Pregranulosa Cells Research Articles

Single-cell and bulk transcriptional profiling of mouse ovaries reveals novel genes and pathways associated with DNA damage response in oocytes

Immature oocytes enclosed in primordial follicles stored in female ovaries are under constant threat of DNA damage induced by endogenous and exogenous factors. Checkpoint kinase 2 (CHEK2) is a key mediator of the DNA damage response (DDR) in all cells. Genetic studies have shown that CHEK2 and its downstream targets, p53, and TAp63, regulate primordial follicle elimination in response to DNA damage. However, the mechanism leading to their demise is still poorly characterized. Single-cell and bulk RNA sequencing were used to determine the DDR in wild-type and Chek2-deficient ovaries. A low but oocyte-lethal dose of ionizing radiation induces ovarian DDR that is solely dependent on CHEK2. DNA damage activates multiple response pathways related to apoptosis, p53, interferon signaling, inflammation, cell adhesion, and intercellular communication. These pathways are differentially employed by different ovarian cell types, with oocytes disproportionately affected by radiation. Novel genes and pathways are induced by radiation specifically in oocytes, shedding light on their sensitivity to DNA damage, and implicating a coordinated response between oocytes and pregranulosa cells within the follicle. These findings provide a foundation for future studies on the specific mechanisms regulating oocyte survival in the context of aging, therapeutic and environmental genotoxic exposures.

Protein phosphatase 4 maintains the survival of primordial follicles by regulating autophagy in oocytes

In mammalian ovary, the primordial follicle pool serves as the source of developing follicles and fertilizable ova. To maintain the normal length of female reproductive life, the primordial follicles must have adequate number and be kept in a quiescent state before menopause. However, the molecular mechanisms underlying primordial follicle survival are poorly understood. Here, we provide genetic evidence showing that lacking protein phosphatase 4 (PPP4) in oocytes, a member of PP2A-like subfamily, results in infertility in female mice. A large quantity of primordial follicles has been depleted around the primordial follicle pool formation phase and the ovarian reserve is exhausted at about 7 months old. Further investigation demonstrates that depletion of PPP4 causes the abnormal activation of mTOR, which suppresses autophagy in primordial follicle oocytes. The abnormal primordial follicle oocytes are eventually erased by pregranulosa cells in the manner of lysosome invading. These results show that autophagy prevents primordial follicles over loss and PPP4-mTOR pathway governs autophagy during the primordial follicle formation and dormant period.

The Differentiation Fate of Granulosa Cells and the Regulatory Mechanism in Ovary.

Granulosa cells (GCs) are important drives of the reproductive process, not only the supporting cells for nutrition, but also cells with endocrine functions. Their differentiation and development parallel the entire menstruation period and even during pregnancy, making it tightly linked to the fate of the follicle. To elucidate the underlying mechanism is of great significance for related researches. The life course of GCs is briefly divided into five stages, from epithelial cells to pre-granulosa cells, GCs, mural and cumulus cells, lutein cells, and eventually disappear. A wide variety of genes and transcription factors participate in the regulation of different stages, and more importantly, various hormones secreted by the pituitary gland and GCs themselves play a leading role. These endogenous and exogenous signalling molecules interact to form a cross-linked communication network, promoting the development of GCs. Together with oocytes, theca cells and other functional cells in the ovary, GCs drive one of the most vital biological processes in women.

Single-cell sequencing reveals the transcriptional alternations of 17β-estradiol suppressing primordial follicle formation in neonatal mouse ovaries.

Estrogen has been implicated in multiple biological processes, but the variation underlying estrogen-mediated primordial follicle (PF) formation remains unclear. Here, we show that 17β-estradiol (E2) treatment of neonatal mice led to the inhibition of PF formation and cell proliferation. Single-cell RNA sequencing (scRNA-seq) revealed that E2 treatment caused significant changes in the transcriptome of oocytes and somatic cells. E2 treatment disrupted the synchronised development of oocytes, pre-granulosa (PG) cells and stromal cells. Mechanistically, E2 treatment disrupted several signalling pathways critical to PF formation, especially down-regulating the Kitl and Smad1/3/4/5/7 expression, reducing the frequency and number of cell communication. In addition, E2 treatment influenced key gene expression, mitochondrial function of oocytes, the recruitment and maintenance of PG cells, the cell proliferation of somatic cells, as well as disordered the ovarian microenvironment. This study not only revealed insights into the regulatory role of estrogen during PF formation, but also filled in knowledge of dramatic changes in perinatal hormones, which are critical for the physiological significance of understanding hormone changes and reproductive protection.

Interaction of the transforming growth factor-β and Notch signaling pathways in the regulation of granulosa cell proliferation.

The Notch and transforming growth factor (TGF)-β signalling pathways play an important role in granulosa cell proliferation. However, the mechanisms underlying the cross-talk between these two signalling pathways are unknown. Herein we demonstrated a functional synergism between Notch and TGF-β signalling in the regulation of preantral granulosa cell (PAGC) proliferation. Activation of TGF-β signalling increased hairy/enhancer-of-split related with YRPW motif 2 gene (Hey2) expression (one of the target genes of the Notch pathway) in PAGCs, and suppression of TGF-β signalling by Smad3 knockdown reduced Hey2 expression. Inhibition of the proliferation of PAGCs by N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butylester (DAPT), an inhibitor of Notch signalling, was rescued by both the addition of ActA and overexpression of Smad3, indicating an interaction between the TGF-β and Notch signalling pathways. Co-immunoprecipitation (CoIP) and chromatin immunoprecipitation (ChIP) assays were performed to identify the point of interaction between the two signalling pathways. CoIP showed direct protein-protein interaction between Smad3 and Notch2 intracellular domain (NICD2), whereas ChIP showed that Smad3 could be recruited to the promoter regions of Notch target genes as a transcription factor. Therefore, the findings of the present study support the idea that nuclear Smad3 protein can integrate with NICD2 to form a complex that acts as a transcription factor to bind specific DNA motifs in Notch target genes, such as Hey1 and Hey2, and thus participates in the transcriptional regulation of Notch target genes, as well as regulation of the proliferation of PAGCs.

P-649 Unconventional action of Luteinizing Hormone

Abstract Study question Has Luteinizing Hormone (LH) a role in the activation/survival of follicles of the primary ovarian reserve? Summary answer LH promotes survival and perhaps activation/growth of primordial and primary follicles, proliferation of granulosa cells of secondary follicles, reduces follicle atresia at later growing stages. What is known already In the mammalian ovary, Primordial Follicles (PMFs) constitutes the primary ovarian reserve, which declines over time due to physiological events such as atresia, recruitment, and ovulation. While in the adult, the role of LH in steroidogenesis and ovulation is well established, recent work from our laboratory has shown that under chemotherapeutic treatments, LH supports PMFs survival in prepuberal mouse ovary. Study design, size, duration LH effects on follicle dynamics and some activities of pre-granulosa cells were investigated in vitro and in vivo. In vitroexperiments were performed on PD4 GFP/Kit mice ovarian fragments and on PD8 CD1 mice ovarian somatic cells cultured up to six days in continuous presence of 0.2, 0.5, 1IU LH. In vivo experiments were performed on 9 PD4 and 8 PD28 CD1 mice receiving an intra-peritoneal injection for eight consecutive days of 1-5IU LH. Participants/materials, setting, methods Number and diameters of the GFP stained oocyte were counted in the ovarian fragments. Kit-Ligand and LH-R mRNA expression levels were analyzed by qPCR in pre-granulosa cells. For in vivo experiments, ovarian volume was measured by Micro-Ct and follicle counts were performed on histological sections. Estrous cycle phases were monitored by vaginal smear during the 8 days of treatment. Statistics was performed using Student’s t-test. Main results and the role of chance In fragments of PD4 mouse ovaries cultured in vitro for six days, 1IU LH significantly increased the number of oocytes with a diameter >40 mm (CTRL 17.7% vs LH 36.07%, p < 0,05), suggesting activation of PMFs. Moreover, the hormone increased the expression of mRNA for LHR and KL in the somatic ovarian cells and promoted their proliferation in vitro. The effects of 1IU LH on PD4 females can be summarized as follows: 1. Ovary volume increase (CTRL 0.3 mm3 vs LH 0.36 mm3, p = 0.07), 2. higher percent of PMFs (CTRL 68.227% vs LH 78.29%, p < 0,05) associated with lower percent of primary (CTRL 7.2% vs LH 4.05%, p < 0.04) and growing follicles (CTRL 14.18% vs LH 8.56%, p = 0.1), 3. higher number of granulosa cell layers (> three layers) in secondary follicles (CTRL 42.8% vs LH 54.6%), and 4. lower percent of atretic follicles (CTRL 2.64% vs LH 1.72%, p = 0.1). Similar results were obtained on 28 dpp mice. Morever, we observed that corpora lutea were present only in ovaries of LH-treated females and that all such females were in estrus. Limitations, reasons for caution The study was conducted only in mice. Wider implications of the findings These studies have highlighted new possible roles of LH on follicle classes (primordial/primary) whose development is considered gonadotropin independent. The results, if confirmed in humans, imply that LH might be used as a treatment for patients with ovarian disorders involved the survival/dynamics of the ovarian reserve. Trial registration number NA

The effect of bisphenol A on the Notch (Notch2 and Jagged2) signaling pathway in the follicular development of the neonatal rat ovary

ABSTRACT The formation of primordial follicles determines the pool size of follicles in the ovary, and is crucial for female reproductivity. Oocyte nest breakdown, and the formation of primordial follicles, largely depend upon the communication between oocytes and the surrounding pregranulosa cells. The neurogenic locus notch homolog protein (Notch) signaling pathway is the key player for this cell-to-cell communication, and is responsible for primordial folliculogenesis. However, different endocrine disruptors, including bisphenol A (BPA; a plasticizer and a constituent of reusable plastic containers) may affect the Notch signaling pathway, and might induce ovary dysfunction via Notch signaling. Consequently, we investigated the possible influence of BPA treatment on the proportional distribution of the follicular stages, follicle numbers, levels of apoptosis, and on Notch2 and Jagged2 expressions in the ovary. BPA was administered at doses of either 50 µg/kg/day or 50 mg/kg/day, at different time intervals, during neonatal and fetal periods in vivo. After collecting the ovaries from the various experimental groups, follicles were counted, and frequency of apoptosis was determined by TUNEL assay. In addition, Notch2 and Jagged2 expressions were assessed by immunohistochemical staining and qPCR. In summary, BPA treatment affected the follicle numbers and apoptosis level, and Notch2 and Jagged2 expressions varied with follicular stage. It was also observed that these parameters were dose and time dependent with respect to BPA exposure.

Prenatal acetaminophen exposure and the developing ovary: Time, dose, and course consequences for fetal mice

Acetaminophen is an emerging endocrine disrupting chemical and has been detected in various natural matrices. Numerous studies have documented developmental toxicity associated with prenatal acetaminophen exposure (PAcE). In this study, we established a PAcE Kunming mouse model at different time (middle pregnancy and third trimester), doses (low, middle, high) and courses (single or multi-) to systematically investigate their effects on fetal ovarian development. The findings indicated PAcE affected ovarian development, reduced fetal ovarian oocyte number and inhibited cell proliferation. A reduction in mRNA expression was observed for genes associated with oocyte markers (NOBOX and Figlα), follicular development markers (BMP15 and GDF9), and pre-granulosa cell steroid synthase (SF1 and StAR). Notably, exposure in middle pregnancy, high dose, multi-course resulted in the most pronounced inhibition of oocyte development; exposure in third trimester, high dose and multi-course led to the most pronounced inhibition of follicular development; and in third trimester, low dose and single course, the inhibition of pre-granulosa cell function was most pronounced. Mechanistic investigations revealed that PAcE had the most pronounced suppression of the ovarian Notch signaling pathway. Overall, PAcE caused fetal ovarian multicellular toxicity and inhibited follicular development with time, dose and course differences.

The effects of prenatal azithromycin exposure on offspring ovarian development at different stages, doses, and courses

Azithromycin, a commonly used macrolide antibiotic for treating chlamydial infections during pregnancy, has sparked investigations into its potential effects on offspring development. Despite these inquiries, there remains uncertainty about the specific impact of prenatal azithromycin exposure (PAzE) on offspring ovarian development and the precise "effect window". Pregnant mice, following clinical guidelines for azithromycin dosing, were orally administered azithromycin at different gestational stages [(gestational day, GD) 10–12 or GD 15–17], doses (50, 100, or 200 mg/kg·d), and courses (single or multiple). On GD 18, we collected offspring blood and ovaries to examine changes in fetal serum estradiol (E2) levels, fetal ovarian morphology, pre-granulosa cell function, and oocyte development. Multiple courses of PAzE resulted in abnormal fetal ovarian morphological development, disorganized germ cell nests, enhanced ovarian cell proliferation, and reduced apoptosis. Simultaneously, multiple courses of PAzE significantly increased fetal serum E2 levels, elevated ovarian steroidogenic function (indicated by Star, 3β-hsd, and Cyp19 expression), disrupted oocyte development (indicated by Figlα and Nobox expression), and led to alterations in the MAPK signal pathway in fetal ovaries, particularly in the high-dose treatment group. In contrast, a single course of PAzE reduced fetal ovarian cell proliferation, decreased steroidogenic function, and inhibited oocyte development, particularly through the downregulation of Mek2 expression in the MAPK signal pathway. These findings suggest that PAzE can influence various aspects of fetal mouse ovarian cell development. Multiple courses enhance pre-granulosa cell estrogen synthesis function and advance germ cell development, while a single terminal gestation dose inhibits germ cell development. These differential effects may be associated with changes in the MAPK signal pathway.

COUP-TFII regulates early bipotential gonad signaling and commitment to ovarian progenitors

BackgroundThe absence of expression of the Y-chromosome linked testis-determining gene SRY in early supporting gonadal cells (ESGC) leads bipotential gonads into ovarian development. However, genetic variants in NR2F2, encoding three isoforms of the transcription factor COUP-TFII, represent a novel cause of SRY-negative 46,XX testicular/ovotesticular differences of sex development (T/OT-DSD). Thus, we hypothesized that COUP-TFII is part of the ovarian developmental network. COUP-TFII is known to be expressed in interstitial/mesenchymal cells giving rise to steroidogenic cells in fetal gonads, however its expression and function in ESGCs have yet to be explored.ResultsBy differentiating induced pluripotent stem cells into bipotential gonad-like cells in vitro and by analyzing single cell RNA-sequencing datasets of human fetal gonads, we identified that NR2F2 expression is highly upregulated during bipotential gonad development along with markers of bipotential state. NR2F2 expression was detected in early cell populations that precede the steroidogenic cell emergence and that retain a multipotent state in the undifferentiated gonad. The ESGCs differentiating into fetal Sertoli cells lost NR2F2 expression, whereas pre-granulosa cells remained NR2F2-positive. When examining the NR2F2 transcript variants individually, we demonstrated that the canonical isoform A, disrupted by frameshift variants previously reported in 46,XX T/OT-DSD patients, is nearly 1000-fold more highly expressed than other isoforms in bipotential gonad-like cells. To investigate the genetic network under COUP-TFII regulation in human gonadal cell context, we generated a NR2F2 knockout (KO) in the human granulosa-like cell line COV434 and studied NR2F2-KO COV434 cell transcriptome. NR2F2 ablation downregulated markers of ESGC and pre-granulosa cells. NR2F2-KO COV434 cells lost the enrichment for female-supporting gonadal progenitor and acquired gene signatures more similar to gonadal interstitial cells.ConclusionsOur findings suggest that COUP-TFII has a role in maintaining a multipotent state necessary for commitment to the ovarian development. We propose that COUP-TFII regulates cell fate during gonad development and impairment of its function may disrupt the transcriptional plasticity of ESGCs. During early gonad development, disruption of ESGC plasticity may drive them into commitment to the testicular pathway, as observed in 46,XX OT-DSD patients with NR2F2 haploinsufficiency.

Theophylline derivatives promote primordial follicle activation via cAMP-PI3K/Akt pathway and ameliorate fertility deficits in naturally aged mice.

In elderly women and patients with premature ovarian insufficiency (POI), activating their remaining dormant primordial follicles in vivo is challenging. In this study, we found that phosphodiesterase (PDE) subtypes were expressed mainly in primordial follicle oocytes. The specific PDE inhibitors and theophylline derivatives (aminophylline, dyphylline, and enprofylline) activated primordial follicles in neonatal mice by ovary culture and intraperitoneal injection. These inhibitors also increased the levels of ovarian cyclic adenosine monophosphate (cAMP) and oocyte phosphorylated protein kinase B (p-Akt). The blockade of gap junctions using carbenoxolone (CBX) increased the levels of ovarian cAMP and pre-granulosa cell phosphorylated mammalian target of rapamycin (p-mTOR), suggesting that oocyte PDEs hydrolyze cAMP from pre-granulosa cells through gap junctions to maintain primordial follicle dormancy. Importantly, oral aminophylline improved ovulated oocyte quantity and quality, and increased offspring numbers in naturally aged mice. In addition, theophylline derivatives also activated human primordial follicles and increased p-Akt levels. Thus, theophylline derivatives activate primordial follicles by accumulating cAMP levels and activating phosphatidylinositol 3-kinase (PI3K)/Akt pathway in oocytes, and oral aminophylline increased fertility in naturally aged female mice by improving ovulated oocyte quantity and quality. As oral medications, theophylline derivatives may be used to improve fertility in elderly women and patients with POI.

The -KTS splice variant of WT1 is essential for ovarian determination in mice.

Sex determination in mammals depends on the differentiation of the supporting lineage of the gonads into Sertoli or pregranulosa cells that govern testis and ovary development, respectively. Although the Y-linked testis-determining gene Sry has been identified, the ovarian-determining factor remains unknown. In this study, we identified -KTS, a major, alternatively spliced isoform of the Wilms tumor suppressor WT1, as a key determinant of female sex determination. Loss of -KTS variants blocked gonadal differentiation in mice, whereas increased expression, as found in Frasier syndrome, induced precocious differentiation of ovaries independently of their genetic sex. In XY embryos, this antagonized Sry expression, resulting in male-to-female sex reversal. Our results identify -KTS as an ovarian-determining factor and demonstrate that its time of activation is critical in gonadal sex differentiation.

Reconstituted ovaries self-assemble without an ovarian surface epithelium

Three-dimensional (3D) stem cell models of the ovary have the potential to benefit women's reproductive health research. One such model, the reconstituted ovary (rOvary) self-assembles with pluripotent stem cell-derived germ cells creating a 3D ovarian mimic competent to support the differentiation of functional oocytes inside follicles. In this study, we evaluated the cellular composition of the rOvary revealing the capacity to generate multiple follicles surrounded by NR2F2+ stroma cells. However, the rOvary does not develop a surface epithelium, the source of second-wave pre-granulosa cells, or steroidogenic theca. Therefore, the rOvary models represent the self-assembly of activated follicles in a pre-pubertal ovary poised but not yet competent for hormone production.

Single-cell analysis of the developing human ovary defines distinct insights into ovarian somatic and germline progenitors.

Formation of either an ovary or a testis during human embryonic life is one of the most important sex-specific events leading to the emergence of secondary sexual characteristics and sex assignment of babies at birth. Our study focused on the sex-specific and sex-indifferent characteristics of the prenatal ovarian stromal cells, cortical cords, and germline, with the discovery that the ovarian mesenchymal cells of the stroma are transcriptionally indistinguishable from the mesenchymal cells of the testicular interstitium. We found that first-wave pre-granulosa cells emerge at week 7 from early supporting gonadal cells with stromal identity and are spatially defined by KRT19 levels. We also identified rare transient state f0 spermatogonia cells within the ovarian cords between weeks 10 and 16. Taken together, our work illustrates a unique plasticity of the embryonic ovary during human development.

Cell-cell interactions during the formation of primordial follicles in humans.

Gametogenesis is a complex and sex-specific multistep process during which the gonadal somatic niche plays an essential regulatory role. One of the most crucial steps during human female gametogenesis is the formation of primordial follicles, the functional unit of the ovary that constitutes the pool of follicles available at birth during the entire reproductive life. However, the relation between human fetal germ cells (hFGCs) and gonadal somatic cells during the formation of the primordial follicles remains largely unexplored. We have discovered that hFGCs can form multinucleated syncytia, some connected via interconnecting intercellular bridges, and that not all nuclei in hFGC-syncytia were synchronous regarding meiotic stage. As hFGCs progressed in development, pre-granulosa cells formed protrusions that seemed to progressively constrict individual hFGCs, perhaps contributing to separate them from the multinucleated syncytia. Our findings highlighted the cell-cell interaction and molecular dynamics between hFGCs and (pre)granulosa cells during the formation of primordial follicles in humans. Knowledge on how the pool of primordial follicle is formed is important to understand human infertility.

Current progress on in vitro differentiation of ovarian follicles from pluripotent stem cells.

Mammalian female reproduction requires a functional ovary. Competence of the ovary is determined by the quality of its basic unit-ovarian follicles. A normal follicle consists of an oocyte enclosed within ovarian follicular cells. In humans and mice, the ovarian follicles are formed at the foetal and the early neonatal stage respectively, and their renewal at the adult stage is controversial. Extensive research emerges recently to produce ovarian follicles in-vitro from different species. Previous reports demonstrated the differentiation of mouse and human pluripotent stem cells into germline cells, termed primordial germ cell-like cells (PGCLCs). The germ cell-specific gene expressions and epigenetic features including global DNA demethylation and histone modifications of the pluripotent stem cells-derived PGCLCs were extensively characterized. The PGCLCs hold potential for forming ovarian follicles or organoids upon cocultured with ovarian somatic cells. Intriguingly, the oocytes isolated from the organoids could be fertilized in-vitro. Based on the knowledge of in-vivo derived pre-granulosa cells, the generation of these cells from pluripotent stem cells termed foetal ovarian somatic cell-like cells was also reported recently. Despite successful in-vitro folliculogenesis from pluripotent stem cells, the efficiency remains low, mainly due to the lack of information on the interaction between PGCLCs and pre-granulosa cells. The establishment of in-vitro pluripotent stem cell-based models paves the way for understanding the critical signalling pathways and molecules during folliculogenesis. This article aims to review the developmental events during in-vivo follicular development and discuss the current progress of generation of PGCLCs, pre-granulosa and theca cells in-vitro.

Pregranulosa cell–derived FGF23 protects oocytes from premature apoptosis during primordial follicle formation by inhibiting p38 MAPK in mice

A large number of oocytes in the perinatal ovary in rodents get lost for unknown reasons. The granulosa cell-oocyte mutual communication is pivotal for directing formation of the primordial follicle; however, little is known if paracrine factors participate in modulating programmed oocyte death perinatally. We report here that pregranulosa cell-derived fibroblast growth factor 23 (FGF23) functioned in preventing oocyte apoptosis in the perinatal mouse ovary. Our results showed that FGF23 was exclusively expressed in pregranulosa cells, while fibroblast growth factor receptors (FGFRs) were specifically expressed in the oocytes in perinatal ovaries. FGFR1 was one of the representative receptors in mediating FGF23 signaling during the formation of the primordial follicle. In cultured ovaries, the number of live oocytes declines significantly, accompanied by the activation of the p38 mitogen-activated protein kinase signaling pathway, under the condition of FGFR1 disruption by specific inhibitors of FGFR1 or silencing of Fgf23. As a result, oocyte apoptosis increased and eventually led to a decrease in the number of germ cells in perinatal ovaries following the treatments. In the perinatal mouse ovary, pregranulosa cell-derived FGF23 binds to FGFR1 and activates at least the p38 mitogen-activated protein kinase signaling pathway, thereby regulating the level of apoptosis during primordial follicle formation. This study reemphasizes the importance of granulosa cell-oocyte mutual communication in modulating primordial follicle formation and supporting oocyte survival under physiological conditions.

Toxic effect window of ovarian development in female offspring mice induced by prenatal prednisone exposure with different doses and time

BackgroundPrednisone is one of the most used synthetic glucocorticoids during pregnancy. Epidemiological investigations suggested that prenatal prednisone therapy could affect fetal development, but systematic studies on its effects on ovarian development and the “toxic effect window” remained scarce.MethodsIn this study, by simulating clinical application characteristics, Kunming mice were given prednisone by oral gavage with different doses (0.25 or 1.0 mg/kg·d) or at different time gestational days (GD) (GD0-9, GD10-18, or GD0-18). Blood and ovaries of fetal mice were collected on GD18, and the serum estradiol level and the related function indexes of ovarian granulosa cells and oocytes were detected.ResultsCompared with the control group, prenatal prednisone exposure (PPE) induced pathological injury and enhanced cell proliferation in fetal mice ovary. Furthermore, the expression of steroid synthesis functional genes in pre-granulosa cells, the oocyte function markers, and developmentally related genes was enhanced with different doses or at different time of PPE. The Hippo signaling was activated in the fetal ovary of PPE groups. The above changes were most significant in the low or high-dose and full-term PPE groups.ConclusionPPE caused various cell developmental toxicity in the fetal ovary, especially in the low or high-dose, full-term exposure groups. The potential mechanism might be related to the activation of the Hippo signaling pathway.

DMRT1 regulation of TOX3 modulates expansion of the gonadal steroidogenic cell lineage in the chicken embryo.

During gonadal sex determination, the supporting cell lineage differentiates into Sertoli cells in males and pre-granulosa cells in females. Recently, single cell RNA-seq data indicate that that chicken steroidogenic cells, derive from differentiated supporting cells. This differentiation process is achieved by a sequential upregulation of steroidogenic genes and down-regulation of supporting cell markers. The exact mechanism regulating this differentiation process remains unknown. We identified the gene TOX3 as a novel transcription factor expressed in embryonic Sertoli cells of the chicken testis. TOX3 knockdown in males resulted in increased CYP17A1 positive Leydig cells. TOX3 over-expression in male and female gonads resulted in a significant decline in CYP17A1 positive steroidogenic cells. In ovo knock-down of the testis determinant, DMRT1, in male gonads resulted in a down-regulation of TOX3 expression. Conversely, DMRT1 over-expression caused an increase in TOX3 expression. Taken together, this data indicates that DMRT1 regulation of TOX3 modulates expansion of the steroidogenic lineage, either directly, via cell lineage allocation, or indirectly via signaling from the supporting to steroidogenic cell populations.

Somatic FGFR2 is Required for Germ Cell Maintenance in the Mouse Ovary.

During sex determination in the mouse, fibroblast growth factor 9 signals through the fibroblast growth factor receptor 2c isoform (FGFR2c) to trigger Sertoli cell and testis development from 11.5 days post coitum (dpc). In the XX gonad, the FOXL2 and WNT4/RSPO1 pathways drive granulosa cell and ovarian development. The function of FGFR2 in the developing ovary, and whether FGFR2 is required in the testis after sex determination, is not clear. In fetal mouse gonads from 12.5 dpc, FGFR2 shows sexually dimorphic expression. In XX gonads, FGFR2c is coexpressed with FOXL2 in pregranulosa cells, whereas XY gonads show FGFR2b expression in germ cells. Deletion of Fgfr2c in XX mice led to a marked decrease/absence of germ cells by 13.5 dpc in the ovary. This indicates that FGFR2c in the somatic pregranulosa cells is required for the maintenance of germ cells. Surprisingly, on the Fgfr2c-/- background, the germ cell phenotype could be rescued by ablation of Foxl2, suggesting a novel mechanism whereby FGFR2 and FOXL2 act antagonistically during germ cell development. Consistent with low/absent FGFR2 expression in the Sertoli cells of 12.5 and 13.5 dpc XY gonads, XY AMH:Cre; Fgfr2flox/flox mice showed normal testis morphology and structures during fetal development and in adulthood. Thus, FGFR2 is not essential for maintaining Sertoli cell fate after sex determination. Combined, these data show that FGFR2 is not necessary for Sertoli cell function after sex determination but does play an important role in the ovary.