The Regulation Of Transcriptomes In Oocyte And Their Roles In Follicle Maintenance And Preimplantational Development

The mammalian ovary is made up of ovarian follicles at different developmental stages, including primordial follicles, primary follicles, secondary follicles and antral follicles. The primordial folliclesare generated early in life and form a reservoirof female germ cells.In adult ovaries, no new oocytes can be generated and the primordial follicles are gradually activated. To ensure a sufficientlylong reproductive period, some primordial folliclesmust survive in a resting state for months(mice) or decades (humans). However, themolecular mechanisms that control the balancebetween primordial follicle survival and loss arenot well known. In this study, we used oocyte-expressed Cre to specifically delete genes in oocytes to investigate the dynamics of transcriptomes as well as functions of epigenetic regulators in oogenesis, oocyte meiotic maturation and preimplatational development, to demonstrate the mechanisms of oocyte maintenance and female fertility. Therefore, we abolished the expression of E3 ubiquitin ligase CRL4 complex, CpG binding protein CFP1 and transcriptional co-activator YAP in oocytes, and analyzed their functions in these processes. By these mouse models, we clarified the regulation of transcriptomes in oocytes and early embryos, and found that the maintenance of oocyte transcriptome is crucial for oocyte survival and reprogramming after fertilization.We found that in oocytes, CRL4 complex activates the activity of DNA demethylase TET proteins to regulate expression of oocyte-specific genes and the paternal DNA demethylation in fertilized eggs. This mechanism is crucial for female fertility, especially the maintenance of primordial follicles and reprogramming of zygotes.Oocyte-specific deletion of CRL41inker protein DDB1 and the substrate adaptor DCAF1 caused rapid oocyte loss and premature ovarian failure.CRL4DCAF1 was required for oocyte-specific gene expressions essential for primordial follicle survival and ovulation, and also functioned as a zygotic reprogramming factor. In DDB1/DCAF1-deleted oocytes, the promoters of many oocyte-specific genes and imprinting sequences are hypermethylated. CRL4DCAF1 activates the TET methylcytosinedioxygenases, which are involved in female germ cell development and zygote genome reprogramming.Hence, CRL4DCAF1 ubiquitin ligase is a guardian of female reproductive life in germ cells, and a maternal reprogramming factor after fertilization.CpG binding protein CFP1, also known as CGBP or CXXC1, is a component of SET1 histone methylase complex, which binds to unmethylated CpG and directs the trimethylation on the lysine 4 of Histone H3 (H3K4me3) and regulates the expression of target genes. We found that deletion of CFP1 in oocytes causes female infertility. CFP1 regulates the transcription of downstream genes and knockout of Cxxc1 in oocytes caused defects in maternal mRNA accumulation, especially oocyte-expressed genes. These CFP1-deleted oocytes failed to clear maternal mRNA during meiotic maturation and exhibited 1- to 2-cell arrest after fertilization. POF is arised in these Cxxc1fvfl;Gdf9-Cre females when grow to about 8-month-old. Our results demonstrated that the hypermethylation after CRL4 deletion and decreased transcription in CFP1-deleted oocytes severely affected the maintenance of oocyte and the ability to reprogramming paternal DNA, which caused POF and female infertility. Therefore, the regulation of transcriptomes in oocyte is crucial for the maintenance of female fertility.On the other hand, we also investigated the functions of YAP transcripton co-activator in zygotic genome activation (ZGA).We generated maternal and paternal mouse models with Yap1-deletion, and elucidated that maternally accumulated YAP in oocyte is crucial for zygotic genome activation. Maternal Yap1-knockout embryos had a prolonged 2-cell stage and developed into the 4-cell stage at a much slower rate when compared to that of wild-type controls.We profiled transcriptomes of these embryos, andidentified YAP target genes in early blastomeres. By targeting the expression of key early zygotic genes, particularly Rpl13 and Rrm2, maternal YAP renders developmental competence to preimplantation embryos. The physiological YAP activator, lysophosphatidic acid (LPA), stimulates early embryonic development both in oviduct and in culture. Adding LPA to culture medium remarkably increased the development rates of wild-type, but not maternal Yap1-knockoutembryos.These observations provide insights into the mechanisms of zygotic genome activation, and suggest potential implications of YAP activators in improving the developmental competence of cultured embryos in human assisted reproduction and animal biotechnology.