Apoptosis In Ovarian Follicles Research Articles

Immune Checkpoint Inhibitors Induce Apoptosis in Ovarian Follicles.

Checkpoint inhibitor immunotherapy can harm ovarian follicles and decrease oocyte quality in mice.

Effects of Levofloxacin on Expression of Apoptosis Genes of Ovarian Follicles in NMRI Mouse in Vitro and in Vivo

Background and Objectives: Levofloxacin is a fluoroquinolone. Long-term use of antibiotics, including fluoroquinolone antibiotics, causes dystrophic changes in the ovaries. In this study, the effects of levofloxacin on the development of ovarian follicles and their apoptosis in the in vitro and in vivo conditions of NMRI Mouse were investigated. Methods: In the vitro study, isolated ovaries of animals were treated with levofloxacin at concentrations of 1, 2, 5, 10 µg/ml for 6 days and in the vivo study were treated with levofloxacin at concentrations of 100, 200, 400 and 800 mg/kg. After 24 days, animals were sacrificed in the in vivo groups and ovary samples were obtained. Hematoxylin and eosin (H&E) stains for histological studies also Real Time PCR techniques for study of expression rate of Bax and Bcl-2 genes were performed in both groups. Results: The results of the in vitro study showed that the dose of 2 μg/ml induced apoptosis in the monolayer primary follicles. Doses of 5 and 10 μg/ml increased the induction of apoptosis in the multilayer primary follicle. The number of secondary follicles at the 5 μg/ml dose had the most decrease. While at the dose of 10 μg/ml, the number of mature follicles had the most increase. As the antibiotic concentration increases, the expression of the Bax gene decreases and Bcl-2 gene increases significantly. The results of the in vitro study showed that the number of primary and secondary follicles decreased dose-dependently. The number of atretic follicles increased significantly with increasing dose. The expression of the Bax and Bcl-2 genes increased with increasing levofloxacin antibiotic concentration. Conclusion: This study demonstrates that levofloxacin induces apoptosis in ovarian follicles

Effect of resveratrol on mouse ovarian vitrification and transplantation

BackgroundAfter ovarian tissue transplantation, ischemia-reperfusion injury and free radicals cause follicle depletion and apoptosis. Therefore, the use of antioxidants to reduce the production of free radicals is an important method to address the consequences of ischemia-reperfusion injury. Resveratrol is a natural active polyphenol compound with anti-inflammatory, antitumor, strong antioxidant and anti-free radical properties. The aim of this study was to investigate whether resveratrol could improve the effect of autologous ovarian transplantation after cryopreserve-thawn mouse ovarian tissue.MethodsWhole-ovary vitrification and autotransplantation models were used to investigate the effects of resveratrol. Six-week-old female mice from the Institute of Cancer Research (ICR) were subjected to vitrification. All ovaries were preserved in liquid nitrogen for 1 week before being thawed. After thawing, ovarian tissues were autotransplanted in the bilateral kidney capsules. Mice (n = 72) were randomly divided into four groups to determine the optimal concentration of resveratrol (experiment I).Treatments were given as follows: saline, 5 mg/kg resveratrol, 15 mg/kg resveratrol and 45 mg/kg resveratrol, which were administered orally for one week. Grafted ovaries were collected for analysis on days 3, 7, and 21 after transplantation. Ovarian follicle morphology was assessed by hematoxylin and eosin staining. Serum FSH and E2 levels were measured to estimate the transplanted ovarian reserve and endocrine function. Other mice were randomly divided into two groups—saline and 45 mg/kg resveratrol to further evaluate the effect of resveratrol and explore the mechanisms underlying this effect (experiment II). Ovarian follicle apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays. Immunohistochemistry, qRT-PCR and western blotting (MDA, SOD, NF-κB, IL-6 and SIRT1) were used to explore the mechanisms of resveratrol. Moreover, oocytes derived from autotransplanted ovaries at 21 days were cultured and fertilized in vitro.ResultsThe proportions of morphologically normal (G1) follicles at 3, 7 and 21 days were significantly higher in the 45 mg/kg resveratrol group than in the saline group. The TUNEL-stained follicles (%) at 7 days were significantly decreased in the 45 mg/kg resveratrol group compared with the saline group. Western blot analysis revealed that SOD2 and SIRT1 levels were significantly higher in the 45 mg/kg resveratrol group than in the saline group at day 7 and that MDA and NF-κB levels were lower in the saline group on day 3. Likewise, IL-6 was lower in the saline group on day 7. These results are basically consistent with the qRT-PCR results. In addition, the mean number of retrieved oocytes and fertilization and cleavage were significantly increased in the 45 mg/kg resveratrol group compared with the saline group.ConclusionsAdministration of resveratrol could improve the quality of cryopreserved mouse ovarian tissue after transplantation and the embryo outcome, through anti-inflammatory and antioxidative mechanisms.

The effects of CLP-induced sepsis on proliferation and apoptosis of granulosa and theca cells in rat ovary: A histochemical and ultrastructural study

Sepsis is defined as a systemic inflammatory response to infection. This study is aimed to evaluate the effects of experimental sepsis on the proliferation and apoptosis of granulosa and theca cells in the rat ovary.28-day-old immature Wistar-Albino female rats were treated with pregnant mare serum gonadotrophin to develop the first generation of preovulatory follicles. Sepsis was induced by cecal ligation and puncture (CLP). Following in vivo 5-Bromo-2-deoxyuridine (BrdU) labeling, animals were sacrificed and ovaries were embedded in paraffin and Epon. Besides electron microscopic evaluation, BrdU, cleaved caspase-3, p27 immunostaining, and TUNEL labeling were performed.In CLP-operated animals, cleaved caspase-3 immunoreactivity was significantly increased in Graafian follicles. TUNEL and BrdU labeling in the ovarian follicles were not statistically different between CLP and sham-operated rats. In septic animals, p27 immunoreactivity was increased significantly in the nuclei of oocytes and decreased in the cytoplasm of granulosa and theca cells in multilaminar primary follicles compared to the sham group. In ultrastructural evaluation, increased apoptosis was observed in theca interna and granulosa cells in both the early and late stages of follicles in the CLP group.In conclusion, experimentally-induced sepsis leads to apoptosis in ovarian follicles at advanced stages of development. Our data suggest that although sepsis may not cause a potential threat to developing follicles at least in the short term, more severe damage may occur during advanced stages of follicle development.

The Impact of a Long-Acting Oral Sphingosine-1-Phosphate Analogue on Ovarian Aging in a Rat Model.

In animal studies, intravenous continuous infusion or peritoneal injection of sphingosine-1-phosphate (S1P) has been shown to decrease chemotherapy- and radiotherapy-induced apoptosis on primordial follicles. Although a long-acting oral form of an S1P analogue (FTY720, fingolimod) has been recently developed and utilized in women with multiple sclerosis, there are no data exploring its ability to avoid spontaneous follicle apoptosis. Thirty 10-month-old female rats were randomly assigned to 3 groups to investigate whether fingolimod would be able to decrease the spontaneous ovarian follicle apoptosis ratio. An oral analogue form of S1P was administered for 60 days at a dose of 0.1 mg/kg (n = 10) or dose of 1 mg/kg (n = 10) per day. The control group (n = 10) received physiological serum via an orogastric feeding tube. The main outcome measures were anti-Müllerian hormone (AMH) level and nonapoptotic follicle ratio. While low-dose S1P group had comparable AMH levels to high-dose S1P group and controls, high-dose S1P group had higher mean levels of AMH, reaching marginal significance with controls (5.72 ± 0.61 vs 4.81 ± 0.85 ng/mL, P = .050). For the nonapoptotic primordial follicle ratio, both low-dose S1P group (67.0% ± 16.4% vs 29.9% ± 19.5%, P < .001) and high-dose S1P group (51.1% ± 11.5% vs 29.9% ± 19.5%, P = .023) had superior rates when compared with controls. Interestingly, low-dose S1P groups also had a statistically higher nonapoptotic primordial follicle ratio than high-dose S1P group ( P = .047). Our findings suggest that a long-acting oral analogue of S1P might decrease spontaneous follicular apoptosis based on the nonapoptotic primordial follicle ratio and AMH levels when compared with placebo.

Charged-Iron-Particles Found in Galactic Cosmic Rays are Potent Inducers of Epithelial Ovarian Tumors

Astronauts traveling in deep space are exposed to high-charge and energy (HZE) particles from galactic cosmic rays. We have previously determined that irradiation of adult female mice with iron HZE particles induces DNA double-strand breaks, oxidative damage and apoptosis in ovarian follicles, causing premature ovarian failure. These effects occur at lower doses than with conventional photon irradiation. Ovarian failure with resultant loss of negative feedback and elevated levels of gonadotropin hormones is thought to play a role in the pathophysiology of ovarian cancer. Therefore, we hypothesized that charged-iron-particle irradiation induces ovarian tumorigenesis in mice. In this study, three-month-old female mice were exposed to 0 cGy (sham) or 50 cGy iron ions and aged to 18 months. The 50 cGy irradiated mice had increased weight gain with age and lack of estrous cycling, consistent with ovarian failure. A total of 47% and 7% of mice irradiated with 50 cGy had unilateral and bilateral ovarian tumors, respectively, whereas 14% of mice in the 0 cGy group had unilateral tumors. The tumors contained multiple tubular structures, which were lined with cells positive for the epithelial marker cytokeratin, and had few proliferating cells. In some tumors, packets of cells between the tubular structures were immunopositive for the granulosa cell marker FOXL2. Based on these findings, tumors were diagnosed as tubular adenomas or mixed tubular adenoma/granulosa cell tumors. In conclusion, charged-iron-particle-radiation induces ovarian tumors in mice, raising concerns about ovarian tumors as late sequelae of deep space travel in female astronauts.

CGMP and epigenetic factor in the suppression of apoptosis in ovarian follicles

The role of cGMP (cyclic guanosine monophosphate) in the suppression of apoptosis in preantral and preovulatory follicles has been established. But the underlying mechanisms have not yet been established. The present study has been focused on revealing the mechanisms involved in the cGMP mediated suppression of apoptosis and in survival of ovarian follicle. cGMP reduces calcium burden of the cell by mediating Ca2+ channels, BK (Big Potassium) channels, phosphatidylinositol 3-kinase (PI3K)/Akt pathway thus, prevents the mitochondrial membrane pore formation and apoptosis. cGMP induces the expression of Trx (thioredoxin) protein and prevents the TNF-α (Tumour Necrosis factor) induced apoptosis. cGMP also induces the expression of cytoskeleton proteins which are involved in heterogeneous distribution of mitochondria and there by reduces the Ca2+ burden and apoptosis. Histone 3 lysine 4 methyltransferase (MLL2) is an epigenetic factor inhibits the transcription of genes involved apoptosis which acts as co-activator of Estrogen receptor-α. In conclusion the present review predicts some mechanisms underlying the cGMP mediated suppression of apoptosis in ovarian follicles.

Sphingosine-1-phosphate, a protector against chemotherapy-induced ovarian follicle apoptosis, does not diminish the effectiveness of chemotherapy

It has recently been reported that S1P (sphingosine-1-phosphate), a ceramide-induced death pathway inhibitor, prevents the human primordial follicles from apoptosis during chemotherapy 1. We conducted this study to address the concern that Sphingosine-1 Phosphate (S1P) may shield the tumor, along with the primordial follicles, from cytotoxic effects of chemotherapy drugs.

Does tubal sterilization affect the ovarian follicle apoptosis? an experimental rat model

Does tubal sterilization affect the ovarian follicle apoptosis? an experimental rat model

Antiapoptotic and proliferative activity of curcumin on ovarian follicles in mice exposed to whole body ionizing radiation

The aim of this study was to evaluate the antiapoptotic and proliferative activity of curcumin (Cur) on the ovarian follicles in mice exposed to whole body ionizing radiation (Rd). The mice were exposed to 8.3 gray whole body Rd, and Cur groups were given as a daily dose of 100mg/kg of Cur for 10 days (10 days before Rd). The ovaries were collected 3 and 12h after irradiation. To date, no such studies have been performed on antiapoptotic and proliferative activity of Cur on the ovarian follicles in mice exposed to whole body Rd. Analysis of mice ovary after exposure to Rd by terminal-deoxynucleotidyl-transferase-mediated dUTP nick end labeling showed that there were apoptotic cells both in the follicular wall and the antrum, and that the number of follicles showing early atresic features was high 3h after Rd. On the other hand, analysis of mice ovary 12h after exposure to Rd showed that the number of follicles containing apoptotic cells with advanced atresic features was significantly higher when compared to the 3-h Rd exposure group. The proliferating cell nuclear antigen -positive granulosa cells were decreased in association with follicular atresia. The groups given treatment were observed to have some benefit from Cur against the damage caused by Rd. The results of this study demonstrate that Cur prevents follicular atresia in Rd-induced apoptosis in ovarian follicles.

Premature Ovarian Failure and Abnormal Preimplantation Development in Female Mice Deficient in Glutathione Synthesis.

Glutathione (GSH) is the most abundant intracellular non-protein thiol and an important regulator of cellular redox status. Mice that lack the modifier subunit of glutamate cysteine ligase (Gclm), the rate limiting enzyme in GSH synthesis, have decreased tissue levels of GSH. Our and others previous work supports roles for GSH in inhibiting apoptosis in ovarian follicles and in preimplantation embryonic development. We hypothesized that Gclm-/- mice have accelerated age-related declines in ovarian follicles and increased preimplantation embryonic demise. Ovarian total GSH concentrations in Gclm-/- mice were 27% of those in Gclm+/+ littermates. The ratio of reduced to oxidized glutathione was decreased in Gclm-/- ovaries, consistent with a state of relative ovarian oxidative stress. At 2 and 21 days of age, there were no differences in the numbers of oocytes and follicles in the ovaries of Gclm-/- and Gclm+/+ mice. However, by 3 months of age, there were significantly fewer follicles in Gclm-/- ovaries, and this difference became more pronounced with increasing age. A more rapid decline in primordial follicle numbers appeared to be driving the accelerated age-related decline in ovarian follicles in Gclm-/- mice. In a continuous breeding assay beginning at 2 months of age and lasting 20 weeks, Gclm-/- females produced similar numbers of litters as Gclm+/+ littermates, but the litters were significantly smaller. The numbers of oocytes ovulated in a natural estrous cycle or upon superovulation did not differ by genotype. A timed breeding study revealed fewer uterine implantation sites in the Gclm-/- females at gestational day 18 and no evidence for increased postimplantation mortality. We next investigated the effects of maternal lack of Gclm on early embryonic development in vivo and in vitro. Prepubertal Gclm-/- and Gclm+/+ females were superovulated with 5 IU eCG followed 46 h later by 5 IU hCG, then mated overnight with a Gclm+/+ male. The following morning, at 0.5 days postcoitum (dpc), Gclm-/- females had significantly lower percentages of zygotes with 2 pronuclei and significantly higher percentages of zygotes with one pronucleus and of unfertilized oocytes than Gclm+/+ females. At 3.5 dpc, a significantly lower percentage of blastocyst stage embryos and higher percentages of 3-cell and 8-cell embryos were recovered from the uteri of Gclm-/- females than of Gclm+/+ females. Total GSH concentrations in oocytes from superovulated Gclm-/- females were less than 20% those of wild type females. To test the effects of low oocyte GSH on in vitro embryo development, superovulated oocytes were harvested from Gclm-/- and Gclm+/+ females and were fertilized in vitro with sperm from wild type males; embryos were cultured for 102 h. There was no effect of Gclm genotype on progression to the 2-3 cell stage by 30 h, but significantly fewer embryos of Gclm-/- females progressed to the blastocyst stage by 102 h after gamete mixing. These results demonstrate critical roles for GSH in regulating the ovarian reserve of follicles during reproductive aging and in supporting normal preimplantation development. Supported by NIH AG032087. (platform)

Chronic cocaine exposure in Drosophila: Life, cell death and oogenesis

Developmental signaling cascades that can be perturbed by cocaine and other drugs of abuse have been difficult to study in humans and vertebrate models. Although numerous direct neural targets of cocaine have been elucidated at the molecular level, little is known about the specific cellular events that are impacted indirectly as a result of the drug's perturbation of neural circuits. We have developed oogenesis in Drosophila melanogaster as a model in which to identify downstream biochemical and/or cellular processes that are disrupted by chronic cocaine exposure. In this model, cocaine feeding resulted not only in expected reductions in viability, but also in unanticipated developmental defects during oogenesis, including aberrant follicle morphogenesis and vitellogenic follicle degeneration. To identify mechanisms through which cocaine exerted its deleterious effects on oogenesis, we examined candidate components of neural and hormonal signaling pathways. Cocaine-induced disruptions in follicle formation were enhanced by juvenile hormone exposure and phenocopied by serotonin feeding, while cocaine-activated follicle apoptosis was enhanced by concomitant dopamine feeding. HPLC analysis of dopamine and serotonin in the ovary suggests that these neurotransmitters could variably mediate cocaine's effects on oogenesis indirectly in the brain and/or directly in the ovary itself. We confirmed the involvement of hormone signaling by measuring ecdysteroids, which increase following cocaine exposure, and by demonstrating suppression of cocaine-induced follicle loss by hormone receptor mutants. Cocaine-induced ovarian follicle apoptosis and adult lethality appear to be caused by modulation of dopamine levels, while morphological defects during follicle formation likely result from perturbing serotonin signaling during cocaine exposure. Our work suggests not only a new role for juvenile hormone and/or serotonin in Drosophila ovarian follicle formation, but also a cocaine-sensitive role for dopamine in modulating hormone levels in the female fly.

Immune stimulation and malaria infection impose reproductive costs in Anopheles gambiae via follicular apoptosis

The employment of defense mechanisms is recognized as a costly life-history trait. In the malaria vector Anopheles gambiae, reproductive costs have been associated with both humoral and cellular innate immune responses and also with malaria infection. The resorption of developing oocytes associated with malaria infection is preceded by the programmed cell death, or apoptosis, of follicular cells. Here we demonstrate that apoptosis in ovarian follicular epithelial cells also occurs when mosquitoes are subjected to artificial immune-elicitors that induce a melanization response or humoral antimicrobial activity. Caspases are key cysteine proteases involved in apoptosis. Caspase-like activity was detected in epithelial cells in approximately 4.0% of the developing ovarian follicles of untreated, blood-fed, mosquitoes. Lipopolysaccharide injection resulted in a significant increase in anti- Micrococcus luteus humoral activity and a significant increase of 257.7% of follicles exhibiting apoptosis compared to results after saline injections. Melanization also triggered follicular apoptosis, which increased by 106.25% or 134.37% in Sephadex C-25 or G-25 bead-inoculated mosquitoes, respectively, compared to that in sham-injected ones. Ovaries from Plasmodium yoelii nigeriensis-infected mosquitoes exhibited a significant increase in follicular apoptosis of 440.9% compared to non-infected ones. Thus, at the time point investigated, infection had a much greater effect than artificial immune-elicitors. Death of follicular epithelial cells has been shown to lead to follicle resorption and hence a decrease in egg production. We propose the trade-off between reproductive fitness and immune defense in A. gambiae operates via the induction of apoptosis in ovarian follicles and that different immune responses impose costs via the same pathway.

Effects of cyclophosphamide and buthionine sulfoximine on ovarian glutathione and apoptosis

Treatment with the anticancer drug cyclophosphamide (CPA) destroys ovarian follicles. The active metabolites of CPA are detoxified by conjugation with glutathione (GSH). We tested the hypotheses that CPA causes apoptosis in ovarian follicles and that suppression of ovarian GSH synthesis before CPA administration enhances CPA-induced apoptosis. Proestrous rats were given two injections, 2 h apart, with (1) saline, then saline; (2) saline, then 50 mg/kg CPA; (3) saline, then 300 mg/kg CPA; or (4) 5 mmol/kg buthionine sulfoximine (BSO) to inhibit glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis, and then 50 mg/kg CPA. Statistically significantly increased DNA fragmentation by agarose gel electrophoresis and granulosa cell apoptosis by TUNEL were observed in the CPA-treated ovaries 24 h after the second injection, but BSO did not enhance the effect of 50 mg/kg CPA. We next tested the hypothesis that CPA depresses ovarian GSH concentration and expression of the rate-limiting enzyme in GSH synthesis, GCL. Proestrous rats were injected with 300 or 50 mg/kg CPA or vehicle and were sacrificed 8 or 24 h later. After CPA treatment, ovarian and hepatic GSH levels decreased significantly, and ovarian GCL subunit mRNA levels increased significantly. There were no significant changes in GCL subunit protein levels. Finally, we tested the hypothesis that GSH depletion causes apoptosis in ovarian follicles. Proestrous or estrous rats were injected with 5 mmol/kg BSO or saline at 0700 and 1900 h. There was a significant increase in the percentage of histologically atretic follicles and a nonsignificant increase in the percentage of apoptotic, TUNEL-positive follicles 24 h after onset of BSO treatment. Our results demonstrate that CPA destroys ovarian follicles by inducing granulosa cell apoptosis and that CPA treatment causes a decline in ovarian GSH levels. More pronounced GSH suppression achieved after BSO treatment did not cause a statistically significant increase in follicular apoptosis. Thus, GSH depletion does not seem to be the mechanism by which CPA causes follicular apoptosis.

Effects of cyclophosphamide and buthionine sulfoximine on ovarian glutathione and apoptosis

Treatment with the anticancer drug cyclophosphamide (CPA) destroys ovarian follicles. The active metabolites of CPA are detoxified by conjugation with glutathione (GSH). We tested the hypotheses that CPA causes apoptosis in ovarian follicles and that suppression of ovarian GSH synthesis before CPA administration enhances CPA-induced apoptosis. Proestrous rats were given two injections, 2 h apart, with (1) saline, then saline; (2) saline, then 50 mg/kg CPA; (3) saline, then 300 mg/kg CPA; or (4) 5 mmol/kg buthionine sulfoximine (BSO) to inhibit glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis, and then 50 mg/kg CPA. Statistically significantly increased DNA fragmentation by agarose gel electrophoresis and granulosa cell apoptosis by TUNEL were observed in the CPA-treated ovaries 24 h after the second injection, but BSO did not enhance the effect of 50 mg/kg CPA. We next tested the hypothesis that CPA depresses ovarian GSH concentration and expression of the rate-limiting enzyme in GSH synthesis, GCL. Proestrous rats were injected with 300 or 50 mg/kg CPA or vehicle and were sacrificed 8 or 24 h later. After CPA treatment, ovarian and hepatic GSH levels decreased significantly, and ovarian GCL subunit mRNA levels increased significantly. There were no significant changes in GCL subunit protein levels. Finally, we tested the hypothesis that GSH depletion causes apoptosis in ovarian follicles. Proestrous or estrous rats were injected with 5 mmol/kg BSO or saline at 0700 and 1900 h. There was a significant increase in the percentage of histologically atretic follicles and a nonsignificant increase in the percentage of apoptotic, TUNEL-positive follicles 24 h after onset of BSO treatment. Our results demonstrate that CPA destroys ovarian follicles by inducing granulosa cell apoptosis and that CPA treatment causes a decline in ovarian GSH levels. More pronounced GSH suppression achieved after BSO treatment did not cause a statistically significant increase in follicular apoptosis. Thus, GSH depletion does not seem to be the mechanism by which CPA causes follicular apoptosis.

Effects of a gonadotropin-releasing hormone agonist on rat ovarian follicle apoptosis: regulation by epidermal growth factor and the expression of Bcl-2-related genes.

The purpose of the present study was to evaluate the in vivo effect of the GnRH analogue leuprolide acetate (LA) on follicular development and apoptosis-related mechanisms in preovulatory ovarian follicles (POF) obtained from prepubertal eCG-treated rats. Serum progesterone and estradiol levels were measured, and a significant decrease in circulating estradiol levels was observed in the LA group, whereas serum progesterone levels remained unchanged. Ovarian histology revealed an inhibitory effect of LA treatment on the follicular development induced by eCG. After 48 h of LA treatment, the numbers of atretic and preantral follicles were increased as compared with controls, whereas the number of antral follicles had decreased. Cells undergoing DNA fragmentation were quantified by performing in situ 3' end labeling of DNA with digoxygenin-dUTP on ovarian sections. LA treatment caused an increase in the percentage of apoptotic cells in preantral and antral follicles. DNA isolated from these POF incubated 24 h in serum-free medium exhibited the typical apoptotic DNA degradation pattern. Treatment of follicles with epidermal growth factor (EGF) suppressed the spontaneous onset of DNA fragmentation, and a similar effect was observed in LA follicles. POF obtained from LA-treated rats showed no changes in Bcl-2 or Bax protein levels. However, a reduction in the Bcl-xL:Bcl-xS ratio was observed, with a greater decrease in Bcl-xL compared with Bcl-xS during the incubation, suggesting a lower stability of the Bcl-xL isoform in the LA group. These results indicate that in vivo GnRH agonist treatment produces an increase in the apoptosis process in POF from eCG-treated rats, and this effect is reversed in vitro by EGF. This GnRH analogue also reduced the stability of the Bcl-xL protein, thus interfering with follicular development by an as yet unknown mechanism.

Ovarian follicle apoptosis at the onset of standing estrus in virgin and repeat-breeder dairy heifers

There is evidence that repeat breeding in dairy cattle can be caused by both extrinsic, environmental factors and intrinsic, animal factors. In repeat-breeder heifers (RBH), disturbed endocrine patterns and estrous events result in a subsequent decreased fertility associated with delayed ovulation. Whether infertility is also due to the presence of an unsuitable follicular environment impairing normal fertilization, remains to be determined. At the onset of standing estrus, ovaries were obtained from 7 strictly defined RBH and 5 virgin heifers (VH) of the Swedish Red and White breed. Detection of apoptosis in the preovulatory and three subordinate follicle walls was done by using the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) technique at light microscopy level. The follicles were histologically assessed for degree of atresia. The ultrastructure of the follicle wall and recovered oocytes was studied using transmission electron microscopy. The overall degree of apoptosis in membrana granulosa and theca interna of preovulatory and subordinate follicles did not differ between RBH and VH, but the numbers of TUNEL-positive cells differed significantly between preovulatory and subordinate follicles in both RBH and VH. There was a strong relationship between density of apoptotic cells and degree of atresia. No differences in follicle wall apoptosis nor morphology were detectable, suggesting that repeat breeder heifers enter standing estrus with the same morphological prerequisites as normal animals, considering follicular structure.

Neuronal apoptosis inhibitory protein (NAIP) may enhance the survival of granulosa cells thus indirectly affecting oocyte survival

In mammals, the postnatal loss of more than 99% of female germ cells is due mainly to the process of ovarian follicular atresia. Atresia is known to be mediated by apoptotic granulosa cell-death. Here we show the involvement of neuronal apoptosis inhibitory protein (NAIP) in ovarian folliculogenesis in which it prevents granulosa cell-death. NAIP has been isolated in association with a neurodegenerative disorder, spinal muscular atrophy (SMA), in which it has been shown to suppress mammalian cell-death induced by a variety of stimuli (Liston et al., 1996, Nature 379:349–353). In an in situ hybridization analysis with mouse ovaries, active expression of NAIP mRNA was localized in the granulosa cells of developing follicles from the primary stage to the Graafian stages, whereas the NAIP gene was not expressed or was weakly expressed in follicles that might be undergoing atresia. Gonadotropin, which is known to inhibit apoptosis in ovarian follicles, caused a 2.4-fold increase in NAIP gene expression in the ovary. To study the role of ovarian NAIP, antisense NAIP oligonucleotides were locally delivered into the ovarian bursa. Suppression of ovarian NAIP expression with antisense oligonucleotides evoked a decrease in the number of morphologically normal ovulated oocytes, implying an indirect involvement of NAIP in germ cell development by enhancing the survival of granulosa cells. These findings suggest that gonadotropin-inducible NAIP may indirectly affect oocyte survival as a result of the inhibition of apoptotic granulosa cell-death during folliculogenesis. Mol. Reprod. Dev. 54:103–111, 1999. © 1999 Wiley-Liss, Inc.

Neuronal apoptosis inhibitory protein (NAIP) may enhance the survival of granulosa cells thus indirectly affecting oocyte survival.

In mammals, the postnatal loss of more than 99% of female germ cells is due mainly to the process of ovarian follicular atresia. Atresia is known to be mediated by apoptotic granulosa cell-death. Here we show the involvement of neuronal apoptosis inhibitory protein (NAIP) in ovarian folliculogenesis in which it prevents granulosa cell-death. NAIP has been isolated in association with a neurodegenerative disorder, spinal muscular atrophy (SMA), in which it has been shown to suppress mammalian cell-death induced by a variety of stimuli (Liston et al., 1996, Nature 379:349-353). In an in situ hybridization analysis with mouse ovaries, active expression of NAIP mRNA was localized in the granulosa cells of developing follicles from the primary stage to the Graafian stages, whereas the NAIP gene was not expressed or was weakly expressed in follicles that might be undergoing atresia. Gonadotropin, which is known to inhibit apoptosis in ovarian follicles, caused a 2.4-fold increase in NAIP gene expression in the ovary. To study the role of ovarian NAIP, antisense NAIP oligonucleotides were locally delivered into the ovarian bursa. Suppression of ovarian NAIP expression with antisense oligonucleotides evoked a decrease in the number of morphologically normal ovulated oocytes, implying an indirect involvement of NAIP in germ cell development by enhancing the survival of granulosa cells. These findings suggest that gonadotropin-inducible NAIP may indirectly affect oocyte survival as a result of the inhibition of apoptotic granulosa cell-death during folliculogenesis.

Paracrine mechanisms of ovarian follicle apoptosis

It has become evident that the physiological removal of cells through apoptosis during embryonic and postnatal development of multicellular organisms is a mandatory process to maintain a homeostatic state of the individual. In the ovary, massive cell death occurs during neonatal and postnatal life as an integral part of the normal ovarian development. At birth, mammalian ovaries are endowed with a fixed number of non-growing follicles that will be gradually recruited into a growing pool during reproductive life. Once follicles start growth they are either selected for ovulation or, for the majority of them, removed by apoptosis. Thus, removal of excess ovarian cells by apoptosis is necessary for normal development of the ovary. Despite the important role of follicle atresia in the maintenance of normal follicle development, studies on the hormonal control of follicle cell demise during follicle growth have not been possible until the recent development of apoptosis detection methods. Recent biochemical analysis has revealed the occurrence of internucleosomal DNA fragmentation, a hallmark of apoptosis, in atretic follicles and has facilitated the investigation into the intra-ovarian hormonal regulation of follicle atresia. This review summarizes the recent advances in the intra-ovarian hormonal mechanisms that control follicle apoptosis.