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Female germ cell development is a highly complex process that includes meiosis initiation, oocyte growth recruitment, oocyte meiosis retardation and resumption and final meiotic maturation. A series of coordinated molecular signaling factors ensure successful oogenesis. The recent rapid development of high-throughput sequencing technologies allows for the dynamic omics in female germ cells, which is essential for further understanding the regulatory mechanisms of molecular events comprehensively. In this review, we summarize the current literature of multi-omics sequenced by epigenome-, transcriptome- and proteome-associated technologies, which provide valuable information for understanding the regulation of key events during female germ cell development.

Protein phosphatase 4 (PPP4) is a protein phosphatase that, although highly expressed in the testis, currently has an unclear physiological role in this tissue. Here, we show that deletion of PPP4 catalytic subunit gene Ppp4c in the mouse causes male-specific infertility. Loss of PPP4C, when assessed by light microscopy, did not obviously affect many aspects of the morphology of spermatogenesis, including acrosome formation, nuclear condensation and elongation, mitochondrial sheaths arrangement and ‘9 + 2’ flagellar structure assembly. However, the PPP4C mutant had sperm tail bending defects (head-bent-back), low sperm count, poor sperm motility and had cytoplasmic remnants attached to the middle piece of the tail. The cytoplasmic remnants were further investigated by transmission electron microscopy to reveal that a defect in cytoplasm removal appeared to play a significant role in the observed spermiogenesis failure and resulting male infertility. A lack of PPP4 during spermatogenesis causes defects that are reminiscent of oligoasthenoteratospermia (OAT), which is a common cause of male infertility in humans. Like the lack of functional PPP4 in the mouse model, OAT is characterized by abnormal sperm morphology, low sperm count and poor sperm motility. Although the causes of OAT are probably heterogeneous, including mutation of various genes and environmentally induced defects, the detailed molecular mechanism(s) has remained unclear. Our discovery that the PPP4C-deficient mouse model shares features with human OAT might offer a useful model for further studies of this currently poorly understood disorder.

DNA methylation and demethylation are crucial for modulating gene expression and regulating cell differentiation. Functions and mechanisms of DNA methylation/demethylation in mammalian embryos are still far from being understood clearly. In this review we firstly describe new insights into DNA demethylation mechanisms, and secondly introduce the differences in active DNA methylation patterns in zygotes and early embryos in various mammalian species. Thirdly, we attempt to clarify the functions of DNA demethylation in early embryos. Most importantly we summarize the importance of active DNA demethylation and its possible relevance to human IVF clinics. Finally research perspectives regarding DNA demethylation are also discussed.

STUDY QUESTION: There is an unexplored physiological role of N-WASP (neural Wiskott-Aldrich syndrome protein) in oocyte maturation that prevents completion of second meiosis. SUMMARY ANSWER: In mice, N-WASP deletion did not affect oocyte polarity and asymmetric meiotic division in first meiosis, but did impair midbody formation and second meiosis completion. WHAT IS KNOWN ALREADY: N-WASP regulates actin dynamics and participates in various cell activities through the RHO-GTPase-Arp2/3 (actin-related protein 2/3 complex) pathway, and specifically the Cdc42 (cell division cycle 42)-N-WASP-Arp2/3 pathway. Differences in the functions of Cdc42 have been obtained from in vitro compared to in vivo studies. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: By conditional knockout of N-WASP in mouse oocytes, we analyzed its in vivo functions by employing a variety of different methods including oocyte culture, immunofluorescent staining and live oocyte imaging. Each experiment was repeated at least three times, and data were analyzed by paired-samples t-test. MAIN RESULTS AND THE ROLE OF CHANCE: Oocyte-specific deletion of N-WASP did not affect the process of oocyte maturation including spindle formation, spindle migration, polarity establishment and maintenance, and homologous chromosome or sister chromatid segregation, but caused failure of cytokinesis completion during second meiosis (P

Previous studies by others and ourselves have suggested that the methylation pattern of imprinted genes in oocytes is altered during postovulatory aging. The purpose of the current study was to evaluate the effects of postovulatory aging of mouse oocytes on methylation and expression of imprinted genes at the mid-gestation development stages. Proestrous females were artificially inseminated at 13 h (fresh-oocyte group) or 22 h (aged-oocyte group) post-hCG. Estrous females were mated with males as a control group. On dpc (day post coitus) 10.5 of development, embryos and placentas were collected and DNA and RNA were extracted, respectively. Methylation and total expression of Igf2r and H19 was investigated by quantitative analysis of methylation by PCR and quantitative real-time RT-PCR, respectively. Our results showed no significant changes of methylation in the differentially methylated region (DMR) and total expression of Igf2r in embryos and placentas, and no significant changes in methylation of H19 in embryos at dpc 10.5 of development compared with the control group regardless of artificial insemination of fresh or aged oocytes. In contrast, placentas of the aged-oocyte group exhibited significantly lower methylation levels in the H19 DMR. Furthermore, we observed that the increased expression of H19 in placentas of the aged-oocyte group was associated with significant hypomethylation of H19 DMR. These results suggest that postovulatory aging of mouse oocytes has adversed effects on methylation and expression of H19 in placentas at the mid-gestation development stage.

The oocyte's meiotic spindle is a dynamic structure that relies on microtubule organization and regulation by centrosomes. Disorganization of centrosomal proteins, including the nuclear mitotic apparatus (NuMA) protein and the molecular motor complex dynein/ dynactin, can lead to chromosomal instability and developmental abnormalities. The present study reports the distribution and function of these proteins in human oocytes, zygotes and early embryos. A total of 239 oocytes, 90 zygotes and discarded embryos were fixed and analyzed with confocal microscopy for NuMA and dynactin distribution together with microtubules and chromatin. Microtubule-associated dynein-dependent transport functions were explored by inhibiting phosphatase and ATPase activity with sodium-orthovanadate (SOV). At germinal vesicle (GV) stages, NuMA was dispersed across the nucleoplasm. After GV breaks down, NuMA became cytoplasmic before loca- lizing at the spindle poles in metaphase I and II oocytes. Aberrant NuMA localization patterns were found during oocyte in vitro maturation. After fertilization, normal and abnormal pronuclear stage zygotes and embryos displayed translocation of NuMA to interphase nuclei. SOV treatment for up to 2 h induced lower maturation rates with chromosomal scattering and ectopic localization of NuMA. Accurate distri- bution of NuMA is important for oocyte maturation, zygote and embryo development in humans. Proper assembly of NuMA is likely necess- ary for bipolar spindle organization and human oocyte developmental competence.

The cause of polycystic ovary syndrome (PCOS), a complex endocrine disorder, is unknown, but its familial aggregation implies underlying genetic influences. Hyperandrogenemia is regarded as a major endocrine character of the PCOS. In this study, we employed bisulfite sequencing and bisulfite restriction analysis to investigate the DNA methylation status of LHR, AR, FSHR and H19 in dehydroepiandrosterone (DHEA)-induced mouse PCOS model. The result showed that methylation of LHR was lost in ovary from induced PCOS mouse. However, AR, FSHR and H19 had similar methylation pattern in DHEA-treated group and control groups. These data provide evidence for close linkage between DNA demethylation of LHR and PCOS.

Unlike the well-established roles of androgen and androgen receptor (AR) in males, the functions of this steroid and its receptor in the ovary are still unclear. For decades, androgen and AR have long been considered to play a negative (at least not a positive) role in mammalian oocyte maturation. However, recent studies by us and others showed their positive influence in promoting meiotic maturation. On the other hand, rapid non-genomic effects of androgens have been observed and are now generally accepted as contributing to the physiological effects of the steroids and their related receptors in somatic cells, and this has stimulated us to explore the complex roles of AR in the ovary. Based on the classic dogma and new findings, we collected evidence to propose that the expression of AR shifts from the oocytes to the theca cells and finally disappears in the oocytes during evolution. It is suggested that the non-genomic pathway involving androgen and AR in the mammalian oocytes, unlike somatic cells, cells will undergo elimination. The function of androgen and AR in promoting meiotic maturation may have been replaced gradually by gonadotrophins. Moreover, a possible relationship between AR and polycystic ovary syndrome is also discussed, which might provide a clue for the pathology of the disease.

Centrosome integrity is critically important for successful fertilization and embryo development. In humans, the sperm contributes the dominant centrosomal material containing centrioles and centrosomal components onto which oocyte centrosomal proteins assemble after sperm incorporation to form the sperm aster that is essential for uniting sperm and oocyte pronuclei. Increasingly, dysfunctional sperm centrosomes have been identified as a factor for sperm-derived infertility and heterologous Intracytoplasmic Sperm Injection (ICSI) has been used to assess centrosome and sperm aster formation and clearly established a relationship between infertility and sperm centrosomal dysfunction. ICSI has been used successfully to provide novel treatment to overcome male factor infertility and it may open up new possibilities to correct specific sperm-related centrosome dysfunctions at molecular levels. New data indicate that it is now possible to replace dysfunctional centrosomes with functional donor sperm centrosomes which may provide new treatment for couples in which infertility is a result of centrosome-related sperm dysfunctions.