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.     

Activation of follicle development: the primordial follicle

Investigations of primordial follicle formation and growth are fundamental to our understanding of female gamete production. In all mammalian females the full complement of oocytes is established during fetal development. This store of primordial follicles is not renewable and serves the entire reproductive life span of the adult. The correct programming of fetal ovarian development and the number of primordial follicles formed will therefore limit the fecundity of the ovary. Primordial follicles are characterized by the presence of a single oocyte surrounded by a varying number of pregranulosa cells. The relatively small size, undifferentiated status and large numbers of primordial follicles make them prime candidates for use in basic and applied research in animal production, gene transfer and cloning. Furthermore, the development of cell culture systems that use primordial follicles as a source of oocytes for in vitro growth and maturation will enable us to maximize the potential of high genetic merit females and to shorten generation intervals. Despite these possibilities, primordial follicles are the least understood of all stages of follicle development. The factor(s) responsible for maintaining the primordial pool or, conversely, for activating primordial follicle growth remain elusive.     

Pathways coordinating oocyte attrition and abundance during mammalian ovarian reserve establishment

The mammalian ovarian reserve is comprised of a finite pool of primordial follicles, representing the lifetime reproductive capacity of females. In most mammals, the reserve is produced during embryonic and early postnatal development with oocyte numbers peaking during mid‐to‐late gestation, and then experiencing a dramatic decline continuing until shortly after birth. Oocytes remaining after the bulk of this attrition are subsequently surrounded by a layer of somatic pre‐granulosa cells with these units then referred to as “primordial follicles.” The complex and varied cell death mechanisms intrinsic to this process are not only characteristic of, but also essential for, the proper formation of this pool of follicles, and as a result must be immaculately balanced to ensure long‐term fertility and reproductive health. Too few follicles can lead to Primary Ovarian Insufficiency, resulting in fertility loss and other features of aging, such as an overall shorter lifespan. On the other hand, whereas an excess of follicles might extend reproductive lifespan, this might also be the underlying etiology of other ovarian pathologies. The last decade, in particular, has vastly expanded our understanding of oocyte attrition and determinants of ovarian reserve abundance. By continuing to decipher the intricacies underlying the cell death processes and development of the initial primordial follicle pool, we may be in a much better position to understand idiopathic cases of premature follicle depletion and improve ovarian health in reproductive‐age women.     

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.     

Follistatin288 Regulates Germ Cell Cyst Breakdown and Primordial Follicle Assembly in the Mouse Ovary

In mammals, the primordial follicle pool represents the entire reproductive potential of a female. The transforming growth factor-β (TGF-β) family member activin (ACT) contributes to folliculogenesis, although the exact mechanism is not known. The role of FST288, the strongest ACT-neutralizing isoform of follistatin (FST), during cyst breakdown and primordial follicle formation in the fetal mice ovary was assessed using an in vitro culture system. FST was continuously expressed in the oocytes as well as the cuboidal granulosa cells of growing follicles in perinatal mouse ovaries. Treatment with FST288 delayed germ cell nest breakdown, particularly near the periphery of the ovary, and dramatically decreased the percentage of primordial follicles. In addition, there was a dramatic decrease in proliferation of granulosa cells and somatic cell expression of Notch signaling was impaired. In conclusion, FST288 impacts germ cell nest breakdown and primordial follicle assembly by inhibiting somatic cell proliferation.     

Fate of the initial follicle pool: empirical and mathematical evidence supporting its sufficiency for adult fertility

The importance of the initial follicle pool in fertility in female adult mammals has recently been debated. Utilizing a mathematical model of the dynamics of follicle progression (primordial to primary to secondary), we examined whether the initial follicle pool is sufficient for adult fertility through reproductive senescence in CD1 mice. Follicles in each stage were counted from postnatal day 6 through 12 months and data were fit to a series of first-order differential equations representing two mechanisms: an initial pool of primordial follicles as the only follicle source (fixed pool model), or an initial primordial follicle pool supplemented by germline stem cells (stem cell model). The fixed pool model fit the experimental data, accurately representing the maximum observed primary follicle number reached by 4-6 months of age. Although no germline stem cells could be identified by SSEA-1 immunostaining, the stem cell model was tested using a range of de novo primordial follicle production rates. The stem cell model failed to describe the observed decreases in follicles over time and did not parallel the accumulation and subsequent reduction in primary follicles during the early fertile lifespan of the mouse. Our results agree with established dogma that the initial endowment of ovarian follicles is not supplemented by an appreciable number of stem cells; rather, it is sufficient to ensure the fertility needs of the adult mouse.     

Postnatal regulation of germ cells by activin: the establishment of the initial follicle pool

Mammalian females enter puberty with follicular reserves that exceed the number needed for ovulation during a single lifetime. Follicular depletion occurs throughout reproductive life and ends in menopause, or reproductive senescence, when the follicle pool is exhausted. The mechanisms regulating the production of a species-specific initial follicle pool are not well understood. However, the establishment of a follicular reserve is critical to defining the length of reproductive cyclicity. Here we show that activin A (rh-ActA), a known regulator of follicle formation and growth in vitro, increased the number of postnatal mouse primordial follicles by 30% when administered to neonatal animals during the time of germline cyst breakdown and follicle assembly. This expansion in the initial follicle pool was characterized by a significant increase in both germ cell and granulosa cell proliferation. However, the excess follicles formed shortly after birth did not persist into puberty and both adult rh-ActA- and vehicle-treated animals demonstrated normal fertility. A follicle atresia kinetic constant (k(A)) was modeled for the two groups of animals, and consistent with the empirical data, the k(A) for rh-ActA-treated was twice that of vehicle-treated animals. Kinetic constants for follicle formation, follicle loss and follicle expansion from birth to postnatal day 19 were also derived for vehicle and rh-ActA treatment conditions. Importantly, introduction of exogenous rh-ActA revealed an intrinsic ovarian quorum sensing mechanism that controls the number of follicles available at puberty. We propose that there is an optimal number of oocytes present at puberty, and when the follicle number is exceeded, it occurs at the expense of oocyte quality. The proposed mechanism provides a means by which the ovary eliminates excess follicles containing oocytes of poor quality prior to puberty, thus maintaining fertility in the face of abnormal hormonal stimuli in the prepubertal period.     

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.     

Autophagy participates in cyst breakdown and primordial folliculogenesis by reducing reactive oxygen species levels in perinatal mouse ovaries

The reserve of primordial follicles, which serves all oocytes for the female reproductive lifespan, is established a few days after birth in mice. During this process, more than half of the oocytes are primarily eliminated by apoptosis. Autophagy, the conserved intracellular process maintaining cellular homeostasis, serves as a protective mechanism for oocyte survival. In the current study, we speculate a new role for autophagy during primordial folliculogenesis. Active autophagy was observed in perinatal ovaries from 16.5 days post coitus to 3 days post parturition. The inhibition of autophagy by 3-methyladenine (3-MA) increased the number of cyst oocytes and delayed follicle formation in vivo and in organ cultures. Furthermore, the reactive oxygen species (ROS) level was elevated in ovaries treated with 3-MA, while N-acetylcysteine, an oxidant, alleviated the inhibitory effect of 3-MA on primordial folliculogenesis. Additionally, the expression of growth differentiation factor 9 and transforming growth factor β1, which regulates follicle activation, was decreased after 3-MA treatment. These data suggest that the physiological level of autophagy in perinatal ovaries regulates germ cell cyst breakdown and primordial follicle assembly by ROS clearance and exerts extensive effects on further follicular development.     

Effect of bcl-2 on the primordial follicle endowment in the mouse ovary

Little is known about the embryonic factors that regulate the size of the primordial follicle endowment at birth. A few studies suggest that members of the B-cell lymphoma/leukemia-2 (bcl-2) family of protooncogenes may be important determinants. Thus, the purpose of this study was to test whether bcl-2 regulates the size of the primordial follicle pool at birth. To test this hypothesis, three lines of transgenic mice (c-kit/bcl-2 mice) were generated that overexpress human bcl-2 in an effort to reduce prenatal oocyte loss. The overexpression was targeted to the ovary and appropriate embryonic time period with the use of a 4.8-kilobase c-kit promoter. This promoter provided two to three times more expression of bcl-2 in the ovaries with minimal or no overexpression in most nongonadal tissues. On Postnatal Days 8-60, ovaries were collected from homozygous c-kit/bcl-2 and nontransgenic littermates (controls) and processed for histological evaluation of follicle numbers. All lines of c-kit/bcl-2 mice were born with significantly more primordial follicles than control mice (P < or = 0.05). By Postnatal Days 30-60, however, there were no significant differences in follicle numbers between c-kit/bcl-2 and control mice. These results indicate that bcl-2 overexpression increases the number of primordial follicles at birth, but that the surfeit of primordial follicles is not maintained in postnatal life. These data suggest that it is possible that the ovary may contain a census mechanism by which excess numbers of primordial follicles at birth are detected and removed from the ovary by adulthood.     

Lanosterol metabolic product(s) is involved in primordial folliculogenesis and establishment of primordial follicle pool in mouse fetal ovary

The primordial follicles present in neonatal ovary represent the fecundity of a female throughout her reproductive life. Germ cell meiosis and apoptosis are two important events during primordial folliculogenesis. In this study, through focusing on the cytochrome P450 lanosterol 14 alphademethylase (CYP51) and its lanosterol metabolic product(s), we explored the possible regulatory mechanism of the initiation of germ cell meiosis and primordial follicle formation. The expression of CYP51 could be detected in both oocytes and granulosa cells during primordial folliculogenesis by immunochemistry. RS21745, which leads to the reduction of lanosterol metabolic product(s) level, inhibited the primordial follicle formation in a dose-dependent manner, and thus postpone the establishment of the primordial follicle pool when the mouse fetal ovaries were cultured in serum-free medium. In contrast, the number of primordial follicle increased significantly with the accumulation of the lanosterol metabolic products caused by 0.025, 0.0625, and 0.125 microM AY9944-A-7 supplements. AY9944-A-7 also up-regulated the expression of meiotic diplotene stage marker gene msy2 and primordial follicle formation regulatory gene fig-alpha. Furthermore, AY9944-A-7 decreased the expression of apoptosis gene bax and significantly prevented oocyte apoptosis from 15.37 +/- 1.97% to 3.68 +/- 0.27% (P < 0.01) in neonatal ovary in vitro. In conclusion, our results indicate that lanosterol metabolic product(s) is involved in the primordial folliculogenesis by regulating the oocyte meiosis and apoptosis.     

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     

Low expression of SEMA6C accelerates the primordial follicle activation in the neonatal mouse ovary

The primordial follicle assembly, activation and the subsequent development are critical processes for female reproduction. A limited number of primordial follicles are activated to enter the growing follicle pool each wave, and the primordial follicle pool progressively diminishes over a woman's life‐time. The number of remaining primordial follicles represents the ovarian reserve. Identification and functional investigation of the factors involved in follicular initial recruitment will be of great significance to the understanding of the female reproduction process and ovarian ageing. In this study, we aimed to study whether and how semaphorin 6C (Sema6c) regulated the primordial follicle activation in the neonatal mouse ovary. The attenuation of SEMA6C expression by SiRNA accelerated the primordial follicle activation in the in vitro ovary culture system. PI3K‐AKT‐rpS6 pathway was activated when SEMA6C expression was down‐regulated. And the LY294002 could reverse the effect of low SEMA6C expression on primordial follicle activation. Our findings revealed that Sema6c was involved in the activation of primordial follicles, and the down‐regulation of SEMA6C led to massive primordial follicle activation by interacting with the PI3K‐AKT‐rpS6 pathway, which might also provide valuable information for understanding premature ovarian failure and ovarian ageing.     

Activation of bovine and baboon primordial follicles in vitro

Mammalian ovaries contain a large pool of non-growing, primordial follicles. The ability to initiate growth of this pool of resting follicles in vitro and to maintain follicular growth to a stage when the oocyte could be matured and fertilized would increase the reproductive potential of valuable domestic animals, endangered species and infertile women. This paper summarizes our progress to date in activating primordial follicles of cattle and baboons. Pieces of ovarian cortex, rich in primordial follicles, were obtained from fetal bovine and baboon ovaries during late gestation. Pieces were maintained in organ culture in serum-free medium containing ITS+ (insulin-transferrin-selenium-linoleic acid-BSA) for up to 20 days and at various times during culture some pieces were fixed for histological morphometry. As early as 2 days of culture, the number of primordial follicles had decreased by 88% or 55%, whereas the number of primary follicles had increased 2.5- or 5-fold, compared to tissue freshly isolated from bovine or baboon ovaries, respectively (P < 0.01). In baboon cortical pieces a small number of secondary follicles developed during a 20-day culture period. The development of primary and secondary follicles was accompanied by an increase in diameter of both the granulosa cell layer and the oocyte. The addition of FSH (1, 10, or 100 ng/ml) had no effect on the development of follicles in bovine cortical pieces after 7 or 14 days of culture, relative to control cultures without FSH. These results show that a high percentage of primordial follicles from cattle and baboons can be activated to grow in serum-free medium in the absence of gonadotropins. Conditions that will support further growth in vitro of follicles from these species remain to be elucidated. The culture system we have developed could be used to develop such conditions and to explore factors that regulate the movement of primordial follicles into the pool of growing follicles.     

Tumor necrosis factor alpha enhances oocyte/follicle apoptosis in the neonatal rat ovary.

Tumor necrosis factor alpha (TNFalpha) is a multifunctional cytokine present in oocytes and macrophages in the neonatal rat ovary. The presence of both TNFalpha and its receptors in the neonatal rat ovary suggests a potential role for it in follicle assembly or oocyte atresia. Previous studies have provided support for effects of TNFalpha on isolated granulosa and theca cells and intact follicles; however, to our knowledge, this is the first study to investigate the effects of TNFalpha on the earliest stages of follicular development. Effects of TNFalpha on oocyte/follicle number and apoptosis were investigated using an ovarian organ-culture system that supported assembly of primordial follicles in vitro. Ovaries were collected on the day of birth and treated with TNFalpha (0, 0.1, 1.0, 10, or 50 ng/ml), a function-blocking TNFalpha antibody (5 microg/ml), or control immunoglobulin (Ig) G. At 1 ng/ml, TNFalpha decreased follicle and oocyte numbers during 3 days of culture, whereas higher (10 and 50 ng/ml) or lower (0.1 ng/ml) doses had no effect. Treatment with TNFalpha antibodies increased the number of oocytes and follicles compared to nonspecific IgG control. To determine whether the decreased oocyte/follicle numbers were due to an apoptotic effect of TNFalpha, apoptosis was examined by DNA laddering. At 1 ng/ml, TNFalpha increased apoptotic DNA laddering twofold, with no significant effect from lower or higher doses. The cells undergoing apoptosis, as determined by in situ end-labeling, were oocytes, interstitial cells, and granulosa cells. These findings suggest that TNFalpha may be involved in oocyte atresia that normally occurs during the perinatal period.     

Development of primordial and prenatal follicles from undifferentiated somatic cells and oocytes in the hamster prenatal ovary in vitro: effect of insulin

Fetal hamster ovaries were cultured for up to 16 days in the presence or absence of various dosages of insulin to evaluate the induction of folliculogenesis in vitro. In the absence of insulin, a few primordial follicle-like structures appeared by the 4th day, and distinct primary follicles (stage 1) appeared by the 12th day of culture. The organelles in the oocytes and adjacent granulosa cells developed along with follicular growth. Moreover, gap junctions between the oocyte and somatic cell plasma membrane also developed as early as 8 days in culture. In the presence of 0.2 microg/ml insulin, primary follicles developed after 8 days, and approximately 4% secondary follicles with 2-3 layers of granulosa cells appeared after 16 days of culture. However, higher dosages (> 0.2 microg/ml) of insulin retarded primary follicle formation and induced the formation of primordial follicles with larger oocytes. An increased number of larger oocytes with a few granulosa cells accumulated at the periphery of the ovary. The results indicate that although primordial and primary follicles can develop after 12 days in vitro in the absence of exogenous insulin, the latter is required for timely progression of follicular development through primary and secondary stages.     

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.     

Rapamycin preserves the follicle pool reserve and prolongs the ovarian lifespan of female rats via modulating mTOR activation and sirtuin expression

To maintain the normal length of female reproductive life, the majority of primordial follicles must be maintained in a quiescent state for later use. In this study, we aimed to study the effects of rapamycin on primordial follicle development and investigate the role of mTOR and sirtuin signaling. Rats were treated every other day with an intraperitoneal injection of rapamycin (5 mg/kg) or vehicle. After 10 weeks of treatment, ovaries were harvested for hematoxylin and eosin (HE) staining, and analysis by immunohistochemistry and Western blotting. HE staining showed that the number and percentage of primordial follicles in the rapamycin-treated group were twice the control group (P < 0.001). Immunohistochemical analysis showed that mTOR and phosphorylated-p70S6K were extensively expressed in surviving follicles with strong staining observed in the cytoplasm of the oocyte. Western blotting showed decreased expression of phosphorylated mTOR and phosphorylated p70S6K in the rapamycin-treated group, and increased the expression of both SIRT1 and SIRT6 compared to the control group (P < 0.05). Taken together, these results suggest that rapamycin may inhibit the transition from primordial to developing follicles and preserve the follicle pool reserve, thus extending the ovarian lifespan of female rats via the modulation of mTOR and sirtuin signalings.