothy R Westwood, J Timothy Sidhu, Sachdev S Morris, Quaid Lipshitz, Howard D Smibert, Craig A

Brain tumor (BRAT) is a Drosophila member of the TRIM-NHL protein family. This family is conserved among metazoans and its members function as post-transcriptional regulators. BRAT was thought to be recruited to mRNAs indirectly through interaction with the RNA-binding protein Pumilio (PUM). However, it has recently been demonstrated that BRAT directly binds to RNA. The precise sequence recognized by BRAT, the extent of BRAT-mediated regulation, and the exact roles of PUM and BRAT in post-transcriptional regulation are unknown. Genome-wide identification of transcripts associated with BRAT or with PUM in Drosophila embryos shows that they bind largely non-overlapping sets of mRNAs. BRAT binds mRNAs that encode proteins associated with a variety of functions, many of which are distinct from those implemented by PUM-associated transcripts. Computational analysis of in vitro and in vivo data identified a novel RNA motif recognized by BRAT that confers BRAT-mediated regulation in tissue culture cells. The regulatory status of BRAT-associated mRNAs suggests a prominent role for BRAT in post-transcriptional regulation, including a previously unidentified role in transcript degradation. Transcriptomic analysis of embryos lacking functional BRAT reveals an important role in mediating the decay of hundreds of maternal mRNAs during the maternal-to- zygotic transition. Our results represent the first genome-wide analysis of the mRNAs associated with a TRIM-NHL protein and the first identification of an RNA motif bound by this protein family. BRAT is a prominent post-transcriptional regulator in the early embryo through mechanisms that are largely independent of PUM.

Hamm, Danielle C Bondra, Eliana R Harrison, Melissa M

Delayed transcriptional activation of the zygotic genome is a nearly universal phenomenon in metazoans. Immediately following fertilization, development is controlled by maternally deposited products, and it is not until later stages that widespread activation of the zygotic genome occurs. Although the mechanisms driving this genome activation are currently unknown, the transcriptional activator Zelda (ZLD) has been shown to be instrumental in driving this process in Drosophila melanogaster. Here we define functional domains of ZLD required for both DNA binding and transcriptional activation . We show that the C-terminal cluster of four zinc fingers mediates binding to TAGteam DNA elements in the promoters of early expressed genes. All four zinc fingers are required for this activity , and splice isoforms lacking three of the four zinc fingers fail to activate transcription. These truncated splice isoforms dominantly suppress activation by the full-length, embryonically expressed isoform. We map the transcriptional activation domain of ZLD to a central region characterized by low complexity. Despite relatively little sequence conservation within this domain, ZLD orthologs from Drosophila virilis, Anopheles gambiae, and Nasonia vitripennis activate transcription in D. melanogaster cells. Transcriptional activation by these ZLD orthologs suggests that ZLD functions through conserved interactions with a protein cofactor(s). We have identified distinct DNA-binding and activation domains within the critical transcription factor ZLD that controls the initial activation of the zygotic genome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Antoine, A F Faure, J E Cordeiro, S Dumas, C Rougier, M Feijó, J A

In this paper, we report direct measurement of an influx of extracellular Ca(2+) induced by gamete fusion in flowering plants. This result was obtained during maize in vitro fertilization with the use of an extracellular Ca(2+)-selective vibrating probe. Ca(2+) influx recorded at the surface of isolated egg cells, with or without adhesion of a male sperm cell, was close to zero and stable over time. Gamete fusion, however, triggered a Ca(2+) influx in the vicinity of the sperm entry site with a delay of 1.8 +/- 0.6 sec. The Ca(2+) influx spread subsequently through the whole egg cell plasma membrane as a wavefront, progressing at an estimated rate of 1.13 micrometer.(-1). Once established, Ca(2+) influx intensities were sustained, monotonic and homogeneous over the whole egg cell, with an average peak influx of 14.92 pmol .cm(-2).(-1) and an average duration of 24.4 min. The wavefront spread of channel activation correlates well with the cytological modifications induced by fertilization, such as egg cell contraction, and with the cytosolic Ca(2+) ((c)[Ca(2+)]) elevation previously reported. Calcium influx was inhibited effectively by gadolinium, possibly implicating mechanosensitive channels. Furthermore, artificial influxes created by incubation with Ca(2+) ionophores mimicked some aspects of egg activation . Taken together, these results suggest that, during fertilization in higher plants, gamete membrane fusion starts the first embryonic events by channel opening and Ca(2+) influx. In turn, (c)[Ca(2+)] may work as a trigger and possibly a space and time coordinator of many aspects of egg activation .

Meyer, Axel Begemann, Gerrit

In poeciliid fish the male anal fin has been transformed into a gonopodium, an intromittent organ required for internal fertilization. Elevated testosterone levels induce metamorphosis of a subset of anal fin rays to grow and form the specialized terminal structures of the gonopodium. The molecular mechanisms underlying these processes are largely unknown. Here, we investigated whether retinoic acid (RA) signaling is involved in gonopodium development in the swordtail Xiphophorus hellerii. We showed that aldh 1 a 2 , a RA synthesizing enzyme, and the RA receptors, rar-ga and rar-gb, are expressed in anal fins during metamorphosis. aldh 1 a 2 expression is regulated by testosterone in a concentration-dependent manner and is up-regulated in both hormone-induced and naturally developing gonopodia. Androgen receptor (ar), a putative regulator of gonopodial development, is co-expressed with aldh 1 a 2 and the RA receptors in gonopodial rays. Importantly, experimental increase of RA signaling promoted growth of the gonopodium and increased the number of new segments. Based on gene expression analyses and pharmacological manipulation of gonopodium development, we show that the RA signaling pathway is activated in response to androgen signaling and promotes fin ray growth and development during the metamorphosis of the anal fin into the gonopodium. PMID:24204880

Salvini, Mariangela Fambrini, Marco Giorgetti, Lucia Pugliesi, Claudio

The link HaWUS/ HaL1L , the opposite transcriptional behavior, and the decrease/increase in positive histone marks bond to both genes suggest an inhibitory effect of WUS on HaL1L in sunflower zygotic embryos. In Arabidopsis, a group of transcription factors implicated in the earliest events of embryogenesis is the WUSCHEL-RELATED HOMEOBOX (WOX) protein family including WUSCHEL (WUS) and other 14 WOX protein, some of which contain a conserved WUS-box domain in addition to the homeodomain. WUS transcripts appear very early in embryogenesis, at the 16-cell embryo stage, but gradually become restricted to the center of the developing shoot apical meristem (SAM) primordium and continues to be expressed in cells of the niche/organizing center of SAM and floral meristems to maintain stem cell population. Moreover, WUS has decisive roles in the embryonic program presumably promoting the vegetative-to-embryonic transition and/or maintaining the identity of the embryonic stem cells. However, data on the direct interaction between WUS and key genes for seed development (as LEC1 and L1L) are not collected. The novelty of this report consists in the characterization of Helianthus annuus WUS (HaWUS) gene and in its analysis regarding the pattern of the methylated lysine 4 (K4) of the Histone H3 and of the acetylated histone H3 during the zygotic embryo development. Also, a parallel investigation was performed for HaL1L gene since two copies of the WUS-binding site (WUSATA), previously identified on HaL1L nucleotide sequence, were able to be bound by the HaWUS recombinant protein suggesting a not described effect of HaWUS on HaL1L transcription.

Anil, V S Harmon, A C Rao, K S

Western-blot analysis and protein kinase assays identified two Ca(2+)-dependent protein kinases (CDPKs) of 55 to 60 kD in soluble protein extracts of embryogenic cultures of sandalwood (Santalum album L.). However, these sandalwood CDPKs (swCDPKs) were absent in plantlets regenerated from somatic embryos. swCDPKs exhibited differential expression (monitored at the level of the protein) and activity in different developmental stages. Zygotic embryos, seedlings, and endosperm showed high accumulation of swCDPK, but the enzyme was not detected in the soluble proteins of shoots and flowers. swCDPK exhibited a temporal pattern of expression in endosperm, showing high accumulation and activity in mature fruit and germinating stages the enzyme was localized strongly in the storage bodies of the endosperm cells. The study also reports for the first time to our knowledge a post-translational inhibition/inactivation of swCDPK in zygotic embryos during seed dormancy and early stages of germination. The temporal expression of swCDPK during somatic/ zygotic embryogenesis, seed maturation, and germination suggests involvement of the enzyme in these developmental processes.

In the sea urchin, some other marine invertebrates, and the frog, Xenopus, egg activation at fertilization is accompanied by an increase in intracellular pH (pHi). We measured pHi in germinal vesicle (GV)-intact mouse oocytes, ovulated eggs, and in vivo fertilized zygotes using the pH indicator dye, SNARF-1. The mean pH, was 6.96 +/- 0.004 (+/- SEM) in GV-intact oocytes, 7.00 +/- 0.01 in ovulated, unfertilized eggs, and 7.02 +/- 0.01 in fertilized zygotes , indicating no sustained changes in pHi after germinal vesicle breakdown (GVBD) or fertilization. To examine whether transient changes in pHi occur shortly after egg activation , mouse eggs were parthenogenetically activated by 7% ethanol in phosphate buffered saline (PBS) no significant change in pHi followed ethanol activation . Since increased Na+/H+ antiporter activity is responsible for pHi increase in the sea urchin, pHi was measured in the absence of added bicarbonate or CO2 (a condition under which the antiporter would be the only major pHi regulatory mechanism able to operate, since the others were bicarbonate-dependent) in GV-intact oocytes, ovulated eggs, and in vivo fertilized zygotes to determine whether a Na+/H+ antiporter was activated . There was no physiologically significant difference in pHi after GVBD or fertilization, when pHi was measured in bicarbonate-free medium, nor any change upon parthenogenetic activation . Thus, a change in pHi is not a feature of egg activation in the mouse.

Zhang, Zhenbin Sun, Liangliang Zhu, Guijie Cox, Olivia F Huber, Paul W Dovichi, Norman J

A sulfonate-silica hybrid strong cation exchange monolith microreactor was synthesized and coupled to a linear polyacrylamide coated capillary for online sample preparation and capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) bottom-up proteomic analysis. The protein sample was loaded onto the microreactor in an acidic buffer. After online reduction, alkylation, and digestion with trypsin, the digests were eluted with 200 mM ammonium bicarbonate at pH 8.2 for CZE-MS/MS analysis using 1 M acetic acid as the background electrolyte. This combination of basic elution and acidic background electrolytes results in both sample stacking and formation of a dynamic pH junction. 369 protein groups and 1274 peptides were identified from 50 ng of Xenopus laevis zygote homogenate, which is comparable with an offline sample preparation method, but the time required for sample preparation was decreased from over 24 h to less than 40 min. Dramatically improved performance was produced by coupling the reactor to a longer separation capillary (∼100 cm) and a Q Exactive HF mass spectrometer. 975 protein groups and 3749 peptides were identified from 50 ng of Xenopus protein using the online sample preparation method.

Anil, Veena S. Harmon, Alice C. Rao, K. Sankara

Western-blot analysis and protein kinase assays identified two Ca2+-dependent protein kinases (CDPKs) of 55 to 60 kD in soluble protein extracts of embryogenic cultures of sandalwood (Santalum album L.). However, these sandalwood CDPKs (swCDPKs) were absent in plantlets regenerated from somatic embryos. swCDPKs exhibited differential expression (monitored at the level of the protein) and activity in different developmental stages. Zygotic embryos, seedlings, and endosperm showed high accumulation of swCDPK, but the enzyme was not detected in the soluble proteins of shoots and flowers. swCDPK exhibited a temporal pattern of expression in endosperm, showing high accumulation and activity in mature fruit and germinating stages the enzyme was localized strongly in the storage bodies of the endosperm cells. The study also reports for the first time to our knowledge a post-translational inhibition/inactivation of swCDPK in zygotic embryos during seed dormancy and early stages of germination. The temporal expression of swCDPK during somatic/ zygotic embryogenesis, seed maturation, and germination suggests involvement of the enzyme in these developmental processes. PMID:10759499

Zhang, Yuan-Ming Zhang, Yinghao Guo, Mingyue

Wang's et al. article [1] is the first to integrate game theory (especially evolutionary game theory) with epigenetic modification of zygotic genomes. They described and assessed a modeling framework based on evolutionary game theory to quantify, how sperms and oocytes interact through epigenetic processes, to determine embryo development. They also studied the internal mechanisms for normal embryo development: 1) evolutionary interactions between DNA methylation of the paternal and maternal genomes, and 2) the application of game theory to formulate and quantify how different genes compete or cooperate to regulate embryogenesis through methylation. Although it is not very comprehensive and profound regarding game theory modeling, this article bridges the gap between evolutionary game theory and the epigenetic control of embryo development by powerful ordinary differential equations (ODEs). The epiGame framework includes four aspects: 1) characterizing how epigenetic game theory works by the strategy matrix, in which the pattern and relative magnitude of the methylation effects on embryogenesis, are described by the cooperation and competition mechanisms, 2) quantifying the game that the direction and degree of P-M interactions over embryo development can be explained by the sign and magnitude of interaction parameters in model (2), 3) modeling epigenetic interactions within the morula, especially for two coupled nonlinear ODEs, with explicit functions in model (4), which provide a good fit to the observed data for the two sexes (adjusted R2 = 0.956), and 4) revealing multifactorial interactions in embryogenesis from the coupled ODEs in model (2) to triplet ODEs in model (6). Clearly, this article extends game theory from evolutionary game theory to epigenetic game theory.

Shi, Junchao Zhang, Xudong Liu, Ying Chen, Qi

In their interesting article [1] Wang et al. proposed a mathematical model based on evolutionary game theory [2] to tackle the fundamental question in embryo development, that how sperm and egg interact with each other, through epigenetic processes, to form a zygote and direct successful embryo development. This work is based on the premise that epigenetic reprogramming (referring to the erasure and reconstruction of epigenetic marks, such as DNA methylation and histone modifications) after fertilization might be of paramount importance to maintain the normal development of embryos, a premise we fully agree, given the compelling experimental evidence reported [3]. Wang et al. have specifically chosen to employ the well-studied DNA methylation reprogramming process during mammalian early embryo development, as a basis to develop their mathematical model, namely epigenetic game theory (epiGame). They concluded that the DNA methylation pattern in mammalian early embryo could be formulated and quantified, and their model can be further used to quantify the interactions, such as competition and/or cooperation of expressed genes that maximize the fitness of embryos. The efforts by Wang et al. in quantitatively and systematically analyzing the beginning of life apparently hold value and represent a novel direction for future embryo development research from both theoretical and experimental biologists. On the other hand, we see their theory still at its infancy, because there are plenty more parameters to consider and there are spaces for debates, such as the cases of haploid embryo development [4]. Here, we briefly comment on the dynamic process of epigenetic reprogramming that goes beyond DNA methylation, a dynamic interplay that involves histone modifications, non-coding RNAs, transposable elements et al., as well as the potential input of the various types of 'hereditary' epigenetic information in the gametes - a game that has started before the fertilization.

Hamaji, Takashi Lopez, David Pellegrini, Matteo

Upon fertilization Chlamydomonas reinhardtii zygotes undergo a program of differentiation into a diploid zygospore that is accompanied by transcription of hundreds of zygote -specific genes. We identified a distinct sequence motif we term a zygotic response element (ZYRE) that is highly enriched in promoter regions of C. reinhardtii early zygotic genes. A luciferase reporter assay was used to show that native ZYRE motifs within the promoter of zygotic gene ZYS3 or intron of zygotic gene DMT4 are necessary for zygotic induction. A synthetic luciferase reporter with a minimal promoter was used to show that ZYRE motifs introduced upstream are sufficient tomore » confer zygotic upregulation, and that ZYRE-controlled zygotic transcription is dependent on the homeodomain transcription factor GSP1. Furthermore, we predict that ZYRE motifs will correspond to binding sites for the homeodomain proteins GSP1-GSM1 that heterodimerize and activate zygotic gene expression in early zygotes .« less

Forsberg, Lars A. Rasi, Chiara Pekar, Gyula Davies, Hanna Piotrowski, Arkadiusz Absher, Devin Razzaghian, Hamid Reza Ambicka, Aleksandra Halaszka, Krzysztof Przewoźnik, Marcin Kruczak, Anna Mandava, Geeta Pasupulati, Saichand Hacker, Julia Prakash, K. Reddy Dasari, Ravi Chandra Lau, Joey Penagos-Tafurt, Nelly Olofsson, Helena M. Hallberg, Gunilla Skotnicki, Piotr Mituś, Jerzy Skokowski, Jaroslaw Jankowski, Michal Śrutek, Ewa Zegarski, Wojciech Tiensuu Janson, Eva Ryś, Janusz Tot, Tibor Dumanski, Jan P.

Sporadic breast cancer (SBC) is a common disease without robust means of early risk prediction in the population. We studied 282 females with SBC, focusing on copy number aberrations in cancer-free breast tissue (uninvolved margin, UM) outside the primary tumor (PT). In total, 1162 UMs (1–14 per breast) were studied. Comparative analysis between UM(s), PT(s), and blood/skin from the same patient as a control is the core of the study design. We identified 108 patients with at least one aberrant UM, representing 38.3% of cases. Gains in gene copy number were the principal type of mutations in microscopically normal breast cells, suggesting that oncogenic activation of genes via increased gene copy number is a predominant mechanism for initiation of SBC pathogenesis. The gain of ERBB2, with overexpression of HER2 protein, was the most common aberration in normal cells. Five additional growth factor receptor genes (EGFR, FGFR1, IGF1R, LIFR, and NGFR) also showed recurrent gains, and these were occasionally present in combination with the gain of ERBB2. All the aberrations found in the normal breast cells were previously described in cancer literature, suggesting their causative, driving role in pathogenesis of SBC. We demonstrate that analysis of normal cells from cancer patients leads to identification of signatures that may increase risk of SBC and our results could influence the choice of surgical intervention to remove all predisposing cells. Early detection of copy number gains suggesting a predisposition toward cancer development, long before detectable tumors are formed, is a key to the anticipated shift into a preventive paradigm of personalized medicine for breast cancer. PMID:26430163

Biolistic transformation of cotton meristems, isolated from mature seed is detailed in this book chapter. This method is simple and avoids the necessity to use genotype-dependent regenerable cell cultures. However, identification of germ line transformation using this method is laborious and time-c.

Wang, Yingchun Wang, Fangfei Sun, Tong Trostinskaia, Anna Wygle, Dana Puscheck, Elizabeth Rappolee, Daniel A

To understand how mitogenic signals are transduced into the trophoblasts in preimplantation embryos, the expression of mitogen- activated protein kinase (MAPK) pathway molecules was tested. We used immunocytochemical means and reverse transcriptase-polymerase chain reaction to test whether MAPK pathway molecule gene products exist at the protein and phosphoprotein level in the zygote and the RNA level in the egg and zygote . In addition, all antibodies detected the correct-sized major band in Westerns of placental cell lines representing the most prevalent cell type in preimplantation embryos. A majority of mRNA transcripts of MAPK pathway genes were detected in unfertilized eggs, and all were expressed in the zygote . We found that the MAPK pathway protein set consisting of the following gene products was present: FRS2 alpha, GRB2, GAB1, SOS1, Ha-ras, Raf1/RafB, MEK1,2,5, MAPK/ERK1,2, MAPK/ERK5, and RSK1,2,3 (see abbreviations). These proteins were detected in trophoblasts in embryonic day (E) 3.5 embryos when they could mediate mitogenic fibroblast growth factor signals from the embryo or colony stimulating factor-1 signals from the uterus. The phosphorylation state and position of the phosphoproteins in the cells suggested that they might function in mediating mitogenic signals. Interestingly, a subtle transition from maternal MAPK function to zygotic function was suggested by the localization for three MAPK pathway enzymes between E2.5 and E3.5, Raf1 phospho is largely cell membrane-localized at E2.5 and E3.5, and MEK1,2 phospho accumulates in the nucleus on E2.5 and E3.5. However, MAPK phospho shifts from nuclear accumulation at E2.5 to cytoplasmic accumulation at E3.5. This finding is similar to the cytoplasmic MAPK phospho localization reported in fibroblast growth factor signaling fields in postimplantation embryos (Corson et al. [2003] Development 130:4527-4537). This spatial and temporal expression study lays a foundation to plan and analyze perturbation

The material presented is designed to help students explore geometric patterns involving Fibonnaci numbers and the golden ratio, and to aid in review of basic geometry skills. Worksheet masters intended for duplication are provided. Suggestions are made of possible classroom extensions to the initial activities . (MP)

Chouvenc, Thomas Helmick, Ericka E Su, Nan-Yao

While hybridization of an invasive species with a native species is a common occurrence, hybridization between two invasive species is rare. Formosan subterranean termites (Coptotermes formosanus) and Asian subterranean termites (C. gestroi) are both ecologically successful and are the two most economically important termite pests in the world. Both species have spread throughout many areas of the world due to human activity however, their distributions overlap in only three narrow areas because of distinct ecological requirements. In south Florida, where C. formosanus and C. gestroi are both invasive, the dispersal flight seasons of both species overlapped for the first time on record in 2013 and 2014. Pairings of heterospecific individuals were readily observed in the field and C. gestroi males preferentially engaged in mating behavior with C. formosanus females rather than females from their own species. In the laboratory, heterospecific and conspecific pairings had an equal colony establishment rate, but heterospecific incipient colonies had twice the growth rate of conspecific incipient colonies, suggesting a potential case of hybrid vigor. As all pre- zygotic barriers were lifted between the two species in the field, the apparent absence of post- zygotic barriers in the laboratory raises the possibility for introgressive hybridization in south Florida. While laboratory observations remain to be confirmed in the field, and the alate hybrid fertility is currently unknown, our results raise a tangible concern about the hybridization of two major destructive pest species. Such hybridization would likely be associated with a new economic impact.

Background The wide use of gametocytocidal artemisinin-based combination therapy (ACT) lead to a reduction of Plasmodium falciparum transmission in several African endemic settings. An increased impact on malaria burden may be achieved through the development of improved transmission-blocking formulations, including molecules complementing the gametocytocidal effects of artemisinin derivatives and/or acting on Plasmodium stages developing in the vector. Azadirachtin, a limonoid (tetranortriterpenoid) abundant in neem (Azadirachta indica, Meliaceae) seeds, is a promising candidate, inhibiting Plasmodium exflagellation in vitro at low concentrations. This work aimed at assessing the transmission-blocking potential of NeemAzal®, an azadirachtin-enriched extract of neem seeds, using the rodent malaria in vivo model Plasmodium berghei/Anopheles stephensi. Methods Anopheles stephensi females were offered a blood-meal on P. berghei infected, gametocytaemic BALB/c mice, treated intraperitoneally with NeemAzal, one hour before feeding. The transmission-blocking activity of the product was evaluated by assessing oocyst prevalence, oocyst density and capacity to infect healthy mice. To characterize the anti-plasmodial effects of NeemAzal® on early midgut stages, i.e. zygotes and ookinetes, Giemsa-stained mosquito midgut smears were examined. Results NeemAzal® completely blocked P. berghei development in the vector, at an azadirachtin dose of 50 mg/kg mouse body weight. The totally 138 examined, treated mosquitoes (three experimental replications) did not reveal any oocyst and none of the healthy mice exposed to their bites developed parasitaemia. The examination of midgut content smears revealed a reduced number of zygotes and post- zygotic forms and the absence of mature ookinetes in treated mosquitoes. Post- zygotic forms showed several morphological alterations, compatible with the hypothesis of an azadirachtin interference with the functionality of the microtubule

Sharma, Dipika Kinsey, William H.

Fertilization begins with binding and fusion of a sperm with the oocyte, a process that triggers a high amplitude calcium transient which propagates through the oocyte and stimulates a series of preprogrammed signal transduction events critical for zygote development. Identification of the pathways downstream of this calcium transient remains an important step in understanding the basis of zygote quality. The present study demonstrates that the calcium-calmodulin sensitive protein tyrosine kinase PYK2 is a target of the fertilization-induced calcium transient in the zebrafish oocyte and that it plays an important role in actin-mediated events critical for sperm incorporation. At fertilization, PYK2 was activated initially at the site of sperm-oocyte interaction and was closely associated with actin filaments forming the fertilization cone. Later PYK2 activation was evident throughout the entire oocyte cortex, however activation was most intense over the animal hemisphere. Fertilization-induced PYK2 activation could be blocked by suppressing calcium transients in the ooplasm via injection of BAPTA as a calcium chelator. PYK2 activation could be artificially induced in unfertilized oocytes by injection of IP3 at concentrations sufficient to induce calcium release. Functionally, suppression of PYK2 activity by chemical inhibition or by injection of a dominant-negative construct encoding the N-terminal ERM domain of PKY2 inhibited formation of an organized fertilization cone and reduced the frequency of successful sperm incorporation. Together, the above findings support a model in which PYK2 responds to the fertilization-induced calcium transient by promoting reorganization of the cortical actin cytoskeleton to form the fertilization cone. PMID:23084926

Sershen Berjak, Patricia Pammenter, N W Wesley-Smith, James

Effects of sequential procedures required for cryopreservation of embryos excised from the recalcitrant seeds of Haemanthus montanus were assessed ultrastructurally and in conjunction with respiratory activity and the rate of protein synthesis. Fresh material (water content, 5.05 ± 0.92 g g(-1) dry mass) afforded ultrastructural evidence of considerable metabolic activity , borne out by respiratory rates. Neither exposure to glycerol nor sucrose as penetrating and non-penetrating cryoprotectants, respectively, brought about degradative changes, although increased vacuolation and autophagy accompanied both, while respiratory and protein synthetic activity were not adversely affected. Glycerol-cryoprotected embryos flash dried to water contents >0.4 g g(-1) showed organised ultrastructural features and considerable autophagy consistent with metabolic activity , and although respiratory activity was lower, protein synthesis rate was enhanced relative to fresh material. However, at water contents 0.4 g g(-1)-in which the degree of vacuolation remained moderate-were rapidly cooled. The outcomes of this study are considered particularly in terms of the stresses imposed by prolonged, relatively slow dehydration and ultimate water contents, on embryos showing considerable metabolic activity .

Kong, Qingran Banaszynski, Laura A Geng, Fuqiang Zhang, Xiaolei Zhang, Jiaming Zhang, Heng O'Neill, Claire L Yan, Peidong Liu, Zhonghua Shido, Koji Palermo, Gianpiero D Allis, C David Rafii, Shahin Rosenwaks, Zev Wen, Duancheng

Derepression of chromatin-mediated transcriptional repression of paternal and maternal genomes is considered the first major step that initiates zygotic gene expression after fertilization. The histone variant H3.3 is present in both male and female gametes and is thought to be important for remodeling the paternal and maternal genomes for activation during both fertilization and embryogenesis. However, the underlying mechanisms remain poorly understood. Using our H3.3B-HA-tagged mouse model, engineered to report H3.3 expression in live animals and to distinguish different sources of H3.3 protein in embryos, we show here that sperm-derived H3.3 (sH3.3) protein is removed from the sperm genome shortly after fertilization and extruded from the zygotes via the second polar bodies (PBII) during embryogenesis. We also found that the maternal H3.3 (mH3.3) protein is incorporated into the paternal genome as early as 2 h postfertilization and is detectable in the paternal genome until the morula stage. Knockdown of maternal H3.3 resulted in compromised embryonic development both of fertilized embryos and of androgenetic haploid embryos. Furthermore, we report that mH3.3 depletion in oocytes impairs both activation of the Oct4 pluripotency marker gene and global de novo transcription from the paternal genome important for early embryonic development. Our results suggest that H3.3-mediated paternal chromatin remodeling is essential for the development of preimplantation embryos and the activation of the paternal genome during embryogenesis. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Epigenetics typically refers to changes in the structure of a chromosome that affect gene activity and expression. Genomic imprinting is a special type of epigenetic phenomenon in which the expression of an allele depends on its parental origin. When an allele inherited from the mother (or father) is imprinted (i.e., silent), it is termed as maternal (or paternal) imprinting. Imprinting is often resulted from DNA methylation and tends to cluster together in the genome [1]. It has been shown to play a key role in many genetic disorders in humans [2]. Imprinting is heritable and undergoes a reprogramming process in gametes before and after fertilization [1]. Sometimes the reprogramming process is not reversible, leading to the loss of imprinting [3]. Although efforts have been made to experimentally or computationally infer imprinting genes, the underlying molecular mechanism that leads to unbalanced allelic expression is still largely unknown.

I isolated a Ciona intestinalis homolog of p53, Ci-p53/p73-a, in a microarray screen of rapidly degraded maternal mRNA by comparing the transcriptomes of unfertilized eggs and 32-cell stage embryos. Higher expression of the gene in eggs and lower expression in later embryonic stages were confirmed by whole-mount in situ hybridization (WISH) and quantitative reverse transcription-PCR (qRT-PCR) expression was ubiquitous in eggs and early embryos. Knockdown of Ci-p53/p73-a by injection of antisense morpholino oligonucleotides (MOs) severely perturbed gastrulation cell movements and expression of notochord marker genes. A key regulator of notochord differentiation in Ciona embryos is Brachyury (Ci-Bra), which is directly activated by a zic-like gene (Ci-ZicL). The expression of Ci-ZicL and Ci-Bra in A-line notochord precursors was downregulated in Ci-p53/p73-a knockdown embryos. Maternal expression of Ci-p53/p73-b, a homolog of Ci-p53/p73-a, was also detected. In Ci-p53/p73-b knockdown embryos, gastrulation cell movements, expression of Ci-ZicL and Ci-Bra in A-line notochord precursors, and expression of notochord marker gene at later stages were perturbed. The upstream region of Ci-ZicL contains putative p53-binding sites. Cis-regulatory analysis of Ci-ZicL showed that these sites are involved in expression of Ci-ZicL in A-line notochord precursors at the 32-cell and early gastrula stages. These results suggest that p53 genes are maternal factors that play a crucial role in A-line notochord differentiation in C. intestinalis embryos by regulating Ci-ZicL expression. Copyright © 2011 Elsevier Inc. All rights reserved.

Witty, Alec D. Mihic, Anton Tam, Roger Y. Fisher, Stephanie A. Mikryukov, Alexander Shoichet, Molly S. Li, Ren-Ke Kattman, Steven J. Keller, Gordon

The epicardium supports cardiomyocyte proliferation early in development and provides fibroblasts and vascular smooth muscle cells to the developing heart. The epicardium has been shown to play an important role during tissue remodeling after cardiac injury, making access to this cell lineage necessary for the study of regenerative medicine. Here we describe the generation of epicardial lineage cells from human pluripotent stem cells by stage-specific activation of the BMP and WNT signaling pathways. These cells display morphological characteristics and express markers of the epicardial lineage, including the transcription factors WT1 and TBX18 and the retinoic acid–producing enzyme ALDH 1 A 2 . When induced to undergo epicardial-tomesenchymal transition, the cells give rise to populations that display characteristics of the fibroblast and vascular smooth muscle lineages. These findings identify BMP and WNT as key regulators of the epicardial lineage in vitro and provide a model for investigating epicardial function in human development and disease. PMID:25240927

Miklavcic, John Janez Flaman, Paul

The fields of biology, medicine, and embryology have described the developmental milestones of humans throughout gestation in great detail. It is less clear as to when humans are recognized as people, persons, or beings with rights that are protected by legislation. The practice of law is irrevocably intertwined with that of ethical conduct and the time at which a human life is considered a person has implications that extend to health care, legislation on abortion, and autonomy of individuals. This article reviews the economical position that fertilization is the moment that personhood of the conceptus begins. Alternate positions proposing that personhood begins at other possible times after fertilization are presented and contrasted to the economical hypothesis. Summary : This article is an original work critically analyzing the various arguments for human personhood at fertilization and thereafter. The various positions on human personhood are compared and contrasted herein. The time of the human lifespan at which personhood is conferred has important implications for health care, legislation, and personal autonomy.

Tartakoff, Alan Michael Jaiswal, Purnima

When haploid cells of Saccharomyces cerevisiae are crossed, parental nuclei congress and fuse with each other. To investigate underlying mechanisms, we have developed assays that evaluate the impact of drugs and mutations. Nuclear congression is inhibited by drugs that perturb the actin and tubulin cytoskeletons. Nuclear envelope (NE) fusion consists of at least five steps in which preliminary modifications are followed by controlled flux of first outer and then inner membrane proteins, all before visible dilation of the waist of the nucleus or coalescence of the parental spindle pole bodies. Flux of nuclear pore complexes occurs after dilation. Karyogamy requires both the Sec18p/NSF ATPase and ER/NE luminal homeostasis. After fusion, chromosome tethering keeps tagged parental genomes separate from each other. The process of NE fusion and evidence of genome independence in yeast provide a prototype for understanding related events in higher eukaryotes.

Hu, Zhilian Holzschuh, Jochen Driever, Wolfgang

DNA damage-binding protein 1 (DDB1) is a large subunit of the heterodimeric DDB complex that recognizes DNA lesions and initiates the nucleotide excision repair process. DDB1 is also a component of the CUL4 E3 ligase complex involved in a broad spectrum of cellular processes by targeted ubiquitination of key regulators. Functions of DDB1 in development have been addressed in several model organisms, however, are not fully understood so far. Here we report an ENU induced mutant ddb1 allele (ddb1m863) identified in zebrafish (Danio rerio), and analyze its effects on development. Zebrafish ddb1 is expressed broadly, both maternally and zygotically , with enhanced expression in proliferation zones. The (ddb1m863 mutant allele affects the splice acceptor site of exon 20, causing a splicing defect that results in truncation of the 1140 amino acid protein after residue 800, lacking part of the β-propeller domain BPC and the C-terminal helical domain CTD. ddb1m863 zygotic mutant embryos have a pleiotropic phenotype, including smaller and abnormally shaped brain, head skeleton, eyes, jaw, and branchial arches, as well as reduced dopaminergic neuron groups. However, early forming tissues develop normally in zygotic ddb1m863 mutant embryos, which may be due to maternal rescue. In ddb1m863 mutant embryos, pcna-expressing proliferating cell populations were reduced, concurrent with increased apoptosis. We also observed a concomitant strong up-regulation of transcripts of the tumor suppressor p53 (tp53) and the cell cycle inhibitor cdkn1a (p21a/bCIP1/WAF1) in proliferating tissues. In addition, transcription of cyclin genes ccna2 and ccnd1 was deregulated in ddb1m863 mutants. Reduction of p53 activity by anti-sense morpholinos alleviated the apoptotic phenotype in ddb1m863 mutants. These results imply that Ddb1 may be involved in maintaining proper cell cycle progression and viability of dividing cells during development through transcriptional mechanisms regulating genes

Bchini, Raphaël Vasiliou, Vasilis Branlant, Guy Talfournier, François Rahuel-Clermont, Sophie

Retinoic acid (RA), a metabolite of vitamin A, exerts pleiotropic effects throughout life in vertebrate organisms. Thus, RA action must be tightly regulated through the coordinated action of biosynthetic and degradating enzymes. The last step of retinoic acid biosynthesis is irreversibly catalyzed by the NAD-dependent retinal dehydrogenases (RALDH), which are members of the aldehyde dehydrogenase (ALDH) superfamily. Low intracellular retinal concentrations imply efficient substrate molecular recognition to ensure high affinity and specificity of RALDHs for retinal. This study addresses the molecular basis of retinal recognition in human ALDH1A1 (or RALDH1) and rat ALDH 1 A 2 (or RALDH2), through the comparison of the catalytic behavior of retinal analogs and use of the fluorescence properties of retinol. We show that, in contrast to long chain unsaturated substrates, the rate-limiting step of retinal oxidation by RALDHs is associated with acylation. Use of the fluorescence resonance energy transfer upon retinol interaction with RALDHs provides evidence that retinal recognition occurs in two steps: binding into the substrate access channel, and a slower structural reorganization with a rate constant of the same magnitude as the kcat for retinal oxidation: 0.18 vs. 0.07 s−1 and 0.25 vs. 0.1 s−1 for ALDH1A1 and ALDH 1 A 2 , respectively. This suggests that the conformational transition of the RALDH-retinal complex significantly contributes to the rate-limiting step that controls the kinetics of retinal oxidation, as a prerequisite for the formation of a catalytically competent Michaelis complex. This conclusion is consistent with the general notion that structural flexibility within the active site of ALDH enzymes has been shown to be an integral component of catalysis. PMID:23220587

Kang, Hee Jung Hwang, Soo Jin Yoon, Jung Ah Jun, Jin Hyun Lim, Hyunjung Jade Yoon, Tae Ki Song, Haengseok

Prostaglandins participate in a variety of female reproductive processes, including ovulation, fertilization, embryo implantation and parturition. In particular, maternal prostacyclin (PGI(2)) is critical for embryo implantation and the action of PGI(2) is not mediated via its G-protein-coupled membrane receptor, IP, but its nuclear receptor, peroxisome-proliferator- activated receptor δ (PPARδ). Recently, several studies have shown that PGI(2) enhances blastocyst development and/or hatching rate in vitro, and subsequently implantation and live birth rates in mice. However, the mechanism by which PGI(2) improves preimplantation embryo development in vitro remains unclear. Using molecular, pharmacologic and genetic approaches, we show that PGI(2)-induced PPARδ activation accelerates blastocyst hatching in mice. mRNAs for PPARδ, retinoid X receptor (heterodimeric partners of PPARδ) and PGI(2) synthase (PGIS) are temporally induced after zygotic gene activation , and their expression reaches maximum levels at the blastocyst stage, suggesting that functional complex of PPARδ can be formed in the blastocyst. Carbaprostacyclin (a stable analogue of PGI(2)) and GW501516 (a PPARδ selective agonist) significantly accelerated blastocyst hatching but did not increase total cell number of cultured blastocysts. Whereas U51605 (a PGIS inhibitor) interfered with blastocyst hatching, GW501516 restored U51605-induced retarded hatching. In contrast to the improvement of blastocyst hatching by PPARδ agonists, PPAR antagonists significantly inhibited blastocyst hatching. Furthermore, deletion of PPARδ at early stages of preimplantation mouse embryos caused delay of blastocyst hatching, but did not impair blastocyst development. Taken together, PGI(2)-induced PPARδ activation accelerates blastocyst hatching in mice.

Kisielewska, J. Philipova, R. Huang, J.-Y. Whitaker, M.

Sea urchins provide an excellent model for studying cell cycle control mechanisms governing DNA replication in vivo. Fertilization and cell cycle progression are tightly coordinated by Ca2+ signals, but the mechanisms underlying the onset of DNA replication after fertilization remain less clear. In this study we demonstrate that calcium-dependent activation of ERK1 promotes accumulation of cyclinE/cdk2 into the male and female pronucleus and entry into first S-phase. We show that cdk2 activity rises quickly after fertilization to a maximum at 4 min, corresponding in timing to the early ERK1 activity peak. Abolishing MAP kinase activity after fertilization with MEK inhibitor, U0126, substantially reduces the early peak of cdk2 activity and prevents cyclinE and cdk2 accumulation in both sperm pronucleus and zygote nucleus in vivo. Both p27kip1 and roscovitine, cdk2 inhibitors, prevented DNA replication suggesting cdk2 involvement in this process in sea urchin. Inhibition of cdk2 activity using p27kip1 had no effect on the phosphorylation of MBP by ERK, but completely abolished phosphorylation of retinoblastoma protein, a cdk2 substrate, indicating that cdk2 activity is downstream of ERK1 activation . This pattern of regulation of DNA synthesis conforms to the pattern observed in mammalian somatic cells. PMID:19665013

Bahia, Ana C Dong, Yuemei Blumberg, Benjamin J Mlambo, Godfree Tripathi, Abhai BenMarzouk-Hidalgo, Omar J Chandra, Ramesh Dimopoulos, George

SUMMARY Malaria parasite transmission requires the successful development of Plasmodium gametocytes into flagellated microgametes upon mosquito blood ingestion, and the subsequent fertilization of microgametes and macrogametes for the development of motile zygotes , called ookinetes, which invade and transverse the Anopheles vector mosquito midgut at around 18-36 h after blood ingestion. Within the mosquito midgut, the malaria parasite has to withstand the mosquito's innate immune response and the detrimental effect of its commensal bacterial flora. We have assessed the midgut colonization capacity of 5 gut bacterial isolates from field-derived, and 2 from laboratory colony, mosquitoes and their effect on Plasmodium development in vivo and in vitro, along with their impact on mosquito survival. Some bacterial isolates activated the mosquito's immune system, affected the mosquito's life span, and were capable of blocking Plasmodium development. We have also shown that the ability of these bacteria to inhibit the parasites is likely to involve different mechanisms and factors. A Serratia marcescens isolate was particularly efficient in colonizing the mosquitoes’ gut, compromising mosquito survival, and inhibiting both sexual- and asexual-stage Plasmodium through secreted factors, thereby rendering it a potential candidate for the development of a malaria transmission intervention strategy. PMID:24428613

Foe, Victoria E. von Dassow, George

The cytokinetic furrow arises from spatial and temporal regulation of cortical contractility. To test the role microtubules play in furrow specification, we studied myosin II activation in echinoderm zygotes by assessing serine19-phosphorylated regulatory light chain (pRLC) localization after precisely timed drug treatments. Cortical pRLC was globally depressed before cytokinesis, then elevated only at the equator. We implicated cell cycle biochemistry (not microtubules) in pRLC depression, and differential microtubule stability in localizing the subsequent myosin activation . With no microtubules, pRLC accumulation occurred globally instead of equatorially, and loss of just dynamic microtubules increased equatorial pRLC recruitment. Nocodazole treatment revealed a population of stable astral microtubules that formed during anaphase among these, those aimed toward the equator grew longer, and their tips coincided with cortical pRLC accumulation. Shrinking the mitotic apparatus with colchicine revealed pRLC suppression near dynamic microtubule arrays. We conclude that opposite effects of stable versus dynamic microtubules focuses myosin activation to the cell equator during cytokinesis. PMID:18955555

Ouellette, Marie-Hélène Martin, Emmanuel Lacoste-Caron, Germain Hamiche, Karim Jenna, Sarah

Collective epithelial cell migration requires the maintenance of cell-cell junctions while enabling the generation of actin-rich protrusions at the leading edge of migrating cells. Ventral enclosure of Caenorhabditis elegans embryos depends on the collective migration of anterior-positioned leading hypodermal cells towards the ventral midline where they form new junctions with their contralateral neighbours. In this study, we characterized the zygotic function of RGA-7/SPV-1, a CDC-42/Cdc42 and RHO-1/RhoA-specific Rho GTPase- activating protein, which controls the formation of actin-rich protrusions at the leading edge of leading hypodermal cells and the formation of new junctions between contralateral cells. We show that RGA-7 controls these processes in an antagonistic manner with the CDC-42's effector WSP-1/N-WASP and the CDC-42-binding proteins TOCA-1/2/TOCA1. RGA-7 is recruited to spatially distinct locations at junctions between adjacent leading cells, where it promotes the accumulation of clusters of activated CDC-42. It also inhibits the spreading of these clusters towards the leading edge of the junctions and regulates their accumulation and distribution at new junctions formed between contralateral leading cells. Our study suggests that RGA-7 controls collective migration and junction formation between epithelial cells by spatially restricting active CDC-42 within cell-cell junctions. © The Author (2015). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

Lian, Hua-Yu Jiao, Guang-Zhong Wang, Hui-Li Tan, Xiu-Wen Wang, Tian-Yang Zheng, Liang-Liang Kong, Qiao-Qiao Tan, Jing-He

Although fusion of nucleoli was observed during pronuclear development of zygotes and the behavior of nucleoli in pronuclei has been suggested as an indicator of embryonic developmental potential, the mechanism for nucleolar fusion is unclear. Although both cytoskeleton and the nucleolus are important cellular entities, there are no special reports on the relationship between the two. Role of cytoskeleton in regulating fusion of nucleoli was studied using the activated mouse oocyte model. Mouse oocytes were cultured for 6 h in activating medium (Ca²⁺-free CZB medium containing 10 mM SrCl₂) supplemented with or without inhibitors for cytoskeleton or protein synthesis before pronuclear formation, nucleolar fusion, and the activity of maturation-promoting factor (MPF) were examined. Whereas treatment with microfilament inhibitor cytochalasin D or B or intermediate filament inhibitor acrylamide suppressed nucleolar fusion efficiently, treatment with microtubule inhibitor demecolcine or nocodazole or protein synthesis inhibitor cycloheximide had no effect. The cytochalasin D- or acrylamide-sensitive temporal window coincided well with the reported temporal window for nucleolar fusion in activated oocytes. Whereas a continuous incubation with demecolcine prevented pronuclear formation, pronuclei formed normally when demecolcine was excluded during the first hour of activation treatment when the MPF activity dropped dramatically. The results suggest that 1) microfilaments and intermediate filaments but not microtubules support nucleolar fusion, 2) proteins required for nucleolar fusion including microfilaments and intermediate filaments are not de novo synthesized, and 3) microtubule disruption prevents pronuclear formation by activating MPF. © 2014 by the Society for the Study of Reproduction, Inc.

Matsukuma, S Nakatsuru, Y Nakagawa, K Utakoji, T Sugano, H Kataoka, H Sekiguchi, M Ishikawa, T

The E. coli ada gene encodes O6-methylguanine DNA methyltransferase (O6MTase) which repairs the methylation of guanine at the O6 position in DNA. After recombination with a Chinese hamster metallothionein I gene promoter, the ada gene was microinjected into C3H/HeN mouse zygotes . Eventually, transgenic mice containing the ada fusion DNA were generated. The integrated ada DNA complex was transmitted to the progeny in a mode conforming to tandem integration at a single chromosome site, and homozygotes were also obtained from an inter-transgenic mouse cross. RNA transcripts of the chimeric ada gene were identified in the livers of these transgenic mice using dot and Northern blot analyses. O6MTase activity was increased in the liver of transgenic mice of line No. 708, and was more than 3 times the activity found in non-transgenic mice, especially in the transgenic homozygotes. The ada gene product was detected in the liver of a transgenic homozygote by immunoblot analysis. These transgenic mice have great potential for analysis of the role played by O6MTase in chemical carcinogenesis.

Bryńska, Anita Lipińska, Elzbieta Matelska, Monika

Repetitive and stereotyped behaviours in the form of stereotyped interests or specific routine activities are one ofthe diagnostic criteria in pervasive developmental disorders. The occurrence of repetitive behaviours in patients with pervasive developmental disorders is a starting point for questions about the type and classification criteria of such behaviours. The aim of the article is to present case studies of patients with pervasive developmental disorders and co-morbid symptoms in the form of routine activities , tics, obsessive-compulsive symptoms or stereotyped behaviours. The first case study describes a patient with Asperger's syndrome and obsessive compulsive symptoms. The diagnostic problems regarding complex motor tics are discussed in the second case study which describes a patient with Asperger's syndrome and Gilles de la Tourette syndrome. The third and fourth case study describes mono- zygotic twins with so called High Functioning Autism whose repetitive activities point to either obsessive compulsive symptoms, stereotypic movements, need for absolute consistency or echopraxia. The possible comorbidity of pervasive developmental disorders and symptoms in the form of repetitive behaviours, possible interactions as well as diagnostic challenges is discussed in the article.

Chen, Yuting Lu, Wenqing Gao, Na Long, Yi Shao, Yanjiao Liu, Meizhen Chen, Huaqing Ye, Shixin Ma, Xueyun Liu, Mingyao Li, Dali

The laboratory rat is a valuable mammalian model organism for basic research and drug discovery. Here we demonstrate an efficient methodology by applying transcription activator -like effector nucleases (TALENs) technology to generate Leptin receptor (Lepr) knockout rats on the Sprague Dawley (SD) genetic background. Through direct injection of in vitro transcribed mRNA of TALEN pairs into SD rat zygotes , somatic mutations were induced in two of three resulting pups. One of the founders carrying bi-allelic mutation exhibited early onset of obesity and infertility. The other founder carried a chimeric mutation which was efficiently transmitted to the progenies. Through phenotyping of the resulting three lines of rats bearing distinct mutations in the Lepr locus, we found that the strains with a frame-shifted or premature stop codon mutation led to obesity and metabolic disorders. However, no obvious defect was observed in a strain with an in-frame 57 bp deletion in the extracellular domain of Lepr. This suggests the deleted amino acids do not significantly affect Lepr structure and function. This is the first report of generating the Lepr mutant obese rat model in SD strain through a reverse genetic approach. This suggests that TALEN is an efficient and powerful gene editing technology for the generation of disease models.

King, Ryan S. Maiden, Stephanie L. Hawkins, Nancy C. Kidd, Ambrose R. Kimble, Judith Hardin, Jeff Walston, Timothy D.

Dishevelleds are modular proteins that lie at the crossroads of divergent Wnt signaling pathways. The DIX domain of dishevelleds modulates a β-catenin destruction complex, and thereby mediates cell fate decisions through differential activation of Tcf transcription factors. The DEP domain of dishevelleds mediates planar polarity of cells within a sheet through regulation of actin modulators. In Caenorhabditis elegans asymmetric cell fate decisions are regulated by asymmetric localization of signaling components in a pathway termed the Wnt/β-catenin asymmetry pathway. Which domain(s) of Disheveled regulate this pathway is unknown. We show that C. elegans embryos from dsh-2(or302) mutant mothers fail to successfully undergo morphogenesis, but transgenes containing either the DIX or the DEP domain of DSH-2 are sufficient to rescue the mutant phenotype. Embryos lacking zygotic function of SYS-1/β-catenin, WRM-1/β-catenin, or POP-1/Tcf show defects similar to dsh-2 mutants, including a loss of asymmetry in some cell fate decisions. Removal of two dishevelleds (dsh-2 and mig-5) leads to a global loss of POP-1 asymmetry, which can be rescued by addition of transgenes containing either the DIX or DEP domain of DSH-2. These results indicate that either the DIX or DEP domain of DSH-2 is capable of activating the Wnt/β-catenin asymmetry pathway and regulating anterior–posterior fate decisions required for proper morphogenesis. PMID:19298786

Perisé-Barrios, Ana J. Gómez, Rafael Corbí, Angel L. de La Mata, Javier Domínguez-Soto, Angeles Muñoz-Fernandez, María A.

Tumor microenvironment favors the escape from immunosurveillance by promoting immunosuppression and blunting pro-inflammatory responses. Since most tumor-associated macrophages (TAM) exhibit an M2-like tumor cell growth promoting polarization, we have studied the role of 2G-03NN24 carbosilane dendrimer in M2 macrophage polarization to evaluate the potential application of dendrimers in tumor immunotherapy. We found that the 2G-03NN24 dendrimer decreases LPS-induced IL-10 production from in vitro generated monocyte-derived M2 macrophages, and also switches their gene expression profile towards the acquisition of M1 polarization markers (INHBA, SERPINE1, FLT1, EGLN3 and ALDH 1 A 2 ) and the loss of M2 polarization-associated markers (EMR1, IGF1, FOLR2 and SLC40A1). Furthermore, 2G-03NN24 dendrimer decreases STAT3 activation . Our results indicate that the 2G-03NN24 dendrimer can be a useful tool for antitumor therapy by virtue of its potential ability to limit the M2-like polarization of TAM.Tumor microenvironment favors the escape from immunosurveillance by promoting immunosuppression and blunting pro-inflammatory responses. Since most tumor-associated macrophages (TAM) exhibit an M2-like tumor cell growth promoting polarization, we have studied the role of 2G-03NN24 carbosilane dendrimer in M2 macrophage polarization to evaluate the potential application of dendrimers in tumor immunotherapy. We found that the 2G-03NN24 dendrimer decreases LPS-induced IL-10 production from in vitro generated monocyte-derived M2 macrophages, and also switches their gene expression profile towards the acquisition of M1 polarization markers (INHBA, SERPINE1, FLT1, EGLN3 and ALDH 1 A 2 ) and the loss of M2 polarization-associated markers (EMR1, IGF1, FOLR2 and SLC40A1). Furthermore, 2G-03NN24 dendrimer decreases STAT3 activation . Our results indicate that the 2G-03NN24 dendrimer can be a useful tool for antitumor therapy by virtue of its potential ability to limit the M2-like polarization of TAM

Simmons, Michael J Haley, Kevin J Grimes, Craig D Raymond, John D Fong, Joseph C L

Fusions between the Drosophila hsp70 promoter and three different incomplete P elements, KP, SP, and BP1, were inserted into the Drosophila genome by means of hobo transformation vectors and the resulting transgenic stocks were tested for repression of P-element transposase activity . Only the H(hsp/KP) transgenes repressed transposase activity , and the degree of repression was comparable to that of a naturally occurring KP element. The KP transgenes repressed transposase activity both with and without heat-shock treatments. Both the KP element and H(hsp/KP) transgenes repressed the transposase activity encoded by the modified P element in the P(ry(+), Delta2-3)99B transgene more effectively than that encoded by the complete P element in the H(hsp/CP)2 transgene even though the P(ry(+), Delta2-3)99B transgene was the stronger transposase source. Repression of both transposase sources appeared to be due to a zygotic effect of the KP element or transgene. There was no evidence for repression by a strictly maternal effect nor was there any evidence for enhancement of KP repression by the joint maternal transmission of H(hsp/KP) and H(hsp/CP) transgenes. These results are consistent with the idea that KP-mediated repression of P-element activity involves a KP-repressor polypeptide that is not maternally transmitted and that KP-mediated repression is not strengthened by the 66-kD repressor produced by complete P elements through alternate splicing of their RNA. PMID:12019235

An operational amplifier-type active filter in which the only capacitor in the circuit is the compensating capacitance of the operational amplifiers, the various feedback and coupling elements being essentially solely resistive.

Itou, Junji Akiyama, Ryutaro Pehoski, Steve Yu, Xiaodan Kawakami, Hiroko Kawakami, Yasuhiko

Background The zebrafish heart regenerates after various severe injuries. Common processes of heart regeneration are cardiomyocyte proliferation, activation of epicardial tissue and neovascularization. In order to further characterize heart regeneration processes, we introduced milder injuries and compared responses to those induced by ventricular apex resection, a widely used injury method. We used scratching of the ventricular surface and puncturing of the ventricle with a fine tungsten needle as injury inducing techniques. Results Scratching the ventricular surface induced subtle cardiomyocyte proliferation and responses of the epicardium. Endothelial cell accumulation was limited to the surface of the heart. Ventricular puncture induced cardiomyocyte proliferation, endocardial and epicardial activation and neo-vascularization, similar to the resection method. However, the degree of the responses was milder, correlating with milder injury. Sham operation induced epicardial aldh 1 a 2 expression but not tbx18 and WT1. Conclusions Puncturing the ventricle induces responses equivalent to resection at milder degrees in a shorter time frame and would be used as simple injury model. Scratching the ventricle did not induce heart regeneration and would be used for studying wound responses to epicardium. PMID:25074230

Nakajima, Tadaaki Iguchi, Taisen Sato, Tomomi

The Müllerian duct develops into the oviduct, uterus, and vagina, all of which are quite distinct in their morphology and function. The epithelial fate of these female reproductive organs in developing mice is determined by factors secreted from the stroma however, how stromal differentiation occurs in the female reproductive organs derived from the Müllerian duct is still unclear. In the present study, roles of retinoic acid (RA) signaling in developing female reproductive tracts were investigated. Retinol dehydrogenase 10 (RDH10) and aldehyde dehydrogenase family 1 subfamily A2 ( ALDH 1 A 2 ) mRNAs and proteins and transactivation activity of endogenous RA were found in the stroma of proximal Müllerian ducts and gradually decreased from the proximal to caudal regions in fetal mice. In organ-cultured Müllerian ducts, retinaldehyde or RA treatment induced uterine epithelial differentiation, defined as a layer of columnar epithelial cells negative for oviductal and vaginal epithelial markers. In contrast, inhibition of RA receptor (RAR) signaling induced vaginal epithelial differentiation, characterized as vaginal epithelial marker genes–positive stratified epithelium. Grafting experiments of the organ-cultured Müllerian duct revealed irreversible epithelial fate determination. Although RAR did not directly bind to the homeobox A10 (Hoxa10) promoter region, RA–RAR signaling stimulated Hoxa10 expression. Thus, RA–RAR signaling in the Müllerian duct determines the fate of stroma to form the future uterus and vagina. PMID:27911779

Troncoso-Ponce, Manuel Adrián Barthole, Guillaume Tremblais, Geoffrey

In angiosperms, double fertilization of the embryo sac initiates the development of the embryo and the endosperm. In Arabidopsis thaliana, an exalbuminous species, the endosperm is reduced to one cell layer during seed maturation and reserves such as oil are massively deposited in the enlarging embryo. Here, we consider the strikingly different fatty acid (FA) compositions of the oils stored in the two zygotic tissues. Endosperm oil is enriched in ω-7 monounsaturated FAs, that represent more than 20 mol% of total FAs, whereas these molecular species are 10-fold less abundant in the embryo. Two closely related transcription factors, MYB118 and MYB115, are transcriptionally induced at the onset of the maturation phase in the endosperm and share a set of transcriptional targets. Interestingly, the endosperm oil of myb115 myb118 double mutants lacks ω-7 FAs. The identification of two Δ9 palmitoyl-ACP desaturases responsible for ω-7 FA biosynthesis, which are activated by MYB115 and MYB118 in the endosperm, allows us to propose a model for the transcriptional control of oil FA composition in this tissue. In addition, an initial characterization of the structure-function relationship for these desaturases reveals that their particular substrate specificity is conferred by amino acid residues lining their substrate pocket that distinguish them from the archetype Δ9 stearoyl-ACP desaturase. PMID:27681170

Wang, Qian Gosik, Kirk Xing, Sujuan Jiang, Libo Sun, Lidan Chinchilli, Vernon M. Wu, Rongling

Epigenetic reprogramming is thought to play a critical role in maintaining the normal development of embryos. How the methylation state of paternal and maternal genomes regulates embryogenesis depends on the interaction and coordination of the gametes of two sexes. While there is abundant research in exploring the epigenetic interactions of sperms and oocytes, a knowledge gap exists in the mechanistic quantitation of these interactions and their impact on embryo development. This review aims at formulating a modeling framework to address this gap through the integration and synthesis of evolutionary game theory and the latest discoveries of the epigenetic control of embryo development by next-generation sequencing. This framework, named epigenetic game theory or epiGame, views embryogenesis as an ecological system in which two highly distinct and specialized gametes coordinate through either cooperation or competition, or both, to maximize the fitness of embryos under Darwinian selection. By implementing a system of ordinary differential equations, epiGame quantifies the pattern and relative magnitude of the methylation effects on embryogenesis by the mechanisms of cooperation and competition. epiGame may gain new insight into reproductive biology and can be potentially applied to design personalized medicines for genetic disorder intervention.

Most oocytes eliminate their centrioles during meiotic divisions through unclear mechanisms. In this issue, Borrego-Pinto et al. (2016. J Cell. Biol. http://dx.doi.org/10.1083/jcb.201510083) show that mother centrioles need to be eliminated from starfish oocytes by extrusion into the polar bodies for successful embryo development. © 2016 Verlhac.

Evidence for spatial variations in cytoplasmic free Ca 2+ in tip-growing cells is briefly summarized. Methods are described for detecting such gradients using fura-2 with dual wavelength excitation fluorescence microscopy. Results so far indicate that gradients of Ca 2+ are present in growing rhizoid cells but physiologically significant gradients have not yet been detected in the early stages of polarization.

The domestic pig is an important “dual purpose” animal model for agricultural and biomedical applications. There is an emerging consensus in the biomedical community that even though mouse is a powerhouse genetic model, there is a requirement for large animal models such as pigs that can either ser.

Wang, Qian Gosik, Kirk Xing, Sujuan Jiang, Libo Sun, Lidan Chinchilli, Vernon M Wu, Rongling

Epigenetic reprogramming is thought to play a critical role in maintaining the normal development of embryos. How the methylation state of paternal and maternal genomes regulates embryogenesis depends on the interaction and coordination of the gametes of two sexes. While there is abundant research in exploring the epigenetic interactions of sperms and oocytes, a knowledge gap exists in the mechanistic quantitation of these interactions and their impact on embryo development. This review aims at formulating a modeling framework to address this gap through the integration and synthesis of evolutionary game theory and the latest discoveries of the epigenetic control of embryo development by next-generation sequencing. This framework, named epigenetic game theory or epiGame, views embryogenesis as an ecological system in which two highly distinct and specialized gametes coordinate through either cooperation or competition, or both, to maximize the fitness of embryos under Darwinian selection. By implementing a system of ordinary differential equations, epiGame quantifies the pattern and relative magnitude of the methylation effects on embryogenesis by the mechanisms of cooperation and competition. epiGame may gain new insight into reproductive biology and can be potentially applied to design personalized medicines for genetic disorder intervention. Copyright © 2016 Elsevier B.V. All rights reserved.

Two nonoverlapping autosomal inversions defined unusual neo-sex chromosomes in the Hessian fly (Mayetiola destructor). Like other neo-sex chromosomes, these were normally heterozygous, present only in one sex, and suppressed recombination around a sex-determining master switch. Their unusual propert.

Maheshwari, Shamoni Tan, Ek Han West, Allan Franklin, F. Chris H. Comai, Luca

The point of attachment of spindle microtubules to metaphase chromosomes is known as the centromere. Plant and animal centromeres are epigenetically specified by a centromere-specific variant of Histone H3, CENH3 (a.k.a. CENP-A). Unlike canonical histones that are invariant, CENH3 proteins are accumulating substitutions at an accelerated rate. This diversification of CENH3 is a conundrum since its role as the key determinant of centromere identity remains a constant across species. Here, we ask whether naturally occurring divergence in CENH3 has functional consequences. We performed functional complementation assays on cenh3-1, a null mutation in Arabidopsis thaliana, using untagged CENH3s from increasingly distant relatives. Contrary to previous results using GFP-tagged CENH3, we find that the essential functions of CENH3 are conserved across a broad evolutionary landscape. CENH3 from a species as distant as the monocot Zea mays can functionally replace A. thaliana CENH3. Plants expressing variant CENH3s that are fertile when selfed show dramatic segregation errors when crossed to a wild-type individual. The progeny of this cross include hybrid diploids, aneuploids with novel genetic rearrangements and haploids that inherit only the genome of the wild-type parent. Importantly, it is always chromosomes from the plant expressing the divergent CENH3 that missegregate. Using chimeras, we show that it is divergence in the fast-evolving N-terminal tail of CENH3 that is causing segregation errors and genome elimination. Furthermore, we analyzed N-terminal tail sequences from plant CENH3s and discovered a modular pattern of sequence conservation. From this we hypothesize that while the essential functions of CENH3 are largely conserved, the N-terminal tail is evolving to adapt to lineage-specific centromeric constraints. Our results demonstrate that this lineage-specific evolution of CENH3 causes inviability and sterility of progeny in crosses, at the same time producing karyotypic variation. Thus, CENH3 evolution can contribute to postzygotic reproductive barriers. PMID:25622028

Špírek, Mário Poláková, Silvia Jatzová, Katarína Sulo, Pavol

Nucleo-mitochondrial interactions, particularly those determining the primary divergence of biological species, can be studied by means of xenomitochondrial cybrids, which are cells where the original mitochondria are substituted by their counterparts from related species. Saccharomyces cerevisiae cybrids are prepared simply by the mating of the ρ0 strain with impaired karyogamy and germinating spores from other Saccharomyces species and fall into three categories. Cybrids with compatible mitochondrial DNA (mtDNA) from Saccharomyces paradoxus CBS 432 and Saccharomyces cariocanus CBS 7994 are metabolically and genetically similar to cybrids containing mtDNA from various S. cerevisiae. Cybrids with mtDNA from other S. paradoxus strains, S. cariocanus, Saccharomyces kudriavzevii, and Saccharomyces mikatae require a period of adaptation to establish efficient oxidative phosphorylation. They exhibit a temperature-sensitive phenotype, slower growth rate on a non-fermentable carbon source and a long lag phase after the shift from glucose. Their decreased respiration capacity and reduced cytochrome aa3 content is associated with the inefficient splicing of cox1I3β, the intron found in all Saccharomyces species but not in S. cerevisiae. The splicing defect is compensated in cybrids by nuclear gain-of-function and can be alternatively suppressed by overexpression of MRP13 gene for mitochondrial ribosomal protein or the MRS2, MRS3, and MRS4 genes involved in intron splicing. S. cerevisiae with Saccharomyces bayanus mtDNA is unable to respire and the growth on ethanol–glycerol can be restored only after mating to some mit− strains. The nucleo-mitochondrial compatibility limit of S. cerevisiae and other Saccharomyces was set between S. kudriavzevii and S. bayanus at the divergence from S. cerevisiae about 15 MYA. The MRS1-cox1 S. cerevisiae/S. paradoxus cytonuclear Dobzhansky–Muller pair has a neglible impact on the separation of species since its imperfection is compensated for by gain-of-function mutation. PMID:25628643

Describes a science activity on the importance of meiosis for variability. Uses a coin flip to demonstrate the random arrangement of genetic materials and explains how this results in zygotes with a new DNA combination. (YDS)

Alcaín, Francisco J Villalba, José M

Sirtuin 1-7 (SIRT1-7) are deacetylases that are dependent on NAD(+) for their activity . SIRT1 down-regulates p53 activity , increasing lifespan, cell survival, and neuroprotection it also deacetylates peroxisome proliferator- activated receptor-gamma and its coactivator 1alpha, promoting fat mobilization, increasing mitochondrial size and number, and positively regulating insulin secretion. Sirtuins link nutrient availability and energy metabolism. Calorie restriction, which increases lifespan and is beneficial in age-related disorders, activates sirtuin. Major efforts are thus focused to developing sirtuin activators . After discussing the potential involvement of sirtuins in pathophysiological processes, this review looks at new, synthetic sirtuin activators . To date, resveratrol is the most potent natural compound able to activate SIRT1, mimicking the positive effect of calorie restriction. Resveratrol might help in the treatment or prevention of obesity and in preventing the aging-related decline in heart function and neuronal loss. As resveratrol has low bioavailability and interacts with multiple molecular targets, the development of new molecules with better bioavailability and targeting sirtuin at lower concentrations is a promising field of the medicinal chemistry. New SIRT1 activators that are up to 1000 times more effective than resveratrol have recently been identified. These improve the response to insulin and increase the number and activity of mitochondria in obese mice. Human trials with a formulation of resveratrol with improved bioavailability and with a synthetic SIRT1 activator are in progress.

McAdon, Mark H. Nickias, Peter N. Marks, Tobin J. Schwartz, David J.

A catalyst activator particularly adapted for use in the activation of metal complexes of metals of Group 3-10 for polymerization of ethylenically unsaturated polymerizable monomers, especially olefins, comprising two Group 13 metal or metalloid atoms and a ligand structure including at least one bridging group connecting ligands on the two Group 13 metal or metalloid atoms.

Minneapolis Independent School District 275, Minn.

Twenty-four activities suitable for outdoor use by elementary school children are outlined. Activities designed to make children aware of their environment include soil painting, burr collecting, insect and pond water collecting, studies of insect galls and field mice, succession studies, and a model of natural selection using dyed toothpicks. A…

This document consists of activities and references for teaching astronomy. The activities (which include objectives, list of materials needed, and procedures) focus on: observing the Big Dipper and locating the North Star examining the Big Dipper's stars making and using an astrolabe examining retograde motion of Mars measuring the Sun's…

. is used to treat poisonings, reduce intestinal gas (flatulence), lower cholesterol levels, prevent hangover, and treat bile . lower cholesterol levels in the blood. Decreasing gas (flatulence). Some studies show that activated charcoal is effective .

. Health Benefits 1 of 8 sections The Basics: Health Benefits What are the benefits of physical activity ? Physical . a disability . Next section Get Started Previous section Health Benefits 3 of 8 sections Take Action! Take Action: .

Tan, Xiu-Wen Ji, Chang-Li Zheng, Liang-Liang Zhang, Jie Yuan, Hong-Jie Gong, Shuai Zhu, Jiang Tan, Jing-He

What are the mechanisms by which corticotrophin-releasing hormone (CRH) and corticosterone impair the development of preimplantation embryos in the oviduct. CRH and corticosterone do not affect preimplantation embryos directly, but impair their development indirectly by triggering apoptosis of oviductal epithelial cells (OECs) through activation of the Fas system. Studies report that stress impairs embryo development with facilitated secretion of CRH and glucocorticoids. Although an in vivo study demonstrated that preimplantation stress impaired embryo development in conjunction with oviductal apoptosis and activation of the Fas system, whether CRH or glucocorticoids damage embryos directly or indirectly by way of oviductal cells remains to be clarified. Mice of Kunming strain, the generalized lymphoproliferative disorder (gld) mice with a germline mutation F273L in Fas ligand in a C57BL/6J genomic background and the wild-type C57BL/6J mice were used. Female mice were used 8-10 weeks after birth. While some female mice were killed 48 h after being injected with equine CG to collect oviducts and prepare OECs, others were killed to recover zygotes after mating with males following superovulation with eCG and hCG. The zygotes obtained were cultured with or without CRH or corticosterone (CRH/Cort) either in Chatot-Ziomek-Bavister (CZB) medium with or without OECs or in conditioned medium (CM) conditioned with OECs pretreated or not with CRH/Cort. Preimplantation development, levels of redox potential and apoptosis, and expression of CRH receptor 1 (CRHR1), glucocorticoid receptor (GR), Fas and 11β-hydroxysteroid dehydrogenase (HSD) were observed in embryos recovered at different times of in vitro culture. After culture of OECs with or without CRH/Cort, levels of redox potential and apoptosis, mRNA and protein expression of growth factors, and protein expression of CRHR1, GR and Fas were examined in OECs and the level of FasL was measured in CM. The gld mice were used

Mornet, René Theiler, Jane B. Leonard, Nelson J. Schmitz, Ruth Y. Moore, F. Hardy Skoog, Folke

Four series of azidopurines have been synthesized and tested for cytokinin activity in the tobacco callus bioassay: 2- and 8-azido-N6-benzyladenines, -N6-(Δ2-isopentenyl)adenines, and -zeatins, and N6-(2- and 4-azidobenzyl)adenines. The compounds having 2-azido substitution on the adenine ring are as active as the corresponding parent compounds, while those with 8-azido substitution are about 10 or more times as active . The 8-azidozeatin, which is the most active cytokinin observed, exhibited higher than minimal detectable activity at 1.2 × 10−5 micromolar, the lowest concentration tested. The shape of the growth curve indicates that even a concentration as low as 5 × 10−6 micromolar would probably be effective. By comparison, the lowest active concentration ever reported for zeatin has been 5 × 10−5 micromolar, representing a sensitivity rarely attained. All of the azido compounds have been submitted to photolysis in aqueous ethanol, and the photoproducts have been detected and identified by low and high resolution mass spectrometry. They are rationalized as products of abstraction and insertion reactions of the intermediate nitrenes. The potential of the major released products as cytokinins was also assessed by bioassay. 2-Azido-N6-(Δ2-isopentenyl)adenine competed with [14C]kinetin for the cytokinin-binding protein isolated from wheat germ. When the azido compound was photolysed in the presence of this protein, its attachment effectively blocked the binding of [14C]kinetin. PMID:16661017

Research on the use of active microwaves in remote sensing, presented during plenary and poster sessions, is summarized. The main highlights are: calibration techniques are well understood innovative modeling approaches have been developed which increase active microwave applications (segmentation prior to model inversion, use of ERS-1 scatterometer, simulations) polarization angle and frequency diversity improves characterization of ice sheets, vegetation, and determination of soil moisture (X band sensor study) SAR (Synthetic Aperture Radar) interferometry potential is emerging use of multiple sensors/extended spectral signatures is important (increase emphasis).

Presents the 1978 literature review of wastewater treatment. This review covers: (1) activated sludge process (2) process control (3) oxygen uptake and transfer (4) phosphorus removal (5) nitrification (6) industrial wastewater and (7) aerobic digestion. A list of 136 references is also presented. (HM)

Leaf activities can provide a means of using basic concepts of outdoor education to learn in elementary level subject areas. Equipment needed includes leaves, a clipboard with paper, and a pencil. A bag of leaves may be brought into the classroom if weather conditions or time do not permit going outdoors. Each student should pick a leaf, examine…

Nickel, Kai Pardàs, Montse Stiefelhagen, Rainer Canton, Cristian Landabaso, José Luis Casas, Josep R.

When a person enters a room, he or she immediately develops a mental concept about “what is going on” in the room for example, people may be working in the room, people may be engaged in a conversation, or the room may be empty. The CHIL services depend on just the same kind of semantic description, which is termed activity in the following. The “Connector” or the “Memory Jog”, for example, could provide support that is appropriate for the given context if it knew about the current activity at the user’s place. This kind of higher-level understanding of human interaction processes could then be used, e.g., for rating the user’s current availability in a certain situation.

A review such as this can do no more than provide an indication of the issues involved in using autonomic activity as a means of providing a "window on the brain." Several points arise. One of the most important is that of careful and appropriate use of techniques available. One well-known textbook of experimental psychology published some time ago advocated the use of two dimes applied to the palm of the hand for the measurement of electrodermal activity . It was this sort of recommendation that led to the use of psychophysiological measurement falling into disrepute. As indicated in the second section, it is important to understand fully the peripheral mechanisms involved before measurement of electrodermal activity can be usefully carried out. Appropriate use of silver/silver chloride electrodes and physiologically appropriate levels of saline in the electrolyte medium can lead to accurate and repeatable measurement where artefact is not carelessly introduced. Equally important is the context in which studies are carried out. The psychological invasiveness of the technique is important to recognize, and it is here that measurement of autonomic activity probably scores over other methods that are available insofar as very little restriction of the subject is required and the number of transducers that must be applied is minimal. The measurement of autonomic activity within the totality of the experimental context is all important. As an example Dawson and Schell investigated the SCR to words which had previously been associated with shock. When these words were presented to the ear to which attention was not directed in a dichotic listening paradigm, an SCR could be elicited although the subject was unaware of the presentation of the stimulus. The importance of the Dawson and Schell study was the care that they took to make sure that the subject really was unaware of the critical stimulus and had not momentarily switched attention from the attended ear. More

Analgesics are agents which selectively relieve pain by acting in the CNS and peripheral pain mediators without changing consciousness. Analgesics may be narcotic or non-narcotic. The study of pain in animals raises ethical, philosophical, and technical problems. Both peripheral and central pain models are included to make the test more evident for the analgesic property of the plant. This chapter highlights methods such as hot plate and formalin and acetic acid-induced pain models to check the analgesic activity of medicinal plants.

This study is part of a series of Studies in Geophysics that have been undertaken for the Geophysics Research Forum by the Geophysics Study Committee. One purpose of each study is to provide assessments from the scientific community to aid policymakers in decisions on societal problems that involve geophysics. An important part of such assessments is an evaluation of the adequacy of current geophysical knowledge and the appropriateness of current research programs as a source of information required for those decisions. The study addresses our current scientific understanding of active tectonics --- particularly the patterns and rates of ongoing tectonicmore » processes. Many of these processes cannot be described reasonably using the limited instrumental or historical records however, most can be described adequately for practical purposes using the geologic record of the past 500,000 years. A program of fundamental research focusing especially on Quaternary tectonic geology and geomorphology, paleoseismology, neotectonics, and geodesy is recommended to better understand ongoing, active tectonic processes. This volume contains 16 papers. Individual papers are indexed separately on the Energy Database.« less

Wani, Nisar A Hong, Seungbum

Experiments were conducted to investigate the development of in vitro matured camel oocytes after their intra-cytoplasmic sperm injection (ICSI) with epididymal sperm collected from slaughtered male camels. Ovaries and testes were collected from a local slaughterhouse in normal saline solution (NSS) at 37 °C and on ice (0-1 °C), respectively. Cumulus-oocyte complexes (COCs) aspirated from the follicles were randomly distributed to 4-well culture plates (20-25 COCs/well) containing 500 μL of maturation medium and cultured at 38.5 °C in an atmosphere of 5% CO 2 in air for about 30 h. Spermatozoa were collected from the cauda epididymites in syringes containing 2-3 mL of tris-tes egg yolk extender. They were cooled down slowly and stored at refrigeration (4 °C) temperature. On the day of use, spermatozoa were prepared by the swim up technique before use in ICSI. The injected oocytes were either activated by ionomycin and roscovitine or put into the culture without any chemical activation . In Experiment 1, presumptive zygotes were fixed and stained with Hoechst 33342 for evaluation of fertilization after 18 h of culture, while, in Experiment 2, they were cultured in 500 μL of the culture medium at 38.5 °C in an atmosphere of 5% CO 2, 5% O 2 and 90% N 2 in air for 7 days to evaluate their development. The proportion of oocytes activated when ICSI was followed by chemical activation was significantly higher (P activated ones. In experiment 2, a higher number of oocytes cleaved (59 vs. 35%) and developed to blastocysts (20 vs. 7%) in the group with post-ICSI activation when compared with the group without chemical activation , respectively. In conclusion, to the best of our knowledge, this is the first report where embryos were produced by ICSI in camels. Chemical activation of oocytes by ionomycin and roscovitine, post -ICSI, enhanced their cleavage and development to blastocyst stage. Copyright © 2018

Mishra, Ajay Aloimonos, Yiannis

The human visual system observes and understands a scene/image by making a series of fixations. Every fixation point lies inside a particular region of arbitrary shape and size in the scene which can either be an object or just a part of it. We define as a basic segmentation problem the task of segmenting that region containing the fixation point. Segmenting the region containing the fixation is equivalent to finding the enclosing contour- a connected set of boundary edge fragments in the edge map of the scene - around the fixation. This enclosing contour should be a depth boundary.We present here a novel algorithm that finds this bounding contour and achieves the segmentation of one object, given the fixation. The proposed segmentation framework combines monocular cues (color/intensity/texture) with stereo and/or motion, in a cue independent manner. The semantic robots of the immediate future will be able to use this algorithm to automatically find objects in any environment. The capability of automatically segmenting objects in their visual field can bring the visual processing to the next level. Our approach is different from current approaches. While existing work attempts to segment the whole scene at once into many areas, we segment only one image region, specifically the one containing the fixation point. Experiments with real imagery collected by our active robot and from the known databases 1 demonstrate the promise of the approach.

Hojaev, Alisher S. Ibragimova, Elvira M.

It’s well known, astronomy in Uzbekistan has ancient roots and traditions (e.g., Mirzo Ulugh Beg, Abū al-Rayhān al-Bīrūnī, Abū ‘Abdallāh al-Khwārizmī) and astronomical heritage carefully preserved. Nowadays uzbek astronomers play a key role in scientific research but also in OAD and Decadal Plan activity in the Central Asia region. International Aerospace School (IASS) is an amazing and wonderful event held annually about 30 years. IASS is unique project in the region, and at the beginning we spent the Summer and Winter Schools. At present in the summer camp we gather about 50 teenage and undergraduate students over the country and abroad (France, Malaysia, Turkey, Azerbaijan, Pakistan, Russia, etc.). They are selected on the basis of tests of astronomy and space issues. During two weeks of IASS camp the invited scientists, cosmonauts and astronauts as well as other specialists give lectures and engage in practical exercises with IASS students in astronomy, including daily observations of the Sun and night sky observations with meniscus telescope, space research and exploration, aerospace modelling, preparation and presentation of original projects. This is important that IASS gives not theoretical grounds only but also practically train the students and the hands-on training is the major aims of IASS. Lectures and practice in the field of astronomy carried out with the direct involvement and generous assistance of Uranoscope Association (Paris, France). The current 26-th IASS is planned to held in July 2015.

Hinrichs, Arne Kessler, Barbara Kurome, Mayuko Blutke, Andreas Kemter, Elisabeth Bernau, Maren Scholz, Armin M Rathkolb, Birgit Renner, Simone Bultmann, Sebastian Leonhardt, Heinrich de Angelis, Martin Hrabĕ Nagashima, Hiroshi Hoeflich, Andreas Blum, Werner F Bidlingmaier, Martin Wanke, Rüdiger Dahlhoff, Maik Wolf, Eckhard

Laron syndrome (LS) is a rare, autosomal recessive disorder in humans caused by loss-of-function mutations of the growth hormone receptor (GHR) gene. To establish a large animal model for LS, pigs with GHR knockout (KO) mutations were generated and characterized. CRISPR/Cas9 technology was applied to mutate exon 3 of the GHR gene in porcine zygotes . Two heterozygous founder sows with a 1-bp or 7-bp insertion in GHR exon 3 were obtained, and their heterozygous F1 offspring were intercrossed to produce GHR-KO, heterozygous GHR mutant, and wild-type pigs. Since the latter two groups were not significantly different in any parameter investigated, they were pooled as the GHR expressing control group. The characterization program included body and organ growth, body composition, endocrine and clinical-chemical parameters, as well as signaling studies in liver tissue. GHR-KO pigs lacked GHR and had markedly reduced serum insulin-like growth factor 1 (IGF1) levels and reduced IGF-binding protein 3 (IGFBP3) activity but increased IGFBP2 levels. Serum GH concentrations were significantly elevated compared with control pigs. GHR-KO pigs had a normal birth weight. Growth retardation became significant at the age of five weeks. At the age of six months, the body weight of GHR-KO pigs was reduced by 60% compared with controls. Most organ weights of GHR-KO pigs were reduced proportionally to body weight. However, the weights of liver, kidneys, and heart were disproportionately reduced, while the relative brain weight was almost doubled. GHR-KO pigs had a markedly increased percentage of total body fat relative to body weight and displayed transient juvenile hypoglycemia along with decreased serum triglyceride and cholesterol levels. Analysis of insulin receptor related signaling in the liver of adult fasted pigs revealed increased phosphorylation of IRS1 and PI3K. In agreement with the loss of GHR, phosphorylation of STAT5 was significantly reduced. In contrast, phosphorylation

Teaming is so common in today's project management environment that most of us assume it comes naturally. We further assume that when presented with meaningful and challenging work, project teams will naturally engage in productive activity to complete their tasks. This assumption is expressed in the simple (but false) equation: Team + Work = Teamwork. Although this equation appears simple and straightforward, it is far from true for most project organizations whose reality is a complex web of institutional norms based on individual achievement and rewards. This is illustrated by the very first successful team experience from my early Air Force career. As a young lieutenant, I was sent to Squadron Officer School, which was the first in the series of Air Force professional military education courses I was required to complete during my career. We were immediately formed into teams of twelve officers. Much of the course featured competition between these teams. As the most junior member of my team, I quickly observed the tremendous pressure to show individual leadership capability. At one point early in the course, almost everyone in our group was vying to become the team leader. This conflict was so intense that it caused us to fail miserably in our first outdoor team building exercise. We spent so much time fighting over leadership that we were unable to complete any of the events on the outdoor obstacle course. This complete lack of success was so disheartening to me that I gave our team little hope for future success. What followed was a very intense period of bickering, conflict, and even shouting matches as our dysfunctional team tried to cope with our early failures and find some way to succeed. British physician and researcher Wilfred Bion (Experiences in Groups, 1961) discovered that there are powerful psychological forces inherent in all groups that divert from accomplishing their primary tasks. To overcome these restraining forces and use the potential

Ma, Jun Flemr, Matyas Strnad, Hynek Svoboda, Petr Schultz, Richard M.

ABSTRACT The oocyte-to- zygote transition entails transforming a highly differentiated oocyte into totipotent blastomeres and represents one of the earliest obstacles that must be successfully hurdled for continued development. Degradation of maternal mRNAs, which likely lies at the heart of this transition, is characterized by a transition from mRNA stability to instability during oocyte maturation. Although phosphorylation of the oocyte-specific RNA-binding protein MSY2 during maturation is implicated in making maternal mRNAs more susceptible to degradation, mechanisms underlying mRNA degradation during oocyte maturation remain poorly understood. We report that DCP1A and DCP2, proteins responsible for decapping mRNA, are encoded by maternal mRNAs recruited for translation during maturation via cytoplasmic polyadenylation elements located in their 3′ untranslated regions. Both DCP1A and DCP2 are phosphorylated during maturation, with CDC2A being the kinase likely responsible for both, although MAPK may be involved in DCP1A phosphorylation. Inhibiting accumulation of DCP1A and DCP2 by RNA interference or morpholinos decreases not only degradation of mRNAs during meiotic maturation but also transcription of the zygotic genome. The results indicate that maternally recruited DCP1A and DCP2 are critical players in the transition from mRNA stability to instability during meiotic maturation and that proper maternal mRNA degradation must be successful to execute the oocyte-to- zygote transition. PMID:23136299

Betekhtin, Alexander Milewska-Hendel, Anna Lusinska, Joanna Chajec, Lukasz Kurczynska, Ewa Hasterok, Robert

The plant cell wall shows a great diversity regarding its chemical composition, which may vary significantly even during different developmental stages. In this study, we analysed the distribution of several cell wall epitopes in embryos of Brachypodium distachyon (Brachypodium). We also described the variations in the nucleus shape and the number of nucleoli that occurred in some embryo cells. The use of transmission electron microscopy, and histological and immunolocalisation techniques permitted the distribution of selected arabinogalactan proteins, extensins, pectins, and hemicelluloses on the embryo surface, internal cell compartments, and in the context of the cell wall ultrastructure to be demonstrated. We revealed that the majority of arabinogalactan proteins and extensins were distributed on the cell surface and that pectins were the main component of the seed coat and other parts, such as the mesocotyl cell walls and the radicula. Hemicelluloses were localised in the cell wall and outside of the radicula protodermis, respectively. The specific arrangement of those components may indicate their significance during embryo development and seed germination, thus suggesting the importance of their protective functions. Despite the differences in the cell wall composition, we found that some of the antibodies can be used as markers to identify specific cells and the parts of the developing Brachypodium embryo.

Wang, Xiaolong Yu, Honghao Lei, Anmin Zhou, Jiankui Zeng, Wenxian Zhu, Haijing Dong, Zhiming Niu, Yiyuan Shi, Bingbo Cai, Bei Liu, Jinwang Huang, Shuai Yan, Hailong Zhao, Xiaoe Zhou, Guangxian He, Xiaoling Chen, Xiaoxu Yang, Yuxin Jiang, Yu Shi, Lei Tian, Xiue Wang, Yongjun Ma, Baohua Huang, Xingxu Qu, Lei Chen, Yulin

Recent advances in the study of the CRISPR/Cas9 system have provided a precise and versatile approach for genome editing in various species. However, the applicability and efficiency of this method in large animal models, such as the goat, have not been extensively studied. Here, by co-injection of one-cell stage embryos with Cas9 mRNA and sgRNAs targeting two functional genes (MSTN and FGF5), we successfully produced gene-modified goats with either one or both genes disrupted. The targeting efficiency of MSTN and FGF5 in cultured primary fibroblasts was as high as 60%, while the efficiency of disrupting MSTN and FGF5 in 98 tested animals was 15% and 21% respectively, and 10% for double gene modifications. The on- and off-target mutations of the target genes in fibroblasts, as well as in somatic tissues and testis of founder and dead animals, were carefully analyzed. The results showed that simultaneous editing of several sites was achieved in large animals, demonstrating that the CRISPR/Cas9 system has the potential to become a robust and efficient gene engineering tool in farm animals, and therefore will be critically important and applicable for breeding. PMID:26354037

The distinct reproductive program of orchids provides a unique evolutionary model with pollination-triggered ovule development and megasporogenesis, a modified embryogenesis program resulting in seeds with immature embryos, and mycorrhiza-induced seed germination. However, the molecular mechanisms that have evolved to establish these unparalleled developmental programs are largely unclear. Here, we conducted comparative studies of genome-wide gene expression of various reproductive tissues and captured the molecular events associated with distinct reproductive programs in Phalaenopsis aphrodite. Importantly, our data provide evidence to demonstrate that protocorm-like body (PLB) regeneration (the clonal regeneration practice used in the orchid industry) does not follow the embryogenesis program. Instead, we propose that SHOOT MERISTEMLESS, a class I KNOTTED-LIKE HOMEOBOX gene, is likely to play a role in PLB regeneration. Our studies challenge the current understanding of the embryonic identity of PLBs. Taken together, the data obtained establish a fundamental framework for orchid reproductive development and provide a valuable new resource to enable the prediction of gene regulatory networks that is required for specialized developmental programs of orchid species. PMID:27338813



Comments:

  1. Jermane

    I think you are wrong. I'm sure. I can defend my position. Email me at PM.

  2. Bell

    My mother used to say that God gave a man two heads, but there is so little blood that you can only think of them one by one. Once upon a time there was an ordinary Swedish family: mother, father, brother, sister and Kid who wanted a dog. Virginity is not a vice, but sexual illiteracy. I trust, but I check



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