Primordial Follicle Formation Research Articles

The earliest stages of follicular development: Follicle formation and activation

The formation of primordial follicles to establish a reservoir of resting follicles and the gradual depletion of that reservoir to provide a succession of growing follicles are key to female fertility, but little is known about the regulation of these early stages of follicular development. This review summarizes the efforts of our laboratory to elucidate these critical processes in cattle. Primordial follicles first appear in fetal ovaries around the end of the first trimester of pregnancy (Day 90), during a decline in fetal ovarian production of estradiol and progesterone. In ovarian cortical pieces from 90 to 120-day-old fetuses, follicles form in vitro and estradiol or progesterone inhibits follicle formation, whereas the non-aromatizable androgen 5a-dihydrotestosterone (DHT) does not. Newly formed bovine follicles are not capable of activating within 2 days in vitro, but they can acquire the capacity to activate during a longer culture; estradiol and progesterone inhibit the acquisition of their capacity to activate. When primordial follicles first form in cattle, their oocytes are not yet in meiotic arrest and acquisition of competence to activate is correlated with their progression to meiotic arrest at the diplotene stage of first prophase. After they acquire the competence to activate, bovine primordial follicles can be stimulated to activate in vitro by insulin or kit ligand, whereas anti- Mullerian hormone (AMH) is inhibitory. Although few follicles progress to the secondary stage in vitro, addition of testosterone or vascular endothelial growth factor (VEGF) dramatically increased the incidence of that transition. Regulation of the earliest stages of follicular development is complex and far from understood; better understanding could lead to new interventions to enhance fertility.

Proteomic analysis of mouse ovaries during the prepubertal stages

Postnatal folliculogenesis, primordial follicle activation and follicular development at early stage are important for normal ovarian function and fertility, and a comprehensive understanding of this process under physiological condition is necessary. To observe the regulation and mechanism of ovarian follicle development during the prepubertal stages, we collected the mouse ovaries from three time points, including 1 day, 7 days, and 4 weeks after birth. We then performed a proteomic analysis using tandem mass tags (TMT) labeling combined with a two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) technique. A total of 706 proteins were determined to be significant differential abundance (P-SDA). Sixty upregulated proteins and 12 downregulated proteins that were P-SDA and 3 significant KEGG pathways (P < 0.05) were found at 7 days vs. 1 day after birth, while 237 upregulated proteins, 271 downregulated proteins and 42 significant KEGG pathways were found for 4 weeks vs. 7 days after birth. Some vital genes (Figla, Ooep, Padi6, Zp3, Hsd3b1, cyp11a1), key pathways (ECM-receptor interaction, focal adhesion, ovarian steroidogenesis, complement and coagulation cascades, PI3K/Akt/mTOR), and metabolic regulation (energy metabolism, lipid metabolism, metal ion metabolism) were found to be related to the postnatal folliculogenesis, primordial follicle activation and follicular development. Finally, qRT-PCR and western blotting verified some vital genes and further elucidated the developmental process of follicles, and the results may contribute to the understanding of the formation and activation of primordial follicle and follicular development.Significance: This study offers the first proteomic insights into mechanisms of follicle development under physiological condition during the prepubertal stages. By comparing P-SDA of mouse ovaries during various period of age, our data reveals that the regulation of primordial follicle formation and activation is significantly different from that of follicular development. These findings demonstrate that many unique molecular mechanisms underlie ovarian development could be used for ovarian disease research.

Interaction of Follicle-Stimulating Hormone and Stem Cell Factor to Promote Primordial Follicle Assembly in the Chicken.

Follicle-stimulating hormone (FSH) and KIT signaling are required for ovarian development. In this study the interactive effect of FSH and stem cell factor (SCF) on folliculogenesis was investigated in the chicken. Correlated changes between the FSH receptor and the expression of KIT signaling genes were seen to be involved in the formation of the chicken primordial follicles. Follicle-stimulating hormone and SCF displayed a reciprocal stimulating effect in the promotion of folliculogenesis involving elevated phosphorylation of mitogen-activated protein kinases (MAPK) and protein kinase B (AKT) proteins. Knockdown of c-KIT or SCF reduced the stimulatory effect of FSH on KIT signaling as well as upon MAPK and AKT phosphorylation. Treatment of FSH and SCF in combination enhanced ovarian cell proliferation and N-cadherin expression, but inhibited cell apoptosis and E-cadherin expression. Overall, the reciprocal stimulating effect of FSH and SCF in promoting chicken follicle assembly involving accelerated ovarian cell proliferation, N-cadherin expression, inhibited cell apoptosis, and E-cadherin expression is demonstrated.

In vitro and in vivo mouse follicle development in ovaries and reaggregated ovaries.

Follicle development requires complex and coordinated interactions between both the oocyte and its associated somatic cells. In ovarian dysfunction, follicle development may be abnormal due to defective somatic cell function; for example, premature ovarian insufficiency or malignancies. Replacing defective somatic cells, using the reaggregated ovary (RO) technique, may ‘rescue’ follicle development. ROs containing mature follicles have been generated when transplanted to a host mouse to develop. We have developed a RO culture technique and the aims were to determine how follicle development differed between transplanted and cultured ROs, and the influence of ovarian age (P2 vs P6). Mouse ROs were cultured for 14 days; P2 and P6 ovaries cultured as Controls. Follicle development was compared to ROs transplanted for 14 days and ovaries from P16 and P20 mice. ROs generated from either P2 or P6 exhibited similar follicle development in culture whereas in vivo follicle development was more advanced in P6 ROs. Follicles were more developed in cultured ROs than transplanted ROs. However, follicles in cultured ROs and ovaries had smaller oocytes with fewer theca and granulosa cells than in vivo counterparts. Our results demonstrate the fluidity of follicle development despite ovary dissociation and that environment is more important to basal lamina formation and theca cell development. Furthermore, follicle development within cultured ROs appears to be independent of oocyte nest breakdown and primordial follicle formation in source ovaries. Our results highlight the need for understanding follicle development in vitro, particularly in the development of the RO technique as a potential fertility treatment.

Retinoic acid-induced CYP51 nuclear translocation promotes meiosis prophase I process and is correlated to the expression of REC8 and STAG3 in mice

ABSTRACTLanosterol 14 α-demethylase (CYP51) plays a crucial role in cholesterol biosynthesis. In gamete development, CYP51 is involved in initiating meiosis resumption in oocytes through its product, meiosis activating sterol (MAS). In this study, CYP51 was observed to localize within the nucleus of germ cells undergoing meiotic prophase I. Following the addition of retinoic acid (RA) to induce meiosis or the RA receptor pan-antagonist AGN193109 to block meiosis in fetal ovaries, the translocation of CYP51 into the nucleus of oocytes was advanced or delayed, respectively. In addition, treatment with Cyp51-siRNA or RS21745, a specific CYP51 inhibitor, significantly delayed the meiotic progression of oocytes in the ovary, with most oocytes arresting at the zygotene stage, and likewise, significantly reduced perinatal primordial follicle formation. Furthermore, inhibition of CYP51 is correlated to significantly decreased expression of REC8 and STAG3, both of which are meiosis-specific cohesin subunits. To sum up, RA-induced CYP51 nuclear translocation is critical for oocytes meiotic progression, and consequently folliculogenesis, which might act through impacting the expression of meiosis-specific cohesins REC8 and STAG3.

Formation and activation induction of primordial follicles using granulosa and cumulus cells conditioned media

Fertility preservation of prepubertal girls subjected to invasive cancer therapy necessitates defining protocols for activation of isolated primordial follicles. Granulosa (GCs) and cumulus cells (CCs) play pivotal role in oocyte development. Although GCs and CCs share some similarities, they differ in growth factors production. The current study was conducted to evaluate the effects of GCs, CCs and their conditioned media on mice primordial follicles activation. One-day-old mice ovaries were subjected to 6-day culture with base medium (BM), GC conditioned medium (GCCM), GC coculture (GCCC), CC conditioned medium (CCCM) or CC coculture (CCCC). Follicular growth and primordial to primary follicle transition was observed during 6-day culture, and follicular activation rate tended to be greater in GCCM than other groups (0.05 <P < 0.10). On Day 6, the expression of phosphatase and tensin homolog (PTEN) in GCCM group was lower than that in BM group (P = 0.020), the expression of phosphoinositide-3-kinase was higher in CCCC group than BM, GCCM and CCCM groups (P < 0.05), and the expression of connexin 37 was greater in the CCCM group as compared with BM, GCCC, and CCCC groups (P < 0.01). In conclusion, the current study showed that condition medium of GCs could enhance in vitro activation of primordial follicles, probably through downregulation of PTEN.

Morphological evaluation of the primordial follicle formation process in neonatal mice that are activated autophagy

Morphological evaluation of the primordial follicle formation process in neonatal mice that are activated autophagy

CAV1 regulates primordial follicle formation via the Notch2 signalling pathway and is associated with premature ovarian insufficiency in humans.

What is the function of CAV1 in folliculogenesis and female reproduction? CAV1 regulates germline cyst breakdown and primordial follicle (PF) formation in mice, and CAV1 mutation may be related to premature ovarian insufficiency (POI). Pre-granulosa cells are essential for the establishment of the PF pool, which determines female fertility and reproductive lifespan. Cav1 participates in vascularization in fetal mouse ovaries. However, the role of CAV1 in early folliculogenesis and POI pathogenesis remains unclear. Cav1 function was investigated in mice and Human Embryonic Kidney 293 cells. Ovaries (six per group) were randomly assigned to Cav1-vivo-morpholino, control and control-morpholino groups, and all experiments were repeated at least three times. To investigate CAV1 mutations in women, 200 Chinese women with POI and 200 control individuals with regular menstrual cycles and normal endocrine profiles were recruited from the Center for Reproductive Medicine of Shandong University between September 2012 and December 2013. Wild-type CD1 mice, Lgr5-EGFP-ires-CreERT2 (Lgr5-KI) reporter mice and Human Embryonic Kidney 293 cells were used for these experiments. Protein expression was detected by Western blot, and quantitative RT-PCR was used to detect gene expression. The expression pattern of CAV1 in mouse ovaries and the phenotype of Cav1 deficiency in mice were detected by immunofluorescence. Pre-granulosa cell proliferation in ovaries was detected by bromodeoxyuridine (BrdU) assay and immunofluorescence. The coding region of the CAV1 gene was sequenced in 200 women with POI and 200 controls. The functional effect of the novel mutation c.142 G > C (p.Glu48Gln) was investigated by Cell Counting Kit-8 (CCK8) assays and Western blot. We confirmed that Cav1 deficiency in mouse ovary induced by CAV1-vivo-morpholino resulted in more multi-oocyte follicles than in the control and control-morpholino groups (P < 0.01). Suppression of Cav1 decreased Leucine rich repeat containing G protein coupled receptor 5 (Lgr5)-positive cell proliferation (P < 0.01) and reduced the number of Lgr5 and Forkhead box L2 (Foxl2) double-positive cells (P < 0.01). Furthermore, suppression of Cav1 inhibited ovarian epithelial Lgr5-positive cell proliferation and differentiation through the Notch2 signalling pathway. Two of the POI women carried novel CAV1 mutations (c.45 C > G synonymous and c.142 G > C [Glu48Gln]). The deleterious effect of p.Glu48Gln was corroborated by showing that it adversely affected the function of CAV1 in cell proliferation and NOTCH2 expression in HEK293FT cells. N/A. The novel Glu48Gln mutation was only detected in one of 200 POI patients and we were unable to investigate its effects in the ovary. The identification of CAV1 as a potentially causative gene for POI provides a theoretical basis to devise treatments for POI in women. This work was supported by the National Basic Research Program of China (973 Programs: 2012CB944700; 2013CB945501; 2013CB911400; 2014CB943202), the National Key Research and Development Program of China (2016YFC1000604, 2017YFC1001301), the State Key Program of National Natural Science Foundation of China (81430029), and the National Natural Science Foundation of China (31571540, 81522018, 81471509, 81601245, 81701406, 81571406). The authors declare no competing financial interests.

Dynamic expression patterns of Irx3 and Irx5 during germline nest breakdown and primordial follicle formation promote follicle survival in mouse ovaries.

Women and other mammalian females are born with a finite supply of oocytes that determine their reproductive lifespan. During fetal development, individual oocytes are enclosed by a protective layer of granulosa cells to form primordial follicles that will grow, mature, and eventually release the oocyte for potential fertilization. Despite the knowledge that follicles are dysfunctional and will die without granulosa cell-oocyte interactions, the mechanisms by which these cells establish communication is unknown. We previously identified that two members of the Iroquois homeobox transcription factor gene family, Irx3 and Irx5, are expressed within developing ovaries but not testes. Deletion of both factors (Irx3-Irx5EGFP/Irx3-Irx5EGFP) disrupted granulosa cell-oocyte contact during early follicle development leading to oocyte death. Thus, we hypothesized that Irx3 and Irx5 are required to develop cell-cell communication networks to maintain follicle integrity and female fertility. A series of Irx3 and Irx5 mutant mouse models were generated to assess roles for each factor. While both Irx3 and Irx5 single mutant females were subfertile, their breeding outcomes and ovary histology indicated distinct causes. Careful analysis of Irx3- and Irx5-reporter mice linked the cause of this disparity to dynamic spatio-temporal changes in their expression patterns. Both factors marked the progenitor pre-granulosa cell population in fetal ovaries. At the critical phase of germline nest breakdown and primordial follicle formation however, Irx3 and Irx5 transitioned to oocyte- and granulosa cell-specific expression respectively. Further investigation into the cause of follicle death in Irx3-Irx5EGFP/Irx3-Irx5EGFP ovaries uncovered specific defects in both granulosa cells and oocytes. Granulosa cell defects included poor contributions to basement membrane deposition and mis-localization of gap junction proteins. Granulosa cells and oocytes both presented fewer cell projections resulting in compromised cell-cell communication. Altogether, we conclude that Irx3 and Irx5 first work together to define the pregranulosa cell population of germline nests. During primordial follicle formation, they transition to oocyte- and granulosa cell-specific expression patterns where they cooperate in neighboring cells to build the foundation for follicle integrity. This foundation is left as their legacy of the essential oocyte-granulosa cell communication network that ensures and ultimately optimizes the integrity of the ovarian reserve and therefore, the female reproductive lifespan.

Differentially Expressed Genes Associated with Primordial Follicle Formation and Transformation to Primary Follicles

Objective: Primordial follicle assembly and the transition from primordial follicle to primary follicle are critical processes in ovarian biology. These processes determine the size of the primordial follicle pool, which may limit the reproductive lifespan of a female. When the follicle pool is depleted, reproduction ceases and females enter menopause. In this study, we aimed to explore the role of differentially expressed genes (DEGs) in the processes of primordial follicle assembly and the transformation from primordial to primary follicle. Methods: The gene chip GSE9300 was downloaded from the Gene Expression Omnibus database. DEGs between ovaries on postnatal day 0 and ovaries on postnatal day 4, as well as ovaries on postnatal day 4 and ovaries cultured for 7 days from postnatal day 0 were identified. Gene ontology (GO), pathway enrichment analysis, and protein–protein interaction network construction were performed. Results: In total, 99 upregulated genes and 123 downregulated genes were identified in the process of primordial follicle assembly, and 101 upregulated genes and 98 downregulated genes were identified in the process of transformation from primordial to primary follicle. The GO analysis showed that response to estradiol was associated with two of the processes of assembly and subsequent development of primordial follicle, but the genes involved in response to estradiol between the two processes were significantly different. Furthermore, the two groups of DEGs were all associated with metabolic pathways. Conclusions: DEGs may provide new insights into the exploration of mechanisms related to primordial follicle assembly and subsequent development.

Caracterização estrutural e papel do fator de crescimento derivado de plaquetas (PDGF) na foliculogênese ovariana

Background: : : : The platelet-derived growth factor (PDGF) is expressed in a wide variety of cell types, exerts a potent mitogenic role and acts on the growth, differentiation and cell chemotaxis. Studies have shown that during folliculogenesis, PDGF and their receptors are expressed in oocytes, granulosa cells and thecal cells of ovarian follicles at different developmental stages in several species. Although exist many information about its expression sites, as well as about its action in different cells types, the role of PDGF on ovarian folliculogenesis remains understudied. Thus, this article aims to review issues related to PDGF, suggesting the involvement of this mitogenic factor during follicular development. Review: Along this work, it was shown aspects related to structural characterization of PDGF and its receptors, as well as PDGF expression in different cells types, emphasizing its importance to follicular development. PDGF family is composed by four polypeptide chains (each encoded by a different gene), which are synthesized in the form of inactive pro-proteins. After a proteolytic processing, these chains undergo homo or heterodimerization, resulting in five isoforms (PDGF-AA, -BB, -AB, -CC e -DD). The cellular effects of these different PDGF isoforms are mediated by binding, with different specificities, to three transmembrane receptors isoforms of type tyrosine kinase generated by the association of subunits ? e ? (PDGFR- ??, - ??, - ??). PDGF was initially purified from platelets, being later verified its production by many varieties of cell types. It acts as an important mitogenic factor, especially to cells of mesenchymal origin as fibroblasts and smooth muscle cells, and also exerts a role on cellular migration and survival. Studies with PDGF and its receptors have demonstrated its importance as a paracrine growth factor for normal embryo development and angiogenesis. Although the action of this growth factor has been shown in several cellular responses, its role in ovarian folliculogenesis of mammals is understudied. Folliculogenesis, event initiated at pre-natal life in most species, can be defined as the process of follicular formation, growth and maturation, beggining with the primordial follicle formation and resulting in preovulatory stage. Until the present moment, the different PDGF types and their receptors were detected in ovarian follicles of murines, rodents, swines and humans according to developmental stage. Evidences suggest that this growth factor may acts as a potent regulator of ovarian function, increasing DNA synthesis in granulosa cells and stimulating theca cells growth in swines. In addition, PDGF has shown benefic effects in in vitro maturation of bovine oocytes when associated with follicle stimulating hormone (FSH). Conclusion: Nevertheless, the specific actions of PDGF on ovarian follicles development have not been described due to lack of data available in the literature about this subject. Therefore, this review was performed to clarify the involvement of this growth factor in the regulation of ovarian function in mammals. However, it is necessary to perform additional studies that may provide a greater understanding about the importance of PDGF during folicullogenesis.

Requirement of the 3'-UTR-dependent suppression of DAZL in oocytes for pre-implantation mouse development.

Functional oocytes are produced through complex molecular and cellular processes. In particular, the contribution of post-transcriptional gene regulation mediated by RNA-binding proteins (RBPs) is crucial for controlling proper gene expression during this process. DAZL (deleted in azoospermia-like) is one of the RBPs required for the sexual differentiation of primordial germ cells and for the progression of meiosis in ovulated oocytes. However, the involvement of DAZL in the development of follicular oocytes is still unknown. Here, we show that Dazl is translationally suppressed in a 3′-UTR-dependent manner in follicular oocytes, and this suppression is required for normal pre-implantation development. We found that suppression of DAZL occurred in postnatal oocytes concomitant with the formation of primordial follicles, whereas Dazl mRNA was continuously expressed throughout oocyte development, raising the possibility that DAZL is dispensable for the survival and growth of follicular oocytes. Indeed, follicular oocyte-specific knockout of Dazl resulted in the production of normal number of pups. On the other hand, genetically modified female mice that overexpress DAZL produced fewer numbers of pups than the control due to defective pre-implantation development. Our data suggest that post-transcriptional suppression of DAZL in oocytes is an important mechanism controlling gene expression in the development of functional oocytes.

Sex Steroid Hormones and Related Substances Involved in Primordial Follicle Activation

Abstract: Primordial follicles maintain dormancy in the ovary and only a small number of them are activated towards ovulation every day. Several signaling pathways in oocytes and the surrounding flattened pre-granulosa cells have been shown to play crucial roles in primordial follicle activations and sex steroid hormones are also known to affect it. Intrinsic estrogen, estradiol, has been reported to suppress primordial follicle formation and the later primordial follicle activation in rodent neonatal ovaries. Conversely, some phytoestrogens and endocrine disruptors which possess intrinsic estrogen-like biological activity with different binding affinities to estrogen receptors, can activate primordial follicles. Testosterone and dehydroepiandrosterone have been used in fertility treatments in the expectation that they would activate primordial follicle, although evidence of their efficacy is inconclusive. Progesterone suppresses primordial follicle formation and the later primordial follicle activation i...

Vasorin: a newly identified regulator of ovarian folliculogenesis.

Members of the TGF-β superfamily take part in the control of folliculogenesis. Vasorin (Vasn) is a newly identified negative regulator of TGF-β signaling whose possible involvement in ovarian physiology has never been studied. Here, we demonstrate that Vasn is expressed in the ovary by somatic cells of follicles, and that its expression is up-regulated by LH. We established a conditional knockout (cKO) mouse model in which Vasn is deleted specifically in granulosa cells of growing follicles from the secondary stage onwards. Using this model, we show that, upon hormonal stimulation, follicle ovulation size is almost 2-fold higher. This enhanced ovulatory response is associated with overactivation of the TGF-β signaling pathway and a lower number of atretic antral follicles. Of importance, we demonstrate that the number of primordial follicles is reduced in prepubertal cKO mouse ovaries, which suggests that the production of VASN by growing follicles protects the ovarian reserve. Finally, analysis of systemic KO mice revealed that the ovarian reserve is almost 2.5-fold higher, which implies that Vasn may also play a role in primordial follicle formation. Overall, our findings reveal that Vasn is a new regulator that exerts an effect on several key ovarian functions, including folliculogenesis, maintenance of the ovarian reserve, and ovulation.-Rimon-Dahari, N., Heinemann-Yerushalmi, L., Hadas, R., Kalich-Philosoph, L., Ketter, D., Nevo, N., Galiani, D., Dekel, N. Vasorin: a newly identified regulator of ovarian folliculogenesis.

Administration of Tempehethanol Extract on Prenatal Until Weaning Period Inhibit the Ovary Follicles Developing of Little Wistar Rats

Background and objective: soybean tempeh contains isoflavones that are able to bind to estrogen receptors, so they have estrogenic properties. This study aims to prove that the administration of tempeh soybean extract during periods of perception can inhibit the formation of ovarian follicles. Method: posttest only control group design, using female rats aged 12-13 weeks with BB 150 g, a research site in Udayana University’s integrated laboratory. Result: the results showed that there were significant differences in the mean numberof follicles in the three groups, namely primordial, primary, secondary was p <0.01; atretic follicles wasp <0.05. Conclusion: the administration of ethanol extract of tempeh during the perimenopause period can inhibit the formation of primordial follicles, primary follicles, and secondary follicles. High doses can increase the average number of atretic follicles. Keywords: ethanol extract tempeh, perimenopause period, ovarian follicles

Non-suckling starvation of neonatal mice promotes primordial follicle formation with activation of ovarian autophagy.

Around the time of oocyte meiotic arrest, germ cell nest breakdown occurs, and primordial follicle (PF) formation is initiated at the perinatal stage. Recently, autophagy was implicated in this process. Autophagy is induced by nutrient starvation. This study was conducted to understand how starvation affects PF formation and autophagy induction during neonatal life. Suckling of neonatal female mice was blocked immediately after birth for 12–36 h to induce starvation. The numbers of PFs at each stage were subsequently counted from serial sections of ovaries. The expression of autophagy-related proteins was also evaluated. The number of PFs peaked at 60 h after birth in the control group. The numbers for the starvation groups were significantly higher than those for the control groups at 12 and 36 h. LC3B was clearly present in the oocyte cytoplasm. At 36 h after birth, the starvation group showed a higher rate of LC3II/LC3-I expression as a marker for autophagy. Moreover, the expression of p62 as a selective substrate for autophagy decreased compared to the control group. The expression of caspase-9 as a marker for apoptosis tended to be lower at 36 h in the starvation groups. These results indicate that starvation promotes PF formation with a concomitant activation of autophagy in early neonatal ovaries, suggesting that autophagy induction during follicle assembly might increase the number of PFs.

Stimulation of primordial follicle assembly by estradiol-17\u03b2 requires the action of bone morphogenetic protein-2 (BMP2)

Primordial follicle (PF) pool determines the availability of follicles for ovulation in all mammals. Premature depletion of the PF reserve leads to subfertility or infertility. Bone morphogenetic protein 2 (BMP2) promotes PF formation by facilitating oocyte and granulosa cell development. Estradiol-17β (E2) upregulates PF formation in developing hamster ovaries. However, if BMP2 mediates E2 effect is not known. We hypothesize that E2 facilitates the effect of BMP2 on somatic to granulosa cell transition. BMP2 and E2 together significantly upregulated the percentage of PFs in hamster fetal ovaries in vitro compared with either of the treatments alone. E2 also promoted BMP2 expression in vivo. Inhibition of BMP2 receptors suppressed E2-stimulation of PF formation while knockdown of BMP2 in vitro significantly suppressed the E2 effect. In contrast, estrogen receptor blocker did not affect BMP2 action. Inhibition of the activity of E2 or BMP2 receptors, either alone or combined during the last two days of the culture (C6-C8) resulted in a significant decrease in PF formation by C8, suggesting that both BMP2 and E2 action is essential for somatic cell differentiation for PF formation. Together, the results suggest that E2 activates BMP2-BMPR system leading to the formation of primordial follicles.

Bisphenol A accelerates meiotic progression in embryonic chickens via the estrogen receptor β signaling pathway

Bisphenol A (BPA) as an endocrine-disrupting chemical with weak estrogenic activity affects formation of primordial follicles. This study aimed to identify the potential effects and molecular mechanisms of BPA on meiosis and primordial follicle formation in chickens. The results suggest that the cortical layer was thickened and the number of germ cells that entered into meiosis was increased in BPA-treated ovaries. The percentage of γH2AX-positive cells increased significantly. In addition, up-regulated mRNA expression of meiotic genes, including stimulated by retinoic acid gene 8 (Stra8), disrupted meiotic cDNA 1 homologue (Dmc1) and synaptonemal complex protein 3 (Scp3) were observed in BPA-treated ovaries. Therefore, progression to meiosis prophase I was accelerated by exposure to BPA. Furthermore, the results demonstrated that injection of BPA resulted in hypomethylation of Dazl (Deleted in A Zoospermia-Like gene) and Stra8 and up-regulation mRNA expression of Dazl and Stra8 during meiotic onset. Finally, the relationship between estrogen receptor (ER) expression and BPA-induced meiosis was revealed using an in vitro ovarian culture system. BPA enhanced ERβ expression at the levels of mRNA and protein, while BPA exerted no significant effect on ERα and membrane-bound estrogen receptor (GPR30) expression. The inducing effects of BPA on meiosis were blocked by ER inhibitor. Collectively, these results demonstrate the dynamic ovarian response to BPA exposure, which indicate that BPA affects the formation of primordial follicles by promoting meiotic progression of oocytes via hypomethylation of Dazl and Stra8 and ERβ signaling pathways.

Nrf2 regulates female germ cell meiosis initiation

Meiosis initiation of the female germ cell is critical for primordial follicle formation and ovarian reserve. Nuclear factor E2-related factor 2 (Nrf2) is a vital transcriptional factor for antioxidation. However, little is known about the role of Nrf2 in female fertility. Our aim of this study was to investigate the effect of Nrf2 on meiosis initiation and early oogenesis, hoping to find a new way to improve ovarian reserve. CD-1 mice were maintained in a temperature- and light-controlled facility with free access to water and food. Mating was timed overnight and the appearance of the vaginal plug was considered as E0.5 in the next morning. Female genital ridges (GRs) were collected from E11.5 to E14.5 fetal mice. GRs were either used directly to analyzed Nrf2 expression or incubated with inhibitors for 48 h to test the effect of Nrf2 on meiosis initiation and cell cycle. After additional culture with normal medium, the effect of Nrf2 on oocyte progression and primordial follicle formation was tested. Then, p53 inhibitor was used to analyzed the effect of p53-p21 pathway in this process by retesting the above indexes. Brusatol was used as Nrf2 inhibitor, while PFTa as p53 inhibitor. mRNA and protein levels of genes were tested by real-time PCR and western blot, respectively. Meiotic prophase I stages of oocytes were evaluated by oocyte cytospread. The expression pattern of Scp3 was used as a marker of different oocyte progression stages. Serial paraffin section and HE staining was used for the analysis of primordial follicle formation. Consistent with the previous study1, Brusatol was also confirmed as an effective inhibitor of Nrf2 in GR, as protein level of Nrf2 and mRNA levels of the downstream factors were all inhibited by Brusatol. Nrf2 was dynamically expressed in E11.5-14.5 GR, and reached peak in E12.5, suggesting Nrf2 might be involved in meiosis initiation. Inhibiting the expression of Nrf2 with Brusatol downregulated mRNA levels of meiosis genes (Stra8, Scp3, Dmc1, Dazl, Rec8), changed the expression of cell cycle-related genes (p53, p21, cyclin E), delayed oocyte progression in lepetotene, and decreased the number of primordial follicles. Further studies showed the effect of Brusatol on meiosis initiation and follicle formation was significantly rescued by the addition of p53 inhibitor. Nrf2 regulated meiosis initiation and early oogenesis in female germ cell via p53-p21 pathway.

Erratum to: Mechanisms controlling germline cyst breakdown and primordial follicle formation.

Erratum to: Mechanisms controlling germline cyst breakdown and primordial follicle formation.