Neonatal exposure to genistein induces estrogen receptor (ER)alpha expression and multioocyte follicles in the maturing mouse ovary: evidence for ERbeta-mediated and nonestrogenic actions

Outbred CD-1 mice were treated neonatally on Days 1-5 with the phytoestrogen, genistein (1, 10, or 100 micro g per pup per day), and ovaries were collected on Days 5, 12, and 19. Ribonuclease protection assay analysis of ovarian mRNA showed that estrogen receptor beta (ERbeta) predominated over ERalpha in controls and increased with age. Genistein treatment did not alter ERbeta expression, however, ERalpha expression was higher on Days 5 and 12. ERbeta was immunolocalized in granulosa cells, whereas ERalpha was immunolocalized in interstitial and thecal cells. Genistein treatment caused a dramatic increase in ERalpha in granulosa cells. Genistein-treated ERbeta knockout mice showed a similar induction of ERalpha, which is seen in CD-1 mice, suggesting that ERbeta does not mediate this effect. Similar ERalpha induction in granulosa cells was seen in CD-1 mice treated with lavendustin A, a tyrosine kinase inhibitor that has no known estrogenic actions, which suggests that this property of genistein may be responsible. As a functional analysis, genistein-treated mice were superovulated and the number of oocytes was counted. A statistically significant increase in the number of ovulated oocytes was observed with the lowest dose, whereas a decrease was observed with the two higher doses. This increase in ovulatory capacity with the low dose coincided with higher ERalpha expression. Histological evaluations on Day 19 revealed a dose-related increase in multioocyte follicles (MOFs) in genistein-treated mice. Tyrosine kinase inhibition was apparently not responsible for MOFs because they were not present in mice that had been treated with lavendustin; however, ERbeta must play a role, because mice lacking ERbeta showed no MOFs. These data taken together demonstrate alterations in the ovary following neonatal exposure to genistein. Given that human infants are exposed to high levels of genistein in soy-based foods, this study indicates that the effects of such exposure on the developing reproductive tract warrant further investigation.     

Relationship between Numerous Mast Cells and Early Follicular Development in Neonatal MRL/MpJ Mouse Ovaries

In the neonatal mouse ovary, clusters of oocytes called nests break into smaller cysts and subsequently form individual follicles. During this period, we found numerous mast cells in the ovary of MRL/MpJ mice and investigated their appearance and morphology with follicular development. The ovarian mast cells, which were already present at postnatal day 0, tended to localize adjacent to the surface epithelium. Among 11 different mouse strains, MRL/MpJ mice possessed the greatest number of ovarian mast cells. Ovarian mast cells were also found in DBA/1, BALB/c, NZW, and DBA/2 mice but rarely in C57BL/6, NZB, AKR, C3H/He, CBA, and ICR mice. The ovarian mast cells expressed connective tissue mast cell markers, although mast cells around the surface epithelium also expressed a mucosal mast cell marker in MRL/MpJ mice. Some ovarian mast cells migrated into the oocyte nests and directly contacted the compressed and degenerated oocytes. In MRL/MpJ mice, the number of oocytes in the nest was significantly lower than in the other strains, and the number of oocytes showed a positive correlation with the number of ovarian mast cells. The gene expression of a mast cell marker also correlated with the expression of an oocyte nest marker, suggesting a link between the appearance of ovarian ? 4mast cells and early follicular development. Furthermore, the expression of follicle developmental markers was significantly higher in MRL/MpJ mice than in C57BL/6 mice. These results indicate that the appearance of ovarian mast cells is a unique phenotype of neonatal MRL/MpJ mice, and that ovarian mast cells participate in early follicular development, especially nest breakdown.     

Forskolin and mouse oocyte maturation in vitro

Oocytes isolated from mature follicles undergo spontaneous maturation when cultured in vitro. Forskolin, an adenylate cyclase stimulator, inhibited resumption of meiosis of cumulus-free mouse oocytes in vitro. Germinal vesicle breakdown (GVBD) was prevented in more than 85% of the oocytes treated by forskolin at concentrations of 20 micrograms/ml and higher. The inhibiting effect of forskolin was dose-dependent and reversible. FSH, LH, FSH plus LH, estrogen, progesterone, and estrogen plus progesterone did not reverse the block induced by forskolin in cumulus-free and cumulus-enclosed oocytes. The present results suggest that intracellular cAMP may play a role in the regulation of oocyte maturation.     

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 follicular cells,IGF-I and tyrosine kinase blockers on oocyte maturation

The aim of this study was to test the following hypotheses: (i) that oocyte maturation is controlled by surrounding follicular cells; (ii) that a meiosis-regulating factor of follicular origin is not species-specific; (iii) that one of the follicular regulators of oocyte maturation is IGF-I; and, (iv) that Cumulus oophorus and tyrosine kinase-dependent intracellular mechanisms do not mediate IGF-I action on oocytes. It was found that co-culture of cumulus-enclosed bovine oocytes with isolated bovine ovarian follicles or with isolated porcine ovarian follicles significantly increased the proportion of matured oocytes (at metaphase II of meiosis) after culture. Porcine oocytes without cumulus investments had lower maturation rates than cumulus-enclosed oocytes. Co-culture with isolated porcine ovarian follicles resulted in stimulation of maturation of both cumulus-free and cumulus-enclosed porcine oocytes. These observations suggest that follicular cells (whole follicles or Cumulus oophorus) support bovine and porcine oocyte maturation, and that follicular maturation-promoting factor is not species-specific. The release of significant amounts of IGF-I by cultured bovine and porcine isolated follicles and granulosa cells was demonstrated. Addition of IGF-I to culture medium at 10 or 100 (but not 1000) ng/ml stimulated meiotic maturation of both cumulus-enclosed and cumulus-free porcine oocytes. Neither of the tyrosine kinase blockers, genistein or lavendustin (100 ng/ml medium), changed the stimulating effect of IGF-I on porcine oocytes. The present data suggest that at least one of the follicular stimulators of oocyte nuclear maturation is IGF-I, and that its effect is probably not mediated by cumulus investment or by tyrosine kinase-dependent intracellular mechanisms.     

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.     

Intact fetal ovarian cord formation promotes mouse oocyte survival and development

Background  

Female reproductive potential, or the ability to propagate life, is limited in mammals with the majority of oocytes lost before birth. In mice, surviving perinatal oocytes are enclosed in ovarian follicles for subsequent oocyte development and function in the adult. Before birth, fetal germ cells of both sexes develop in clusters, or germline cysts, in the undifferentiated gonad. Upon sex determination of the fetal gonad, germ cell cysts become organized into testicular or ovarian cord-like structures and begin to interact with gonadal somatic cells. Although germline cysts and testicular cords are required for spermatogenesis, the role of cyst and ovarian cord formation in mammalian oocyte development and female fertility has not been determined.     

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.     

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.     

From primordial germ cell to primordial follicle: mammalian female germ cell development

In mammals, the final number of oocytes available for reproduction of the next generation is defined at birth. Establishment of this oocyte pool is essential for fertility. Mammalian primordial germ cells form and migrate to the gonad during embryonic development. After arriving at the gonad, the germ cells are called oogonia and develop in clusters of cells called germ line cysts or oocyte nests. Subsequently, the oogonia enter meiosis and become oocytes. The oocyte nests break apart into individual cells and become packaged into primordial follicles. During this time, only a subset of oocytes ultimately survive and the remaining immature eggs die by programmed cell death. This phase of oocyte differentiation is poorly understood but molecules and mechanisms that regulate oocyte development are beginning to be identified. This review focuses on these early stages of female germ cell development.     

TAF4b, a TBP associated factor, is required for oocyte development and function

Development of a fertilizable oocyte is a complex process that relies on the precise temporal and spatial expression of specific genes in germ cells and in surrounding somatic cells. Since female mice null for Taf4b, a TBP associated factor, are sterile, we sought to determine when during follicular development this phenotype was first observed. At postnatal day 3, ovaries of Taf4b null females contained fewer (P < 0.01) oocytes than ovaries of wild type and heterozygous Taf4b mice. However, expression of only one somatic cell marker Foxl2 was reduced in ovaries at day 15. Despite the reduced number of follicles, many proceed to the antral stage, multiple genes associated with granulosa cell differentiation and oocyte maturation were expressed in a normal pattern, and immature Taf4b null females could be hormonally primed to ovulate and mate. However, the ovulated cumulus oocyte complexes from the Taf4b null mice had fewer (P < 0.01) cumulus cells, and the oocytes were functionally abnormal. GVBD and polar body extrusion were reduced significantly (P < 0.01). The few oocytes that were fertilized failed to progress beyond the two-cell stage of development. Thus, infertility in Taf4b null female mice is associated with defects in early follicle formation, oocyte maturation, and zygotic cleavage following ovulation and fertilization.     

Origin of germ cells and formation of new primary follicles in adult human ovaries

Recent reports indicate that functional mouse oocytes and sperm can be derived in vitro from somatic cell lines. We hypothesize that in adult human ovaries, mesenchymal cells in the tunica albuginea (TA) are bipotent progenitors with a commitment for both primitive granulosa and germ cells. We investigated ovaries of twelve adult women (mean age 32.8 ± 4.1 SD, range 27–38 years) by single, double, and triple color immunohistochemistry. We show that cytokeratin (CK)+ mesenchymal cells in ovarian TA differentiate into surface epithelium (SE) cells by a mesenchymal-epithelial transition. Segments of SE directly associated with ovarian cortex are overgrown by TA, forming solid epithelial cords, which fragment into small (20 micron) epithelial nests descending into the lower ovarian cortex, before assembling with zona pellucida (ZP)+ oocytes. Germ cells can originate from SE cells which cover the TA. Small (10 micron) germ-like cells showing PS1 meiotically expressed oocyte carbohydrate protein are derived from SE cells via asymmetric division. They show nuclear MAPK immunoexpression, subsequently divide symmetrically, and enter adjacent cortical vessels. During vascular transport, the putative germ cells increase to oocyte size, and are picked-up by epithelial nests associated with the vessels. During follicle formation, extensions of granulosa cells enter the oocyte cytoplasm, forming a single paranuclear CK+ Balbiani body supplying all the mitochondria of the oocyte. In the ovarian medulla, occasional vessels show an accumulation of ZP+ oocytes (25–30 microns) or their remnants, suggesting that some oocytes degenerate. In contrast to males, adult human female gonads do not preserve germline type stem cells. This study expands our previous observations on the formation of germ cells in adult human ovaries. Differentiation of primitive granulosa and germ cells from the bipotent mesenchymal cell precursors of TA in adult human ovaries represents a most sophisticated adaptive mechanism created during the evolution of female reproduction. Our data indicate that the pool of primary follicles in adult human ovaries does not represent a static but a dynamic population of differentiating and regressing structures. An essential mission of such follicular turnover might be elimination of spontaneous or environmentally induced genetic alterations of oocytes in resting primary follicles.     

Inhibition of nuclear maturation in fully grown porcine and mouse oocytes after their fusion with growing porcine oocytes

Porcine ovarian oocytes, isolated from follicles of 5 mm in diameter (large oocytes), were fused either together or with oocytes isolated from follicles of 0.5 mm in diameter (small oocytes). In giant cells composed of two large oocytes (control) germinal vesicle breakdown (GVBD) occurred and two metaphase I chromosome sets (M I) were observed 24 to 30 h after fusion. By contrast, in giant cells composed of one large and one small porcine oocyte, both germinal vesicles (GVs) remained well conserved after 24-30 h of culture. An identical situation was observed after fusion and cultivation of small porcine and large mouse oocytes isolated from preovulatory follicles. The results demonstrate the presence of inhibiting activity in the ooplasm of small porcine oocytes that prevents nuclear maturation of large porcine and mouse oocytes fused to them. This maturation inhibiting activity can be overcome by preincubating large porcine oocytes for more than 14 h before fusion with small oocytes. During preincubation the ooplasm produces sufficient amount of maturation promoting factor (MPF) to overcome the inhibiting activity present in small porcine oocytes thus inducing GVBD and chromatin condensation both in small and large oocytes.     

Live offspring produced by mouse oocytes derived from premeiotic fetal germ cells

Mature mouse oocytes currently can be generated in vitro from the primary oocytes of primordial follicles but not from premeiotic fetal germ cells. In this study we established a simple, efficient method that can be used to obtain mature oocytes from the premeiotic germ cells of a fetal mouse 12.5 days postcoitum (dpc). Mouse 12.5-dpc fetal ovaries were transplanted under the kidney capsule of recipient mice to initiate oocyte growth from the premeiotic germ cell stage, and they were recovered after 14 days. Subsequently, the primary and early secondary follicles generated in the ovarian grafts were isolated and cultured for 16 days in vitro. The mature oocytes ovulated from these follicles were able to fertilize in vitro to produce live offspring. We further show that the in vitro fertilization offspring were normal and able to successfully mate with both females and males, and the patterns of the methylated sites of the in vitro mature oocytes were similar to those of normal mice. This is the first report describing premeiotic fetal germ cells able to enter a second meiosis and support embryonic development to term by a combination of in vivo transplantation and in vitro culture. In addition, we have shown that the whole process of oogenesis, from premeiotic germ cells to germinal vesicle (GV)-stage oocytes, can be carried out under the kidney capsule.     

Mouse oocytes derived from fetal germ cells are competent to support the development of embryos by in vitro fertilization

Mouse oocyte development in vitro has been studied in the past several years, but no evidence showed that the fertilizable oocytes could be obtained from the fetal mouse germ cells before the formation of the primordial follicles. In this study, an efficient and simple method has been established to obtain the mature oocytes from the fetal mouse germ cells at 16.5 days post-coitum (dpc). For the initial of follicular formation, fetal mouse 16.5 dpc ovaries were transplanted to the recipient under the kidney capsule, and the ovaries were recovered after 14 days. Subsequently, the growing preantral follicles in the ovarian grafts were isolated and cultured in vitro for 12 days. Practically, the mature oocytes ovulated from the antral follicles were able to be fertilized in vitro and support the embryonic development. The results demonstrate that the fetal mouse 16.5 dpc germ cells are able to form primordial follicles with the ovarian pregranulosa cells during the period of transplantation in the ectopic site, and the oocytes within the growing follicles are able to mature in vitro, then are able to support the embryonic development.     

Meiotic resumption in response to luteinizing hormone is independent of a Gi family G protein or calcium in the mouse oocyte

The signaling pathway by which luteinizing hormone (LH) acts on the somatic cells of vertebrate ovarian follicles to stimulate meiotic resumption in the oocyte requires a decrease in cAMP in the oocyte, but how cAMP is decreased is unknown. Activation of Gi family G proteins can lower cAMP by inhibiting adenylate cyclase or stimulating a cyclic nucleotide phosphodiesterase, but we show here that inhibition of this class of G proteins by injection of pertussis toxin into follicle-enclosed mouse oocytes does not prevent meiotic resumption in response to LH. Likewise, elevation of Ca2+ can lower cAMP through its action on Ca2+-sensitive adenylate cyclases or phosphodiesterases, but inhibition of a Ca2+ rise by injection of EGTA into follicle-enclosed mouse oocytes does not inhibit the LH response. Thus, neither of these well-known mechanisms of cAMP regulation can account for LH signaling to the oocyte in the mouse ovary.     

Development of mouse and rat oocytes in chimeric reaggregated ovaries after interspecific exchange of somatic and germ cell components

The germ cell and somatic cell compartments of newborn rat and mouse ovaries, which contain only primordial stage follicles, were completely exchanged and reaggregated to produce xenogeneic chimeric ovaries. The reaggregated ovaries were grafted beneath the renal capsules of ovariectomized SCID mice to develop for periods up to 21 days. Xenogeneic follicles developed with essentially normal morphological characteristics. Both rat and mouse oocytes with species-specific characteristics grew within follicles that were composed of somatic cells exclusively of the alternative species. Rat oocytes grown in mouse follicles became competent to resume meiosis, and progressed to metaphase II when they were removed from follicles and cultured. In addition, mouse oocytes grown in rat follicles underwent fertilization and preimplantation development in vitro, and developed to term after embryos were transferred to pseudopregnant mouse foster mothers. Therefore, despite an estimated 11 million years of divergent evolution, oocytes and somatic cells of rat and mouse ovaries can be exchanged and can produce functional oocytes. It is concluded that factors involved in oocyte-somatic cell interactions necessary to support oocyte development and appropriate differentiation of the oocyte-associated granulosa cells are conserved between rats and mice. Moreover, although granulosa cells play important roles in oocyte development, the development of species-specific characteristics of oocytes occurs without apparent modification by a xenogeneic follicular environment.     

Targeted expression of Bcl-2 in mouse oocytes inhibits ovarian follicle atresia and prevents spontaneous and chemotherapy-induced oocyte apoptosis in vitro.

Members of the Bcl-2 family serve as central checkpoints for cell death regulation, and overexpression of Bcl-2 is known to inhibit apoptosis in many cell types. To determine whether targeted expression of Bcl-2 could be used to protect female germ cells from apoptosis, we generated transgenic mice expressing fully functional human Bcl-2 protein only in oocytes. Transgenic mice were produced using a previously characterized 480-bp fragment of the mouse zona pellucida protein-3 (ZP3) gene 5'-flanking region to direct oocyte-specific expression of a human bcl-2 complementary DNA. Immunohistochemical analyses using a human Bcl-2-specific antibody showed that transgene expression was restricted to growing oocytes and was not observed in the surrounding ovarian somatic cells or in any other nonovarian tissues. Histomorphometric analyses revealed that ovaries collected from transgenic female mice possessed significantly fewer atretic small preantral follicles compared with wild-type sisters, resulting in a larger population of healthy maturing follicles per ovary. However, the number of oocytes ovulated in response to exogenous gonadotropin priming and the number of pups per litter were not significantly different among wild-type vs. transgenic female mice. Nonetheless, oocytes obtained from transgenic mice and cultured in vitro were found to be resistant to spontaneous and anticancer drug-induced apoptosis. We conclude that targeted expression of Bcl-2 only in oocytes can be achieved as a means to convey resistance of the female germ line to naturally occurring and chemotherapy-induced apoptosis.