Primordial Follicle Formation Research Articles

FOXL2 interaction with different binding partners regulates the dynamics of ovarian development.

The transcription factor FOXL2 is required in ovarian somatic cells for female fertility. Differential timing of Foxl2 deletion, in embryonic versus adult mouse ovary, leads to distinctive outcomes, suggesting different roles across development. Here, we comprehensively investigated FOXL2's role through a multi-omics approach to characterize gene expression dynamics and chromatin accessibility changes, coupled with genome-wide identification of FOXL2 targets and on-chromatin interacting partners in somatic cells across ovarian development. We found that FOXL2 regulates more targets postnatally, through interaction with factors regulating primordial follicle formation and steroidogenesis. Deletion of one interactor, ubiquitin-specific protease 7 (Usp7), results in impairment of somatic cell differentiation, germ cell nest breakdown, and ovarian development, leading to sterility. Our datasets constitute a comprehensive resource for exploration of the molecular mechanisms of ovarian development and causes of female infertility.

Single-cell RNA-seq identified novel genes involved in primordial follicle formation.

The number of primordial follicles (PFs) in mammals determines the ovarian reserve, and impairment of primordial follicle formation (PFF) will cause premature ovarian insufficiency (POI). By analyzing public single-cell RNA sequencing performed during PFF on mice and human ovaries, we identified novel functional genes and novel ligand-receptor interaction during PFF. Based on immunofluorescence and in vitro ovarian culture, we confirmed mechanisms of genes and ligand-receptor interaction in PFF. We also applied whole exome sequencing (WES) in 93 cases with POI and whole genome sequencing (WGS) in 465 controls. Variants in POI patients were further investigated by in silico analysis and functional verification. We revealed ANXA7 (annexin A7) and GTF2F1 (general transcription factor IIF subunit 1) in germ cells to be novel potentially genes in promoting PFF. Ligand Mdk (midkine) in germ cells and its receptor Sdc1 (syndecan 1) in granulosa cells are novel interaction crucial for PFF. Based on immunofluorescence, we confirmed significant up-regulation of ANXA7 in PFs compared with germline cysts, and uniform expression of GTF2F1, MDK and SDC1 during PFF, in 25 weeks human fetal ovary. In vitro investigation indicated that Anxa7 and Gtf2f1 are vital for mice PFF by regulating Jak/Stat3 and Jnk signaling pathways, respectively. Ligand-receptor (Mdk-Sdc1) are crucial for PFF by regulating Pi3k-akt signaling pathway. Two heterozygous variants in GTF2F1, and one heterozygous variants in SDC1 were identified in cases, but no variant were identified in controls. The protein level of GTF2F1 or SDC1 in POI cases are significantly lower than that of controls, indicating the pathogenic effects of the two genes on ovarian function were dosage dependent. Our study identified novel genes and novel ligand-receptor interaction during PFF, and further expanding the genetic architecture of POI.

Melatonin alleviates the toxic effect of di(2-ethylhexyl) phthalate on oocyte quality resulting from CEBPB suppression during primordial follicle formation

Presently, the exposure of plasticizers to humans and animals occurs daily, which pose a potential threat to reproductive health. In the present study, a pregnant mouse model exposed to di(2-ethylhexyl) phthalate (DEHP, one of the most common plasticizers) and melatonin was established, and the single-cell transcriptome technology was applied to investigate the effects of melatonin in ovarian cells against DEHP. Results showed that DEHP markedly altered the gene expression pattern of ovarian cells, and severely weakened the histone methylation modification of oocytes. The administration of melatonin recovered the expression of LHX8 and SOHLH1 proteins that essential for primordial follicle formation, and increased the expression of CEBPB, as well as key genes of histone methylation modification (such as Smyd3 and Kdm5a). In addition, the ovarian damage caused by DEHP was also relieved after the overexpression of CEBPB, which suggested melatonin could improve primordial follicle formation progress via enhancing CEBPB expression in mice. Besides, the apoptosis of ovarian cells induced by DEHP also was diminished by melatonin. The study provides evidence of melatonin preventing the damage mediated by plasticizers on the reproductive system in females and CEBPB may serve as a downstream target factor of melatonin in the process.

Maternal vitamin B1 is a determinant for the fate of primordial follicle formation in offspring

The mediation of maternal-embryonic cross-talk via nutrition and metabolism impacts greatly on offspring health. However, the underlying key interfaces remain elusive. Here, we determined that maternal high-fat diet during pregnancy in mice impaired preservation of the ovarian primordial follicle pool in female offspring, which was concomitant with mitochondrial dysfunction of germ cells. Furthermore, this occurred through a reduction in maternal gut microbiota-related vitamin B1 while the defects were restored via vitamin B1 supplementation. Intriguingly, vitamin B1 promoted acetyl-CoA metabolism in offspring ovaries, contributing to histone acetylation and chromatin accessibility at the promoters of cell cycle-related genes, enhancement of mitochondrial function, and improvement of granulosa cell proliferation. In humans, vitamin B1 is downregulated in the serum of women with gestational diabetes mellitus. In this work, these findings uncover the role of the non-gamete transmission of maternal high-fat diet in influencing offspring oogenic fate. Vitamin B1 could be a promising therapeutic approach for protecting offspring health.

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.

Dynamic role of Scd1 gene during mouse oocyte growth and maturation

Mammalian reproductive ability is regulated by many factors, among which the fatty acid metabolism network provides energy for oocyte growth and primordial follicle formation during early mouse oogenesis. But the mechanism behind that is still unknown. Stearoyl-CoA desaturase 1 (Scd1) gene expression is increased during the oogenesis process, supporting the oocyte's healthy growth. Taking advantage of gene-edited mice lacking stearoyl-Coenzyme A desaturase 1 gene (Scd1−/−), we analyzed relative gene expression in perinatal ovaries from wildtype, and Scd1−/− mice. Scd1 deficiency dysregulates expression of meiosis-related genes (e.g., Sycp1, Sycp2, Sycp3, Rad51, Ddx4) and a variety of genes (e.g., Nobox, Lhx8, Bmp15, Ybx2, Dppa3, Oct4, Sohlh1, Zp3) associated with oocyte growth and differentiation, leading to a lower oocyte maturation rate. The absence of Scd1 significantly impedes meiotic progression, causes DNA damage, and inhibits damage repair in Scd1−/− ovaries. Moreover, we find that Scd1 absense dramatically disrupts the abundance of fatty acid metabolism genes (e.g., Fasn, Srebp1, Acaca) and the lipid droplet content. Thus, our findings substantiate a major role for Scd1 as a multifunctional regulator of fatty acid networks necessary for oocyte maintenance and differentiation during early follicular genesis.

Expression pattern and subcellular localization of p62/SQSTM 1 during mouse oocyte maturation

Objective: Autophagy is a survival mechanism, and it is initiated by several factors such as oxidative stress. Cells give autophagy response against oxidative stress through Nrf2-Keap1-p62 pathway. p62 or Sequestosome 1 (SQSTM 1) which takes place during autophagy, and it is showed that p62 plays critical role during primordial follicle formation. During oocyte maturation, Germinal Vesicle (GV) oocytes resume meiosis with the beginning of puberty and progress through Metaphase I and Metaphase II stages. Metaphase II (MII) oocytes are ready to be fertilized and until fertilization they are arrested at metaphase II stages. It is known that oxidative stress is increasing during oocyte maturation. However, it is not shown that spatial and temporal expression of p62 throughout oocyte maturation. Thus, the aim of the study was to reveal expression pattern and the subcellular localization of the p62 protein in mouse GV, MI and MII oocytes. Methods: GV oocytes were received from female Balb/C mice at 4 weeks aged and GV oocytes were cultured for 8h to develop into MI oocytes and for 14h to mature into MII oocytes. Then, expression of p62 protein was observed in oocytes at GV, MI and MII maturation stages by using immunofluorescence method. Results: Our results showed that there is a significant increasing p62 expression when GV oocytes are compared to MI and MII oocytes, but there is no difference between MI and MII oocytes. Moreover, we revealed that p62 is localized in cytoplasm of GV oocytes, while it is localized around chromosomes in MI and MII oocytes. Conclusion: In conclusion, our results indicate that further study is mandatory to understand the participation of p62 during meiosis, and the impact of p62 during oocyte maturation.

In utero bisphenol A exposure disturbs germ cell cyst breakdown through the PI3k/Akt signaling pathway and BDNF expression

PurposeTo determine the influence of the environmental endocrine disruptor bisphenol A (BPA) on germ cell cyst breakdown and explore the possible mechanisms regulating this activity. MethodsBPA (2 μg/kg/d or 20 μg/kg/d) or tocopherol-stripped corn oil (vehicle control) was administered to pregnant mice by gavage at gestational day 11, and the offspring (prenatally treated mice) were sacrificed and ovariectomized at postnatal day (PND) 4 and PND22. Ovarian morphology was documented in the first filial (F1) generation female offspring, and the follicles were analyzed and classified morphologically on PND 4. To discover differentially expressed genes and associated target pathways, we used RNA-seq, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and Gene Ontology (GO) analysis. The mRNA expression of key steroid hormone synthesis-related genes was evaluated by Q-PCR in forskolin-induced KGN cells. Western blotting (WB) and qRTPCR were used to determine the protein and gene expression levels of brain-derived neurotrophic factor (BDNF). ResultsBPA, a typical endocrine disrupting chemical (EDC), decreased the expression of the key steroid hormone synthesis-related genes P450scc and aromatase, while the expression of Star increased significantly and caused no significant difference in the expression of Cyp17a1 or HSD3β in forskolin-induced KGN cells. Moreover, we confirmed that in utero exposure to environmentally relevant concentrations of BPA (2 μg/kg/d and 20 μg/kg/d) could significantly disrupt germ cell cyst breakdown, leading to the generation of fewer primordial follicles than in the control group. The factors mediating the inhibitory effects included the PI3K-Akt signaling pathway and a significant downregulation of BDNF. ConclusionsThese findings indicate that in utero exposure to BPA at low doses, which are lower than recommended as ‘safe’ dosages, may influence the formation of primordial follicles by inhibiting the expression of steroid hormone synthesis-related genes and partly by regulating the BDNF-mediated PI3K/Akt pathway.

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.

SRSF1 regulates primordial follicle formation and number determination during meiotic prophase I

BackgroundOvarian folliculogenesis is a tightly regulated process leading to the formation of functional oocytes and involving successive quality control mechanisms that monitor chromosomal DNA integrity and meiotic recombination. A number of factors and mechanisms have been suggested to be involved in folliculogenesis and associated with premature ovarian insufficiency, including abnormal alternative splicing (AS) of pre-mRNAs. Serine/arginine-rich splicing factor 1 (SRSF1; previously SF2/ASF) is a pivotal posttranscriptional regulator of gene expression in various biological processes. However, the physiological roles and mechanism of SRSF1 action in mouse early-stage oocytes remain elusive. Here, we show that SRSF1 is essential for primordial follicle formation and number determination during meiotic prophase I.ResultsThe conditional knockout (cKO) of Srsf1 in mouse oocytes impairs primordial follicle formation and leads to primary ovarian insufficiency (POI). Oocyte-specific genes that regulate primordial follicle formation (e.g., Lhx8, Nobox, Sohlh1, Sohlh2, Figla, Kit, Jag1, and Rac1) are suppressed in newborn Stra8-GFPCre Srsf1Fl/Fl mouse ovaries. However, meiotic defects are the leading cause of abnormal primordial follicle formation. Immunofluorescence analyses suggest that failed synapsis and an inability to undergo recombination result in fewer homologous DNA crossovers (COs) in the Srsf1 cKO mouse ovaries. Moreover, SRSF1 directly binds and regulates the expression of the POI-related genes Six6os1 and Msh5 via AS to implement the meiotic prophase I program.ConclusionsAltogether, our data reveal the critical role of an SRSF1-mediated posttranscriptional regulatory mechanism in the mouse oocyte meiotic prophase I program, providing a framework to elucidate the molecular mechanisms of the posttranscriptional network underlying primordial follicle formation.

CAMP controls the balance between dormancy and activation of primordial follicles in mouse ovaries.

In mammalian ovaries, the balance between dormancy and activation of primordial follicles determines the female fecundity and endocrine homeostasis. Recently, several functional molecules and pathways have been reported to be involved in the activation of primordial follicles. However, the homeostasis regulatory mechanisms of primordial follicle activation are still scant. Our previous study has proved that a relatively higher concentration of cyclic AMP (cAMP) is required for primordial follicle formation. Here, we identified that cAMP also plays a vital role in the balance between dormancy and activation of primordial follicles. Our results showed that the concentration of cAMP remained stable in neonatal mouse ovaries, which is due to ADCY3, the synthetase of cAMP, and PDE3A, the hydrolytic enzyme of cAMP, were synchronously increased during the activation of primordial follicles in mouse ovaries. Once the concentration of cAMP in neonatal ovaries was either elevated or reduced in vitro, the activation of primordial follicles was either accelerated or decelerated accordingly. In addition, a higher concentration of cAMP in the ovaries of puberty mice improved primordial follicle activation in vivo. Finally, cAMP promoted primordial follicle activation via canonical mTORC1-PI3K signaling cascades and PKA signaling. In conclusion, our findings reveal that the concentration of cAMP acts as a key regulator in balancing the dormancy and activation of primordial follicles in the mouse ovary.

SP1 impacts the primordial to primary follicle transition by regulating cholesterol metabolism in granulosa cells.

The primordial to primary follicle transition (PPT) in the ovary is critical to maintain sustainable reproductive resources in female mammals. However, it is unclear how granulosa cells (GCs) of the primary follicle participate in regulating PPT. This study focused on exploring the role of transcription factor Sp1 (SP1)in regulating PPT based on the fact that SP1 is pivotal for pregranulosa cell proliferation before primordial follicle formation. The results showed that mice fertility was prolonged when Sp1 was specifically depleted from GCs (GC- Sp1 -/- ). Besides, the PPT in GC- Sp1 -/- mice was reduced, resulting in more primordial follicles being preserved. Single-cell RNA-seq also indicated that the level of cholesterol metabolism was downregulated in GC- Sp1 -/- mice. Additionally, the PPT was promoted by either overexpression of ferredoxin-1 (FDX1), one of the key genes in mediating cholesterol metabolism or supplementing cholesterol for cultured fetal ovaries. Collectively, SP1 in GCs participates in the metabolism of cholesterol partially by regulating the transcription of Fdx1 during the PPT.

H3K4me3 as a target of di(2-ethylhexyl) phthalate (DEHP) impairing primordial follicle assembly

Di (2-ethylhexyl) phthalate (DEHP) is a widely used plastics additive that growing evidence indicates as endocrine disruptor able to negatively affect various reproductive processes both in female and male animals, including humans. However, the precise molecular mechanism of such actions is not completely understood. In the present study, scRNA-seq was performed on the ovaries of offspring from mothers exposed to DEHP from 16.5 days post coitum to 3 days post-partum, when the primordial follicle (PF) stockpile is established. While the histological observations of the offspring ovaries from DEHP exposed mothers confirmed previous data about a distinct reduction of oocytes enclosed in PFs. Focusing on oocytes, scRNA-seq analyses showed that the genes that mostly changed by DEHP were enriched GO terms related to histone H3–K4 methylation. Moreover, we observed H3K4me3 level, an epigenetics modification of H3 that is crucial for chromatin transcription, decreased by 40.28% (P < 0.01) in DEHP-treated group compared with control. When the newborn ovaries were cultured in vitro, the DEHP effects were abolished by tamoxifen (an estrogen receptor antagonist) or overexpression of Smyd3 (one specific methyltransferase of H3K4me3), in particular, the percentage of oocyte enclosed in PF was increased by 15.39% in DEHP plus Smyd3 overexpression group than of DEHP group (P < 0.01), which was accompanied by the upregulation of H3K4me3. Collectively, the present results discover Smyd3-H3K4me3 as a novel target of the deleterious ER-mediated effect of DEHP on PF formation during early folliculogenesis in the mouse and highlight epigenetics changes as prominent targets of endocrine disruptors like DEHP.

Maternal ochratoxin A exposure impairs meiosis progression and primordial follicle formation of F1 offspring.

Ochratoxin A (OTA), a mycotoxin produced by Aspergillus and Penicillium fungi, widely contaminates feed, food and their raw materials. OTA has been proved to have hepatotoxicity and nephrotoxicity. Its reproductive toxicity needs to be further explored. We found that OTA inhibited the progression of meiosis, keeping more germ cells at leptotene and zygotene. Furthermore, OTA impaired primordial follicle formation, keeping more germ cells in cysts. Increased γH2AX suggested that DNA damage occurred both at the stages of meiosis and primordial follicle formation. The expression of RAD51 increased with the concentration of OTA at the stage of meiosis, while decreased later, suggesting the activated DNA repair induced by DNA damage then inhibited by persistent and excessive stress of DNA damage, which further induced apoptosis. DEGs caused by OTA were also mainly enriched in DNA damage and repair through RNA-seq analysis. Higher level of reactive oxygen species (ROS) and increased degree of oxidative damage marker 8-OHdG were both found in the ovaries exposed to OTA. We concluded that maternal OTA exposure affected meiosis progression and primordial follicle formation via oxidative damage and DNA repair. Clarification of the mechanism of OTA will contribute to the development of more effective detoxification strategies.

Oocyte Casein kinase 1α deletion causes defects in primordial follicle formation and oocyte loss by impairing oocyte meiosis and enhancing autophagy in developing mouse ovary

Casein kinase 1α is a member of CK1 family, which is ubiquitously expressed and plays multiple functions, including its potential roles in regulating cell division. But the functions of CK1α in mammalian oogenesis and folliculogenesis remain elusive. In this study, we assayed the cell type of CK1α expression in the developing mouse ovary and confirmed that CK1α was highly expressed in ovaries after birth. The oocyte-specific CK1α knockout (cKO) mouse model was then established by crossing Ddx4-Cre mice with Csnk1a1-floxp mice, and the effects of CK1α deletion on oogenesis and folliculogenesis were identified. The results showed that oocyte CK1α deletion impaired the progression of oocyte meiosis and primordial follicle formation during meiotic prophase I, which subsequently caused oocyte loss and mouse infertility. Further, the in vivo CK1α deletion and in vitro inhibition of CK1 activity resulted in the defects of DNA double-strand break (DSB) repair, whereas apoptosis and autophagy were enhanced in the developing ovary. These may contribute to oocyte loss and infertility in cKO mice. It is thus concluded that CK1α is essential for mouse oogenesis and folliculogenesis by involving in regulating the processes of oocyte meiosis and DNA DSB repair during meiotic prophase I of mouse oocytes. However, the related signaling pathway and molecular mechanisms need to be elucidated further.

The programmed death of fetal oocytes and the correlated surveillance mechanisms

Gamete production is essential for mammalian reproduction. In the ovaries, the primordial follicle, which is the basic reproductive unit, is formed either perinatally or during the second pregnancy stage in humans. However, some oocytes die before the establishment of the primordial follicle pool. Consequently, it is essential to uncover how the size of the primordial follicle pool is determined and how the programmed cell death of oocytes is performed under potential surveillance. According to recent studies, the fate of oocytes in the fetal ovary seems to be determined by different protective strategies through the timely control of apoptosis or autophagy. In this review, we discuss at least three oocyte-derived protective biomarkers, glycogen synthase kinase 3 beta, X-linked inhibitor of apoptosis, and Lysine-specific demethylase 1 (also known as KDM1A), responsible for surveilling the developmental quality of fetal oocytes to coordinate primordial follicle formation in the fetal ovary. This review contributes to a better understanding of the secrets of the female reproductive reserve under physiological conditions.

O-233 Cell-cell interactions within germ cell nests reveal the mechanism of primordial follicle formation in humans

Abstract Study question How interactions between human fetal female germ cells (hFGCs) and pre-granulosa cells (pre-GCs) affect the hFGCs transition from ovarian cord to primordial follicles (PFs). Summary answer Progressive engulfment of hFGCs by pre-GCs and establishment of cell-cell junctions and cell-cell adhesions between hFGCs and pre-GCs leads to PF formation in humans. What is known already In mice, development of FGCs is synchronized due to formation of cysts. Cysts comprise of sister FGCs that remain connected via intercellular bridges until germ cell nest breakdown is initiated and PFs are formed. In contrast, hFGCs differentiate asynchronously. In second trimester human ovaries, mitotic, RA-responsive, meiotic and oogonia hFGCs are present in designated parts of the ovary that corresponds to their differentiation stage. Meiotic hFGCs enter prophase I that is divided into 4 substages: leptotene, zygotene, pachytene, diplotene/dictyate. hFGCs in diplotene are ready to form a PF with pre-GCs and become dormant. Study design, size, duration We collected in total 17 second trimester human fetal ovaries between 14 and 22 weeks of gestation from elective abortions. We have analyzed cellular structures of hFGC and pre-GCs by transmission electron microscopy and investigated different cell adhesion and cell junction proteins by immunostaining. Participants/materials, setting, methods Materials: human fetal ovaries between 14 and 22 weeks of gestation. Methods: characterization of tissue morphology, immunofluorescence staining, whole mount staining, confocal microscopy, super-resolution microscopy, transmission electron microscopy (TEM), cell counting Main results and the role of chance Meiotic hFGCs formed germ cell cysts consisting of multiple sister hFGCs connected by intercellular bridges. Furthermore, we discovered that a subset of hFGCs formed syncytia containing several nuclei (2–4 nuclei). Syncytium formation was caused by cytokinesis failure when an in intercellular bridge failed to be formed. In addition, in some cases hFGC syncytia were connected to other syncytia or mono-nucleated hFGCs by an intercellular bridges. Those multinucleated syncytia did not display signs of apoptosis or morphological abnormalities. Investigation of physical interactions between hFGCs and pre-GCs revealed that as hFGCs enter prophase I, pre-GCs (FOXL2+, VIM+, CDH1+) form membrane protrusions (MPs) and progressively engulf them. First, CDH1+ and VIM+ domains within a pre-GC are mixed, but as engulfment progresses two distinct domains are formed. MP formation is possible due to mesenchymal-epithelial hybrid state and active Hippo pathway in pre-GCs. Inside the cords, hFGCs highly express CDH2, while CDH2 is lowly expressed in pre-GCs. At diplotene, hFGCs (TP63+) downregulate CDH2 and upregulate CDH1. Expression of CDH1 in both diplotene hFGC and pre-GCs causes increase of cell-cell adhesion and enables PF formation. In addition, progressive formation of tight junctions (TJP1+) and cellular interdigitations between pre-GCs and meiotic hFGCs reinforce PF structure. Limitations, reasons for caution Due to technical limitations and lack of in vitro culture protocols that support meiotic hFGCs development and survival as well as human PF formation in vitro, we cannot experimentally validate roles of cadherins and other cell junction and cell adhesion proteins in PF formation Wider implications of the findings In depth understanding of PF formation process in terms of physical interactions between hFGCs and pre-GCs will be crucial to design in vitro conditions that will support PF formation and our findings will be key to achieve assembling of PFs in artificial human oocytes (gametes) created from pluripotent stem cells. Trial registration number not applicable

Bone morphogenetic protein 2- and estradiol-17β-induced changes in ovarian transcriptome during primordial follicle formation†.

Estradiol-17β has been shown to promote primordial follicle formation and to involve bone morphogenetic protein 2 (BMP2) as a downstream effector to promote primordial follicle in hamsters. However, the molecular mechanism whereby these factors regulate ovarian somatic cells to pre-granulosa cells transition leading to primordial follicle formation remains unclear. The objective of this study was to determine whether BMP2 and/or estradiol-17β would regulate the expression of specific ovarian transcriptome during pre-granulosa cells transition and primordial follicle formation in the mouse ovary. BMP2 mRNA level increased during the period of primordial follicle formation with the concurrent presence of BMP2 protein in ovarian somatic cells. Estradiol-17β but not BMP2 exposure led to increased expression of ovarian BMP2 messenger RNA (mRNA), and the effect of estradiol-17β could not be suppressed by 4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline dihydrochloride (LDN) 193189. BMP2 or estradiol-17β stimulated primordial follicle formation without inducing apoptosis. Ribonucleic acid-sequence analysis (RNA-seq) of ovaries exposed to exogenous BMP2 or estradiol-17β revealed differential expression of several thousand genes. Most of the differentially expressed genes, which were common between BMP2 or estradiol-17β treatment demonstrated concordant changes, suggesting that estradiol-17β and BMP2 affected the same set of genes during primordial follicle formation. Further, we have identified that estradiol-17β, in cooperation with BMP2, could affect the expression of three major transcription factors, GATA binding protein 2, GATA binding protein 4 and Early growth response 2, and one serine protease, hepsin, in pre-granulosa cells during primordial follicle formation. Taken together, results of this study suggest that estradiol-17β and BMP2 may regulate ovarian gene expression that promote somatic cells to pre-granulosa cells transition and primordial follicle formation in the mouse ovary.

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 Hdac2in 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.

High steroid content in conditioned medium of granulosa cells may disrupt primordial follicles formation in in vitro cultured one-day-old murine ovaries

This study was conducted to investigate the main and interactive effects of two methods of culture medium preparation [base medium vs granulosa cells conditioned medium (GCCM)] and two nutrient supplements [fetal bovine serum (FBS) vs knock-out serum replacement (KSR)] on formation and activation of primordial follicles and gene expression of corresponding factors during a seven-day culture period. One-day-old mouse ovaries were cultured with four different culture media including base medium containing FBS (BMF), base medium containing KSR (BMK), GCCM prepared with FBS (CMF) and GCCM prepared with KSR (CMK), and samples for histological and molecular assessments were collected on days 3 and 7 of culture. Further, steroid content of media was measured. Histological examination showed that KSR enhanced follicular formation and the number of follicular count was greater in BMK than CMF group (P < 0.05). Moreover, follicular activation was higher in CMK group than BMK and CMF groups (P < 0.05). Additionally, RT-PCR revealed that KSR upregulated Gdf9 gene expression (P < 0.05), while GCCM diminished expression of Gdf9, Bmp15, Notch2, Figla and Foxl2 (P < 0.05). GCCM decreased expression of Pten and increased expression of Pi3k (P < 0.05). Besides, hormonal assays indicated higher concentrations of estradiol and progesterone in GCCM compared with base media (P < 0.0001). In conclusion, the present study showed base medium containing KSR could serve as a proper medium for in vitro culture of neonatal mouse ovary since it could better support formation of primordial follicles. Yet BMK did not promote follicular activation as well as GCCM prepared with KSR did, and therefore, requires modifications.