Primordial follicle assembly in the mouse occurs during perinatal ages and largely determines the ovarian reserve that will be available to support the reproductive life span. The development of primordial follicles is controlled by a complex network of interactions between oocytes and ovarian somatic cells that remain poorly understood. In the present research, using single-cell RNA sequencing performed over a time series on murine ovaries, coupled with several bioinformatics analyses, the complete dynamic genetic programs of germ and granulosa cells from E16.5 to postnatal day (PD) 3 were reported. Along with confirming the previously reported expression of genes by germ cells and granulosa cells, our analyses identified 5 distinct cell clusters associated with germ cells and 6 with granulosa cells. Consequently, several new genes expressed at significant levels at each investigated stage were assigned. By building single-cell pseudotemporal trajectories, 3 states and 1 branch point of fate transition for the germ cells were revealed, as well as for the granulosa cells. Moreover, Gene Ontology (GO) term enrichment enabled identification of the biological process most represented in germ cells and granulosa cells or common to both cell types at each specific stage, and the interactions of germ cells and granulosa cells basing on known and novel pathway were presented. Finally, by using single-cell regulatory network inference and clustering (SCENIC) algorithm, we were able to establish a network of regulons that can be postulated as likely candidates for sustaining germ cell-specific transcription programs throughout the period of investigation. Above all, this study provides the whole transcriptome landscape of ovarian cells and unearths new insights during primordial follicle assembly in mice.

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Rationale: Accumulated evidence indicates that environmental plasticizers are a threat to human and animal fertility. Di (2-ethylhexyl) phthalate (DEHP), a plasticizer to which humans are exposed daily, can trigger reproductive toxicity by acting as an endocrine-disrupting chemical. In mammals, the female primordial follicle pool forms the lifetime available ovarian reserve, which does not undergo regeneration once it is established during the fetal and neonatal period. It is therefore critical to examine the toxicity of DEHP regarding the establishment of the ovarian reserve as it has not been well investigated. Methods: The ovarian cells of postnatal pups, following maternal DEHP exposure, were prepared for single cell-RNA sequencing, and the effects of DEHP on primordial follicle formation were revealed using gene differential expression analysis and single-cell developmental trajectory. In addition, further biochemical experiments, including immunohistochemical staining, apoptosis detection, and Western blotting, were performed to verify the dataset results. Results: Using single-cell RNA sequencing, we revealed the gene expression dynamics of female germ cells and granulosa cells following exposure to DEHP in mice. Regarding germ cells: DEHP impeded the progression of follicle assembly and interfered with their developmental status, while key genes such as Lhx8 , Figla, and others, strongly evidenced the reduction. As for granulosa cells: DEHP likely inhibited their proliferative activity, and activated the regulation of cell death. Furthermore, the interaction between ovarian cells mediated by transforming growth factor-beta signaling, was disrupted by DEHP exposure, since the expression of GDF9, BMPR1A, and SMAD3 was affected. In addition, DNA damage and apoptosis were elevated in germ cells and/or somatic cells. Conclusion: These findings offer substantial novel insights into the reproductive toxicity of DEHP exposure during murine germ cell cyst breakdown and primordial follicle formation. These results may enhance the understanding of DEHP exposure on reproductive health.

Precise control of mammalian oogenesis has been a traditional focus of reproductive and developmental biology research. Recently, new reports have introduced the possibility of obtaining functional gametes derived in vitro from stem cells. The potential to produce functional gametes from stem cells has exciting applications for regenerative medicine though still remains challenging. In mammalian females ovulation and fertilization is a privilege reserved for a small number of oocytes. In reality the vast majority of oocytes formed from primordial germ cells (PGCs) will undergo apoptosis, or other forms of cell death. Removal occurs during germ cell cyst breakdown and the establishment of the primordial follicle (PF) pool, during the long dormancy at the PF stage, or through follicular atresia prior to reaching the ovulatory stage. A way to solve this limitation could be to produce large numbers of oocytes, in vitro, from stem cells. However, to recapitulate mammalian oogenesis and produce fertilizable oocytes in vitro is a complex process involving several different cell types, precise follicular cell–oocyte reciprocal interactions, a variety of nutrients and combinations of cytokines, and precise growth factors and hormones depending on the developmental stage. In 2016, two papers published by Morohaku et al. and Hikabe et al. reported in vitro procedures that appear to reproduce efficiently these conditions allowing for the production, completely in a dish, of a relatively large number of oocytes that are fertilizable and capable of giving rise to viable offspring in the mouse. The present article offers a critical overview of these results as well as other previous work performed mainly in mouse attempting to reproduce oogenesis completely in vitro and considers some perspectives for the potential to adapt the methods to produce functional human oocytes.

Although it is becoming increasingly evident that maternal starvation during pregnancy can have permanent effects on a range of physiological processes in the offspring, scant information is available about the consequence of such condition for oogenesis and hence for lifetime reproductive success of progeny in mammals. In the present study, we address this topic by starving pregnant mice at the time of ovarian differentiation (12.5 days post coitum (dpc)) for three consecutive days and analyzed the consequence first on the survival of the fetal oocytes and their capability to progress throughout the stages of meiotic prophase I (MPI) and then on the postnatal folliculogenesis of the offspring. The results showed that maternal starvation increased apoptosis in the fetal ovaries, resulting in reduction of the oocyte number. Moreover, MPI progression was slowed down in the surviving oocytes and the expression of DNA repair players in the starved ovaries increased. Transcriptome analysis identified 61 differentially expressed genes between control and starved ovaries, the most part of these being involved in metabolic processes. A significant decrease in the percentage of oocytes enclosed in primordial follicles and the expression of oocyte genes critically involved in folliculogenesis such as Nobox, Lhx8 and Sohlh2 in the 3 days post partum (dpp) starved ovaries were found. Finally, at the time of juvenile period (21 dpp), the number of oocytes and antral follicles resulted significantly lower in the ovaries of the offspring from starved mothers in comparison to controls. Our findings support the notion that maternal starvation can affect ovary development in the offspring that could adversely affect their reproductive success in the adult life.

Long non-coding RNA (lncRNA) is involved in many biological processes, and it has been closely investigated. However, research into the role of lncRNA in ovine ovarian development is scant and poorly understood, particularly in relation to the molecular mechanisms of ovine oocyte maturation. In the current study, RNA sequencing was performed with germinal vesicle (GV) and in vitro matured metaphase II (MII) stage oocytes, isolated from ewes. Through the use of bioinformatic analysis, abundant candidate lncRNAs in stage-specific ovine oocytes were identified, and their trans-and cis-regulatory effects were deeply dissected using computational prediction software. Functional enrichment analysis of these lncRNAs revealed that they were involved in the regulation of many key signaling pathways during ovine oocyte development, which was reflected by their targeted genes. From this study, multiple lncRNA-mRNA networks were presumed to be involved in key signaling pathways regarding ovine oocyte maturation and meiotic resumption. In particular, one novel lncRNA (MSTRG.17927) appeared to mediate the regulation of phosphatidylinositol 3-kinase signaling (PI3K) signaling during ovine oocyte maturation. Therefore, this research offers novel insights into the molecular mechanisms underlying ovine oocyte meiotic maturation regulated by lncRNA-mRNA networks from a genome-wide perspective.

Litter size (LS), an important economic trait in livestock, is so complicate that involves many aspects of reproduction, the underlying mechanism of which particularly in goat has always been scanty. To uncover the genetic basis of LS, the genomic sequence of Jining Gray goat groups (one famous breed for high prolificacy in China) with LS 1, 2, and 3 for firstborn was analyzed, obtaining 563.67 Gb sequence data and a total of 31,864,651 high-quality single nucleotide polymorphisms loci were identified. Particularly, the increased heterozygosity in higher LS groups, and large continuous homozygous segments associated with lower LS group had been uncovered. Through an integrated analysis of three popular methods for detecting selective sweeps (Fst, nucleotide diversity, and Tajima's D statistic), 111 selected regions and 42 genes associated with LS were scanned genome wide. The candidate genes with highest selective signatures included KIT, KCNH7, and KMT2E in LS2 and PAK1, PRKAA1, and SMAD9 in LS3 group, respectively. Meanwhile, functional terms of programmed cell death involved in cell development and regulation of insulin receptor signaling pathway were mostly enriched with 42 candidate genes, which also included reproduction related terms of steroid metabolic process and cellular response to hormone stimulus. In conclusion, our study identified novel candidate genes involving in regulation of LS in goat, which expand our understanding of genetic fundament of reproductive ability, and the novel insights regarding to LS would be potentially applied to improve reproductive performance.

The new technology of high‐throughput single‐cell RNA sequencing (10 × scRNA‐seq) was developed recently with many advantages. However, it was not commonly used in farm animal research. There are few reports for the gene expression of goat ovarian follicle granulosa cells (GCs) during different developmental stages. In the current investigation, the gene expression of follicle GCs at different stages from two populations of Ji'ning grey goats: high litter size (HL; ≥3/L; 2 L) and low litter size (LL; ≤2 /L; 2 L) were analysed by scRNA‐seq. Many GC marker genes were identified, and the pseudo‐time showed that GCs developed during the time course which reflected the follicular development and differentiation trajectory. Moreover, the gene expression difference between the two populations HL versus LL was very clear at different developmental stages. Many marker genes differentially expressed at different developmental stages. ASIP and ASPN were found to be highly expressed in the early stage of GCs, INHA, INHBA, MFGE8 and HSD17B1 were identified to be highly expressed in the growing stage of GCs, while IGFBP2, IGFBP5 and CYP11A1 were found to be highly expressed in late stage. These marker genes could be used as reference genes of goat follicle GC development. This investigation for the first time discovered the gene expression patterns in goat follicle GCs in high‐ or low‐fertility populations (based on litter size) by scRNA‐seq which may be useful for uncovering the oocyte development potential.