Zygotic Gene Research Articles

Multiple gene editing in porcine embryos using a combination of microinjection, electroporation, and transfection methods.

Mosaicism - the presence of both wild-type and mutant alleles - is a serious problem for zygotic gene modification through gene editing using the Clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR/Cas9) system. Different delivery methods, such as microinjection (MI), electroporation (EP), and transfection (TF), can be used to transfer CRISPR/Cas9 components into porcine zygotes. This study aimed to develop a method that combines MI, EP, and TF to improve mutation efficiency mediated through the CRISPR/Cas9 system for a triple-gene knockout in pigs. The study consisted of three groups: The MI group with three simultaneously microinjected guide RNAs (gRNAs) targeting α-1,3-galactosyltransferase (GGTA1), cytidine 32 monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), and β-1,4-N-acetyl-galactosaminyltransferase 2 (B4GALNT2); the MI + EP group with two gRNAs targeting GGTA1 and B4GALNT2 genes delivered into zygotes through MI, followed by EP of gRNA targeting the CMAH 1 h later; and the MI + EP + TF group with MI of gRNA targeting GGTA1 gene into zygotes, followed by EP of gRNA targeting CMAH 1 h later, and then TF of gRNA targeting the B4GALNT2 gene into zona-free zygotes after another hour. The rate of blastocysts carrying mutations in one or two gene(s) was significantly higher in the MI + EP + TF group than in the MI group. However, the blastocyst formation rate of zygotes in the MI + EP + TF group was lower than that of the zygotes in the other treatment groups. The combination of CRISPR/Cas9 delivery methods may improve the mutation efficiency of triple-gene edited porcine blastocysts.

An additive repression mechanism sets the anterior limits of anterior pair-rule stripes 1.

Segments are repeated anatomical units forming the body of insects. In Drosophila, the specification of the body takes place during the blastoderm through the segmentation cascade. Pair-rule genes such as hairy (h), even-skipped (eve), runt (run), and fushi-tarazu (ftz) are of the intermediate level of the cascade and each pair-rule gene is expressed in seven transversal stripes along the antero-posterior axis of the embryo. Stripes are formed by independent cis-regulatory modules (CRMs) under the regulation of transcription factors of maternal source and of gap proteins of the first level of the cascade. The initial blastoderm of Drosophila is a syncytium and it also coincides with the mid-blastula transition when thousands of zygotic genes are transcribed and their products are able to diffuse in the cytoplasm. Thus, we anticipated a complex regulation of the CRMs of the pair-rule stripes. The CRMs of h 1, eve 1, run 1, ftz 1 are able to be activated by bicoid (bcd) throughout the anterior blastoderm and several lines of evidence indicate that they are repressed by the anterior gap genes slp1 (sloppy-paired 1), tll (tailless) and hkb (huckebein). The modest activity of these repressors led to the premise of a combinatorial mechanism regulating the expression of the CRMs of h 1, eve 1, run 1, ftz 1 in more anterior regions of the embryo. We tested this possibility by progressively removing the repression activities of slp1, tll and hkb. In doing so, we were able to expose a mechanism of additive repression limiting the anterior borders of stripes 1. Stripes 1 respond depending on their distance from the anterior end and repressors operating at different levels.

Single-cell multiomics sequencing reveals the reprogramming defects in embryos generated by round spermatid injection.

Round spermatid injection (ROSI) technique holds great promise for clinical treatment of a proportion of infertile men. However, the compromised developmental potential of ROSI embryos largely limits the clinical application, and the mechanisms are not fully understood. Here, we describe the transcriptome, chromatin accessibility, and DNA methylation landscapes of mouse ROSI embryos derived from early-stage round spermatids using a single-cell multiomics sequencing approach. By interrogating these data, we identify the reprogramming defects in ROSI embryos at the pronuclear stages, which are mainly associated with the misexpression of a cohort of minor zygotic genome activation genes. We screen a small compound, A366, that can significantly increase the developmental potential of ROSI embryos, in which A366 can partially overcome the reprogramming defects by amending the epigenetic and transcriptomic states. Collectively, our study uncovers the reprogramming defects in ROSI embryos for understanding the mechanisms underlying compromised developmental potential and offers an avenue for ROSI technique optimization.

ELF4 is critical to zygotic gene activation and epigenetic reprogramming during early embryonic development in pigs.

Zygotic gene activation (ZGA) and epigenetic reprogramming are critical in early embryonic development in mammals, and transcription factors are involved in regulating these events. However, the effects of ELF4 on porcine embryonic development remain unclear. In this study, the expression of ELF4 was detected in early porcine embryos and different tissues. By knocking down ELF4, the changes of H3K9me3 modification, DNA methylation and ZGA-related genes were analyzed. Our results showed that ELF4 was expressed at all stages of early porcine embryos fertilized in vitro (IVF), with the highest expression level at the 8-cell stage. The embryonic developmental competency and blastocyst quality decreased after ELF4 knockdown (20.70% control vs. 17.49% si-scramble vs. 2.40% si-ELF4; p < 0.001). Knockdown of ELF4 induced DNA damage at the 4-cell stage. Interfering with ELF4 resulted in abnormal increases in H3K9me3 and DNA methylation levels at the 4-cell stage and inhibited the expression of genes related to ZGA. These results suggest that ELF4 affects ZGA and embryonic development competency in porcine embryos by maintaining genome integrity and regulating dynamic changes of H3K9me3 and DNA methylation, and correctly activating ZGA-related genes to promote epigenetic reprogramming. These results provide a theoretical basis for further studies on the regulatory mechanisms of ELF4 in porcine embryos.

Shared Transcriptional Machinery at Homologous Alleles Leads to Reduced Transcription in Early Drosophila Embryos.

The mechanism by which transcriptional machinery is recruited to enhancers and promoters to regulate gene expression is one of the most challenging and extensively studied questions in modern biology. We explored the possibility that interallelic interactions between two homologous alleles might affect gene regulation. Using an MS2- and PP7-based, allele-specific live imaging assay, we visualized de novo transcripts of a reporter gene in hemizygous and homozygous Drosophila embryos. Surprisingly, each homozygous allele produced fewer RNAs than the corresponding hemizygous allele, suggesting the possibility of allelic competition in homozygotes. However, the competition was not observed when the enhancer-promoter interaction was weakened by placing the reporter construct in a different chromosome location or by moving the enhancer further away from the promoter. Moreover, the reporter gene showed reduced transcriptional activity when a partial transcription unit (either an enhancer or reporter gene only) was in the homologous position. We propose that the transcriptional machinery that binds both the enhancer and promoter regions, such as RNA Pol II or preinitiation complexes, may be responsible for the allelic competition. We showed that the degree of allelic interference increased over developmental time as more Pol II was needed to activate zygotic genes. Such allelic competition was observed for an endogenous gene as well. Our study provides new insights into the role of 3D interallelic interactions in gene regulation.

USP16-mediated histone H2A lysine-119 deubiquitination during oocyte maturation is a prerequisite for zygotic genome activation.

Maternal-to-zygotic transition (MZT) is the first and key step in the control of animal development and intimately related to changes in chromatin structure and histone modifications. H2AK119ub1, an important epigenetic modification in regulating chromatin configuration and function, is primarily catalyzed by PRC1 and contributes to resistance to transcriptional reprogramming in mouse embryos. In this study, the genome-wide dynamic distribution of H2AK119ub1 during MZT in mice was investigated using chromosome immunoprecipitation and sequencing. The results indicated that H2AK119ub1 accumulated in fully grown oocytes and was enriched at the TSSs of maternal genes, but was promptly declined after meiotic resumption at genome-wide including the TSSs of early zygotic genes, by a previously unidentified mechanism. Genetic evidences indicated that ubiquitin-specific peptidase 16 (USP16) is the major deubiquitinase for H2AK119ub1 in mouse oocytes. Conditional knockout of Usp16 in oocytes did not impair their survival, growth, or meiotic maturation. However, oocytes lacking USP16 have defects when undergoing zygotic genome activation or gaining developmental competence after fertilization, potentially associated with high levels of maternal H2AK119ub1 deposition on the zygotic genomes. Taken together, H2AK119ub1 level is declined during oocyte maturation by an USP16-dependent mechanism, which ensures zygotic genome reprogramming and transcriptional activation of essential early zygotic genes.

Restriction of subapical proteins during cellularization depends on the onset of zygotic transcription and the formin Dia

Cortical domains are characterized by spatially restricted polarity proteins. The pattern of cortical domains is dynamic and changes during cell differentiation and development. Although there is a good understanding for how the cortical pattern is maintained, e. g. by mutual antagonism, less is known about how the initial pattern is established, and its dynamics coordinated with developmental progression. Here we investigate the initial restriction of subapical marker proteins during the syncytial-cellular transition in Drosophila embryos. The subapical markers Canoe/Afadin, the complex ELMO-Sponge, Baz and Arm become initially restricted between apical and lateral domains during cellularization. We define the role of zygotic genome activation as a timer for subapical domain formation. Subapical markers remained widely spread in embryos treated with α-amanitin and became precociously restricted in mutant embryos with premature zygotic transcription. In contrast, remodeling of the nuclear division cycle without cytokinesis to a full cell cycle is not a prerequisite for subapical domain formation, since we observed timely subapical restriction in embryos undergoing an extra nuclear cycle. We provide evidence that earliest subapical markers ELMO-Sponge and Canoe are required for subapical accumulation of Baz. Supporting an important role of cortical F-actin in subapical restriction, we found that the formin Dia was required for Baz restriction, and its distribution depended on the onset of zygotic gene expression. In summary, we define zygotic transcription as a timer, to which subapical markers respond in a dia-dependent mechanism.

Vitrification of Pronuclear Zygotes Perturbs Porcine Zygotic Genome Activation.

Simple SummaryVitrification is a commonly used cryopreservation technique that has played an important role in the preservation of animal genetic resources and assisted reproduction in humans. However, the effect of vitrification on embryonic genome activation remains to be elucidated. This study found that vitrification reduced the developmental efficiency of pig blastocysts. Further studies found that vitrification reduced transcriptional activity during embryonic genome activation and disrupted gene expression. Our findings suggest that vitrification impairs genome activation in pig embryos, and our results can provide new insight into improving the developmental efficiency of vitrified embryos.Zygotic genome activation (ZGA) plays an essential role in early embryonic development. Vitrification is a common assisted reproductive technology that frequently reduces the developmental competence of embryos. However, the effect of vitrification on porcine ZGA and gene expression during ZGA remains largely unclear. Here, we found that vitrification of pronuclear zygotes derived from parthenogenetic activation (PA) and in vitro fertilization (IVF) resulted in a significant reduction in the rates of 2-cell, 4-cell, and blastocysts, but did not affect the quality of blastocysts. Functional research revealed that RNA polymerase II Inhibitor (α-amanitin) treatment significantly reduced global transcriptional activity and developmental efficiency of both 4-cell and 8-cell embryos, implying an essential role of ZGA in porcine early embryonic development. Furthermore, vitrification did not affect the synthesis of nascent mRNA of 2-cell embryos, but significantly inhibited global transcriptional activity of both 4-cell and 8-cell embryos, suggesting an impaired effect of vitrification on porcine ZGA. Correspondingly, the single-cell analysis showed that vitrification caused the downregulation or upregulation expression of maternal genes in 4-cell embryos, also significantly decreased the expression of zygotic genes. Taken together, these results indicated that vitrification of pronuclear zygotes impairs porcine zygotic genome activation.

Transgenerational Genetic Effects Help Explain Latitudinal Variation in Seed Mass and Germination Timing in Plantago lanceolata.

We know little about the underlying genetic control of phenotypic patterns of seed traits across large-scale geographic and environmental gradients. Such knowledge is important for understanding the evolution of populations within species and for improving species conservation. Therefore, to test for genetic variation in Plantago lanceolata, we made reciprocal crosses between northern and southern genotypes that span the species’ range in Europe. The results provide evidence of transgenerational genetic effects on seed mass and germination timing. Northern mothers produced larger seeds with delayed germination, in contrast to southern mothers, which produced smaller seeds with accelerated germination. A maternal latitude affected both the seed coat, solely maternal tissue, and embryo/endosperm tissues. Thus, latitudinal variation in seed size and germination timing can be explained, in part, by the direct influence of maternal genotype, independent of zygotic genes that parents pass directly to the embryo and endosperm. Data suggest that researchers exploring the existence and evolution of large-scale geographic variation within species test for transgenerational genetic effects. In addition, data suggest that transgenerational control of seed traits should be considered when developing procedures designed to facilitate species conservation and restoration.

Triple gene editing in porcine embryos using electroporation alone or in combination with microinjection.

Background and Aim:We previously developed the gene-editing by electroporation (EP) of Cas9 protein method, in which the CRISPR/Cas9 system was introduced into porcine in vitro fertilized (IVF) zygotes through EP to disrupt a target gene. This method should be further developed, and a combination of EP and MI methods should be evaluated in pigs. This study aimed to determine that a combination of microinjection (MI) and EP of CRISPR/Cas9 system could increase the rates of biallelic mutation for triple-gene knockout in porcine blastocysts. We targeted the pancreatic and duodenal homeobox1 (PDX1) gene using cytoplasmic MI 1 h before or after EP, which was used to edit alpha-1,3-galactosyltransferase (GGTA1) and cytidine 32 monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes in porcine zygotes.Materials and Methods:We introduced guide RNAs targeting PDX1, GGTA1, and CMAH with the Cas9 protein into IVF zygotes (one-cell stage) through EP 10 h after the start of IVF (IVF; EP group) or in combination with MI (1 h before, MI-EP group, or after EP treatment EP-MI group) and evaluated the blastocyst formation rate and efficiency of target mutations in the resulting blastocysts.Results:Our results revealed a significant reduction in the rate of blastocyst formation in the two groups that underwent MI before and after EP (MI-EP and EP-MI group), compared with that in the groups treated with EP alone (EP group) (p=0.0224 and p<0.0001, respectively) and control (p=0.0029 and p<0.0001, respectively). There was no significant difference in the total mutation rates among the treatment groups in the resulting blastocysts. As an only positive effect of additional MI treatment, the rate of blastocysts carrying biallelic mutations in at least one target gene was higher in the MI-EP group than in the EP group. However, there was no difference in the rates of embryos carrying biallelic mutations in more than 2 target genes.Conclusion:These results indicate that although a combination of MI and EP does not improve the mutation efficiency or biallelic mutation for triple-gene knockout, MI treatment before EP is better to reduce mortality in porcine zygotic gene editing through a combination of MI and EP.

Polycomb repressive complex 1 initiates and maintains tailless repression in Drosophila embryo

Maternally-deposited morphogens specify the fates of embryonic cells via hierarchically regulating the expression of zygotic genes that encode various classes of developmental regulators. Once the cell fates are determined, Polycomb-group proteins frequently maintain the repressed state of the genes. This study investigates how Polycomb-group proteins repress the expression of tailless, which encodes a developmental regulator in Drosophila embryo. Previous studies have shown that maternal Tramtrack69 facilitates maternal GAGA-binding factor and Heat shock factor binding to the torso response element (tor-RE) to initiate tailless repression in the stage-4 embryo. Chromatin-immunoprecipitation and genetic-interaction studies exhibit that maternally-deposited Polycomb repressive complex 1 (PRC1) recruited by the tor-RE-associated Tramtrack69 represses tailless expression in the stage-4 embryo. A noncanonical Polycomb-group response element (PRE) is mapped to the tailless proximal region. High levels of Bric-a-brac, Tramtrack, and Broad (BTB)-domain proteins are fundamental for maintaining tailless repression in the stage-8 to -10 embryos. Trmtrack69 sporadically distributes in the linear BTB-domain oligomer, which recruits and retains a high level of PRC1 near the GCCAT cluster for repressing tll expression in the stage-14 embryos. Disrupting the retention of PRC1 decreases the levels of PRC1 and Pleiohomeotic protein substantially on the PRE and causes tailless derepression in the stage-14 embryo. Furthermore, the retained PRC1 potentially serves as a second foundation for assembling the well-characterized polymer of the Sterile alpha motif domain in Polyhomeotic protein, which compacts chromatin to maintain the repressed state of tailless in the embryos after stage 14.

Zygotic gene activation in mice: profile and regulation.

The zygotic genome is transcriptionally silent immediately after fertilization. In mice, initial activation of the zygotic genome occurs in the middle of the one-cell stage. At the mid-to-late two-cell stage, a burst of gene activation occurs after the second round of DNA replication, and the profile of transcribed genes changes dramatically. These two phases of gene activation are called minor and major zygotic gene activation (ZGA), respectively. As they mark the beginning of the gene expression program, it is important to elucidate gene expression regulation during these stages. This article reviews the outcomes of studies that have clarified the profiles and regulatory mechanisms of ZGA.

Whole-Genome DNA Methylation Dynamics of Sheep Preimplantation Embryo Investigated by Single-Cell DNA Methylome Sequencing.

The early stages of mammalian embryonic development involve the participation and cooperation of numerous complex processes, including nutritional, genetic, and epigenetic mechanisms. However, in embryos cultured in vitro, a developmental block occurs that affects embryo development and the efficiency of culture. Although the block period is reported to involve the transcriptional repression of maternal genes and transcriptional activation of zygotic genes, how epigenetic factors regulate developmental block is still unclear. In this study, we systematically analyzed whole-genome methylation levels during five stages of sheep oocyte and preimplantation embryo development using single-cell level whole genome bisulphite sequencing (SC-WGBS) technology. Then, we examined several million CpG sites in individual cells at each evaluated developmental stage to identify the methylation changes that take place during the development of sheep preimplantation embryos. Our results showed that two strong waves of methylation changes occurred, namely, demethylation at the 8-cell to 16-cell stage and methylation at the 16-cell to 32-cell stage. Analysis of DNA methylation patterns in different functional regions revealed a stable hypermethylation status in 3′UTRs and gene bodies; however, significant differences were observed in intergenic and promoter regions at different developmental stages. Changes in methylation at different stages of preimplantation embryo development were also compared to investigate the molecular mechanisms involved in sheep embryo development at the methylation level. In conclusion, we report a detailed analysis of the DNA methylation dynamics during the development of sheep preimplantation embryos. Our results provide an explanation for the complex regulatory mechanisms underlying the embryo developmental block based on changes in DNA methylation levels.

Generation and timing of graded responses to morphogen gradients.

ABSTRACTMorphogen gradients are known to subdivide a naive cell field into distinct zones of gene expression. Here, we examine whether morphogens can also induce a graded response within such domains. To this end, we explore the role of the Dorsal protein nuclear gradient along the dorsoventral axis in defining the graded pattern of actomyosin constriction that initiates gastrulation in early Drosophila embryos. Two complementary mechanisms for graded accumulation of mRNAs of crucial zygotic Dorsal target genes were identified. First, activation of target-gene expression expands over time from the ventral-most region of high nuclear Dorsal to lateral regions, where the levels are lower, as a result of a Dorsal-dependent activation probability of transcription sites. Thus, sites that are activated earlier will exhibit more mRNA accumulation. Second, once the sites are activated, the rate of RNA Polymerase II loading is also dependent on Dorsal levels. Morphological restrictions require that translation of the graded mRNA be delayed until completion of embryonic cell formation. Such timing is achieved by large introns, which provide a delay in production of the mature mRNAs. Spatio-temporal regulation of key zygotic genes therefore shapes the pattern of gastrulation.

Single-cell RNA sequencing reveals abnormal fluctuations in human eight-cell embryos associated with blastocyst formation failure.

Infertility has become a global health issue, with the number of people suffering from the disease increasing year by year, and ART offering great promise for infertility treatment. However, the regulation of early embryonic development is complicated and a series of processes takes place, including the maternal-to-zygotic transition. In addition, developmental arrest is frequently observed during human early embryonic development. In this study, we performed single-cell RNA sequencing on a biopsied blastomere from human eight-cell embryos and tracked the developmental potential of the remaining cells. To compare the sequencing results between different eight-cell embryos, we have combined the research data of this project with the data previously shared in the database and found that cells from the same embryo showed a higher correlation. Additionally, the transcriptome of embryos with blastocyst formation failure was significantly different from developed embryos, and the gene expression as well as cell signaling pathways related to embryonic development were also altered. In particular, the expression of some maternal and zygotic genes in the failed blastocyst formation group was significantly altered: the overall expression level of maternal genes was significantly higher in the failed blastocyst than the developed blastocyst group. In general, these findings provide clues for the causes of human embryonic arrest after the eight-cell stage, and they also provide new ideas for improving the success rate of ART in clinical practice.

Application of Domain- and Genotype-Specific Models to Infer Post-Transcriptional Regulation of Segmentation Gene Expression in Drosophila.

Unlike transcriptional regulation, the post-transcriptional mechanisms underlying zygotic segmentation gene expression in early Drosophila embryo have been insufficiently investigated. Condition-specific post-transcriptional regulation plays an important role in the development of many organisms. Our recent study revealed the domain- and genotype-specific differences between mRNA and the protein expression of Drosophila hb, gt, and eve genes in cleavage cycle 14A. Here, we use this dataset and the dynamic mathematical model to recapitulate protein expression from the corresponding mRNA patterns. The condition-specific nonuniformity in parameter values is further interpreted in terms of possible post-transcriptional modifications. For hb expression in wild-type embryos, our results predict the position-specific differences in protein production. The protein synthesis rate parameter is significantly higher in hb anterior domain compared to the posterior domain. The parameter sets describing Gt protein dynamics in wild-type embryos and Kr mutants are genotype-specific. The spatial discrepancy between gt mRNA and protein posterior expression in Kr mutants is well reproduced by the whole axis model, thus rejecting the involvement of post-transcriptional mechanisms. Our models fail to describe the full dynamics of eve expression, presumably due to its complex shape and the variable time delays between mRNA and protein patterns, which likely require a more complex model. Overall, our modeling approach enables the prediction of regulatory scenarios underlying the condition-specific differences between mRNA and protein expression in early embryo.

The F-box protein Bard (CG14317) targets the Smaug RNA-binding protein for destruction during the Drosophila maternal-to-zygotic transition.

During the maternal-to-zygotic transition (MZT), which encompasses the earliest stages of animal embryogenesis, a subset of maternally supplied gene products is cleared, thus permitting activation of zygotic gene expression. In the Drosophila melanogaster embryo, the RNA-binding protein Smaug (SMG) plays an essential role in progression through the MZT by translationally repressing and destabilizing a large number of maternal mRNAs. The SMG protein itself is rapidly cleared at the end of the MZT by a Skp/Cullin/F-box (SCF) E3-ligase complex. Clearance of SMG requires zygotic transcription and is required for an orderly MZT. Here, we show that an F-box protein, which we name Bard (encoded by CG14317), is required for degradation of SMG. Bard is expressed zygotically and physically interacts with SMG at the end of the MZT, coincident with binding of the maternal SCF proteins, SkpA and Cullin1, and with degradation of SMG. shRNA-mediated knock-down of Bard or deletion of the bard gene in the early embryo results in stabilization of SMG protein, a phenotype that is rescued by transgenes expressing Bard. Bard thus times the clearance of SMG at the end of the MZT.

Satb2 acts as a gatekeeper for major developmental transitions during early vertebrate embryogenesis

Zygotic genome activation (ZGA) initiates regionalized transcription underlying distinct cellular identities. ZGA is dependent upon dynamic chromatin architecture sculpted by conserved DNA-binding proteins. However, the direct mechanistic link between the onset of ZGA and the tissue-specific transcription remains unclear. Here, we have addressed the involvement of chromatin organizer Satb2 in orchestrating both processes during zebrafish embryogenesis. Integrative analysis of transcriptome, genome-wide occupancy and chromatin accessibility reveals contrasting molecular activities of maternally deposited and zygotically synthesized Satb2. Maternal Satb2 prevents premature transcription of zygotic genes by influencing the interplay between the pluripotency factors. By contrast, zygotic Satb2 activates transcription of the same group of genes during neural crest development and organogenesis. Thus, our comparative analysis of maternal versus zygotic function of Satb2 underscores how these antithetical activities are temporally coordinated and functionally implemented highlighting the evolutionary implications of the biphasic and bimodal regulation of landmark developmental transitions by a single determinant.

Wenshen Shengjing Decoction Improves Early Embryo Development by Maintaining Low H3K27me3 Levels in Sperm and Pronuclear Embryos of Spermatogenesis Impaired Mice.

Many ingredients in Wenshen Shengjing Decoction (WSSJD) can cause epigenetic changes in the development of different types of cells. It is not yet known whether they can cause epigenetic changes in sperms or early embryos. Here, we investigated the role of WSSJD in epigenetic modifications of sperms or early embryos and early embryo development. A mouse model with spermatogenesis disorders was established with cyclophosphamide (CPA). WSSJD was administrated for 30 days. The male model mice after the treatment were mated with the female mice treated with superovulation. The embryo development rate of each stage was calculated. Immunofluorescence staining was used to detect the expression of H3K27me3 in sperm, pronuclear embryos, and 2-cell embryos. Western blotting was used to detect the expression of histone demethylase KDM6A and methyltransferase EZH2 in 2-cell embryos with developmental arrest. The expressions of zygotic genome activation genes (ZSCAN4, E1F1AX, HSPA1A, ERV4-2, and MYC) in 2-cell embryos with developmental arrest were analyzed with qRT-PCR. Comparing with the control group, CPA destroyed the development of seminiferous epithelium, significantly increased the expression level of H3K27me3 in sperm, reduced the expression ratio of H3K27me3 in female and male pronuclei, delayed the development of 2-cell embryos, and increased the developmental arrest rate and degeneration rate of 2-cell embryos. Moreover, the expressions of EZH2 and H3K27me3 were significantly increased in the 2-cell embryos with developmental arrest, and the expression of zygotic genome activation genes (ZSCAN4, E1F1AX, HSPA1A, ERV4-2, and MYC) was significantly decreased. Compared with the CPA group, WSSJD promoted the development of seminiferous epithelium, maintained a low level of H3K27me3 modification in sperm and male pronucleus, significantly increased the development rate of 2-cell embryos and 3-4 cell embryos, and reduced the developmental arrest rate and degeneration rate of 2-cell embryos. WSSJD may promote early embryonic development by maintaining a low level of H3K27me3 modification in sperm and male pronucleus and regulating the zygotic genome activation in mice with spermatogenesis disorders induced by CPA.

Krüppel-like factor gene function in the ctenophore Mnemiopsis leidyi assessed by CRISPR/Cas9-mediated genome editing.

The Krüppel-like factor (Klf) gene family encodes transcription factors that play an important role in the regulation of stem cell proliferation, cell differentiation and development in bilaterians. Although Klf genes have been shown to specify functionally various cell types in non-bilaterian animals, their role in early-diverging animal lineages has not been assessed. Thus, the ancestral activity of these transcription factors in animal development is not well understood. The ctenophore Mnemiopsis leidyi has emerged as an important non-bilaterian model system for understanding early animal evolution. Here, we characterize the expression and functional role of Klf genes during M. leidyi embryogenesis. Zygotic Klf gene function was assessed with both CRISPR/Cas9-mediated genome editing and splice-blocking morpholino oligonucleotide knockdown approaches. Abrogation of zygotic Klf expression during M. leidyi embryogenesis resulted in abnormal development of several organs, including the pharynx, tentacle bulbs and apical organ. Our data suggest an ancient role for Klf genes in regulating endodermal patterning, possibly through regulation of cell proliferation.