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.

Recent studies have suggested that leptin has a central role in female reproduction, including ovarian function. The leptin receptor (Ob-R) has six isoforms and can signal through either the MAPK or the Janus-activated kinase/signal transducer and activator of transcription signal-transduction pathway, depending on the isoform. Expression of Ob-R has been reported in human and mouse oocytes; however, the physiological role of leptin during follicular development and oocyte maturation is largely unknown. In the current study, expression of Ob-R during oocyte growth and maturation was investigated in porcine oocytes from small, medium, and large follicles and in oocytes in the germinal vesicle (GV), GV breakdown, and metaphase II (MII) stages at both the mRNA and protein levels. The proportion of oocytes expressing Ob-R was maximal in oocytes from medium follicles and at the GV breakdown stage (P < 0.05), whereas the proportion of oocytes expressing the long isoform, Ob-Rb, was found to be consistently low throughout growth and maturation. When included in oocyte maturation medium, leptin significantly increased the proportion of oocytes reaching MII (P < 0.01), elevated cyclin B1 protein content in MII-stage oocytes (P < 0.05), and enhanced embryo developmental potential (P < 0.05), suggesting that leptin plays a role in both nuclear and cytoplasmic maturation. During oocyte maturation, leptin increased phosphorylated MAPK content by 2.8-fold (P < 0.05), and leptin-stimulated oocyte maturation was blocked when leptin-induced MAPK phosphorylation was suppressed by a specific MAPK activation inhibitor, U0126 (P < 0.01), demonstrating that leptin enhances nuclear maturation via activation of the MAPK pathway.

Di (2-ethylhexyl) phthalate (DEHP), is the most common member of the class of phthalates that are used as plasticizers and have become common environmental contaminants. A number of studies have shown that DEHP exposure impacts reproductive health in both male and female mammals by acting as an estrogen analog. Here, we investigated the effects of DEHP on meiotic progression of fetal mouse oocytes by using an in vitro model of ovarian tissue culture. The results showed that 10 or 100 μM DEHP exposure inhibited the progression of oocytes throughout meiotic prophase I, specifically from the pachytene to diplotene stages. DEHP possibly impairs the ability to repair DNA double-strand breaks induced by meiotic recombination and as a consequence activates a pachytene check point. At later stages, such defects led to an increased number of oocytes showing apoptotic markers (TUNEL staining, expression of pro-apoptotic genes), resulting in reduced oocyte survival, gap junctions, and follicle assembly in the ovarian tissues. Microarray analysis of ovarian tissues exposed to DEHP showed altered expression of several genes including some involved in apoptosis and gonad development. The expression changes of some genes clustered in cell-cell communication and signal transduction, along with plasma membrane, extracellular matrix and ion channel function classes, were dependent on the DEHP concentration. Together, these results bring new support to the notion that exposure to DEHP during gestation might exert deleterious effects on ovary development, perturbing germ cell meiosis and the expression of genes involved in a wide range of biological processes including ovary development.

We have previously demonstrated that stem cells isolated from fetal porcine skin have the potential to form oocyte-like cells (OLCs) in vitro. However, primordial germ cells (PGCs), which must also be specified during the stem cell differentiation to give rise to these putative oocytes at more advanced stages of culture, were not systematically characterized. The current study tested the hypothesis that a morphologically distinct population of cells derived from skin stem cells prior to OLC formation corresponds to putative PGCs, which differentiate further into more mature gametes. When induced to differentiate in an appropriate microenvironment, a subpopulation of morphologically distinct cells, some of which are alkaline phosphatase (AP)-positive, also express Oct4, Fragilis, Stella, Dazl, and Vasa, which are markers indicative of germ cell formation. A known differentially methylated region (DMR) within the H19 gene locus, which is demethylated in oocytes after establishment of the maternal imprint, is hypomethylated in PGC-like cells compared to undifferentiated skin-derived stem cells, suggesting that the putative germ cell population undergoes imprint erasure. Additional evidence supporting the germ cell identity of in vitro-generated PGC-like cells is that, when labeled with a Dazl-GFP reporter, these cells further differentiate into GFP-positive OLCs. The ability to generate germ cell precursors from somatic stem cells may provide an in vitro model to study some of the unanswered questions surrounding early germ cell formation.