Activin signals via SMAD2/3 between germ and somatic cells in the human fetal ovary and regulates kit ligand expression

Ovarian germ cell survival is dependent upon the formation of primordial follicles, which occurs during fetal life in the human. Activin contributes to germ cell proliferation and survival at this time. SMADs2 and 3 are central elements in the activin signalling pathway and thus indicate sites of activin action. We have investigated the expression and localisation of SMADs2 and 3 in the fetal ovary between 14 and 20 weeks gestation, i.e. preceding and during primordial follicle formation. SMAD3 mRNA expression increased 1.9 fold (P = 0.02). SMAD2 and 3 proteins were localised by immunofluorescence to the nuclei of three distinct populations of somatic cells: (a) stromal cells between clusters of germ cells; (b) some somatic cells intermingled with activin βA-expressing germ cells; (c) pre-granulosa cells surrounding primordial follicles. Germ cells did not express SMAD2 or 3. Activin A increased and follistatin decreased phosphorylation of SMAD2/3 in vitro, and activin increased SMAD2 and decreased KITLG mRNA expression. It therefore appears that somatic cells are the targets for activin signalling in the developing ovary. The effects of activin on germ cells are indirect and include mediation by the kit ligand/c-Kit pathway, rather than being an autocrine germ cell effect.     

HDAC6 regulates primordial follicle activation through mTOR signaling pathway

Primordial follicle pool established perinatally is a non-renewable resource which determines the female fecundity in mammals. While the majority of primordial follicles in the primordial follicle pool maintain dormant state, only a few of them are activated into growing follicles in adults in each cycle. Excessive activation of the primordial follicles accelerates follicle pool consumption and leads to premature ovarian failure. Although previous studies including ours have emphasized the importance of keeping the balance between primordial follicle activation and dormancy via molecules within the primordial follicles, such as TGF-β, E-Cadherin, mTOR, and AKT through different mechanisms, the homeostasis regulatory mechanisms of primordial follicle activation remain unclear. Here, we reported that HDAC6 acts as a key negative regulator of mTOR in dormant primordial follicles. In the cytoplasm of both oocytes and granulosa cells of primordial follicles, HDAC6 expressed strong, however in those activated primordial follicles, its expression level is relatively weaker. Inhibition or knockdown of HDAC6 significantly promoted the activation of limited primordial follicles while the size of follicle pool was not affected profoundly in vitro. Importantly, the expression level of mTOR in the follicle and the activity of PI3K in the oocyte of the follicle were simultaneously up-regulated after inhibiting of HDAC6. The up-regulated mTOR leads to not only the growth and differentiation of primordial follicles granulosa cells (pfGCs) into granulosa cells (GCs), but the increased secretion of KITL in these somatic cells. As a result, inhibition of HDAC6 awaked the dormant primordial follicles of mice in vitro. In conclusion, HDAC6 may play an indispensable role in balancing the maintenance and activation of primordial follicles through mTOR signaling in mice. These findings shed new lights on uncovering the epigenetic factors involved physiology of sustaining female reproduction.Subject terms: TOR signalling, Cell proliferation, Endocrine reproductive disorders     

Transforming Growth Factor-β Signaling Participates in the Maintenance of the Primordial Follicle Pool in the Mouse Ovary

Physiologically, only a few primordial follicles are activated to enter the growing follicle pool each wave. Recent studies in knock-out mice show that early follicular activation depends on signaling from the tuberous sclerosis complex, the mammalian target of rapamycin complex 1 (mTORC1), phosphatase and tensin homolog deleted on chromosome 10, and phosphatidylinositol 3-kinase (PI3K) pathways. However, the manner in which these pathways are normally regulated, and whether or not TGF-β acts on them are poorly understood. So, this study aims to identify whether or not TGF-β acts on the process. Ovary organ culture experiments showed that the culture of 18.5 days post-coitus (dpc) ovaries with TGF-β1 reduced the total population of oocytes and activated follicles, accelerated oocyte growth was observed in ovaries treated with TGF-βR1 inhibitor 2-(5-chloro-2-fluorophenyl)pteridin-4-yl]pyridin-4-yl-amine (SD208) compared with control ovaries, the down-regulation of TGF-βR1 gene expression also activated early primordial follicle oocyte growth. We further showed that there was dramatically more proliferation of granulosa cells in SD208-treated ovaries and less proliferation in TGF-β1-treated ovaries. Western blot and morphological analyses indicated that TGF-β signaling manipulated primordial follicle growth through tuberous sclerosis complex/mTORC1 signaling in oocytes, and the mTORC1-specific inhibitor rapamycin could partially reverse the stimulated effect of SD208 on the oocyte growth and decreased the numbers of growing follicles. In conclusion, our results suggest that TGF-β signaling plays an important physiological role in the maintenance of the dormant pool of primordial follicles, which functions through activation of p70 S6 kinase 1 (S6K1)/ribosomal protein S6 (rpS6) signaling in mouse ovaries.     

WNT4/β-Catenin Pathway Maintains Female Germ Cell Survival by Inhibiting Activin βB in the Mouse Fetal Ovary

Female germ cells are essential for organogenesis of the ovary; without them, ovarian follicles do not form and functional and structural characteristics of the ovary are lost. We and others showed previously that when either Wnt4 or β-catenin was inactivated in the fetal ovary, female germ cells underwent degeneration. In this study, we set out to understand whether these two factors belong to the same pathway and how they maintain female germ cell survival. We found that activation of β-catenin in somatic cells in the Wnt4 knockout ovary restored germ cell numbers, placing β-catenin downstream of WNT4. In the absence of Wnt4 or β-catenin, female germ cells entered meiosis properly; however, they underwent apoptosis afterwards. Activin βB (Inhbb), a subunit of activins, was upregulated in the Wnt4 and β-catenin knockout ovaries, suggesting that Inhbb could be the cause for the loss of female germ cells, which are positive for activin receptors. Indeed, removal of Inhbb in the Wnt4 knockout ovaries prevented female germ cells from undergoing degeneration. We conclude that WNT4 maintains female germ cell survival by inhibiting Inhbb expression via β-catenin in the somatic cells. Maintenance of female germ cells hinge upon a delicate balance between positive (WNT4 and β-catenin) and negative (activin βB) regulators derived from the somatic cells in the fetal ovary.     

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime’s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.     

Expression of activin subunits and receptors in the developing human ovary: activin A promotes germ cell survival and proliferation before primordial follicle formation

The formation of the essential functional unit of the ovary, the primordial follicle, occurs during fetal life in humans. Factors regulating oogonial proliferation and interaction with somatic cells before primordial follicle formation are largely unknown. We have investigated the expression, localisation and functional effects of activin and its receptors in the human fetal ovary at 14-21 weeks gestation. Expression of mRNA for the activin betaA and betaB subunits and the activin receptors ActRIIA and ActRIIB was demonstrated by RT-PCR. Expression of betaA mRNA increased 2-fold across the gestational range examined. Activin subunits and receptors were localised by immunohistochemistry. The betaA subunit was expressed by oogonia, and the betaB subunit and activin receptors were expressed by both oogonia and somatic cells. BetaA expression was increased in larger oogonia at later gestations, but was low in oocytes within newly formed primordial follicles. Treatment of ovary fragments with activin A in vitro increased both the number of oogonia present and oogonial proliferation, as detected by bromodeoxyuridine (BrdU) incorporation. These data indicate that activin may be involved in the autocrine and paracrine regulation of germ cell proliferation in the human ovary during the crucial period of development leading up to primordial follicle formation.     

Growth and differentiation factor-9 stimulates progression of early primary but not primordial rat ovarian follicle development

The ovary contains a pool of primordial follicles containing oocytes arrested in meiosis that are the source of developing follicles for the female. Growth and differentiation factor-9 (GDF-9) is a member of the transforming growth factor beta superfamily of growth factors, and follicles of GDF-9 knockout mice arrest in the primary stage of development. The effect of GDF-9 treatment on the primordial to primary follicle transition and on subsequent follicle progression was examined using a rat ovary organ culture system. Ovaries from 4-day-old rats were cultured under serum-free conditions in the absence or presence of growth factors. GDF-9 treatment caused a decrease in the proportion of stage 1 early primary follicles and a concomitant increase in the proportion of stage 2 mature primary follicles. GDF-9 did not effect primordial follicles or stage 0 to stage 1 follicle transition. GDF-9 also did not influence stage 3 or 4 secondary follicle numbers. Isolated antral follicle granulosa and theca cell cultures were used to analyze the actions of GDF-9. GDF-9 treatment did not directly influence either granulosa or theca cell proliferation. The ability of GDF-9 to influence the expression of another growth factor was examined. GDF-9 treatment increased kit ligand (KL) mRNA expression in bovine granulosa cells after 2 days of culture. Ovaries from 4-day-old rats were also cultured with or without GDF-9 treatment, and total ovary expression of KL mRNA was increased by GDF-9. In summary, GDF-9 was found to promote the progression of early primary follicle development but did not influence primordial follicle development. The actions of GDF-9 on specific stages of follicle development may in part be mediated through altering the expression of KL.     

KIT signaling regulates primordial follicle formation in the neonatal mouse ovary

The pool of primordial follicles determines the reproductive lifespan of the mammalian female, and its establishment is highly dependent upon proper oocyte cyst breakdown and regulation of germ cell numbers. The mechanisms controlling these processes remain a mystery. We hypothesized that KIT signaling might play a role in perinatal oocyte cyst breakdown, determination of oocyte numbers and the assembly of primordial follicles. We began by examining the expression of both KIT and KIT ligand in fetal and neonatal ovaries. KIT was expressed only in oocytes during cyst breakdown, but KIT ligand was present in both oocytes and somatic cells as primordial follicles formed. To test whether KIT signaling plays a role in cyst breakdown and primordial follicle formation, we used ovary organ culture to inhibit and activate KIT signaling during the time when these processes occur in the ovary. We found that when KIT was inhibited, there was a reduction in cyst breakdown and an increase in oocyte numbers. Subsequent studies using TUNEL analysis showed that when KIT was inhibited, cell death was reduced. Conversely, when KIT was activated, cyst breakdown was promoted and oocyte numbers decreased. Using Western blotting, we found increased levels of phosphorylated MAP Kinase when KIT ligand was added to culture. Taken together, these results demonstrate a role for KIT signaling in perinatal oocyte cyst breakdown that may be mediated by MAP Kinase downstream of KIT.     

Primordial follicle activation and follicular development in the juvenile rabbit ovary

Of all the stages of mammalian folliculogenesis, the primordial to primary follicle transition is the least understood. In order to gain new insights into this process, we have conducted a comprehensive morphological, morphometric and molecular study of ovarian organisation and early follicle development in the rabbit. The structure of ovaries collected from rabbits aged from 2–12 weeks (a period encompassing primordial follicle formation, activation and the first wave of folliculogenesis in this species) has been analysed by light microscopy and the follicles present have been measured and scored for their developmental stage. To establish useful molecular markers of activation, we have further classified follicles according to their expression of the proliferative marker, proliferating cell nuclear antigen, and the zona pellucida protein, ZPB. The activation of primordial follicles is initiated immediately following their formation in the rabbit ovary and is characterised by oocyte growth, granulosa cell morphogenesis and increased granulosa cell mitosis. Enhanced ZPB protein expression at the oolemma is also associated with follicle activation and development. Few primordial follicles in the juvenile rabbit ovary are lost by atresia, as assessed by the TUNEL assay. The appearance of apoptotic granulosa cells is however coincident with the development of antral follicles. This study thus describes the temporal and spatial regulation of early follicular development in the post-natal rabbit ovary and, for the first time, shows that the primordial to primary transition in the juvenile rabbit is a highly ordered process occurring within quantifiable parameters.K.J.H. was supported by the Pest Animal Control CRC and Post Graduate scholarships from the Australian National University.     

Tumor necrosis factor (TNF) receptor type 2 is an important mediator of TNF alpha function in the mouse ovary

It is believed that a finite pool of primordial follicles is established during embryonic and neonatal life. At birth, the mouse ovary consists of clusters of interconnected oocytes surrounded by pregranulosa cells. Shortly after birth these structures, termed germ cell cysts or nests (GCN), break down to facilitate primordial follicle formation. Tumor necrosis factor alpha (TNF) is a widely expressed protein with myriad functions. TNF is expressed in the ovary and may regulate GCN breakdown in rats. We investigated whether it participates in GCN breakdown and follicle formation in mice by using an in vitro ovary culture system as well as mutant animal models. We found that TNF and both receptors (TNFRSF1A and TNFRSF1B) are expressed in neonatal mouse ovaries and that TNF promotes oocyte death in neonatal ovaries in vitro. However, deletion of either receptor did not affect follicle endowment, suggesting that TNF does not regulate GCN breakdown in vivo. Tnfrsf1b deletion led to an apparent acceleration of follicular growth and a concomitant expansion of the primordial follicle population. This expansion of the primordial follicle population does not appear to be due to decreased primordial follicle atresia, although this cannot be ruled out completely. This study demonstrates that mouse oocytes express both TNF receptors and are sensitive to TNF-induced death. Additionally, TNFRSF1B is demonstrated to be an important mediator of TNF function in the mouse ovary and an important regulator of folliculogenesis.     

Populations of granulosa cells in small follicles of the sheep ovary.

The aim of this study in sheep ovaries was to determine the total number of granulosa cells in primordial follicles and at subsequent stages of growth to early antrum formation. The second aim was to examine the interrelationships among the total number of granulosa cells in the follicles, the number of granulosa cells in the section through the oocyte nucleolus, and the diameter of the oocyte. A third aim was to examine whether proliferating cell nuclear antigen labelling occurred in flattened granulosa cells in primordial follicles or was confined to follicles containing cuboidal granulosa cells. The follicles were classified using the section through the oocyte nucleolus by the configuration of granulosa cells around the oocyte as type 1 (primordial), type 1a (transitory), type 2 (primary), type 3 (small preantral), type 4 (large preantral), and type 5 (small antral). In type 1 follicles, the number of granulosa cells and oocyte diameter were highly variable in both fetal and adult ovaries. Each type of follicle was significantly different from the others (all P < 0.05) with respect to oocyte diameter, number of granulosa cells in the section through the oocyte nucleolus and total number of granulosa cells. Follicles classified as type 2, 3, 4 or 5 each corresponded to two doublings of the total granulosa cell population. The relationships between oocyte diameter and the number of granulosa cells (that is, in the section through the oocyte nucleous or total population per follicle) could all be described by the regression equation loge chi = a + b loge gamma with the correlation coefficients R always > 0.93. For each pair of variables the slopes (b) for each type of follicle were not different from the overall slope for all types of follicle pooled. Immunostaining for proliferating cell nuclear antigen was observed in granulosa cells in type 1 follicles, as well as in the other types of follicle. These findings indicate that 'flattened' granulosa cells in type 1 follicles express an essential nuclear protein involved in cell proliferation before assuming the cuboidal shape. Thus, when considering factors that regulate specific phases of early follicular growth, it is important to consider: (i) the follicle classification system used; (ii) the animal model studied; (iii) whether type 1 follicles are all quiescent; and (iv) the likelihood that each follicle type represents more than one doubling of the population of granulosa cells.     

Primordial follicle assembly was regulated by notch signaling pathway in the mice

Notch signaling pathway, a highly conserved cell signaling system, exists in most multicellular organisms. The objective of this study was to examine Notch signaling pathway in germ cell cyst breakdown and primordial follicle formation. The receptor and ligand genes of Notch pathway (Notch1, Notch2, Jagged1, Jagged2 and Hes1) were extremely down-regulated after newborn mouse ovaries were cultured then exposed to DAPT or L-685,458 in vitro (P < 0.01). Since DAPT or L-685,548 inhibits Notch signaling pathway, the expression of protein LHX8 and NOBOX was significantly reduced during the formation of the primordial follicles. Down-regulated mRNA expression of specific genes including Lhx8, Figla, Sohlh2 and Nobox, were also observed. The percentages of female germ cells in germ cell cysts and primordial follicles were counted after culture of newborn ovaries for 3 days in vitro. The result showed female germ cells in cysts was remarkably up-regulated while as the oocytes in primordial follicles was significantly down-regulated (P < 0.05). In conclusion, Notch signaling pathway may regulate the formation of primordial follicle in mice.     

The ontogeny of immunoreactive, endogenous FSH and LH in the rat ovary during early folliculogenesis

Rabbit antisera to rat pituitary follicle-stimulating hormone (FSH) and to rat luteinizing hormone (LH) were used, in an immunocytochemical probe, to determine the ontogeny and distribution of immunoreactive, endogenous, intraovarian FSH and LH in immature rats. Ovaries from rats 4, 8, 12, and 21 days of age were studied. Both gonadotrophins were first immunodetectable on Day 8. In reactive primordial follicles, LH was restricted to the cytoplasm and nuclei of the surrounding follicle cells. In those follicles possessing both squamous and cuboidal follicle cells, i.e., transitional between primordial and primary, LH was found in both the cytoplasm and nuclei of both follicle cell types. In primary follicles, LH was no longer present in granulosa cells but was concentrated in germ cell cytoplasm. In contrast, in primordial follicles, FSH was restricted to the germ cell but was present in both the oocyte cytoplasm and germinal vesicle. In transitional and primary follicles, FSH remained within the oocyte cytoplasm and germinal vesicle but also became detectable within the cytoplasm and nuclei of granulosa cells. These findings raise some important new questions regarding the role(s) of the gonadotrophins in early follicular development.     

Expression of ErbB3-Binding Protein-1 (EBP1) during Primordial Follicle Formation: Role of Estradiol-17?

The formation of primordial follicles involves the interaction between the oocytes and surrounding somatic cells, which differentiate into granulosa cells. Estradiol-17ß (E) promotes primordial follicle formation in vivo and in vitro; however, the underlying mechanisms are poorly understood. The expression of an ERBB3-binding protein 1 (EBP1) is downregulated in 8-day old hamster ovaries concurrent with the increase in serum estradiol levels and the formation of primordial follicles. The objectives of the present study were to determine the spatio-temporal expression and putative E regulation of EBP1 in ovarian cells during perinatal development with respect to primordial follicle formation. Hamster EBP1 nucleic acid and amino acid sequences were more than 93% and 98% similar, respectively, to those of mouse and human, and contained nucleolar localization signal, RNA-binding domain and several phosphorylation sites. EBP1 protein was present in somatic cells and oocytes from E15, and declined in oocytes by P1 and in somatic cells by P5. Thereafter, EBP1 expression increased through P7 with a transient decline on P8 primarily in interstitial cells. EBP1 mRNA levels mirrored protein expression pattern. E treatment on P1 and P4 upregulated EBP1 expression by P8 whereas E treatment on P4 downregulated it by 72 h suggesting a compensatory upregulation due to E pretreatment. Treatment with an FSH-antiserum, which suppressed primordial follicle formation, prevented the decline in EBP1 levels, and the effect was reversed by E treatment. Therefore, the results provide the first evidence that EBP1 may play an important role in mediating the effect of E in the differentiation of somatic cells into granulosa cells during primordial follicle formation.     

Stem cell factor and c-kit gene expression and protein localization in the sheep ovary during fetal development

The aim of this study was to investigate stem cell factor and c-kit gene expression and protein localization in the mesonephros and ovary of sheep fetuses at different days of gestation, using RNA in situ hybridization and immunohistochemical procedures. At days 24 and 26 of gestation, stem cell factor mRNA and protein were present in cells throughout the developing gonad and mesonephros. From day 28 to day 40 of gestation, stem cell factor mRNA and protein became increasingly localized to the cortical region of the ovary, where most germ cells were present as actively proliferating oogonia. From day 40 to day 90 of gestation, stem cell factor mRNA and protein localization were confined mainly to the ovarian cortex. Moreover, within the cortical region, stem cell factor mRNA was low or absent where follicles were first forming and highest in the outer ovarian cortex, where germ cells were undergoing mitosis or the early stages of meiosis. In contrast, stem cell factor protein was present in newly forming follicles, as well as in mitotic and meiotic germ cells, which is consistent with the presence of both membrane-bound and soluble forms of this ligand. However, by day 90 of gestation, both stem cell factor mRNA and protein were observed in the granulosa cells of most (> 90%) primordial follicles. C-kit mRNA and protein were observed from day 24 of gestation in both germ cells and somatic cells but, with increasing gestational age, preferentially in germ cells (for example, pre-meiotic germ cells and both isolated oocytes and follicle-enclosed oocytes). C-kit protein, but not mRNA, was also observed in germ cells that were undergoing meiosis. The results indicate that the cells containing stem cell factor mRNA within the ovary up to day 90 of gestation originated from the gonadal blastema and from cells that migrated from the mesonephros before day 28 of gestation. Since stem cell factor mRNA was absent in both mesonephric cells migrating after day 28 of gestation and in regions where follicles were first forming, it is suggested that these later migrating mesonephric cells are the progenitors of the granulosa cells in the first forming follicles. In conclusion, during follicle formation, c-kit mRNA is localized to germ cells whereas c-kit, together with stem cell factor protein, is localized to both germ cells and somatic cells, consistent with the hypothesis that the presence of this receptor-ligand pair is essential to prevent apoptosis.     

Consequences of fetal irradiation on follicle histogenesis and early follicle development in rat ovaries

Follicle histogenesis, in which follicles arise from fragmenting ovigerous cords, is a poorly understood mechanism that is strictly dependent upon the presence of germ cells. Our previous studies have shown that severely germ cell-depleted rat ovaries after fetal gamma-irradiation display modifications of follicular endowment and dynamics during the immature period. The primordial follicle stock was absent and the follicles with primary appearance remained quiescent longer than in control ovaries during the neonatal period. The aim of the present work was to analyze the initial steps of follicle histogenesis, and to investigate the etiology of the alterations observed in the development of irradiated ovaries. Just after birth, we observed, in addition to sterile ovigerous cords, the emergence of the first follicles which exhibited several abnormal features as compared to those of control ovaries. Most of the follicles appeared as primary follicles, as they were composed of a layer of cuboidal-shaped granulosa cells surrounding an enlarged oocyte. Interestingly, the granulosa cells of these primary-like follicles did not proliferate and did not express the genes for anti-Müllerian hormone (Amh) or bone morphogenetic protein receptor type II (Bmpr2), both of which are normally expressed from the primary stage onwards. In contrast, the oocytes strongly expressed the gene for growth and differentiation factor 9 (Gdf9), which is normally upregulated from the primary follicle stage onwards, which suggests an uncoupling of granulosa cell development from oocyte development. In addition, irradiated ovaries displayed a higher frequency of follicles that contained 2 or 3 oocytes, which are also referred to as multi-oocyte follicles (MOFs). Examination at the time of follicle histogenesis indicated that MOFs arise from incomplete ovigerous cord breakdown. Taken together, the results of this study indicate that severe perturbations of follicular histogenesis take place following irradiation and massive germ cell depletion during fetal life. In addition to the classically described sterile cords, we have pointed out the differentiation of MOFs and primary-like quiescent follicles, which finally evolve into growing follicles and participate in ovarian function. We propose that these phenotypes are closely correlated to the proportion of granulosa cells to oocytes at the time of neonatal follicle histogenesis.