Primordial Follicle Transition Research Articles

P-407 Artificial ovary construction: short-term in vitro culture of human primordial and primary follicles in a 3D environment on a stromal cell feeder layer

Abstract Study question Do ovarian stromal cells (OSCs) influence viability and growth of human pre-antral follicles in vitro? Summary answer Feeder layer of OSCs advanced growth and transition of primordial follicles to primary/secondary stage while keeping a high proportion of viable follicles. What is known already In the ovary, follicles require the support of ovarian cells through the secretion of essential factors for their survival and development. This was also demonstrated in vitro through the 3D culture of isolated mouse primary and secondary follicles on a feeder layer of ovarian stromal cells. This co-culture significantly increased follicle survival and growth. Study design, size, duration Pre-antral follicles were isolated from human frozen-thawed ovarian tissue (OT) biopsies and encapsulated in 1% alginate scaffolds. Embedded pre-antral follicles were placed directly on the OSCs feeder layer or bottom of a culture dish for a 7-day in vitro culture (control). Follicle viability and growth and hormone production were compared between groups. Participants/materials, setting, methods Primordial and primary follicles were isolated from frozen-thawed OT of cancer patients (n = 6). OSCs were isolated from OT of post-menopausal women and cultured as a feeder layer. Follicle diameter was measured on Days 0 and 7 under an inverted microscope. Viability was assessed by staining a proportion of follicles (n = 87) with calcein AM and ethidium homodimer-I. They were classified (V1/V2: healthy/minimally damaged; V3/V4: damaged/dead follicles) using confocal fluorescence microscopy. Estradiol levels were measured by ELISA. Main results and the role of chance A total of 379 human pre-antral follicles (primordial=360; primary=19) were isolated and embedded in 1% alginate hydrogels and classified according to their viability. Most follicles (96%) were viable after isolation with a diameter of 40.8±9.9 µm (mean±SD). At Day 7, pre-antral follicles have grown significantly in size in both culture conditions: on the OSCs feeder layer and without it (p < 0.0001 for D0 vs. D7). However, no significant difference (p = 0.07) between culture conditions was observed. The mean diameter of follicles cultured on the OSCs layer was 80.6±11.0 μm and without it, 67.3±7.2 μm. Nonetheless, the distribution pattern of different quality grade follicles was significantly different on OSCs (V1: 62%, V2: 25%, V3: 2%, V4: 11%) and follicles without OSCs (V1: 35%, V2: 38%, V3: 0%, V4: 23%; p = 0.03). Additionally, more follicles have been activated and reached a higher developmental stage on OSCs (D0 primordial: 184, primary: 7 vs. D7 primordial: 53, primary/secondary: 93) than without stromal cells (D0 primordial: 186, primary: 4 vs. D7 primordial: 84, primary/secondary: 64; p < 0.001) with 66 and 43 follicles reaching a secondary stage (75<x<200 μm), respectively. Estradiol level was significantly (p = 0.006) higher in media of follicles cultured on the OSCs, 54.1±14.2 pg/ml vs. 29.9±4.0 pg/ml. Limitations, reasons for caution This study was performed only in a short-term culture and no primordial/primary follicles have reached the antral stage. Further in vitro studies on follicular developmental capacity, physiology and steroidogenesis in alginate scaffold with human ovarian stromal cells are needed. Wider implications of the findings Activation and growth of human primordial follicles during in vitro short-term culture to a secondary stage has been a long-lasting challenge. Co-culture with human ovarian stromal cells can help to overcome this limitation. Trial registration number not applicable

Phytoestrogen genistein hinders ovarian oxidative damage and apoptotic cell death-induced by ionizing radiation: co-operative role of ER-\u03b2, TGF-\u03b2, and FOXL-2

Radiotherapy is a well-known cause of premature ovarian failure (POF). Therefore, we investigated the molecular influence of genistein (GEN) on the ovarian reserve of rats exposed to ϒ-radiation. Female Sprague Dawley rats were exposed to a 3.2 Gy γ-radiation to induce POF and/or treated with either GEN (5 mg/kg, i.p.) or Ethinyl estradiol (E2; 0.1 mg/kg, s.c.), once daily for 10 days. GEN was able to conserve primordial follicles stock and population of growing follicles accompanied with reduction in atretic follicles. GEN restored the circulating estradiol and anti-Müllerian hormone levels which were diminished after irradiation. GEN has potent antioxidant activity against radiation-mediated oxidative stress through upregulating endogenous glutathione levels and glutathione peroxidase activity. Mechanistically, GEN inhibited the intrinsic pathway of apoptosis by repressing Bax expression and augmenting Bcl-2 expression resulted in reduced Bax/Bcl-2 ratio with subsequent reduction in cytochrome c and caspase 3 expression. These promising effects of GEN are associated with improving granulosa cells proliferation. On the molecular basis, GEN reversed ovarian apoptosis through up-regulation of ER-β and FOXL-2 with downregulation of TGF-β expression, therefore inhibiting transition of primordial follicles to more growing follicles. GEN may constitute a novel therapeutic modality for safeguarding ovarian function of females’ cancer survivors.

Gremlin-1 and gremlin-2 levels in polycystic ovary syndrome and their clinical correlations

Objective: Gremlin 1 and 2 regulate oocyte primordial follicle transition in animal models. The main objective of this study is to measure the blood levels of Gremlin 1 and 2 in the women with Polycystic Ovary Syndrome (PCOS). We also aimed to evaluate the association of these markers with hormonal and biochemical parameters of PCOS as interrupted folliculogenesis in those women is related to metabolic dysfunction.Material and methods: Fifty women with PCOS were diagnosed according to Rotterdam criteria, and thirty age-matched female controls were included in this prospective study. Gremlin 1 and 2 levels along with hormonal and metabolic parameters were compared between PCOS and control groups.Results: Serum Gremlin 1 levels were significantly higher in the PCOS group than in the control group (p = .001). Gremlin 2 levels were similar between the groups. Besides, there was a significant positive correlation between Gremlin 1 and insulin levels, Homeostasis Model Assessment-Insulin Resistance (HOMA-IR) and waist to hip ratio (WHR) (r = 0.305; r = 0.297; r = 0.303, respectively).Conclusion: Our data suggest that Gremlin 1 may be the key regulator in the pathogenesis of PCOS. In future, Gremlin 1 may be a novel therapeutic target for the treatment of PCOS.

Bisphenol A Initiates Excessive Premature Activation of Primordial Follicles in Mouse Ovaries via the PTEN Signaling Pathway.

The essence of primary ovarian insufficiency (POI) is the premature exhaustion of primordial follicles in the follicle pool, which is caused by the excessive premature activation of primordial follicles after birth. Bisphenol A (BPA) exposure promotes the transition of primordial follicles to primary follicles, thus the number of primordial follicles in the primordial follicle pool decreases significantly. However, the molecular mechanisms underlying abnormal follicle activation are poorly understood. Phosphatase and tensin homologue (PTEN) signal system is a negative regulator of follicle activation, which is called the brake of follicle activation. Besides, BPA induces Michigan Cancer Foundation-7 breast cancer cells proliferation by dysregulating PTEN/serine/threonine kinase/p53 axis. Whether BPA initiates the excessive premature activation of primordial follicles in the mouse ovaries via PTEN signaling pathway is unclear. In this study, we treated 6-week-old female CD-1 mice with different concentrations of BPA to study the effect of BPA on follicular activation and development in vivo, as well as the role of PTEN signaling in this process. We observed that BPA in concentrations from 1 μg/kg to 10 mg/kg groups downregulated PTEN expression and initiated excessive premature activation of primordial follicles in the mouse ovaries, and this effect was partly reversible by PTEN overexpression. Our results improve the understanding of both the effect of BPA in occurrence of POI and molecular mechanisms underlying initiation of primordial follicle pool activation, thus providing insight for POI treatment and theoretical basis for reducing the risk of POI.

Proteomic Analysis of Fetal Ovaries Reveals That Primordial Follicle Formation and Transition Are Differentially Regulated

Primordial follicle formation represents a critical phase of the initiation of embryonic reproductive organ development, while the primordial follicle transition into primary follicle determines whether oestrus or ovulation will occur in female animals. To identify molecular mechanism of new proteins which are involved in ovarian development, we employed 2D-DIGE to compare the protein expression profiles of primordial follicles and primary follicles of fetal ovaries in pigs. Fetal ovaries were collected at distinct time-points of the gestation cycle (g55 and g90). The identified proteins at the g55 time-point are mainly involved in the development of anatomical structures [reticulocalbin-1 (RCN1), reticulocalbin-3 (RCN3)], cell differentiation (actin), and stress response [heterogeneous nuclear ribonucleoprotein K (HNRNPK)]. Meanwhile, at the g90 stage, the isolated proteins with altered expression levels were mainly associated with cell proliferation [major vault protein (MVP)] and stress response [heat shock-related 70 kDa protein 2 (HSPA2)]. In conclusion, our work revealed that primordial follicle formation is regulated by RCN1, RCN3, actin, and HNRNPK, while the primordial follicle transformation to primary follicle is regulated by MVP and HSPA2. Therefore, our results provide further information for the prospective understanding of the molecular mechanism(s) involved in the regulation of the ovarian follicle development.

Effects of gremlin-2 on the transition of primordial follicles during early folliculogenesis in the human ovary

ObjectiveTo investigate the localization and function of gremlin-2 during human ovarian folliculogenesis. Study designOvarian tissue from a gynecologic cancer patient was cultured in the presence or absence of gremlin-2 and then analyzed histologically. Growing follicles were counted by the microscopic observations of ovarian histological sections. Immunocytochemical staining was carried out to detect the expression of bone morphogenetic protein (BMP) 4 and phosphorylated Smad 1/5/8 (p-Smad 1/5/8). ResultsGremlin-2 was detected in human primordial, primary, and early growing follicles before culture. By day 4 of culture, the follicle growth rate in the presence of gremlin-2 (13.7%; 24/175) was significantly lower than that of the control (54.8%; 92/175; p<0.01). BMP4 expression was similar in the presence and absence of gremlin-2, whereas the p-Smad 1/5/8 signal was noticeably stronger in the absence of gremlin-2 in primordial and early-stage growing follicles. ConclusionsGremlin-2 maintains the follicle store as primordial follicles by suppressing Smad 1/5/8 signaling in the human ovary. The data presented here provide potential insight into reproductive medicine for cases of intractable infertility, such as premature ovarian insufficiency and cancer survivors.

Cytokine (IL16) and tyrphostin actions on ovarian primordial follicle development.

An ovarian follicle is composed of an oocyte and surrounding theca and granulosa cells. Oocytes are stored in an arrested state within primordial follicles until they are signaled to re-initiate development by undergoing primordial-to-primary follicle transition. Previous gene bionetwork analyses of primordial follicle development identified a number of critical cytokine signaling pathways and genes potentially involved in the process. In the current study, candidate regulatory genes and pathways from the gene network analyses were tested for their effects on the formation of primordial follicles (follicle assembly) and on primordial follicle transition using whole ovary organ culture experiments. Observations indicate that the tyrphostin inhibitor (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one increased follicle assembly significantly, supporting a role for the MAPK signaling pathway in follicle assembly. The cytokine interleukin 16 (IL16) promotes primordial-to-primary follicle transition as compared with the controls, where as Delta-like ligand 4 (DLL4) and WNT-3A treatments have no effect. Immunohistochemical experiments demonstrated the localization of both the cytokine IL16 and its receptor CD4 in the granulosa cells surrounding each oocyte within the ovarian follicle. The tyrphostin LDN193189 (LDN) is an inhibitor of the bone morphogenic protein receptor 1 within the TGFB signaling pathway and was found to promote the primordial-to-primary follicle transition. Observations support the importance of cytokines (i.e., IL16) and cytokine signaling pathways in the regulation of early follicle development. Insights into regulatory factors affecting early primordial follicle development are provided that may associate with ovarian disease and translate to improved therapy in the future.

Roles of Gremlin 1 and Gremlin 2 in regulating ovarian primordial to primary follicle transition

A network of extracellular signaling factors has previously been shown to act in concert to control the ovarian primordial to primary follicle transition. The current study was designed to investigate the roles of the endogenous bone morphogenetic protein (BMP) inhibitors Gremlin 1 (GREM1) and GREM2 in primordial follicle transition in the rat ovary. GREM1 and GREM2 treatments were found to reverse the effects of anti-Müllerian hormone (AMH) to inhibit follicle transition in a whole-ovary culture system. GREM1 reversed the effect of BMP4 to stimulate primordial follicle transition. Immunohistochemical studies showed that GREM2, but not GREM1, was present in primordial follicles suggesting that GREM2 may regulate primordial follicle transition in vivo. Co-immunoprecipitation studies indicated that GREM2 directly binds to AMH, as well as to BMP4. Transcriptome analyses of ovaries treated with GREM2 or GREM1 yielded negligible numbers of differentially expressed genes, suggesting that the immediate effects of GREM2 or GREM1 appear to be at the level of protein-protein interactions, rather than direct actions on the cells. A number of other ovarian growth factors were found to influence the expression of Grem2. Observations suggest that Grem2 is a part of the signaling network of growth factors that regulate the primordial to primary follicle transition. Insights into the regulatory networks affecting the pool of primordial follicles are important to understand the molecular basis for reproductive diseases such as primary ovarian insufficiency.

The APC/C activator FZR1 is essential for meiotic prophase I in mice

Fizzy-related 1 (FZR1) is an activator of the Anaphase promoting complex/cyclosome (APC/C) and an important regulator of the mitotic cell division cycle. Using a germ-cell-specific conditional knockout model we examined its role in entry into meiosis and early meiotic events in both sexes. Loss of APC/C(FZR1) activity in the male germline led to both a mitotic and a meiotic testicular defect resulting in infertility due to the absence of mature spermatozoa. Spermatogonia in the prepubertal testes of such mice had abnormal proliferation and delayed entry into meiosis. Although early recombination events were initiated, male germ cells failed to progress beyond zygotene and underwent apoptosis. Loss of APC/C(FZR1) activity was associated with raised cyclin B1 levels, suggesting that CDK1 may trigger apoptosis. By contrast, female FZR1Δ mice were subfertile, with premature onset of ovarian failure by 5 months of age. Germ cell loss occurred embryonically in the ovary, around the time of the zygotene-pachytene transition, similar to that observed in males. In addition, the transition of primordial follicles into the growing follicle pool in the neonatal ovary was abnormal, such that the primordial follicles were prematurely depleted. We conclude that APC/C(FZR1) is an essential regulator of spermatogonial proliferation and early meiotic prophase I in both male and female germ cells and is therefore important in establishing the reproductive health of adult male and female mammals.

Changes in ovarian protein expression during primordial follicle formation in the hamster

Although many proteins have been shown to affect the transition of primordial follicles to the primary stage, factors regulating the formation of primordial follicles remains sketchy at best. Differentiation of somatic cells into early granulosa cells during ovarian morphogenesis is the hallmark of primordial follicle formation; hence, critical changes are expected in protein expression. We wanted to identify proteins, the expression of which would correlate with the formation of primordial follicles as a first step to determine their biological function in folliculogenesis. Proteins were extracted from embryonic (E15) and 8-day-old (P8) hamster ovaries and fractionated by two-dimensional gel electrophoresis. Gels were stained with Proteosilver, and images of protein profiles corresponding to E15 and P8 ovaries were overlayed to identify protein spots showing altered expression. Some of the protein spots were extracted from SyproRuby-stained preparative gels, digested with trypsin, and analyzed by mass spectrometry. Both E15 and P8 ovaries had high molecular weight proteins at acidic, basic, and neutral ranges; however, we focused on small molecular weight proteins at 4–7 pH range. Many of those spots might represent post-translational modification. Mass spectrometric analysis revealed the identity of these proteins. The formation of primordial follicles on P8 correlated with many differentially and newly expressed proteins. Whereas Ebp1 expression was downregulated in ovarian somatic cells, Sfrs3 expression was specifically upregulated in newly formed granulosa cells of primordial follicles on P8. The results show for the first time that the morphogenesis of primordial follicles in the hamster coincides with altered and novel expression of proteins involved in cell proliferation, transcriptional regulation, and metabolism. Therefore, formation of primordial follicles is an active process requiring differentiation of somatic cells into early granulosa cells and their interaction with the oocytes.

Testosterone induces activation of porcine primordial follicles in vitro.

The mechanism underlying primordial follicle activation is poorly understood. In this study, in-vitro culture and subsequent xenotransplantation were conducted to determine whether testosterone promotes the activation of porcine primordial follicles. Prepubertal porcine ovarian cortical strips containing primordial follicles were cultured in the presence of testosterone for 7 days, and subsequently transplanted to immunodeficient mice for 2 months. After culture and transplantation, development of follicles was examined histologically. The presence of androgen receptors in oocytes was assessed by use of western blot and immunohistochemical analyses. Testosterone at 10-6 m induced the primordial follicle transition to the intermediate (19 ± 4%) and primary (3 ± 1%) stages after 7-day culture, while 56 ± 5% of primordial follicles remained in the initial pool. Higher concentrations, above 10-5 m, or lower concentrations, below 10-6 m, did not induce follicle transition to the primary stage. After 7-day culture with 10-6 m testosterone, ovarian cortical strips were transplanted to immunodeficient mice. Some of the follicles developed to the secondary (15 ± 3%) and antral (10 ± 3%) stages, whereas 44 ± 7% of primordial follicles remained in the initial pool. In the culture experiment, estradiol-17β (10-7-10-5 m) had no significant effect on follicle activation. The androgen receptor antagonist, cyproterone acetate, inhibited the stimulatory effect of testosterone on primordial follicle activation, suggesting an androgen receptor-mediated action of testosterone. Western blot and immunohistochemical analyses revealed that androgen receptors were present in the oocytes of primordial follicles. These results suggest that testosterone at 10-6 m promotes the activation of porcine primordial follicles in vitro through the androgen receptors in the oocytes.

Kit ligand and basic fibroblast growth factor interactions in the induction of ovarian primordial to primary follicle transition

Ovulated eggs during a female’s reproductive life are derived from a pool of primordial follicles arrested in prophase of the first meiotic division. When follicles leave the resting pool they undergo a primordial to primary follicle transition and will grow and develop until either ovulation occurs or follicles undergo atresia. Several growth factors have been implicated as acting locally within the ovary to regulate the primordial to primary follicle transition. How these growth factors may interact and cooperate to perform this vital function remains to be elucidated. The objective of the current study is to investigate interactions between kit ligand (KL) (i.e. stem cell factor) and basic fibroblast growth factor (bFGF) that promote the primordial to primary follicle transition in rat ovaries. Ovaries were removed from 4-day-old rat pups and cultured for 2 weeks with KL alone or with KL and a neutralizing antibody against bFGF. The ability of KL treatment to increase primordial follicle transition was blocked with a bFGF neutralizing antibody. In addition, ovary cultures were treated with bFGF alone or with bFGF and an anti-c-kit receptor antibody which blocks KL signaling. The ability of bFGF treatment to increase primordial follicle transition was blocked with an anti-c-kit receptor antibody. Observations indicate that both KL and bFGF must be active in order to optimally promote the changes that occur in oocytes, granulosa cells, and stromal/interstitial cells when primordial follicles initiate development. Cultured ovaries were treated with either KL or bFGF for 3 days and then bFGF and KL mRNA expression levels in the whole ovary were measured. KL was not found to regulate bFGF expression. In contrast, bFGF treatment was found to increase KL mRNA expression in cultured ovaries. These observations suggest that one function of the oocyte-derived bFGF is to increase the granulosa derived KL expression and that both KL and bFGF are required to optimally promote primordial to primary follicle transition. Elucidating the cell–cell interactions that mediate this network of specific locally derived growth factors is critical to understanding the physiology of the primordial to primary follicle transition.

Regulation of ovarian primordial follicle assembly and development by estrogen and progesterone: endocrine model of follicle assembly.

The assembly of the developmentally arrested primordial follicle and the subsequent transition of the primordial follicle to the primary follicle are critical processes in normal ovarian physiology that remain to be elucidated. Ovarian follicles do not proliferate and the primordial follicles present in the neonate represent the total number of gametes available to a female throughout her reproductive life. The primordial follicles are oocytes surrounded by less differentiated squamous granulosa cells and are derived from oocyte nests, and primary follicles are oocytes surrounded by a single layer of cuboidal granulosa cells that have initiated follicle development. Abnormalities in primordial follicle assembly, arrest, and development (i.e. primordial to primary follicle transition) can cause pathological conditions such as premature ovarian failure. In this study newborn rat ovaries were cultured for 7 d. The rate of primordial follicle assembly in vivo was identical with the rate in vitro. Interestingly, the rate of primordial follicle transition to the primary follicle was found to be 3 times greater in culture. This abnormal rate of primary follicle development in culture suggests the primordial follicle does not arrest in development as observed in vivo. To investigate this phenomena newborn rat ovaries were cultured in the presence of progesterone, estradiol or calf serum. Estradiol, progesterone, or calf serum significantly reduced the level of initial primordial to primary follicle transition. Approximately 60% of follicles make the primordial to primary follicle transition in control ovaries and about 30% in treated ovaries. Steroids and calf serum had no effect on the primordial to primary follicle transition in ovaries collected and cultured from postnatal 4-d-old rats, suggesting the effects observed are restricted to the initial wave of primordial to primary follicle transition. Interestingly, progesterone was also found to significantly reduce the rate of primordial follicle assembly. All viable oocytes assembled into primordial follicles in control ovaries and approximately 40% remained unassembled in progesterone-treated ovaries. Progesterone was also found to reduce primordial follicle assembly in vivo with 10% of the total follicles remaining unassembled in progesterone injected neonatal animals. Analysis of cellular apoptosis demonstrated that progesterone inhibited the coordinated oocyte apoptosis required for primordial follicle assembly. The hypothesis developed is that high levels of maternal and fetal steroids prevent premature primordial follicle assembly and primordial to primary follicle transition in the embryo. After birth steroid levels fall dramatically and the primordial follicles are free to assemble and initiate development. These observations suggest a novel role for steroids and the maternal-fetal endocrine unit in the control of ovarian primordial follicle assembly and early follicular development.

The initiation of follicle growth: the oocyte or the somatic cells?

The initiation of follicle growth is defined as the transition of primordial follicles from the quiescent to the growth phase. We have shown that follicle growth initiation consists of two distinct, consecutive phases. The first phase is characterized by the transformation of granulosa cells from flattened to cuboidal in shape and by their proliferation, this coincides with follistatin expression. During the second phase, an increase in the number of granulosa cells is accompanied by an increase in the size of the oocyte. Evidence from other species confirms this observation. It seems that a prerequisite for initiation of oocyte growth is the presence of cuboidal granulosa cells. The vast majority of bovine primordial follicles enter the first phase of growth when cultured in serum-free medium. Similar results have been obtained for human primordial follicles. Two proteins of oocyte origin, GDF9 and BMP15, play an essential role in granulosa cell proliferation beyond the primary stage. It can be speculated that the regulation of follicle growth initiation is a two-phase event: the first phase depends on locally produced inhibitory and stimulatory factor(s); in the second phase the follicle become an autonomous unit, whose further development depends on oocyte-produced GDF9/BMP15 and the kit ligand derived from granulosa cells.