The role of Dby mRNA in early development of male mouse zygotes

Ejaculated mammalian spermatozoa contain a complex yet specific population of mRNA. However, the possible roles that mRNA has in early zygotic and embryonic development remain unclear. We found that Dby mRNA is selectively retained in capacitated mouse spermatozoa, and is transferred into the oocyte during fertilization by reverse transcription-polymerase chain reaction even though no DBY protein expression is detected. The cellular location ofDby mRNA is seen in the post-acrosome region, and it comprises nearly half of the mouse spermatozoa in in situ hybridization. In contrast, transcripts of the control gene, Smcy, are not detected in capacitated mouse spermatozoa, although the H-Y antigen encoded by Smcy is expressed on the surface of the spermatozoa. In our microinjection experiment, the zygotic development rate of the as-Dby male pronucleus injection group was significantly lower than that of the as-Smcy male pronucleus injection group （35.9% vs. 95%, P = 0.001） and the as-Dby female pronucleus injection group （35.9% vs. 93.8%, P = 0.001）. The rate of male-developed zygotes was also lower than that of the as-Smcy male pronucleus injection group （17.4% vs. 57.9%, P = 0.002） and the as-Dby female pronucleus injection group （17.4% vs. 54.1%, P = 0.002）. Thus, we conclude that Dby mRNA is selectively retained in capaci- tated mouse spermatozoa, and it has an important role in the early zygotic development of male mouse zygotes. This might imply that spermatozoa mRNA is involved in early zygotic and embryonic stages of reproduction.

Transcriptomic Analysis of Pacific Oyster(Crassostrea gigas) Zygotes Under Hypotonic Triploid Induction

Polyploid breeding is widely used in various marine species. Low salinity treatment is an effective method of inducing triploid of bivalve mollusks. In this study, RNA-seq was performed to determine genes and pathways involved in hyposaline adaption and cell division of Pacific oyster(Crassostrea gigas) zygotes, trying to better understand the possible molecular mechanism of hypo-osmotic induction. A total of 26965 unigenes were generated in the de novo assembly of clean Illumina reads with an average length of 934 bp and N50 of 1721 bp. Of 3024 differentially expressed genes(DEGs), 2501 were up-regulated and 523 were downregulated. GO(Gene Ontology) annotation and KEGG(Kyoto Encyclopedia of Genes and Genomes) pathway analysis of these DEGs revealed that these DEGs participate a variety of biological processes including osmoregulation, cytoskeleton organization, cell survival and death, and substantially modulate cell proliferation and embryonic development. In summery, RNA-seq methodology was applied for the first time to demonstrate hypotonic-induced transcriptomic alteration in oyster zygotes. Our findings not only interpreted the relatively high mortality of induced larvae, but also provided a valuable reference for further investigations on the mechanism of hyposaline induction, thus should aid to the application of low salinity in triploid induction in large scale aquaculture in future.

CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes

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.

A New Micropropagation Technology of Tilia amurensis:In VitroMicropropagation of Mature Zygotic Embryos and the Establishment of a PlantRegeneration System

Tilia amurensis is an economically valuable broadleaf tree species in Northeast China.The production of highqualityT.amurensis varieties at commercial scales has been greatly limited by the low germination rates.Thereis thus a pressing need to develop an organogenesis protocol for in vitro propagation of T.amurensis to alleviate ashortage of high-quality T.amurensis seedlings.Here,we established a rapid in vitro propagation system forT.amurensis from mature zygotic embryos and analyzed the effects of plant growth regulators and culture mediain different stages.We found that Woody plant medium(WPM)was the optimal primary culture medium formature zygotic embryos.The highest callus induction percentage(68.76%)and number of axillary buds induced(3.2)were obtained in WPM+0.89μmol/L 6-benzyladenine(6-BA)+0.46μmol/L kinetin(KT)+0.25μmol/Lindole-3-butryic acid(IBA)+1.44μmol/L gibberellin A_(3)(GA_(3)).The multiple shoot bud development achievedthe highest percentage(83.32%)in the Murashige and Skoog(MS)+2.22μmol/L 6-BA+0.25μmol/L IBA+1.44μmol/L GA_(3).The rooting percentage(96.70%)was highest in 1/2 MS medium+1.48μmol/L IBA.Thesurvival percentage of transplanting plantlets was 82.22%in soil:vermiculite:perlite(5:3:1).Our study is the firstto establish an effective organogenesis protocol for T.amurensis using mature zygotic embryos.

Delivery of Cas9 Protein into Mouse Zygotes through a Series of Electroporation Dramatically Increases the Efficiency of Model Creation

Previously we established Zygote Electroporation of Nucleases（ZEN） technology as an efficient and high-throughput way to generate genetically modified mouse models.However,there were significant variations of the targeting efficiency among different genomic loci using our previously published protocol.In this study,we improved the ZEN technology by delivering Cas9 protein into mouse zygotes through a series of electroporation.Using this approach,we were able to introduce precise nucleotide substitutions,large segment deletion and short segment insertion into targeted loci with high efficiency.

Spatio-Temporal Expression Patterns of Aurora Kinases A, B in Mouse Zygotes during the First Mitosis

Objective To investigate the expression and localization of Aurora kinase A （A URKA ） and Aurora kinase B （A URKB） in mouse zygotes during the process of the first mitosis. Methods Quantitative real-time RT-PCR and Western blotting were performed to analyze the expression of AURKA and AURKB. The subcellular location of AURKA and A URKB was studied by confocal microscopy. Results A URKA and A URKB were increasingly expressed from phase G1 and peaked at phase M. After the entrance into mitosis A URKB became the predominant form both in mRNA and protein levels. The proteins of A URKA and A URKB both distributed in the cytoplasm and were associated with nucleus during the first mitosis of mouse zygotes, with some details in different. Conclusion The expression and localization of Aurora kinases A and B was in a cell- cycle regulated manner during the process of the cleavage of mouse zygotes. This discovery will aid in future investigations on their specific roles and molecular mecha- nisms in the regulation of mammalian early embryonic development.

Somatic embryogenesis and histological analysis from zygotic embryos in Vitis vinifera L.‘Moldova’

We examined the somatic embryogenesis from and histological studies of zygotic embryos of seeds in European Grape ＇Moldova＇ （Vitis vinifera U ＇Moldova＇）. Primary calli were initiated on Nitsch and Nitsch （NN） medium supplemented with 1.0 mg·L^-1 2,4-D and 0.5 mg·L^-1 6-BA. Embryogenic calli were produced upon transfer to a NN medium with 0.5 mg·L^-1 6-BA and 2 mg·L^-1 NAA and somatic embryos were obtained on a half strength MS medium without plant growth regulators. During the somatic embryo germination, an addition of 1.0 mg·L^-1 6-BA in the medium could accelerate somatic embryos to develop into normal plants and increase the conversion rate from 0 to 43.3%. Histological studies of embryogenic calli and somatic embryos demonstrated dynamic changes of proteins and starch grains. The developmental processes of somatic embryos were similar to those of zygotic embryos, including typical epiderma, cotyledon primordium and vascular tissue.

Optimized production of transgenic buffalo embryos and offspring by cytoplasmic zygote injection

Background： Cytoplasmic injection of exogenous DNA into zygotes is a promising technique to generate transgenic livestock. However, it is still relatively inefficient and has not yet been demonstrated to work in buffalo. We sought to improve two key technical parameters of the procedure, namely i） how much linear DNA to inject and ii） when to inject it. For this, we introduced a constitutively expressed enhanced green fluorescent protein （EGFP） plasmid into buffalo zygotes. Results： First, we found that the proportion of EGFP-expressing blastocysts derived from zygotes injected with 20 or 50 ng/pL DNA was significantly higher than from those injected with 5 pg/mL. However, 50 ng/pL exogenous DNA compromised blastocyst development compared to non-injected IVF controls. Therefore the highest net yield of EGFP-positive blastocysts was achieved at 20 ng/pL DNA. Second, zygotes injected early （7-8 h post-insemination [hpi]） developed better than those injected at mid （12-13 hpi） or late （18-19 hpi） time points. Blastocysts derived from early injections were also more frequently EGFP-positive. As a consequence, the net yield of EGFP-expressing blastocysts was more than doubled using early vs late injections （16.4 % vs 7.7 %）. With respect to blastocyst quality, we found no significant difference in cell numbers of EGFP-positive blastocysts vs non-injected blastocysts. Following embryo transfer of six EGFP-positive blastocysts into four recipient animals, two viable buffalo calves were born. Biopsied ear tissues from both buffalo calves were analyzed for transgene presence and expression by Southern blot, PCR and confocal laser scanning microscopy, respectively. This confirmed that both calves were transgenic. Conclusions： Our cytoplasmic injection protocol improved generation of transgenic embryos and resulted in the first transgenic buffalo calves produced by this method.

Embryo culture is an efficient way to conserve a medicinally important endangered forest tree species Strychnos potatorum

The present study reports a protocol for germination of Strychnos potatorum （ver. Tel. Chilla） using zygotic embryo culture as an embryo rescue method. A 100% germination rate was obtained by culturing the embryos on full-strength Murashige and Skoog＇s medium （MS） containing 20 g/L sucrose in comparison to McCown and Lloyd＇s Woody Plant Medium （WPM）. Germination rates decreased when the sucrose concentration was lower or higher than 20 g·L-1 . WPM/MS medium containing glucose at levels 30, 20, 15 g·L-1 showed a smaller percentage of germination and at quarter strength, WPM/MS medium with glucose did not respond. Multiple shoot formation was found at 1.0 2.0 mg/L BAP; 3.0 mg/L Kn; 2.0 mg/L TDZ on MS medium with 20 g·L-1 sucrose. Germination rates improved when the embryos were placed upright （vertically） in the medium. The in vitro germinated seedlings were acclimatized in a walk-in-chamber and maintained in the green house with the survival rate of 65% 75%. These plants were transferred to the field and were found to be phenotypically normal, healthy and similar to donor plants. This protocol will be useful to overcome seed dormancy and for rapid multiplication and conservation of S. potatorum using zygotic embryo culture.

Effect of Semen Quality on the Embryo Development

To investigate the influences of sperm quality on the zygotes and embryos development, as the role of the paternal factor in early human embryogenesis is gaining more attention because of the application of techniques such as intracytoplasmic sperm injection （ICSI） ior the treatment of men infertility. 136 infertility couples with men factors （Group Ⅰ） were included from May 2002 to January 2004. One hundred and seventy-two infertility couples with tube factors （Group Ⅱ） served as controls. The sperm parameters, geminates and embryos quality, implantation rate and pregnant rate in both groups were analyzed. It was found that there was no significant differences in the number of oocytes retrieved, the fertilization rate and number of embryos transferred between two groups. Sperm concentration, percentage of motile sperm and percentage of sperm with normal morphology were significantly lower in group Ⅰ than in group Ⅱ （P〈0.01）. The proportion of good quality zygotes and good quality embryos were significantly lower in,（he male infertility group than in the tubal disease group （P（0.05）. Implantation rate and pregnancy rate were similar in two groups. It was concluded that spermatozoa is involved in the embryo quality, even in the early stages of development, which limited the treatment potency of IVF procedure.

In Vitro Somatic Embryogenesis in Some Oil Yielding Tropical Tree Species

Somatic embryogenesis was achieved in two oil yielding tropical tree species i.e. Simarouba glauca & Azadirachta indica using immature zygotic embryos as explants on Murashige and Skoog (MS) medium supplemented with 0.5 – 1.5 mg/l benzylaminopurine (BA) and 2.0 - 3.0 mg/l NAA (1-napthaleneacetic acid) or 2, 4-D (2,4-dichlorophenoxyacetic acid) and 3% sucrose. MS medium containing 1.0 mg/l BA and 2.0 mg/l NAA was noted to be the most effective in inducing friable embryogenic callus (FEC) in Simarouba glauca;the number of somatic embryos per culture varied in MS medium supplemented with 1.0 – 1.5 mg/l BA and 1.0 mg/l NAA. In Azadirachta indica, somatic embryos developed on MS medium supplemented with 0.5 mg/l BA and 1.5 – 2.0 mg/l 2,4-D which were in various shapes and sizes after the first subculture on MS medium supplemented with 0.25 mg/l abscisic acid. The somatic embryos which developed shoots were isolated and rooted in 1/2 strength MS medium supplemented with 0.25 mg/l abscisic acid and 2% sucrose. About 25% of embryos germinated within 20 days of culture in case of Simarouba glauca and 62% in Azadirachta indica. The somatic embryo-derived plantlets were transferred to the field after being hardened in the climate controlled hardening chamber.

Regeneration of <i>in Vitro</i>Shoot and Root Structure through Hormone Manipulation of Coconut (MATAG F2) Zygotic Embryos

Zygotic embryos tissues have found to be more responsive explants for clonal propagation of coconut. In the present study, the feasibility of using zygotic embryos explants for clonal propagation of a local coconut variety MATAG F2 was assessed. Callus was induced by incorporating of cytokinin and auxin into the medium. The sliced embryos explants were immersed in 1 M maltose for 60 mins, then with 0.05 M maltose for 1 min and followed by 0.01 M maltose for 5 mins was the best for prevention of phenolic compounds excretion. The callus formation depended on the concentration of 2,4-D in the media and the best effect was observed with the high level (2,4-D and BAP) tested. Attempts at inducing multiple shoot from the zygotic embryos callus were unsuccessful. No multiple shoots was present;most of the callus became root structure.

Zygotic combinatorial process in plants

Experimental data that prove the existence of the zygotic combinatorial process occurring in an embryogenesis-entering zygote are presented in the paper. The zygotic combinatorial process is found when analyzing F1 hybrid plants obtained from crossing homozygous forms different, minimum, in two marker enzymes, and it is found in that hybrid plant which, with one marker enzyme heterozygous spectrum, has a homozygous spectrum of the other. The zygotic combinatorial process leads to F1 hybrids uniformity aberration. The zygotic combinatory process revealed in the study is supposed to be conditioned by chromosome polyteny in mother plant cells and diminution of chromatin excess from the embryogenesisentering zygote. An obligatory condition for combinatorial process is the presence of free exchange of chromatides among homological chromosomes in an embryogenesis-entering cell, i.e. the presence of crossing-over analogous to the one proceeding at meiosis.

Sexual Reproduction in Higher Plants I:Fertilization and the Initiation of Zygotic Program

Sexual plant reproduction is a critical developmental step in the life cycle of higher plants, to allow maternal and paternal genes to be transmitted in a highly regulated manner to the next generation. During evolution, a whole set of signal transduction machinery is developed by plants to ensure an error-free recognition between male and female gametes and initiation of zygotic program. In the past few years, the molecular machineries underlying this biological process have been elucidated, particularly on the importance of synergid cells in pollen tube guidance, the Ca^＋＋ spike as the immediate response of fertilization and the epigenetic regulation of parental gene expressions in early zygotic embryogenesis. This review outlines the most recent development in this area.

EMBRYONIC FACTOR 19 Encodes a Pentatricopeptide Repeat Protein that is Essential for the Initiation of Zygotic Embryogenesis in Arabidopsis

Early embryogenesis is the most fundamental developmental process in biology. Screening of ethyl methanesulfonate （EMS）-mutagenized populations of Arabidopsis thaliana led to the identification of a zygote-lethal mutant embryonic factor 19 （fac19） in which embryo development was arrested at the elongated zygote to octant stage. The number of endosperm nuclei decreased significantly in fac19 embryos. Genetic analysis showed fac19 was caused by a single recessive mutation with typical mendelian segregation, suggesting equal maternal and paternal contributions of FAC19 towards zygotic embryogenesis. Positional cloning showed that FAC19 encodes a putative mitochondrial protein with 16 conserved pentatricopeptide repeat （PPR） motifs. The fac19 mutation caused a conversion from hydrophilic serine located in a previously unknown domain to hydrophobic leucine. Crosses between FAC191fac19 and the T-DNA insertion mutants in the same gene failed to complement the fac19 defects, confirming the identity of the gene. This study revealed the critical importance of a PPR protein-mediated mitochondrial function in early embryogenesis.

The Maternal-to-Zygotic Transition in Higher Plants

During early embryogenesis in mammals and higher plants, the maternal- to-zygotic transition （MZT） marks the turnover of developmental control from maternal products to de novo zygotic genome transcripts. Intensive studies in animals indicate that early embryonic development is largely maternally controlled. In recent years, the MZT has drawn the attention of botanists, as it is important for understanding the mechanism of embryogenesis and hybrid vigor. In this study, we present a brief overview of some aspects of the MZT in flowering plants. Based on what we have learned from Nicotiana tabacum, we hypothesize that the MZT occurs before zygotic cell division and that the development of the fertilized egg cell in flowering plants can be divided into two phases： the zygote stage, which is mainly controlled maternally, and the one-celled proembryo stage, in which zygotic genome activation （ZGA） occurs and is required for zygote division.

ZYGOTE-ARREST 3 that encodes the tRNA ligase is essential for zygote division in Arabidopsis

In sexual organisms, division of the zygote initiates a new life cycle. Although several genes involved in zygote division are known in plants, how the zygote is activated to start embryogenesis has remained elusive. Here, we showed that a mutation in ZYGOTE-ARREST 3 （ZYG3） in Arabidopsis led to a tight zygote-lethal phenotype. Map-based cloning revealed that ZYG3 encodes the transfer RNA （tRNA） ligase AtRNL, which is a single-copy gene in the Arabidopsis genome. Expression analyses showed that AtRNL is expressed throughout zygotic embryogenesis, and in meristematic tissues. Using pAtRNL：：cAtRNL-sYFP- complemented zyg3/zyg3 plants, we showed that AtRNL is localized exclusively in the cytoplasm, suggesting that tRNA splicing occurs primarily in the cytoplasm. Analyses using partially rescued embryos showed that mutation in AtRNL compromised splicing of intron-containing tRNA. Mutations of two tRNA endonuclease genes, SEN1 and SEN2, also led to a zygote-lethal phenotype. These results together suggest that tRNA splicing is critical for initiating zygote division in Arabidopsis.

Efficient generation of zebrafish maternal-zygotic mutants through transplantation of ectopically induced and Cas9/gRNA targeted primordial germ cells

The clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 technology has been widely utilized for knocking out genes involved in various biological processes in zebrafish. Despite this technology is efficient for generating different mutations, one of the main drawbacks is low survival rate during embryogenesis when knocking out some embryonic lethal genes. To overcome this problem, we developed a novel strategy using a combination of CRISPR/Cas9 mediated gene knockout with primordial germ cell(PGC) transplantation(PGCT) to facilitate and speed up the process of zebrafish mutant generation, particularly for embryonic lethal genes. Firstly, we optimized the procedure for CRISPR/Cas9 targeted PGCT by increasing the efficiencies of genome mutation in PGCs and induction of PGC fates in donor embryos for PGCT. Secondly, the optimized CRISPR/Cas9 targeted PGCT was utilized for generation of maternal-zygotic(MZ) mutants of tcf7l1a(gene essential for head development), pou5f3(gene essential for zygotic genome activation) and chd(gene essential for dorsal development) at F1 generation with relatively high efficiency. Finally, we revealed some novel phenotypes in MZ mutants of tcf7l1 a and chd, as MZtcf7l1 a showed elevated neural crest development while MZchd had much severer ventralization than its zygotic counterparts. Therefore, this study presents an efficient and powerful method for generating MZ mutants of embryonic lethal genes in zebrafish. It is also feasible to speed up the genome editing in commercial fishes by utilizing a similar approach by surrogate production of CRISPR/Cas9 targeted germ cells.

Molecules and mechanisms controlling the active DNA demethylation of the mammalian zygotic genome

The active DNA demethylation in early embryos is essential for subsequent development. Although the zygotic genome is globally demethylated, the DNA methylation of imprinted regions, part of repeat sequences and some gamete-specific regions are maintained. Recent evidence has shown that multiple proteins and biological pathways participate in the regulation of active DNA demethylation, such as TET proteins, DNA repair pathways and DNA methyltransferases. Here we review the recent understanding regarding proteins associated with active DNA demethylation and the regulatory networks controlling the active DNA demethylation in early embryos.

Developmental and genomic architecture of plant embryogenesis: from model plant to crops

Embryonic development represents an important reproductive phase of sexually reproducing plant species.The fusion of egg and sperm produces the plant zygote,a totipotent cell that,through cell division and cell identity specification in early embryogenesis,establishes the major cell lineages and tissues of the adult plant.The subsequent morphogenesis phase produces the full-sized embryo,while the late embryogenesis maturation process prepares the seed for dormancy and subsequent germination,ensuring continuation of the plant life cycle.In this review on embryogenesis,we compare the model eudicot Arabidopsis thaliana with monocot crops,focusing on genome activation,paternal and maternal regulation of early zygote development,and key organizers of patterning,such as auxin and WOX transcription factors.While the early stages of embryo development are apparently conserved among plant species,embryo maturation programs have diversified between eudicots and monocots.This diversification in crop species reflects the likely effects of domestication on seed quality traits that are determined during embryo maturation,and also assures seed germination in different environmental conditions.This review describes the most important features of embryonic development in plants,and the scope and applications of genomics in plant embryo studies.