Arginine Regulates Zygotic Genome Activation in Porcine Embryos through Promoting Polyamine Synthesis☆

Background:Arginine has a positive effect on preimplantation development in pigs. However, the exact mechanism by which arginine promotes embryonic development to the blastocyst stage is not undefined. Here, single-cell RNA-sequencing technology was applied to porcine in vivo pre-implantation embryos from zygote to morula to determine transcription patterns of arginine metabolism-related genes during preimplantation embryonic development.Results:Transcriptome sequencing showed that arginine metabolism-related genes clearly changed from the 2-cell stage to the 4-cell stage, where zygotic genome activation (ZGA) occurred in porcine embryos. Further analysis of the correlation between arginine metabolism and ZGA shows that arginine metabolism-related genes are significantly correlated with key ZGA genes such as ZSCAN4, DPPA2 and EIF1A, indicating that arginine metabolism may be an indicator of porcine ZGA. To explore the correlation between arginine metabolism and ZGA, embryos cultured in the medium that removes all the amino acids, proteins and pyruvate in the PZM3 medium were employed to generate the ZGA blocked embryo model. The 4-cell arrest rate significantly increased at 72 h after activation, indicating impeded embryonic development. Meanwhile, results of immunofluorescent staining showed that the expression of SIRT1 protein during ZGA was significantly inhibited. Results of quantitative PCR showed that the expression of zygotic genes (ZSCAN4, DPPA2 and EIF1A) was significantly decreased. The above results indicate that the ZGA blocked embryo model was successfully established. Adding of arginine recovered embryonic development, SIRT1 and zygotic genes expression levels and initiated the ZGA. In addition, ROS content significantly increased when ZGA was blocked, and the GSH, ATP and lipid droplet content significantly decreased. After the addition of arginine in the block group, the ROS content significantly decreased, and the GSH, ATP and lipid droplet content significantly increased. Moreover, the ornithine decarboxylase (ODC) inhibitor difluoromethylornithine (DFMO) and arginine were added to the block group at the same time, and the effect of arginine was found to be inhibited. Conclusions: Arginine is essential for ZGA in porcine embryos. Arginine contributes to porcine ZGA by promoting polyamine synthesis in porcine embryos.

Dynamics of Known Long Non-Coding RNAs during the Maternal-to-Zygotic Transition in Rabbit

The control of pre-implantation development in mammals undergoes a maternal-to-zygotic transition (MZT) after fertilization. The transition involves maternal clearance and zygotic genome activation remodeling the terminal differentiated gamete to confer totipotency. In the study, we first determined the profile of long non-coding RNAs (lncRNAs) of mature rabbit oocyte, 2-cell, 4-cell, 8-cell, and morula embryos using RNA-seq. A total of 2673 known rabbit lncRNAs were identified. The lncRNAs exhibited dynamic expression patterns during pre-implantation development. Moreover, 107 differentially expressed lncRNAs (DE lncRNAs) were detected between mature oocyte and 2-cell embryo, while 419 DE lncRNAs were detected between 8-cell embryo and morula, consistent with the occurrence of minor and major zygotic genome activation (ZGA) wave of rabbit pre-implanted embryo. This study then predicted the potential target genes of DE lncRNAs based on the trans-regulation mechanism of lncRNAs. The GO and KEGG analyses showed that lncRNAs with stage-specific expression patterns promoted embryo cleavage and synchronic development by regulating gene transcription and translation, intracellular metabolism and organelle organization, and intercellular signaling transduction. The correlation analysis between mRNAs and lncRNAs identified that lncRNAs ENSOCUG00000034943 and ENSOCUG00000036338 may play a vital role in the late-period pre-implantation development by regulating ILF2 gene. This study also found that the sequential degradation of maternal lncRNAs occurred through maternal and zygotic pathways. Furthermore, the function analysis of the late-degraded lncRNAs suggested that these lncRNAs may play a role in the mRNA degradation in embryos via mRNA surveillance pathway. Therefore, this work provides a global view of known lncRNAs in rabbit pre-implantation development and highlights the role of lncRNAs in embryogenesis regulation.

Maternal Dppa2 and Dppa4 are dispensable for zygotic genome activation but important for offspring survival

ABSTRACT Zygotic genome activation (ZGA) represents the initiation of transcription following fertilisation. Despite its importance, we know little of the molecular events that initiate mammalian ZGA in vivo. Recent in vitro studies in mouse embryonic stem cells have revealed developmental pluripotency associated 2 and 4 (Dppa2/4) as key regulators of ZGA-associated transcription. However, their roles in initiating ZGA in vivo remain unexplored. We reveal that Dppa2/4 proteins are present in the nucleus at all stages of preimplantation development and associate with mitotic chromatin. We generated conditional single and double maternal knockout mouse models to deplete maternal stores of Dppa2/4. Importantly, Dppa2/4 maternal knockout mice were fertile when mated with wild-type males. Immunofluorescence and transcriptome analyses of two-cell embryos revealed that, although ZGA took place, there were subtle defects in embryos that lacked maternal Dppa2/4. Strikingly, heterozygous offspring that inherited the null allele maternally had higher preweaning lethality than those that inherited the null allele paternally. Together, our results show that although Dppa2/4 are dispensable for ZGA transcription, maternal stores have an important role in offspring survival, potentially via epigenetic priming of developmental genes.

DPPA2 and DPPA4 are dispensable for mouse zygotic genome activation and preimplantation development

How maternal factors in oocytes initiate zygotic genome activation (ZGA) remains elusive in mammals, partly due to the challenge of de novo identification of key factors using scarce materials. The 2-cell (2C) embryo like cells has been widely used as an in vitro model to understand mouse ZGA and totipotency given its expression of a group of 2C embryo-specific genes and its simplicity for genetic manipulation. Recent studies indicate that DPPA2 and DPPA4 are required for establishing the 2C-like state in mouse embryonic stem cells (ESCs) in a DUX-dependent manner. These results suggest that DPPA2 and DPPA4 are essential maternal factors that regulate Dux and ZGA in embryos. By analyzing maternal knockout and maternal-zygotic knockout embryos, we unexpectedly found that DPPA2 and DPPA4 are dispensable for Dux activation, ZGA, and preimplantation development. Our study suggests that 2C-like cells do not fully recapitulate 2-cell embryos in terms of 2C-gene regulation and cautions should be taken when studying ZGA and totipotency using 2C-like cells as the model system.

Inhibition of DRP1 Impedes Zygotic Genome Activation and Preimplantation Development in Mice

Mitochondrion plays an indispensable role during preimplantation embryo development. Dynamic-related protein 1 (DRP1) is critical for mitochondrial fission and controls oocyte maturation. However, its role in preimplantation embryo development is still lacking. In this study, we demonstrate that inhibition of DRP1 activity by mitochondrial division inhibitor-1, a small molecule reported to specifically inhibit DRP1 activity, can cause severe developmental arrest of preimplantation embryos in a dose-dependent manner in mice. Meanwhile, DRP1 inhibition resulted in mitochondrial dysfunction including decreased mitochondrial activity, loss of mitochondrial membrane potential, reduced mitochondrial copy number and inadequate ATP by disrupting both expression and activity of DRP1 and mitochondrial complex assembly, leading to excessive ROS production, severe DNA damage and cell cycle arrest at 2-cell embryo stage. Furthermore, reduced transcriptional and translational activity and altered histone modifications in DRP1-inhibited embryos contributed to impeded zygotic genome activation, which prevented early embryos from efficient development beyond 2-cell embryo stage. These results show that DRP1 inhibition has potential cytotoxic effects on mammalian reproduction, and DRP1 inhibitor should be used with caution when it is applied to treat diseases. Additionally, this study improves our understanding of the crosstalk between mitochondrial metabolism and zygotic genome activation.

Histone variant H2A.Z regulates zygotic genome activation

During embryogenesis, the genome shifts from transcriptionally quiescent to extensively active in a process known as Zygotic Genome Activation (ZGA). In Drosophila, the pioneer factor Zelda is known to be essential for the progression of development; still, it regulates the activation of only a small subset of genes at ZGA. However, thousands of genes do not require Zelda, suggesting that other mechanisms exist. By conducting GRO-seq, HiC and ChIP-seq in Drosophila embryos, we demonstrate that up to 65% of zygotically activated genes are enriched for the histone variant H2A.Z. H2A.Z enrichment precedes ZGA and RNA Polymerase II loading onto chromatin. In vivo knockdown of maternally contributed Domino, a histone chaperone and ATPase, reduces H2A.Z deposition at transcription start sites, causes global downregulation of housekeeping genes at ZGA, and compromises the establishment of the 3D chromatin structure. We infer that H2A.Z is essential for the de novo establishment of transcriptional programs during ZGA via chromatin reorganization.

Reciprocal zebrafish-medaka hybrids reveal maternal control of zygotic genome activation timing

After fertilization, the sperm and egg contribute unequally to the newly formed zygote. While the sperm contributes mainly paternal DNA, the egg provides both maternal DNA and the bulk of the future embryonic cytoplasm. Most embryonic processes (like the onset of zygotic transcription) depend on maternally-provided cytoplasmic components, and it is largely unclear whether paternal components besides the centrosome play a role in the regulation of early embryogenesis. Here we report a reciprocal zebrafish-medaka hybrid system as a powerful tool to investigate paternal vs. maternal influence during early development. By combining expression of zebrafish Bouncer on the medaka egg with artificial egg activation, we demonstrate the in vitro generation of paternal zebrafish x maternal medaka (reripes) hybrids. These hybrids complement the previously established paternal medaka x maternal zebrafish (latio) hybrids (Herberg et al., 2018). As proof of concept, we investigated maternal vs. paternal control of zygotic genome activation (ZGA) timing using this reciprocal hybrid system. RNA-seq analysis of the purebred fish species and hybrids revealed that the onset of ZGA is primarily governed by the egg. Overall, our study establishes the reciprocal zebrafish-medaka hybrid system as a versatile tool to dissect parental control mechanisms during early development.

Transposable Element Dynamics and Regulation during Zygotic Genome Activation in Mammalian Embryos and Embryonic Stem Cell Model Systems

Transposable elements (TEs) are mobile genetic sequences capable of duplicating and reintegrating at new regions within the genome. A growing body of evidence has demonstrated that these elements play important roles in host genome evolution, despite being traditionally viewed as parasitic elements. To prevent ectopic activation of TE transposition and transcription, they are epigenetically silenced in most somatic tissues. Intriguingly, a specific class of TEs—retrotransposons—is transiently expressed at discrete phases during mammalian development and has been linked to the establishment of totipotency during zygotic genome activation (ZGA). While mechanisms controlling TE regulation in somatic tissues have been extensively studied, the significance underlying the unique transcriptional reactivation of retrotransposons during ZGA is only beginning to be uncovered. In this review, we summarize the expression dynamics of key retrotransposons during ZGA, focusing on findings from in vivo totipotent embryos and in vitro totipotent-like embryonic stem cells (ESCs). We then dissect the functions of retrotransposons and discuss how their transcriptional activities are finetuned during early stages of mammalian development.

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.

Maternal Dppa2 and Dppa4 are dispensable for zygotic genome activation but important for offspring survival

Zygotic Genome Activation (ZGA) represents the initiation of transcription following fertilisation. Despite its importance in shifting developmental control from primarily maternal stores in the oocyte to the embryo proper, we know little of the molecular events that initiate ZGA in vivo. Recent in vitro studies in mouse embryonic stem cells (ESCs) have revealed Developmental Pluripotency Associated 2 and 4 (Dppa2/4) as key regulators of ZGA-associated transcription. However, their roles in initiating ZGA in vivo remain unexplored. We reveal Dppa2/4 proteins are present in the nucleus at all stages of preimplantation development and associate with mitotic chromatin. We generated single and double maternal knockout mouse models to deplete maternal stores of Dppa2/4. Importantly, while fertile, Dppa2/4 maternal knockout mice had reduced litter sizes, indicating decreased offspring survival. Immunofluorescence and transcriptome analyses of 2-cell embryos revealed while ZGA took place there were subtle defects in embryos lacking maternal Dppa2/4. Strikingly, heterozygous offspring that inherited the null allele maternally had higher preweaning lethality than those that inherited the null allele paternally. Together our results show that while Dppa2/4 are dispensable for ZGA transcription, maternal stores have an important role in offspring survival, potentially via epigenetic priming of developmental genes.