Smad 3 may regulate follicular growth in the mouse ovary

Although Smad 3 is known to serve as a signaling intermediate for the transforming growth factor beta (TGFbeta) family in nonreproductive tissues, its role in the ovary is unknown. Thus, we used a recently generated Smad 3-deficient (Smad 3-/-) mouse model to test the hypothesis that Smad 3 alters female fertility and regulates the growth of ovarian follicles from the primordial stage to the antral stage. In addition, we tested whether Smad 3 affects the levels of proteins that control apoptosis, survival, and proliferation in the ovarian follicle. To test this hypothesis, breeding studies were conducted using Smad 3-/- and wild-type mice. In addition, ovaries were collected from Smad 3-/- and wild-type mice on Postnatal Days 2-90. One ovary from each animal was used to estimate the total number of primordial, primary, and antral follicles. The other ovary was used for immunohistochemical analysis of selected members of the B-cell lymphoma/leukemia-2 family of protooncogenes (Bax, Bcl-2, Bcl-x), proliferating cell nuclear antigen (PCNA), and cyclin-dependent kinase 2 (Cdk-2). The results indicate that Smad 3-/- mice have reduced fertility compared with wild type mice. The results also indicate that Smad 3 may not affect the size of the primordial follicle pool at birth, but it may regulate growth of primordial follicles to the antral stage. Further, the results indicate that Smad 3 may regulate the expression of Bax and Bcl-2, but not Bcl-x, Cdk-2, and PCNA. Collectively, these data suggest that Smad 3 may play an important role in the regulation of ovarian follicle growth and female fertility.     

Effects of methoxychlor on IGF-I signaling pathway in rat ovary

Follicular development and other ovarian functions are regulated by growth factors that can be affected by exogenous agents. Methoxychlor (MXC) is an organochloride pesticide that causes female infertility. We investigated how MXC affects the distribution of developing ovarian follicles in adult rats after treatment between embryonic day (E) 18 and postnatal day (PND) 7. We also measured insulin-like growth factor-I (IGF-I) and its receptor, IGF-IR, expressions in ovarian follicles and investigated whether MXC changed the levels of IGF-I and IGF-IR in the ovary. Using immunohistochemical (IHC) staining, we detected IGF-I expression in oocytes and granulosa cells of the follicles, luteal cells, interstitial cells, theca externa and theca interna, and the smooth muscle of ovarian vessels. IGF-IR was co-localized with IGF-I in the ovary except for the theca externa. IGF-I expression was decreased in granulosa cells of preantral and antral follicles after treatment with MXC compared to granulosa cells of preantral and antral follicles of the control group. We also observed that oocytes of secondary follicles and granulosa cells of secondary and preantral follicles of the MXC treated groups showed increased IGF-IR expression compared to oocytes of secondary follicles and granulosa cells of secondary and preantral follicles of the control group. We also detected more secondary and preantral follicles, and fewer primordial and antral follicles after MXC administration compared to controls. Therefore, the IGF signaling pathway may participate in MXC induced ovary dysfunction and female infertility.     

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.     

Induction of apoptosis by 9,10-dimethyl-1,2-benzanthracene in cultured preovulatory rat follicles is preceded by a rise in reactive oxygen species and is prevented by glutathione

The polycyclic aromatic hydrocarbon (PAH) 9,10-dimethyl-1,2-benzanthracene (DMBA) destroys primordial, primary, and secondary ovarian follicles in rodents, but its effects on antral follicles have received limited attention. PAHs are metabolized to reactive species, some of which can undergo redox cycling to generate reactive oxygen species (ROS). We previously showed that ROS initiate apoptosis in preovulatory follicles cultured without gonadotropin support and that glutathione (GSH) depletion induces apoptosis in the presence of gonadotropins. In the present study, we tested the hypothesis that DMBA induces apoptosis in preovulatory follicles, which is mediated by ROS and prevented by GSH. Preovulatory follicles were isolated from ovaries of 25-day-old rats 48 h after the injection of 10 IU of eCG and were cultured with DMBA in the presence of FSH for 2 to 48 h. DMBA induced granulosa cell (GC) and theca cell (TC) apoptosis at 48 h, as judged by TUNEL and activated caspase-3 immunostaining. DMBA treatment also increased the numbers of GCs and TCs that immunostained for the proapoptotic protein BAX. Follicular ROS levels were significantly increased in DMBA-treated follicles at 12, 24, and 48 h. GSH supplementation protected against and GSH depletion enhanced the induction of apoptosis in GCs and TCs by DMBA. These findings suggest that GSH is a critical protective mechanism against DMBA-induced apoptosis in antral follicles and that ROS generation may mediate DMBA-induced GC apoptosis.     

Recent achievements in in vitro culture and preservation of ovarian follicles in mammals

The mammalian ovary contains a large number of follicles that are in various developmental stages. The largest portion of them are primordial follicles. However, throughout the female reproductive lifespan only a small proportion of these follicles will produce oocytes competent to undergo successful maturation and ovulation. The rest of the ovarian oocytes (>99.9%) undergo atresia. It would be of great practical benefit to rescue some of these follicles by growing them in culture in order to provide an extra source of gametes. There is considerable interest in developing technologies that aim to produce fully-grown, developmentally competent oocytes from a pool of early developmental stages of follicles. Two methods have been used: 1/ long-term in vitro culture of either follicles or oocytes, and 2/ transplantation of ovarian tissue grafts. The development of efficient technologies may provide an additional source of oocytes for livestock production and reproduction in humans and rare or endangered species. The aim of this paper is to present a comprehensive review of recent achievements in the utilization of small ovarian follicles (primordial, preantral and early antral) by long-term in vitro culture and/or transplantation of ovarian tissue grafts (fresh and cryopreserved) in mammals including humans.     

Aspects of ovarian follicle development throughout life

The pool of primordial follicles present in the female ovary reaches its maximum number around 20 weeks of gestational age and then decreases in a logarithmic fashion throughout life until complete depletion occurs around the age of the menopause. Reproductive life is initiated when less than 10% (0.5 million) of primordial follicles are left. The entire growth trajectory of the follicle takes at least 3 months. Follicle growth up to the antral stage occurs during fetal life and infancy. While the role of gonadotropins in early follicular development remains controversial, the last 2 weeks of development are FSH dependent. The intercycle rise in FSH and decreasing levels thereafter are crucial for recruitment of a cohort of healthy, early antral follicles and subsequent single dominant selection. Following puberty, anovulation may persist for years and this may presage the development of adult anovulatory infertility. The menopause is preceded by a period of reduced fertility. The development of reliable and sensitive markers for ovarian ageing will be the challenge of the near future.     

New strategy for in vitro activation of primordial follicles with mTOR and PI3K stimulators

It had been known for decades that primordial follicles in mammalian ovaries are assembled with definite numbers and represent the ovarian reserve throughout the reproductive life. Intra-oocyte PI3K/mTOR pathways have been indicated to play a central role on the activation of primordial follicles. Genetic modified mouse models with chronic activation of PI3K/mTOR signals in primordial oocytes showed premature activation of all primordial follicles and eventually their exhaustion. On the other hand, this may suggest that, unlike chronic activation of PI3K/mTOR, its acute activation in infertility would activate primordial follicles, permitting fertility during the treatment. Previously, PI3K stimulators were reported as a temporary measure to accelerate primordial follicle activation and follicular development in both mouse and human, and were applied in the treatment of infertility in premature ovarian failure (POF) patients. To address whether mTOR stimulators could play similar role in the process, we transiently treated neonatal and aged mouse ovaries with mTOR stimulators-phosphatidic acid (PA) and propranolol. Our results demonstrated the stimulators increased activation of primordial follicles and the production of progeny. Human ovarian cortex cubes were also treated with mTOR or/and PI3K stimulators in vitro. When they were used separately, both of them showed similar promotive effects on primordial follicles. Surprisingly, after joint-treatment with the 2 kinds of stimulators together, synergistic effects on follicular development were observed. Based on increased efficiency of follicular activation in humans, here we propose in vitro transient treatment with mTOR and PI3K stimulators as an optimized protocol for the application in different clinical conditions with limited follicle reserve.     

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.     

mTORC1 Signaling in Oocytes Is Dispensable for the Survival of Primordial Follicles and for Female Fertility

The molecular mechanisms underlying reproductive aging and menopausal age in female mammals are poorly understood. Mechanistic target of rapamycin complex 1 (mTORC1) is a central controller of cell growth and proliferation. To determine whether mTORC1 signaling in oocytes plays a direct role in physiological follicular development and fertility in female mice, we conditionally deleted the specific and essential mTORC1 component Rptor (regulatory-associated protein of mTORC1) from the oocytes of primordial follicles by using transgenic mice expressing growth differentiation factor 9 (Gdf-9) promoter-mediated Cre recombinase. We provide in vivo evidence that deletion of Rptor in the oocytes of both primordial and further-developed follicles leads to the loss of mTORC1 signaling in oocytes as indicated by loss of phosphorylation of S6K1 and 4e-bp1 at T389 and S65, respectively. However, the follicular development and fertility of mice lacking Rptor in oocytes were not affected. Mechanistically, the loss of mTORC1 signaling in Rptor-deleted mouse oocytes led to the elevation of phosphatidylinositol 3-kinase (PI3K) signaling that maintained normal follicular development and fertility. Therefore, this study shows that loss of mTORC1 signaling in oocytes triggers a compensatory activation of the PI3K signaling cascade that maintains normal ovarian follicular development and fertility.     

Oxidative damage increases and antioxidant gene expression decreases with aging in the mouse ovary

Oxidative stress has been implicated in various aspects of aging, but the role of oxidative stress in ovarian aging remains unclear. Our previous studies have shown that the initiation of apoptotic cell death in ovarian follicles and granulosa cells by various stimuli is initiated by increased reactive oxygen species. Herein, we tested the hypothesis that ovarian antioxidant defenses decrease and oxidative damage increases with age in mice. Healthy, wild-type C57BL/6 female mice aged 2, 6, 9, or 12 mo from the National Institute on Aging Aged Rodent Colony were killed on the morning of metestrus. Quantitative real-time RT-PCR was used to measure ovarian mRNA levels of antioxidant genes. Immunostaining using antibodies directed against 4-hydroxynonenal (4-HNE), nitrotyrosine (NTY), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) was used to localize oxidative lipid, protein, and DNA damage, respectively, within the ovaries. TUNEL was used to localize apoptosis. Ovarian expression of glutathione peroxidase 1 (Gpx1) increased and expression of glutaredoxin 1 (Glrx1), glutathione S-transferase mu 2 (Gstm2), peroxiredoxin 3 (Prdx3), and thioredoxin 2 (Txn2) decreased in a statistically significant manner with age. Statistically significant increases in 4-HNE, NTY, and 8-OHdG immunostaining in ovarian interstitial cells and follicles were observed with increasing age. Our data suggest that the decrease in mRNA expression of mitochondrial antioxidants Prdx3 and Txn2 as well as cytosolic antioxidants Glrx1 and Gstm2 may be involved in age-related ovarian oxidative damage to lipid, protein, DNA, and other cellular components vital for maintaining ovarian function and fertility.     

Prenatal and neonatal flutamide administration increases proliferation and reduces apoptosis in large antral follicles of adult pigs

Ovarian follicular atresia is regulated by androgens directly via androgen receptors and indirectly after conversion to estrogens. The balance between proliferation and cell apoptosis is crucial for the physiological functioning of the follicles. The disorder between these processes leads to reproductive failure, such as cyst formation. Recent research suggests maternally or neonatally mediated effects of antiandrogen flutamide on reproductive functions during adulthood. Therefore, the current study was performed to determine whether late gestational or neonatal exposure to flutamide influences proliferation and apoptosis rates in large antral follicles of adult porcine ovary. These periods are critical in ovarian biology and may determine female fertility in adulthood. Flutamide was injected into pregnant gilts between days 80 and 88 of gestation, and into female piglets between days 2 and 10 postnatally. The ovaries were collected from treated and control adult pigs, and healthy large antral follicles were excised. In large antral follicles, granulosa and theca cells revealed elevated (p<0.001) proliferation index measured by immunolocalization of Ki-67 following maternal and neonatal flutamide exposure. The percentage of apoptotic granulosa cells detected using TUNEL assay significantly decreased (p<0.01) after flutamide administration, that paralleled with down-regulation (p<0.01) of caspase-3 protein expression. Moreover, plasma testosterone decreased (p<0.001) in flutamide-treated animals, whereas 17β-estradiol was elevated following flutamide exposure. The present research indicates increased proliferation and diminished apoptosis rates within large antral follicles of adult pigs following prenatal and neonatal flutamide administration, which might influence the normal development of the follicles and pigs fertility as a consequence.     

Retrievable hydrogels for ovarian follicle transplantation and oocyte collection

Cancer survivorship rates have drastically increased due to improved efficacy of oncologic treatments. Consequently, clinical concerns have shifted from solely focusing on survival to quality of life, with fertility preservation as an important consideration. Among fertility preservation strategies for female patients, ovarian tissue cryopreservation and subsequent reimplantation has been the only clinical option available to cancer survivors with cryopreserved tissue. However, follicle atresia after transplantation and risk of reintroducing malignant cells have prevented this procedure from becoming widely adopted in clinics. Herein, we investigated the encapsulation of ovarian follicles in alginate hydrogels that isolate the graft from the host, yet allows for maturation after transplantation at a heterotopic (i.e., subcutaneous) site, a process we termed in vivo follicle maturation. Survival of multiple follicle populations was confirmed via histology, with the notable development of the antral follicles. Collected oocytes (63%) exhibited polar body extrusion and were fertilized by intracytoplasmic sperm injection and standard in vitro fertilization procedures. Successfully fertilized oocytes developed to the pronucleus (14%), two‐cell (36%), and four‐cell (7%) stages. Furthermore, ovarian follicles cotransplanted with metastatic breast cancer cells within the hydrogels allowed for retrieval of the follicles, and no mice developed tumors after removal of the implant, confirming that the hydrogel prevented seeding of disease within the host. Collectively, these findings demonstrate a viable option for safe use of potentially cancer‐laden ovarian donor tissue for in vivo follicle maturation within a retrievable hydrogel and subsequent oocyte collection. Ultimately, this technology may provide novel options to preserve fertility for young female patients with cancer.     

Protective effects of mangafodipir against chemotherapy-induced ovarian damage in mice

Background

Given the seriousness of chemotherapy-induced ovarian injury in female cancer patients, the preservation of fertility, including through the use of cryopreservation technology and pharmaceuticals, requires investigation. Previous studies have shown that damage to the ovaries is related to oxidative stress caused by anticancer drugs. Therefore, superoxide dismutase (SOD) may represent a key factor in the pharmacological protection of the ovaries. The aim of our study was to identify the effects of mangafodipir, a manganese chelate and SOD-mimetic, on suppression of apoptosis in granulosa cells and primordial follicle activation induced by anticancer drugs.

Methods

Cell viability assays using methyltrichlorosilane solutions and immunoblotting for cleaved caspase-3 were performed in in vitro experiments with the simultaneous addition of mangafodipir to human non-luteinized granulosa cell line (HGrC) cultures treated with hydrogen peroxide (H₂O₂), cisplatin, or paclitaxel. Count and morphological analyses of follicles at each developing stage in the ovaries and immunohistochemistry for cleaved caspase-3, Ki67 and 4-hydroxynonenal, a marker for oxidative stress, were also performed using mangafodipir-injected 6-week-old female ICR mice treated with cisplatin or paclitaxel. Further, mangafodipir was injected into 6-week-old female BALB/c mice inoculated with ES-2 to analyze whether mangafodipir inhibits the anti-tumor effects of cisplatin or paclitaxel treatment.

Results

Mangafodipir attenuated apoptosis induced by H₂O₂ and anticancer drugs in vitro. Mangafodipir also decreased the expression of 4-hydroxynonenal and reduced cisplatin- and paclitaxel-induced apoptosis in granulosa cells in vivo. In addition, mangafodipir inhibited the loss of primordial follicles. Tumor xenograft studies in mice showed that mangafodipir did not affect anticancer drug antitumor effects.

Conclusions

Oxidative stress might be one of the mechanisms of cisplatin- and paclitaxel-induced the loss of primordial follicles. Mangafodipir can reduce cisplatin- and paclitaxel-induced apoptosis in granulosa cells and primordial follicle activation partially via its SOD activity. At the same time, mangafodipir might have other potential mechanisms to inhibit the activation of primordial follicles. Further, mangafodipir attenuated the ovarian damage caused by cisplatin and paclitaxel without affecting their antitumor activities. Mangafodipir, therefore, though its efficacy might be limited, may be a new option for the preservation of fertility during anticancer treatment.

     

Immunohistochemical localization of proliferating cell nuclear antigen (PCNA) in the pig ovary

The aim of the study was to determine the expression of proliferating cell nuclear antigen protein (PCNA) in the pig ovary. The localization of PCNA was demonstrated in paraffin sections of pig ovarian tissue using primary mouse monoclonal anti-PCNA antibody. In primordial follicles, no remarkable staining for PCNA either in granulosa cells or in the oocytes was observed. In primary to secondary follicles, positive staining in oocytes and in some granulosa cells was detected. The advanced preantral and particularly actively growing small to large antral follicles showed extensive PCNA labeling in the layers of granulosa and theca cells and in the cumulus cells encircling the oocyte. PCNA labeling was expressed in nuclei of oocytes in preantral and small antral follicles. In atretic follicles, the level of PCNA protein expression was dependent on the stage of atresia. Follicles demonstrating advanced atresia showed only limited or no PCNA labeled granulosa and theca cells. The results of the study demonstrate that follicular growth and development in pig ovary may be effectively monitored by determining the granulosa cell expression of PCNA.     

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.     

Maturation and fertilization of porcine oocytes from primordial follicles by a combination of xenografting and in vitro culture

Our objective was to develop a method of endowing oocytes from porcine primordial follicles with full maturation and fertilizing ability as a model for ovarian xenografting of large mammals. Ovarian tissues from 20-day-old piglets, in which most of the follicles were primordial, were transplanted under the capsules of both kidneys of ovariectomized athymic mice. The host mice were treated with 5 IU of equine chorionic gonadotropin (eCG) for 10 days (eCG-10), 30 days (eCG-30), or 60 days (eCG-60) after detection of cornified epithelial cells in their vaginal smears. Cumulus-oocyte complexes, ovarian grafts, and blood samples were obtained 48 h after eCG treatment. Forty-five to 70 days after grafting, the host mice in all groups for the first time showed vaginal cornification, accompanied by the formation of a small number of antral follicles in the grafts. However, we recovered large numbers of full-sized oocytes only from mice in the eCG-60 group; the numbers of full-sized oocytes in the other groups were low. Peripheral levels of total inhibin were highest in the eCG-60 group; this supports our finding that the most enhanced growth of antral follicles occurred in this eCG-60 group. Of 573 oocytes obtained from the eCG-60 group, 98 (17%) were at the metaphase II stage after in vitro culture for maturation. Moreover, 55% of matured oocytes with the first polar body (n = 20) were fertilized in vitro. These results clearly demonstrate that fertilization of oocytes from porcine primordial follicles is achievable by a combination of xenografting and in vitro culture.     

Characterization of the IGF2 Imprinted Gene Methylation Status in Bovine Oocytes during Folliculogenesis

DNA methylation reprogramming occurs during mammalian gametogenesis and embryogenesis. Sex-specific DNA methylation patterns at specific CpG islands controlling imprinted genes are acquired during this window of development. Characterization of the DNA methylation dynamics of imprinted genes acquired by oocytes during folliculogenesis is essential for understanding the physiological and genetic aspects of female gametogenesis and to determine the parameters for oocyte competence. This knowledge can be used to improve in vitro embryo production (IVP), specifically because oocyte competence is one of the most important aspects determining the success of IVP. Imprinted genes, such as IGF2, play important roles in embryo development, placentation and fetal growth. The aim of this study was to characterize the DNA methylation profile of the CpG island located in IGF2 exon 10 in oocytes during bovine folliculogenesis. The methylation percentages in oocytes from primordial follicles, final secondary follicles, small antral follicles, large antral follicles, MII oocytes and spermatozoa were 73.74 ± 2.88%, 58.70 ± 7.46%, 56.00 ± 5.58%, 65.77 ± 5.10%, 56.35 ± 7.45% and 96.04 ± 0.78%, respectively. Oocytes from primordial follicles showed fewer hypomethylated alleles (15.5%) than MII oocytes (34.6%) (p = 0.039); spermatozoa showed only hypermethylated alleles. Moreover, MII oocytes were less methylated than spermatozoa (p<0.001). Our results showed that the methylation pattern of this region behaves differently between mature oocytes and spermatozoa. However, while this region has a classical imprinted pattern in spermatozoa that is fully methylated, it was variable in mature oocytes, showing hypermethylated and hypomethylated alleles. Furthermore, our results suggest that this CpG island may have received precocious reprogramming, considering that the hypermethylated pattern was already found in growing oocytes from primordial follicles. These results may contribute to our understanding of the reprogramming of imprinted genes during bovine oogenesis.     

Caspase-2 involvement during ionizing radiation-induced oocyte death in the mouse ovary

In mammals, the pool of primordial follicles at birth is determinant for female fertility. Exposure to IR during oogonia proliferation and the diplotene stages of ovarian development induced the virtual disappearance of primordial follicles in the postnatal ovary, while half the follicular reserve remained present after irradiation during the zygotene/pachytene stages. This sensitivity difference was correlated with the level of caspase-2 expression evaluated by immunohistochemistry. At the diplotene stage, Western blot and caspase activity analysis revealed that caspase-2 was activated 2 h after irradiation and a significant increase in the number of oocytes expressing cleaved caspase-9 and -3 occurred 6 h after treatment. Inhibition of caspase-2 activity prevented the cleavage of caspase-9 and partially prevented the loss of oocytes in response to irradiation. Taken together, our results show that caspase-2-dependent activation of the mitochondrial apoptotic pathway is one of the mechanisms involved in the genotoxic stress-induced depletion of the primordial follicle pool.     

Hyal-1 but not Hyal-3 deficiency has an impact on ovarian folliculogenesis and female fertility by altering the follistatin/activin/Smad3 pathway and the apoptotic process

Ovarian follicle development is a process regulated by various endocrine, paracrine and autocrine factors that act coordinately to promote follicle growth. However, the vast majority of follicles does not reach the pre-ovulatory stage but instead, undergo atresia by apoptosis. We have recently described a role for the somatic hyaluronidases (Hyal-1, Hyal-2, and Hyal-3) in ovarian follicular atresia and induction of granulosa cell apoptosis. Herein, we show that Hyal-1 but not Hyal-3 null mice have decreased apoptotic granulosa cells after the induction of atresia and an increased number of retrieved oocytes after stimulation of ovulation. Furthermore, young Hyal-1 null mice had a significantly higher number of primordial follicles than age matched wild-type animals. Recruitment of these follicles at puberty resulted in an increased number of primary and healthy preantral follicles in Hyal-1 null mice. Consequently, older Hyal-1 deficient female mice have prolonged fertility. At the molecular level, immature Hyal-1 null mice have decreased mRNA expression of follistatin and higher levels of phospho-Smad3 protein, resulting in increased levels of phospho-Akt in pubertal mice. Hyal-1 null ovarian follicles did not exhibit hyaluronan accumulation. For Hyal-3 null mice, compensation by Hyal-1 or Hyal-2 might be related to the lack of an ovarian phenotype. In conclusion, our results demonstrate that Hyal-1 plays a key role in the early phases of folliculogenesis by negatively regulating ovarian follicle growth and survival. Our findings add Hyal-1 as an ovarian regulator factor for follicle development, showing for the first time an interrelationship between this enzyme and the follistatin/activin/Smad3 pathway.