Single-cell transcriptome landscape of ovarian cells during primordial follicle assembly in mice.

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.

Impaired primordial follicle assembly in offspring ovaries from zearalenone-exposed mothers involves reduced mitochondrial activity and altered epigenetics in oocytes.

Previous works have shown that zearalenone (ZEA), as an estrogenic pollutant, has adverse effects on mammalian folliculogenesis. In the present study, we found that prolonged exposure of female mice to ZEA around the end of pregnancy caused severe impairment of primordial follicle formation in the ovaries of newborn mice and altered the expression of many genes in oocytes as revealed by single-cell RNA sequencing (scRNA-seq). These changes were associated with morphological and molecular alterations of mitochondria, increased autophagic markers in oocytes, and epigenetic changes in the ovaries of newborn mice from ZEA-exposed mothers. The latter increased expression of HDAC2 deacetylases was leading to decreased levels of H3K9ac and H4K12ac. Most of these modifications were relieved when the expression of  Hdac2 in newborn ovaries was reduced by RNA interference during in vitro culture in the presence of ZEA. Such changes were also alleviated in offspring ovaries from mothers treated with both ZEA and the coenzyme Q10 (CoQ10), which is known to be able to restore mitochondrial activities. We concluded that impaired mitochondrial activities in oocytes caused by ZEA are at the origin of metabolic alterations that modify the expression of genes controlling autophagy and primordial follicle assembly through changes in epigenetic histones.

YAP regulates porcine skin-derived stem cells self-renewal partly by repressing Wnt/ß-catenin signaling pathway.

Skin-derived stem cells (SDSCs) are a class of adult stem cells (ASCs) that have the ability to self-renew and differentiate. The regulation mechanisms involved in the differentiation of SDSCs are a hot topic. In this paper, we explore the link between the transcriptional regulator yes-associated protein (YAP) and the fate of porcine SDSCs (pSDSCs). We found that lysophosphatidylcholine (LPC) activates YAP, promotes pSDSCs pluripotency, and counteracts transdifferentiation of pSDSCs into porcine primordial germ cell-like cells (pPGCLCs). YAP promotes the pluripotent state of pSDSCs by maintaining the high expression of the pluripotency genes Oct4 and Sox2. The overexpression of YAP prevented the differentiation of pSDSCs, and the depletion of YAP by small interfering RNA (siRNAs) suppressed the self-renewal of pSDSCs. In addition, we found that YAP regulates the fate of pSDSCs through a mechanism related to the Wnt/ß-catenin signaling pathway. When an activator of the Wnt/ß-catenin signaling pathway, CHIR99021, was added to pSDSCs overexpressing YAP, the ability of pSDSCs to differentiate was partially restored. Conversely, when XAV939, an inhibitor of the Wnt/ß-catenin signaling pathway, was added to YAP knockdown pSDSCs a higher self-renewal ability resulted. Taken together, our results suggested that YAP and the Wnt/ß-catenin signaling pathway interact to regulate the fate of pSDSCs.

Dissecting the initiation of female meiosis in the mouse at single-cell resolution.

Meiosis is one of the most finely orchestrated events during gametogenesis with distinct developmental patterns in males and females. However, the molecular mechanisms involved in this process remain not well known. Here, we report detailed transcriptome analyses of cell populations present in the mouse female gonadal ridges (E11.5) and the embryonic ovaries from E12.5 to E14.5 using single-cell RNA sequencing (scRNA seq). These periods correspond with the initiation and progression of meiosis throughout the first stage of prophase I. We identified 13 transcriptionally distinct cell populations and 7 transcriptionally distinct germ cell subclusters that correspond to mitotic (3 clusters) and meiotic (4 clusters) germ cells. By analysing cluster-specific gene expression profiles, we found four cell clusters correspond to different cell stages en route to meiosis and characterized their detailed transcriptome dynamics. Our scRNA seq analysis here represents a new important resource for deciphering the molecular pathways driving female meiosis initiation.

Multi-omics analysis reveals that iron deficiency impairs spermatogenesis by gut-hormone synthesis axis.

Considering that research has mainly focussed on how excessive iron supplementation leads to reproductive cytotoxicity, there is a lack of in-depth research on reproductive system disorders caused by iron deficiency. To gain a better understanding of the effects of iron deficiency on the reproductive system, especially spermatogenesis, we first constructed a mouse model of iron deficiency. We employed multi-omic analysis, including transcriptomics, metabolomics, and microbiomics, to comprehensively dissect the impact of iron deficiency on spermatogenesis. Moreover, we verified our findings in detail using western blot, immunofluorescence, immunohistochemistry, qRT-PCR and other techniques. Microbiomic analysis revealed altered gut microbiota in iron-deficient mice, and functional predictive analysis showed that gut microbiota can regulate spermatogenesis. The transcriptomic data indicated that iron deficiency directly alters expression of meiosis-related genes. Transcriptome data also revealed that iron deficiency indirectly regulates spermatogenesis by affecting hormone synthesis, findings confirmed by metabolomic data, western blot and immunofluorescence. Interestingly, competing endogenous RNA networks also play a vital role in regulating spermatogenesis after iron deficiency. Taken together, the data elucidate that iron deficiency impairs spermatogenesis and increases the risk of male infertility by affecting hormone synthesis and promoting gut microbiota imbalance.

The process of ovarian aging: it is not just about oocytes and granulosa cells.

Ovarian age is classically considered the main cause of female reproductive infertility. In women, the process proceeds as an ongoing decline in the primordial follicle stockpile and it is associated with reduced fertility in the mid-thirties, irregular menstruation from the mid-forties, cessation of fertility, and, eventually, menopause in the early fifties. Reproductive aging is historically associated with changes in oocyte quantity and quality. However, besides the oocyte, other cellular as well as environmental factors have been the focus of more recent investigations suggesting that ovarian decay is a complex and multifaceted process. Among these factors, we will consider mitochondria and oxidative stress as related to nutrition, changes in extracellular matrix molecules, and the associated ovarian stromal compartment where immune cells of both the native and adaptive systems seem to play an important role. Understanding such processes is crucial to design treatment strategies to  slow down ovarian aging and consequently prolong reproductive lifespan and, more to this, alleviaingt side effects of menopause on the musculoskeletal, cardiovascular, and nervous systems.

The Beginning of Meiosis in Mammalian Female Germ Cells: A Never-Ending Story of Intrinsic and Extrinsic Factors.

Meiosis is the unique division of germ cells resulting in the recombination of the maternal and paternal genomes and the production of haploid gametes. In mammals, it begins during the fetal life in females and during puberty in males. In both cases, entering meiosis requires a timely switch from the mitotic to the meiotic cell cycle and the transition from a potential pluripotent status to meiotic differentiation. Revealing the molecular mechanisms underlying these interrelated processes represents the essence in understanding the beginning of meiosis. Meiosis facilitates diversity across individuals and acts as a fundamental driver of evolution. Major differences between sexes and among species complicate the understanding of how meiosis begins. Basic meiotic research is further hindered by a current lack of meiotic cell lines. This has been recently partly overcome with the use of primordial-germ-cell-like cells (PGCLCs) generated from pluripotent stem cells. Much of what we know about this process depends on data from model organisms, namely, the mouse; in mice, the process, however, appears to differ in many aspects from that in humans. Identifying the mechanisms and molecules controlling germ cells to enter meiosis has represented and still represents a major challenge for reproductive medicine. In fact, the proper execution of meiosis is essential for fertility, for maintaining the integrity of the genome, and for ensuring the normal development of the offspring. The main clinical consequences of meiotic defects are infertility and, probably, increased susceptibility to some types of germ-cell tumors. In the present work, we report and discuss data mainly concerning the beginning of meiosis in mammalian female germ cells, referring to such process in males only when pertinent. After a brief account of this process in mice and humans and an historical chronicle of the major hypotheses and progress in this topic, the most recent results are reviewed and discussed.

Stimulated by retinoic acid gene 8 (STRA8) interacts with the germ cell specific bHLH factor SOHLH1 and represses c-KIT expression in vitro.

STRA8 (Stimulated by Retinoic Acid Gene 8) controls the crucial decision of germ cells to engage meiotic division up and down-regulating genes involved in the meiotic programme. It has been proven as an amplifier of genes involved in cell cycle control and chromosome events, however, how STRA8 functions as negative regulator are not well understood. In this study, we demonstrate that STRA8 can interact with itself and with other basic Helix-Loop-Helix (bHLH) transcription factors through its HLH domain and that this domain is important for its ability to negatively interfere with the Ebox-mediated transcriptional activity of bHLH transcription factors. Significantly, we show that STRA8 interacts with TCF3/E47, a class I bHLH transcription factors, and with SOHLH1, a gonadal-specific bHLH, in male germ cells obtained from prepuberal mouse testis. We demonstrated that STRA8, indirectly, is able to exert a negative control on the SOHLH1-dependent stimulation of c-KIT expression in late differentiating spermatogonia and preleptotene spermatocytes. Although part of this results were obtained only 'in vitro', they support the notion that STRA8 interacting with different transcription factors, besides its established role as 'amplifier' of meiotic programme, is able to finely modulate the balance between spermatogonia proliferation, differentiation and acquisition of meiotic competence.

Inflammatory cytokines as key players of apoptosis induced by environmental estrogens in the ovary.

Natural and synthetic environmental estrogens (EEs), interfering with the physiological functions of the body's estrogens, are widespread and are rising much concern for their possible deleterious effects on human and animal health, in particular on reproduction. In fact, increasing evidence indicate that EEs can be responsible for a variety of disfunctions of the reproductive system especially in females such as premature ovarian insufficiency (POI). Because of their great structural diversity, the modes of action of EEs are controversial. One important way through which EEs exert their effects on reproduction is the induction of apoptosis in the ovary. In general, EEs can exert pro-and anti-apoptotic effects by agonizing or antagonizing numerous estrogen-dependent signaling pathways. In the present work, results concerning apoptotic pathways and diseases induced by representative EEs (such as zearalenone, bisphenol A and di-2-ethylhexyl phthalate), in ovaries throughout development are presented into an integrated network. By reviewing and elaborating these studies, we propose inflammatory factors, centered on the production of tumor necrosis factor (TNF), as a major cause of the induction of apoptosis by EEs in the mammalian ovary. As a consequence, potential strategies to prevent such EE effect are suggested.

PI3K/PTEN/AKT Signaling Pathways in Germ Cell Development and Their Involvement in Germ Cell Tumors and Ovarian Dysfunctions.

Several studies indicate that the PI3K/PTEN/AKT signaling pathways are critical regulators of ovarian function including the formation of the germ cell precursors, termed primordial germ cells, and the follicular pool maintenance. This article reviews the current state of knowledge of the functional role of the PI3K/PTEN/AKT pathways during primordial germ cell development and the dynamics of the ovarian primordial follicle reserve and how dysregulation of these signaling pathways may contribute to the development of some types of germ cell tumors and ovarian dysfunctions.

To Be or Not to Be a Germ Cell: The Extragonadal Germ Cell Tumor Paradigm.

In the human embryo, the genetic program that orchestrates germ cell specification involves the activation of epigenetic and transcriptional mechanisms that make the germline a unique cell population continuously poised between germness and pluripotency. Germ cell tumors, neoplasias originating from fetal or neonatal germ cells, maintain such dichotomy and can adopt either pluripotent features (embryonal carcinomas) or germness features (seminomas) with a wide range of phenotypes in between these histotypes. Here, we review the basic concepts of cell specification, migration and gonadal colonization of human primordial germ cells (hPGCs) highlighting the analogies of transcriptional/epigenetic programs between these two cell types.

Establishment and depletion of the ovarian reserve: physiology and impact of environmental chemicals.

The reproductive life span in women starts at puberty and ends at menopause, following the exhaustion of the follicle stockpile termed the ovarian reserve. Increasing data from experimental animal models and epidemiological studies indicate that exposure to a number of ubiquitously distributed reproductively toxic environmental chemicals (RTECs) can contribute to earlier menopause and even premature ovarian failure. However, the causative relationship between environmental chemical exposure and earlier menopause in women remains poorly understood. The present work, is an attempt to review the current evidence regarding the effects of RTECs on the main ovarian activities in mammals, focusing on how such compounds can affect the ovarian reserve at any stages of ovarian development. We found that in rodents, strong evidence exists that in utero, neonatal, prepubescent and even adult exposure to RTECs leads to impaired functioning of the ovary and a shortening of the reproductive lifespan. Regarding human, data from cross-sectional surveys suggest that human exposure to certain environmental chemicals can compromise a woman's reproductive health and in some cases, correlate with earlier menopause. In conclusion, evidences exist that exposure to RTECs can compromise a woman's reproductive health. However, human exposures may date back to the developmental stage, while the adverse effects are usually diagnosed decades later, thus making it difficult to determine the association between RTECs exposure and human reproductive health. Therefore, epidemiological surveys and more experimental investigation on humans, or alternatively primates, are needed to determine the direct and indirect effects caused by RTECs exposure on the ovary function, and to characterize their action mechanisms.

Expression and possible roles of extracellular signal-related kinases 1-2 (ERK1-2) in mouse primordial germ cell development.

In the present work, we described the expression and activity of extracellular signal-related kinases 1-2 (ERK1-2) in mouse primordial germ cells (PGCs) from 8.5-14.5 days post coitum (dpc) and investigated whether these kinases play a role in regulating the various processes of PGC development. Using immunofluorescence and immunoblotting to detect the active phosphorylated form of ERK1-2 (p-ERK1-2), we found that the kinases were present in most proliferating 8.5-10.5 dpc PGCs, low in 11.5 dpc PGCs, and progressively increasing between 12.5-14.5 dpc both in female and male PGCs. In vitro culture experiments showed that inhibiting activation of ERK1-2 with the MEK-specific inhibitor U0126 significantly reduced the growth of 8.5 dpc PGCs in culture but had little effect on 11.5-12.5 dpc PGCs. Moreover, we found that the inhibitor did not affect the adhesion of 11.5 dpc PGCs, but it significantly reduced their motility features onto a cell monolayer. Further, while the ability of female PGCs to begin meiosis was not significantly affected by U0126, their progression through meiotic prophase I was slowed down. Notably, the activity of ERK1-2 was necessary for maintaining the correct expression of oocyte-specific genes crucial for germ cells survival and the formation of primordial follicles.

Effects of activin A on the transcriptome of mouse oogenesis in vitro.

From the previous research, it has been supported that activin A (ActA) is conducive to ovarian development in vitro. In the present paper, with the aim to identify the molecular pathways through which ActA can influence processes of the fetal and early postnatal oogenesis, we analyzed the transcriptome of embryonic ovaries (12.5 days postcoitum) in vitro cultured with or without ActA for 6 days, as well as the produced oocytes for 28 days, and further compared the gene expression profile with their in vivo counterparts. With the confirmation of designed test, we found that the addition of ActA to the ovary culture tended, generally, to align oocyte gene expression to the in vivo condition, in particular of a number of genes involved in meiosis and epigenetic modifications of histones. In particular, we identified DNA recombination during the oocyte meiotic prophase I and lysine trimethylation of the histone H3K27 during the oocyte growth phase as molecular pathways modulated by ActA.

Identification of oxidative stress-related Xdh gene as a di(2-ethylhexyl)phthalate (DEHP) target and the use of melatonin to alleviate the DEHP-induced impairments in newborn mouse ovaries.

This study, using an in vitro ovary culture model, investigates the mechanisms through which di(2-ethylhexyl)phthalate (DEHP) impairs germ cell cyst breakdown and primordial follicle assembly. The results indicate the latter effects exerted by 10 or 100 µmol/L DEHP in cultured newborn ovaries were associated with increased levels of reactive oxygen species (ROS) and apoptosis. Based on a transcriptome analysis, we found the expression of the oxidative stress-related gene Xdh (xanthine dehydrogenase) was significantly upregulated in DEHP-cultured ovaries. Two treatments, namely Xdh RNAi or the addition of melatonin to the ovary culture, inhibited the increase in Xdh expression and ROS levels caused by DEHP and, at the same time, reduced apoptosis and the impairment of primordial follicle assembly in the treated ovaries. Together, the results identify Xdh gene as one of the major targets of DEHP in newborn ovaries and that the consequent increased level of ROS is possibly responsible for the increment of apoptosis and primordial follicle assembly impairment. At the same time, they highlight that melatonin alleviates the effects of DEHP as with other endocrine-disrupting compounds on the ovary.

Melatonin protects prepuberal testis from deleterious effects of bisphenol A or diethylhexyl phthalate by preserving H3K9 methylation.

A growing number of couples experience fertility issues with almost half being due to malefactors. The exposure to toxic environmental contaminants, such as endocrine disruptors (EDs), has been shown to negatively affect male fertility. EDs are present in the environment, and exposure to these toxins results in the failure of spermatogenesis. The deleterious effects of EDs on spermatogenesis have been well documented, whereas improvement of infertility associated with spermatogenesis defects remains a great challenge. Herein, we report that in vitro exposure of prepuberal mouse testes to two well-known endocrine disruptors (EDs), bisphenol A (BPA) or diethylhexyl phthalate (DEHP), impairs spermatogenesis with perturbing self-renewal, spermatogonia activity, and meiosis. Evidence indicates that such effects are likely due, at least in part, to decreased G9a-dependent H3K9 di-methylation. Of note, we found that melatonin (MLT) protected the testis from the negative ED impacts with preserving spermatogonia stem and meiotic cells, along with maintaining normal H3K9 di-methylation in these cells. Taken together, this work documents that BPA and EDHP adversely affect prepuberal spermatogenesis and perturb crucial epigenetic activities in male germ cells and highlight the protective ability of MLT.

Gonadal development and germ cell tumors in mouse and humans.

In multicellular organisms, proper development of gonads and germ cells is essential for the transmission of genetic information to the next generations and eventually for the survival of the species. For this reason, germline development is finely regulated to control germ cell proliferation, survival and differentiation. Disruption of such controls can lead to infertility or germ cell tumors (GCTs). GCTs are particularly hideous pathologies since they occur mainly in neonates, infants, and children, rarely in the adults. They arise primarily in the testes and ovaries, though they can also develop in extragonadal sites along the midline of the body and the brain. Many similarities exist between most types of GCTs of the ovary and testis, including a morphological resemblance (often constituting a caricature of normal embryogenesis) and a similar pattern of chromosomal alterations. Furthermore, families with both ovarian and testicular GCTs have been reported, suggesting a possible common genetic etiology. This review focuses on the cellular processes, differentiation events and molecular mechanisms occurring during gonad development in mice and humans whose disturbance can be implicated in GCT formation.

Di (2-ethylhexyl) phthalate impairs steroidogenesis in ovarian follicular cells of prepuberal mice.

Di (2-ethylhexyl) phthalate (DEHP) is a plasticizer which is widely used in the manufacture of plastics. As a common environmental contaminant and recognized endocrine disrupting chemical, DEHP is able to deregulate the functions of a variety of tissues, including the reproductive system both in males and females. In order to investigate the possible effects of DEHP on the first wave of folliculogenesis, occurring in the mouse ovary postnatally, mice were administered 20 or 40 µg/kg DEHP through intraperitoneal injection at days 5, 10 and 15 post partum (dpp). Following DEHP treatment the gene expression profile of control and exposed ovaries was compared by microarray analyses at 20 dpp. We found that in the exposed ovaries DEHP significantly altered the transcript levels of several immune response and steroidogenesis associated genes. In particular, DEHP significantly decreased the expression of genes essential for androgen synthesis by theca cells including Lhcgr, Cyp17a1, Star and Ldlr. Immunohistochemistry and immune flow cytometry confirmed reduced expression of LHCGR and CYP17A1 proteins in the exposed theca cells. These effects were associated to a significant reduction in ovarian concentrations of progesterone, 17ß-estradiol and androstenedione along with a reduction of LH in the serum. Although we did not find a significant reduction of the number of primary, secondary or antral follicles in the DEHP exposed ovaries when compared to controls, we did observe that theca cells showed an altered structure of the nuclear envelope, fewer mitochondria, and mitochondria with a reduced number of cristae. Collectively, these results demonstrate a deleterious effect of DEHP exposure on ovarian steroidogenesis during the first wave of folliculogenesis that could potentially affect the correct establishment of the hypothalamic-pituitary-ovarian axis and the onset of puberty.

Isolation and Purification of Viable PGCs from Mouse Embryos.

In all organisms with sexual reproduction, sperm and oocytes derive from embryonic precursors termed primordial germ cells (PGCs) which pass on genetic information to subsequent generations. Studies aimed to unravel PGC development at molecular level in mammals can be traced at the early 1980s and were hampered by the difficulty in obtaining both sufficient quantities and purity of PGCs. For many laboratories, the isolation and purification methods of PGCs at different stages from embryos are the most shortcut and affordable tool to study many aspects of their development at cellular and molecular levels. In the present chapter, I focus on immunomagnetic cell sorting (MACS) and fluorescence-activated cell sorting (FACS) methods used in my laboratory for the purification of mouse PGCs from 10.5 to 12.5 dpc embryos before their differentiation in oogonia/oocytes in female and prospermatogonia in male.

Sleep deprivation causes gut dysbiosis impacting on systemic metabolomics leading to premature ovarian insufficiency in adolescent mice.

Rationale: Currently, there are occasional reports of health problems caused by sleep deprivation (SD). However, to date, there remains a lack of in-depth research regarding the effects of SD on the growth and development of oocytes in females. The present work aimed to investigate whether SD influences ovarian folliculogenesis in adolescent female mice. Methods: Using a dedicated device, SD conditions were established in 3-week old female mice (a critical stage of follicular development) for 6 weeks and gut microbiota and systemic metabolomics were analyzed. Analyses were related to parameters of folliculogenesis and reproductive performance of SD females. Results: We found that the gut microbiota and systemic metabolomics were severely altered in SD females and that these were associated with parameters of premature ovarian insufficiency (POI). These included increased granulosa cell apoptosis, reduced numbers of primordial follicles (PmFs), correlation with decreased AMH, E2, and increased LH in blood serum, and a parallel increased number of growing follicles and changes in protein expression compatible with PmF activation. SD also reduced oocyte maturation and reproductive performance. Notably, fecal microbial transplantation from SD females into normal females induced POI parameters in the latter while niacinamide (NAM) supplementation alleviated such symptoms in SD females. Conclusion: Gut microbiota and alterations in systemic metabolomics caused by SD induced POI features in juvenile females that could be counteracted with NAM supplementation.