Proliferating cell nuclear antigen (PCNA) regulates primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries

Primordial follicles, providing all the oocytes available to a female throughout her reproductive life, assemble in perinatal ovaries with individual oocytes surrounded by granulosa cells. In mammals including the mouse, most oocytes die by apoptosis during primordial follicle assembly, but factors that regulate oocyte death remain largely unknown. Proliferating cell nuclear antigen (PCNA), a key regulator in many essential cellular processes, was shown to be differentially expressed during these processes in mouse ovaries using 2D-PAGE and MALDI-TOF/TOF methodology. A V-shaped expression pattern of PCNA in both oocytes and somatic cells was observed during the development of fetal and neonatal mouse ovaries, decreasing from 13.5 to 18.5 dpc and increasing from 18.5 dpc to 5 dpp. This was closely correlated with the meiotic prophase I progression from pre-leptotene to pachytene and from pachytene to diplotene when primordial follicles started to assemble. Inhibition of the increase of PCNA expression by RNA interference in cultured 18.5 dpc mouse ovaries strikingly reduced the apoptosis of oocytes, accompanied by down-regulation of known pro-apoptotic genes, e.g. Bax, caspase-3, and TNFα and TNFR2, and up-regulation of Bcl-2, a known anti-apoptotic gene. Moreover, reduced expression of PCNA was observed to significantly increase primordial follicle assembly, but these primordial follicles contained fewer granulosa cells. Similar results were obtained after down-regulation by RNA interference of Ing1b, a PCNA-binding protein in the UV-induced apoptosis regulation. Thus, our results demonstrate that PCNA regulates primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries.     

Expression of GDF-9, BMP-15 and their receptors in mammalian ovary follicles

The synergetic process of folliculogenesis is mainly regulated by GDF-9 and BMP-15 as well as their receptors, such as BMPR2, TβR1 and BMPR1B. Expressions of these factors and the receptors are significant different among species. This study was designed to detect expression of GDF-9, BMP-15 and their receptors in mouse, porcine and human healthy follicles by immunohistochemistry. Three ages of human ovary were studied according to ovarian developmental schedule, i.e. gestational week (GW) 16, puberty (14 year-old) and adult (40 year-old). The results showed that both GDF-9 and BMP-15 were detectable in oocytes from primary follicles onward, besides, BMP-15 also presented in granulosa cells (GCs) and follicular follicle of mature follicles in mouse. However, they were maintained in oocytes and GCs from primordial to mature follicles in porcine except that GDF-9 was undetectable in GCs of mature follicles. For human ovary, GDF-9 presented in oocytes of primordial follicles in all samples, whereas BMP-15 was only observed in primordial follicle of adult ovary. Receptors, BMPR2, TβR1 and BMPR1B were found in oocytes and GCs of all follicles in mouse and porcine. In human, they were stained in oocytes from primordial follices but BMPR1B was not expressed in pubertal primordial follicles. Furthermore, we found that GDF-9, BMP-15 and three receptors distributed in adult corpus lutea. Collectively, our studies suggested that GDF-9, BMP-15 and their receptors might correlate with primordial follicular recruitment in pig and human. Positive expression of the receptors (BMPR2, TβR1 and BMPR1B)in primordial follicles of mouse ovaries indicated that these receptors might interact with others ligands besides GDF-9 and BMP-15 to regulate primordial follicular activity in mouse. Moreover, presence of GDF-9 in oocytes and BMP-15 in oocytes and GCs of mature follicles from mice and porcine elucidated coordinated roles of GDF-9 and BMP-15 in cumulus oophorus expansion. Additionally, expression of these factors in adult human corpus lutea suggested they play roles in corpus luteum activity.     

TAF4b promotes mouse primordial follicle assembly and oocyte survival

Primary ovarian insufficiency (POI) affects 1% of women under the age of 40 and is associated with premature ovarian follicle depletion. TAF4b deficiency in adult female mouse models results in hallmarks of POI including stereotyped gonadotropin alterations indicative of early menopause, poor oocyte quality, and infertility. However, the precise developmental mechanisms underlying these adult deficits remain unknown. Here we show that TAF4b is required for the initial establishment of the primordial follicle reserve at birth. Ovaries derived from TAF4b-deficient mice at birth exhibit delayed germ cell cyst breakdown and a significant increase in Activated Caspase 3 staining compared to control ovaries. Culturing neonatal TAF4b-deficient ovaries with the pan-caspase inhibitor ZVAD-FMK suppresses the excessive loss of these oocytes around the time of birth. These data reveal a novel TAF4b function in orchestrating the correct timing of germ cell cyst breakdown and establishment of the primordial follicle reserve during a critical window of development.     

KIT/KIT ligand in mammalian oogenesis and folliculogenesis: roles in rabbit and murine ovarian follicle activation and oocyte growth

In rodent ovaries Kit ligand (KITL) and its receptor KIT have diverse roles, including the promotion of primordial follicle activation, oocyte growth, and follicle survival. Studies were undertaken to determine whether KITL and KIT carry out similar activities in rabbits.KitlandKitmRNA and protein were localized to oocytes and granulosa cells, respectively, in the rabbit ovary. Ovarian cortical explants from juvenile rabbits and neonatal mouse ovaries were subsequently cultured with recombinant mouse KITL and/or KITL neutralizing antibody. Indices of follicle growth initiation were compared with controls and between treatment groups for each species. Recombinant mouse KITL had no stimulatory effect on primordial follicle recruitment in cultured rabbit ovarian explants. However, the mean diameter of oocytes from primordial, early primary, primary, and growing primary follicles increased significantly in recombinant mouse KITL-treated explants compared with untreated tissues. In contrast, recombinant mouse KITL promoted both primordial follicle activation and an increase in the diameter of oocytes from primordial and early primary follicles in the mouse, and these effects were inhibited by coculture with KITL-neutralizing antibody. Recombinant mouse KITL had no effect on follicle survival for either species. These data demonstrate that KITL promotes the growth of rabbit and mouse oocytes and stimulates primordial follicle activation in the mouse but not in the rabbit. We propose that the physiologic roles of KITL and KIT may differ between species, and this has important implications for the design of in vitro culture systems for folliculogenesis in mammals, including the human.     

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.     

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.     

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.     

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.     

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.     

Follicular expression of c-Kit/SCF and inhibin-alpha in mouse ovary during development.

The mechanism of development of the ovarian follicles has been largely unknown. We performed an immunohistochemical (IHC) study to determine the follicular expressions of c-kit, SCF, and inhibin-alpha at different developmental stages in mouse ovary. Ovaries were obtained from 14 and 16 days post coitum and 2, 7, and 21 days post partum (dpp) mice. IHC for c-kit, SCF, and inhibin-alpha was carried out. c-Kit and SCF were expressed on oogonia regardless of the developmental stage. Immunoreactive c-kit and SCF antigens were expressed on oocytes of primordial and primary follicles of neonate mouse ovaries. In 21 dpp mouse ovary, the expression of c-kit/SCF in oocytes gradually decreased as the follicles developed. c-Kit/SCF was expressed strongly in oocytes of preantral follicles and weakly in granulosa and thecal cells. Inhibin-alpha was mainly expressed on granulosa cells of preantral and early antral follicles of the 21 dpp mouse ovaries. These findings suggest that the IHC expression of c-kit/SCF proteins is specific in all developmental stages of ovarian follicles and is decreased after the follicle starts to grow. The expression of inhibin-alpha is negatively correlated with the expression of c-kit/SCF in the ovarian follicles in mice.     

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.     

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.     

TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary

Although it is well established that both follicular assembly and the initiation of follicle growth in the mammalian ovary occur independently of pituitary hormone support, the factors controlling these processes remain poorly understood. We now report that neurotrophins (NTs) signaling via TrkB receptors are required for the growth of newly formed follicles. Both neurotrophin-4/5 (NT-4) and brain-derived neurotrophic factor (BDNF), the preferred TrkB ligands, are expressed in the infantile mouse ovary. Initially, they are present in oocytes, but this site of expression switches to granulosa cells after the newly assembled primordial follicles develop into growing primary follicles. Full-length kinase domain-containing TrkB receptors are expressed at low and seemingly unchanging levels in the oocytes and granulosa cells of both primordial and growing follicles. In contrast, a truncated TrkB isoform lacking the intracellular domain of the receptor is selectively expressed in oocytes, where it is targeted to the cell membrane as primary follicles initiate growth. Using gene-targeted mice lacking all TrkB isoforms, we show that the ovaries of these mice or those lacking both NT-4 and BDNF suffer a stage-selective deficiency in early follicular development that compromises the ability of follicles to grow beyond the primary stage. Proliferation of granulosa cells-required for this transition-and expression of FSH receptors (FSHR), which reflects the degree of biochemical differentiation of growing follicles, are reduced in trkB-null mice. Ovaries from these animals grafted under the kidney capsule of wild-type mice fail to sustain follicular growth and show a striking loss of follicular organization, preceded by massive oocyte death. These results indicate that TrkB receptors are required for the early growth of ovarian follicles and that they exert this function by primarily supporting oocyte development as well as providing granulosa cells with a proliferative signal that requires oocyte-somatic cell bidirectional communication. The predominance of truncated TrkB receptors in oocytes and their developmental pattern of subcellular expression suggest that a significant number of NT-4/BDNF actions in the developing mammalian ovary are mediated by these receptors.     

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.     

Bone morphogenetic protein-4 acts as an ovarian follicle survival factor and promotes primordial follicle development

The growth and development of follicles within the ovary are highly dependent on autocrine and paracrine signaling involving growth factors from granulosa cells, theca cells, stromal interstitial cells, and the oocytes. The growth factor bone morphogenetic protein-4 (BMP-4) and its receptor (BMPR-IB) have been detected in ovaries, and a mutation in BMPR-IB has been associated with abnormal ovulation rate. The objective of the current study was to examine the role that BMP-4 plays in the early stages of primordial follicle development. Ovaries from 4-day-old rats were placed into a whole-ovary organ culture system for 2 wk to investigate the effect that treatment with exogenous BMP-4 has on early follicle development. BMP-4-treated ovaries had a significantly higher proportion of developing primary follicles and fewer arrested primordial follicles than did untreated controls. This indicates that BMP-4 promotes primordial follicle development and the primordial-to-primary follicle transition. Ovaries were also treated with neutralizing antibody against BMP-4 to determine effects of removing endogenously produced BMP-4. Interestingly, ovaries treated with BMP-4 antibody were markedly smaller than controls. This was associated with a progressive loss of oocytes and primordial follicles, a progressive increase in cellular apoptosis, and an accompanying loss of normal ovarian tissue morphology over time. Immunocytochemistry localized BMP-4 protein to isolated stromal cell populations, selected stromal cells (i.e., pretheca cells) associated with developing primordial follicles, and the basement membrane of follicles. Ovaries were treated with BMP-4 and RNA collected after organ culture to determine whether BMP-4 signaling affects expression of other growth factors. Kit ligand and basic fibroblast growth factor expression was unchanged, but TGFalpha expression was decreased in whole ovaries. Taken together, these data suggest that BMP-4 plays an important role in promoting the survival and development of primordial follicles in the neonatal ovary.