Atresia Of Antral Follicles Research Articles

Acute and Chronic Exposure to Phytoestrogens Disrupts Ovarian Follicle Maturation in Sprague-Dawley Rats.

The consumption of dietary phytoestrogens, such as isoflavones, has grown increasingly popular due to their potential health benefits. These soy products contain the isoflavones genistein, diadzein, and glycitein, which belong to a class of non-steroidal estrogen-like compounds called phytoestrogens. These naturally occurring plant estrogens may provide health-related benefits in some tissues; however, they may present deleterious side effects on estrogen-sensitive reproductive tissue such as the developing ovary. Several studies have documented that an in-utero and lactational exposure to estrogenic chemicals alters normal reproductive tissue development. This study was designed to determine (1) if an in-utero, lactational and chronic exposure to a low or high dose of a mixed isoflavone supplement (LSup and HSup) or a low dose of pure genistein (Gen) exerts detrimental effects on ovarian follicle maturation in the Sprague-Dawley rat; (2) whether estrogen concentrations are altered following acute or chronic exposure; and (3) whether estrous cycles are modulated during chronic exposure. Animals receiving an acute exposure were treated in utero and lactationally from gestational day 7 through postnatal day 28 (weaning). Animals receiving a chronic exposure were treated directly for an additional 65 days. Acute- and chronic-exposure groups were treated daily via oral gavage (LSup, HSup) or subcutaneous injection (Gen). LSup and HSup treatment groups received 2.4 mg/kg/day or 24 mg/kg/day, respectively. The Gen treatment group received 0.2 mg/kg/day. Acutely exposed pups were sacrificed on postnatal day 28 (chosen in order to follow the pups' exposure through lactation). Chronically exposed rats were sacrificed on postnatal day 93. Following sacrifice, the ovaries were excised, fixed for histology, and analyzed. The analysis included a count, and measurement and classification of preantral and antral follicles in the greatest cross-sectional area of the ovary. Blood samples collected at the time of sacrifice were analyzed for estrogen concentrations via radioimmunoassay. No statistical change in estrogen concentrations was noted among acute-treatment groups. Radioimmunoassay results for the chronically exposured groups indicated a significant difference in estrogen concentration between the control group and the other three treatment groups (p<0.001). For both the acute- and chronic-treatment groups, atresia levels and follicular count remained consistent in the control, LSup, and Gen treatment groups; however, increased atresia and decreased follicles were observed in the HSup treatment groups (p<0.05). Modulation of estrous cycles was noted in all four chronic treatment groups. These data support the hypothesis that isoflavones reduce the number of follicles of certain size classes in rats exposed during the critical period of development and during chronic exposure after birth. Atresia of antral follicles may constitute one of the underlying mechanisms by which folliculogenesis is modulated by phytoestrogens. The results of this study begin to elucidate the acute and chronic effects of a soy phytoestrogen on reproductive parameters in the female Sprague-Dawley rat. (platform)

Effect of Angiotensin II on Bovine Follicular Growth and mRNA Encoding Steroidogenic Enzymes, Gonadotrophin Receptors, and Tissue Development Genes.

Angiotensin II (AngII) has been shown to be involved in the regulatory mechanism of ovulation and oocyte nuclear and cytoplasmic maturation in cattle. In rats, AngII induces apoptosis in granulosa cells and atresia of antral follicles during follicular development. However, the angiotensin converting enzyme (ACE) mRNA expression increases at the end of follicular maturation, suggesting a possible modulatory role of AngII for ovulation in cows. In addition, the expression of AngII type 2 receptors (AGTR2) is higher in healthy than in atretic follicles and, in vitro, is stimulated by FSH in bovine granulosa cell culture. Based on this evidence, this study was carried out to establish the role of AngII during the follicular growth and the effect of AngII inhibition on steroid secretion and follicular cell gene expression profile. Cows were pre-synchronized to induce a new follicular wave and the largest follicle was injected with saralasin (AngII inhibitor; 10μM), AngII (10μM) or saline when it reached 7-8mm diameter. The intrafollicular injections were performed through a double-needle system and guided by a 7.5 MHz vaginal ultrasound probe. All follicles were monitored by ultrasonography for 96 hours after intrafollicular injection. Follicles injected with saline (control group) reached ovulatory size and ovulated after a challenge with the GnRH agonist gonadorelin, demonstrating that the intrafollicular injection did not affect follicular growth. Saralasin inhibited follicular growth in all treated cows (4/4), suggesting that AngII is essential for follicular growth in 7-8mm follicles. However, intrafollicular injection of AngII affected neither follicular growth nor the pattern of follicular dynamics, which were similar to control cows. These results imply that bovine ovarian follicles contain sufficient AngII for follicle development. In a second experiment, a single follicle of each cow was injected with saralasin or saline and cows were ovariectomized 24h later. The follicular fluid was aspirated to determine steroid concentrations, and granulosa and theca cells were recovered for gene expression measurement. Estradiol concentration in follicular fluid was determined by radioimmunoassay. Messenger RNA levels were analyzed by real-time RT-PCR and are expressed relative to cyclophilin. The inhibition of AngII action decreased abundance of mRNA encoding aromatase (CYP19), 3β-hydroxysteroid dehydrogenase (3β HSD), LH receptor, SerpinE2 and cyclinD2 in granulosa cells but not StAR, 17β HSD, FSH receptor, growth arrest and DNA damage inducible (GADD45b) or X-linked inhibitor of apoptosis protein (XIAP). In theca cells, the inhibition of AngII decreased the expression of AGTR2 but not the expression of steroidogenic enzymes genes. Taken together, these results show that AngII is essential for follicular growth, and plays important role in regulating genes involved in granulosa cell proliferation and differentiation, which are necessary for development of the dominant follicle. These data suggest that AngII signaling is involved in follicle growth and dominance in cattle. (poster)

Phthalate Esters Affect Expression of Aromatase and Cell Cycle Regulators in Mouse Ovarian Antral Follicles.

Phthalate esters (PEs) are synthetic plasticizers used for a wide range of consumer and industrial products including toys, bottles, household vinyl, automobiles, and medical devices. Previous studies have shown that chronic exposure to PEs is associated with lowered fertility in humans and animal models. For example, chronic exposure to PEs was associated with decreased pregnancy rates in women. Di-(2-ethylhexyl) phthalate (DEHP) caused decreased serum estradiol levels, prolonged estrous cycles, and anovulation in adult cycling rats. Monoethylhexyl phthalate (MEHP), the active metabolite of DEHP, decreased aromatase (Arom) expression in cultured rat granulosa cells. These observations indicate that PEs could target antral follicles in the ovary. Major functions of antral follicles are to produce oocytes and estrogen. Mammalian females are born with a finite number of primordial follicles, of which a small fraction survive and reach the penultimate antral stage, while most die via apoptosis. Any insult that affects the survival of antral follicles can cause fertility problems. Our previous in vitro studies have shown that PEs inhibited antral follicle growth and induced atresia by reducing the levels of estradiol. Further, we have also shown that estradiol supplement partially prevented growth inhibition induced by PEs and fully protected follicles from PE-induced atresia. The mechanism of these PE actions on follicles is unknown. Therefore, the goal of this study was to test the hypothesis that PEs (specifically MEHP and DEHP) act by reducing the expression of aromatase and cell cycle regulators in antral follicles. Antral follicles from adult cycling CD-1 mice were isolated and cultured in α-minimal essential media with or without MEHP (10μg/ml) or DEHP (100 μg/ml) and estradiol (E; 1 and 10 nM) for 96 hr. Dimethyl sulfoxide (DMSO) was used as vehicle control. Follicles without any treatment were also included to monitor the culture conditions. After culture, follicles were processed for qPCR to measure the levels of Arom, cyclin D2 (CCND2), and cyclin dependant kinase 4 (CDK4) genes. We found that DEHP and MEHP significantly reduced the expression of Arom compared to DMSO-treated controls and estradiol restored the expression of Arom in DEHP treated group (DEHP: DMSO=5.77±0.85, DEHP=0.70±0.09. E1nM=5.51±0.21, E10nM=6.89±2.09, E1nM+DEHP=4.09±0.68, and E10nM+DEHP=5.08±0.96; but not in the MEHP-treated group MEHP: DMSO=3.64±0.15, MEHP=0.41±0.05. E1nM=3.23±0.46, E10nM=2.81±0.38, E1nM+MEHP=0.84±0.07, and E10nM+MEHP=0.83±0.09; n=3; p≤0.05). Further, DEHP and MEHP significantly decreased the CCND2 and CDK4 expression compared to DMSO treated controls and estradiol restored the expression of CCND2 and CDK4 in DEHP and MEHP treated groups (CCND2; for DEHP: DMSO=1.96±0.44, DEHP=0.61±0.02. E1nM=2.32±0.15, E10nM=2±0.29, E1nM+DEHP=2.04±0.17, E10nM+DEHP=2.05±0.12, and for MEHP: DMSO=2.67±0.23, MEHP=0.69±0.10. E1nM=2.94±0.72, E10nM=2.91±0.34, E1nM+MEHP=1.89±0.48, and E10nM+MEHP=1.63±0.12; CDK4; for DEHP: DMSO=3.20±0.73, DEHP=0.60±0.09. E1nM=2.35±0.05, E10nM=2.71±0.35, E1nM+DEHP=2.53±0.08, E10nM+DEHP=2.49±0.36, and for MEHP: DMSO=1.75±0.21, MEHP=0.41±0.05. E1nM=2.19±0.15, E10nM=2.29±0.35, E1nM+MEHP=1.96±0.36, E10nM+MEHP=1.55±0.10 n=3; p≤0.05). Collectively, these data indicate that DEHP and MEHP may inhibit the growth and induce atresia of antral follicles by affecting the estradiol biosynthesis and the cell cycle regulators. Support: NIHHD46861 (poster)

Methoxychlor and Its Metabolites Inhibit Growth and Induce Atresia of Baboon Antral Follicles

Methoxychlor (MXC), an organochlorine pesticide, inhibits growth and induces atresia of antral follicles in rodents. MXC metabolites, mono-OH MXC (mono-OH) and bis-OH MXC (HPTE), are thought to be more toxic than the parent compound. Although studies have examined effects of MXC in rodents, few studies have evaluated the effects of MXC in primates. Therefore, the present study tested the hypothesis that MXC, mono-OH, and HPTE inhibit growth and induce atresia of baboon antral follicles. To test this hypothesis, antral follicles were isolated from adult baboon ovaries and cultured with vehicle (dimethylsulfoxide; DMSO), MXC (1-100 micro g/ml), mono-OH (0.1-10 micro g/ml), or HPTE (0.1-10 micro g/ml) for 96 hr. Growth was monitored at 24 hr intervals. After culture, follicles were processed for histological evaluation of atresia. MXC, mono-OH, and HPTE significantly inhibited follicular growth and increased atresia compared to DMSO. Moreover, the adverse effects of MXC and its metabolites on growth and atresia in baboon antral follicles were observed at lower (100-fold) doses than those causing similar effects in rodents. These data suggest that MXC and its metabolites inhibit growth and induce atresia of baboon antral follicles, and that primate follicles are more sensitive to MXC than rodent follicles.

Methoxychlor causes mitochondrial dysfunction and oxidative damage in the mouse ovary☆

Methoxychlor (MXC) is an organochlorine pesticide that reduces fertility in female rodents by causing ovarian atrophy, persistent estrous cyclicity, and antral follicle atresia (apoptotic cell death). Oxidative damage resulting from reactive oxygen species (ROS) generation has been demonstrated to lead to toxicant-induced cell death. Thus, this work tested the hypothesis that MXC causes oxidative damage to the mouse ovary and affects mitochondrial respiration in a manner that stimulates ROS production. For the in vitro experiments, mitochondria were collected from adult cycling mouse ovaries, treated with vehicle (dimethyl sulfoxide; DMSO) or MXC, and subjected to polarographic measurements of respiration. For the in vivo experiments, adult cycling CD-1 mice were dosed with either vehicle (sesame oil) or MXC for 20 days. After treatment, ovarian mitochondria were isolated and subjected to measurements of respiration and fluorimetric measurements of H2O2 production. Some ovaries were also fixed and processed for immunohistochemistry using antibodies for ROS production markers: nitrotyrosine and 8-hydroxy-2'-deoxyguanosine (8-OHG). Ovaries from in vivo experiments were also used to measure the mRNA expression and activity of antioxidants such as Cu/Zn superoxide dismutase (SOD1), glutathione peroxidase (GPX), and catalase (CAT). The results indicate that MXC significantly impairs mitochondrial respiration, increases production of H2O2, causes more staining for nitrotyrosine and 8-OHG in antral follicles, and decreases the expression and activity of SOD1, GPX, and CAT as compared to controls. Collectively, these data indicate that MXC inhibits mitochondrial respiration, causes ROS production, and decreases antioxidant expression and activity in the ovary, specifically in the antral follicles. Therefore, it is possible that MXC causes atresia of ovarian antral follicles by inducing oxidative stress through mitochondrial production of ROS.

Hormonal induction of ovulation stimulates atresia of antral follicles in a vespertilionid bat, Scotophilus heathi

Scotophilus heathi is a seasonally monoestrous subtropical vespertilionid bat found at Varanasi, India. Although the antral follicles remain present in the ovaries of S. heathi from November till March, ovulation is delayed in this species until early March. In order to understand the mechanism of ovulation suppression during this period of delayed ovulation, the effects of human chorionic gonadotropin (hCG), pregnant mare's serum gonadotropin (PMSG), follicle stimulating hormone (FSH) and gonadotropin releasing hormone agonist (GnRH agonist) on ovarian morphology and steroid concentration were investigated. Hormonal treatments were given as a single i.p. dose 24 h after capture. The bats were sacrificed 48 h after the injection. Treatment with hCG, PMSG, FSH and GnRH agonist failed to induce ovulation in S. heathi, although these hormones produced a high degree of ovarian stimulation. The administration of hCG and PMSG induced ovarian enlargement, intense hyperemia, marked changes in the interstitial cells (ICs), development of several antral follicles and a varying degree of abnormalities in the oocytes of most of the antral follicles. In the bats treated with hCG, PMSG and GnRH agonist, androstenedione concentration increased significantly to extraordinarily high levels, whereas estradiol concentration decreased. Administration of FSH caused regression of ICs and pyknosis of granulosa cells in the majority of antral follicles. FSH did not enhance androstenedione concentration. The results of the present study suggest that the failure of hormonal treatments to induce ovulation during the period of delayed ovulation might be due to a seasonal desensitization of ovarian follicles in S. heathi. The hormonal treatment instead stimulated the ICs to produce a high level of androstenedione resulting in atretic changes of the antral follicles.

Methoxychlor Inhibits Growth and Induces Atresia of Antral Follicles through an Oxidative Stress Pathway

The mammalian ovary contains antral follicles, which are responsible for the synthesis and secretion of hormones that regulate estrous cyclicity and fertility. The organochlorine pesticide methoxychlor (MXC) causes atresia (follicle death via apoptosis) of antral follicles, but little is known about the mechanisms by which MXC does so. Oxidative stress is known to cause apoptosis in nonreproductive and reproductive tissues. Thus, we tested the hypothesis that MXC inhibits growth and induces atresia of antral follicles through an oxidative stress pathway. To test this hypothesis, antral follicles isolated from 39-day-old CD-1 mice were cultured with vehicle control (dimethylsulfoxide [DMSO]), MXC (1-100 microg/ml), or MXC + the antioxidant N-acetyl cysteine (NAC) (0.1-10 mM). During culture, growth was monitored daily. At the end of culture, follicles were processed for quantitative real-time polymerase chain reaction of Cu/Zn superoxide dismutase (SOD1), glutathione peroxidase (GPX), and catalase (CAT) mRNA expression or for histological evaluation of atresia. The results indicate that exposure to MXC (1-100 microg/ml) inhibited growth of follicles compared to DMSO controls and that NAC (1-10 mM) blocked the ability of MXC to inhibit growth. MXC induced follicular atresia, whereas NAC (1-10 mM) blocked the ability of MXC to induce atresia. In addition, MXC reduced the expression of SOD1, GPX, and CAT, whereas NAC reduced the effects of MXC on their expression. Collectively, these data indicate MXC causes slow growth and increased atresia by inducing oxidative stress.

Methoxychlor Directly Affects Ovarian Antral Follicle Growth and Atresia through Bcl-2- and Bax-Mediated Pathways

Methoxychlor (MXC) is an organochlorine pesticide and reproductive toxicant. While in vivo studies indicate that MXC exposure increases antral follicle atresia, in part by altering apoptotic regulators (Bcl-2 and Bax), they do not distinguish whether MXC does so via direct or indirect mechanisms. Therefore, we utilized an in vitro follicle culture system to test the hypothesis that MXC is directly toxic to antral follicles, and that overexpression of anti-apoptotic Bcl-2, or deletion of pro-apoptotic Bax, protects antral follicles from MXC-induced toxicity. Antral follicles were isolated from wild-type (WT), Bcl-2 overexpressing (Bcl-2 OE), or Bax deficient (BaxKO) mice, and exposed to dimethylsulfoxide (control) or MXC (1-100 microg/ml) for 96 h. Follicle diameters were measured every 24 h to assess growth. After 96 h, follicles were histologically evaluated for atresia or collected for quantitative PCR analysis of Bcl-2 and Bax mRNA levels. MXC (10-100 microg/ml) significantly inhibited antral follicle growth at 72 and 96 h, and increased atresia (100 microg/ml) compared to controls at 96 h. Furthermore, MXC increased Bax mRNA levels between 48-96 h and decreased Bcl-2 mRNA levels at 96 h. While MXC inhibited growth of WT antral follicles beginning at 72 h, it did not inhibit growth of Bcl-2 OE or BaxKO follicles until 96 h. MXC also increased atresia of small and large WT and BaxKO antral follicles over controls, but it did not increase atresia of large Bcl-2 OE antral follicles over controls. These data suggest that MXC directly inhibits follicle growth partly by Bcl-2 and Bax pathways, and increases atresia partly through Bcl-2 pathways.

Follicular expression of a human β-cell leukaemia/lymphoma-2 (Bcl-2) transgene does not decrease atresia or increase ovulation rate in swine

Transgenic (TG) gilts carrying a human Bcl-2 cDNA transgene driven by mouse inhibin-alpha subunit promoter were produced and evaluated to determine if ectopic expression of Bcl-2 in the ovaries would decrease the frequency of atresia in antral follicles and increase ovulation rate. Immunohistochemical analysis showed that the Bcl-2 transgene protein was expressed in granulosa and theca cells, in 86% of healthy and 54% of atretic follicles analysed in TG prepubertal and Day 50 pregnant gilts combined (n = 24). In contrast, Bcl-2 transgene protein was expressed in only 1.4% of healthy and 0% of atretic follicles in non-TG littermates (n = 13). Real-time reverse transcription-polymerase chain reaction analysis confirmed that human Bcl-2 was expressed in follicles of TG gilts. The atresia rate for the TG and non-TG groups did not differ (P > 0.05) for prepubertal (45 v. 59%) and Day 50 pregnant gilts (53 v. 52%) respectively. The mean +/- s.e.m. ovulation rate did not differ (P > 0.5) between TG (15.9 +/- 0.8, n = 12) and non-TG (16.4 +/- 0.6, n = 7) Day 50 pregnant gilts. The molecular basis of the failure of ectopic Bcl-2 expression to increase the ratio of healthy to atretic follicles is unknown, but it is possible that the activity of the mitochondrial-dependent cell death pathway was not neutralized by ectopic expression of human Bcl-2 or that other cell death pathways compensated for the decreased mitochondrial-dependent cell death.

Methoxychlor may cause ovarian follicular atresia and proliferation of the ovarian epithelium in the mouse.

Methoxychlor (MXC) is currently used to protect agricultural products from insects. Previous studies show that MXC adversely affects the ovary, but the target cells were not revealed by those studies. Therefore, the purpose of this study was to test the hypothesis that MXC induces ovarian changes by adversely affecting the antral follicles and the ovarian surface epithelium in the mouse. To test this hypothesis, cycling female CD-1 mice (39 days) were dosed with MXC (8, 16, or 32 mg/kg/day), kepone (KPN, 8 mg/kg/day, positive control), or sesame oil (vehicle control) via intraperitoneal injection for 10 or 20 days. Estrous cyclicity was evaluated daily via vaginal lavage. After dosing, ovaries were collected for histological evaluation of follicle numbers, atresia, and surface epithelial height. The results indicate that at the 20-day time point, MXC (32 mg/kg) and KPN (8 mg/kg) increased the percentage of atretic antral follicles (n= 4-9,p<or= 0.001). MXC (32 mg/kg) also increased the height of the ovarian surface epithelium compared with controls (n= 7-10,p<or= 0.045), and KPN increased the percentage of days in estrus (n= 6-10,p<or= 0.0001). These data suggest that MXC and KPN increase antral follicle atresia, MXC increases surface epithelial height, and KPN affects vaginal cytology.

Effect of immunization against the amino-terminal peptide (alpha N) of the alpha 43-subunit of inhibin on follicular atresia and expression of tissue inhibitor of matrix metalloproteinase (TIMP-1) in ovarian follicles of sheep.

Ewes actively immunized against alpha N, the N-terminal peptide of inhibin alpha 43 precursor, have lowered fertility associated with ovulation failure, restricted tissue remodelling and reduced matrix metalloproteinase-2 activity in the follicular fluid at the time of expected ovulation. This could be due to altered ratios of matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase (TIMP-1), or to the onset of atresia in antral follicles destined to ovulate. The objectives of the present study were to investigate the effects of immunization against alpha N on the localization of TIMP-1 in ovine follicles, and on follicular growth and atresia in the follicular phase. Ewes were either immunized against alpha N or remained as controls and the ovaries were removed before (0, n = 4) and at 12 h (n = 4) and 24 h (n = 4) after hCG administration in a synchronized follicular phase, 48 h after removal of intravaginal pessaries. Observations were made on a single section taken through the largest follicle present in the ovaries of each ewe. There were no healthy antral follicles > 1 mm in immunized ovaries (0/29) compared with controls (16/31) (P < 0.001), whereas the proportion of healthy antral follicles < 1 mm was the same in each group (9/19 versus 5/12). TIMP-1 immunoactivity was localized in large luteal cells, smooth muscle and endothelial cells, and in all antral follicles, including oocytes. At the time of hCG administration, no TIMP-1 immunoreactivity was detected in the apical region of the follicular wall of large follicles (> 6 mm) compared with the rest of the follicle wall, but staining appeared in the apical granulosa layer 24 h later. In newly formed corpora lutea, TIMP-1 expression was found along the invaginating vascular layer. There was no effect of immunization on the patterns of TIMP-1 immunoreactivity, suggesting that changes in TIMP-1 are not involved in the effects of alpha N. These data are consistent with a paracrine role for alpha N in the selection and atresia of antral follicles, and for TIMP-1 in tissue reorganization and steroidogenesis at the time of ovulation.

Temporal Changes in Inhibin, Steroid Hormones, and Steroidogenic Enzymes during Induced Follicular Atresia in the Hypophysectomized Cyclic Hamster1

The time course for loss of ability of Graafian follicles to secrete inhibin and estradiol was investigated during induced follicular atresia. Cyclic hamsters were hypophysectomized on Day 1 (estrus) and injected s.c. with 30 IU eCG. Thereafter, these animals were given a single i.p. injection of antiserum to eCG on the morning of Day 4 to induce follicular atresia in a rapid and predictable manner. A drastic fall in plasma levels of estradiol and testosterone was noted within 1 h, whereas relatively high levels of plasma inhibin were maintained until 12 h, followed by an abrupt decline by 24 h. The first histological signs of pyknosis in granulosa cells appeared by 4 h, and breakdown of the mural granulosa layer was observed in most follicles by 8-12 h after immunoneutralization of circulating eCG. According to immunohistochemical analysis, inhibin activity was unchanged in granulosa cells at 12 h followed by a slight decline by 24 h, whereas positive reaction for aromatase in these cells rapidly declined by 8 h. Immunoreactivity of 17alpha-hydroxylase/C17,20-lyase (CYP 17) was also reduced in theca cells by 8 h. These results indicate that granulosa cells continue to secrete inhibin during the process of follicular atresia, although these cells quickly shut off the secretion of estradiol, and theca cells shut off the secretion of testosterone. The present results indicate as well that a rapid decline of estradiol and testosterone in plasma is an early sign of atresia in antral follicles. These results, therefore, suggest that the loss of enzymatic activity of aromatase in granulosa cells and CYP 17 in theca cells is a part of the process of follicular atresia.

Expression of messenger ribonucleic acids of insulin-like growth factor binding protein-2, -4, and -5 in the ovine ovary: localization and changes during growth and atresia of antral follicles.

In the sheep as in many mammalian species, growth and atresia of antral follicles are characterized, respectively, by a decrease and a high increase in the intrafollicular levels of insulin-like growth factor binding proteins of less than 40 kDa (IGFBPs < 40 kDa), mainly IGFBP-2, -4, and -5. The objective of this study was to investigate whether such changes are associated with changes in follicular expression of the corresponding mRNA. For this purpose, ovaries were recovered from ewes slaughtered at the end of follicular phase (i.e., 30 h after progestagen sponge removal; control ewes) or at 24 h, 36 h or 72 h after hypophysectomy (hypox) performed 30 h after sponge removal. The expression of mRNA of IGFBPs of less than 40 kDa (IGFBPs < 40 kDa mRNA) was studied in ovine antral follicles from control and hypox ewes by in situ hybridization using [35S]-labeled human IGFBP-2, -4, and -5 cRNA as probes. In control ewes, IGFBP-2 mRNA was mainly expressed in granulosa as a gradient in healthy follicles, the expression being higher in granulosa cells close to the basal membrane than in granulosa cells bordering the antrum and within the cumulus. The level of IGFBP-2 mRNA was lower both in granulosa cells close to the basal membrane and in those bordering the antrum from small follicles than in the corresponding compartments of granulosa cells from large healthy follicles (p < 0.05). In healthy follicles, IGFBP-4 and -5 mRNA were mainly expressed in thecal cells. No change in level of IGFBP-4 mRNA was observed between small and large follicles, whereas the level of IGFBP-5 mRNA tended to be lower in thecal cells from large compared to small follicles (p = 0.055). In atretic follicles, expression of IGFBPs < 40 kDa mRNA strongly increased in granulosa (IGFBP-2 and -5, p < 0.01) and in thecal cells (IGFBP-2 and -4, p < 0.01). In hypox ewes, the chronology of changes in expression of follicular IGFBPs < 40 kDa mRNA and in intrafollicular levels of the corresponding proteins was studied during atresia of large antral follicles. Early atresia of large follicles was associated with a strong decrease in intrafollicular estradiol levels (p < 0.001); an increase in intrafollicular levels of IGFBP-2, -4, and -5 (p < 0.001) an increase in both IGFBP-2 (p < 0.001) and -5 (p < 0.01) mRNA expression in granulosa and thecal cells; but no changed in IGFBP-4 mRNA expression. Late atresia of large follicles was associated with a further decrease in intrafollicular estradiol levels (p < 0.01); a further increase in intrafollicular levels of IGFBP-2, -4, and -5 (p < 0.001); an increase in IGFBP-4 (p < 0.01) and -5 (p < 0.05) mRNA expression in theca and granulosa, respectively; a decrease in IGFBP-5 mRNA expression in theca (p < 0.05); but no further increase in IGFBP-2 mRNA expression. Overall, these data suggest that the decrease and the increase in expression of mRNA of follicular IGFBPs < 40 kDa during follicular growth and atresia, respectively, are involved in the decrease and the increase in intrafollicular levels of the corresponding proteins. Moreover, the increases in expression of follicular IGFBPs < 40 kDa during atresia of large follicles in hypophysectomized ewes followed a specific time course, the increase in IGFBP-2 and -5 mRNA expression being early than the increase in IGFBP-4 mRNA expression.

Hormonal Induction of Ovulation Stimulates Atresia of Antral Follicles in Gilts1

Two breeds of prepubertal gilts known to differ in their ovarian development were used to compare the effect of hormonally stimulated ovulation on follicle numbers and proportion of follicular atresia in size classes other than preovulatory follicles. Hormonal treatments (hCG or eCG-hCG) were given to Meishan gilts (n = 36) at 3, 2, and 1 standard deviations (SD) before the mean age of puberty (51, 64, and 77 days of age) and to Large White gilts (n = 24) at 3 and 1 SD before the mean age of puberty (140 and 166 days of age). Ovarian follicle populations determined in the right ovaries 18 h after hCG injection alone (hCG) or 72 h after eCG injection (eCG-hCG) were compared with those in the left ovaries, which had been removed prior to injections. Follicles with dispersed cumulus cells around the oocytes (expected ovulations) observed after hormonal treatments (right ovaries) were considered for follicular measures and categorized into specific size classes according to their diameter. Human CG alone did not affect any follicular parameters measured at any age in either of the two breeds studied. The overall population of nonatretic follicles was significantly reduced by the eCFG-hCG treatment in the Large White breed when imposed at either 3 or 1 SD before the mean age of puberty (262.6 vs. 158.8; p < 0.01). Among the various follicle size classes studied, eCG-hCG treatment significantly decreased the mean number of follicles in size classes 2 (1.13-2.00 mm in diameter) and 3 (2.01-3.56 mm in diameter) at either 3 or 1 SD before the mean age of puberty in the Large White gilts (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of members of the bcl-2 gene family in the immature rat ovary: equine chorionic gonadotropin-mediated inhibition of granulosa cell apoptosis is associated with decreased bax and constitutive bcl-2 and bcl-xlong messenger ribonucleic acid levels.

The loss of ovarian follicles through atresia, which accounts for greater than 99% of postnatal female germ cell depletion, is mediated through apoptotic cell death. Although recent data have shown that apoptosis in granulosa cells of ovarian follicles can be hormonally manipulated, the molecular mechanisms responsible for transducing external signals remain to be elucidated. Herein we have characterized changes in the expression of the bcl-2 protooncogene (an inhibitor of apoptosis), the bax gene (an inducer of apoptosis), and the bcl-x gene (which encodes both bcl-xlong, an inhibitor of apoptosis, and bcl-xshort, an inducer of apoptosis) during gonadotropin-stimulated follicular development in vivo and during atresia of antral follicles incubated in vitro. Complementary DNA fragments corresponding to rat bcl-2, rat bax, and rat bcl-x coding sequences were obtained by the reverse transcription-polymerase chain reaction (RT-PCR) technique using total RNA prepared from immature rat ovaries. Northern blot analysis of steady-state bcl-2, bax, and bcl-x messenger RNA (mRNA) levels in total RNA prepared from ovaries of immature rats before and after in vivo priming with 10 IU equine CG (eCG) revealed that eCG-induced follicular growth and survival were associated with a relatively constitutive level of bcl-2 and bcl-x expression but markedly reduced levels of bax mRNA (29 +/- 5% of saline-treated control animals). Using the RT-PCR technique coupled with Southern blot hybridization analysis to distinguish the long vs. short forms of bcl-x (which differ in size by 189 base pairs), it was determined that bcl-xlong was the predominant message expressed by granulosa cells of eCG-primed ovaries. The eCG-mediated decrease in bax expression, coupled with a maintenance of bcl-2 and bcl-xlong mRNA levels, were associated with a pronounced reduction in the extent of granulosa cell apoptosis. In contrast, the induction of apoptosis in a homogeneous population of healthy antral follicles by incubation in vitro without tropic support was associated with an significant increase in bax mRNA levels to 220 +/- 10% of those detected in freshly isolated, healthy follicles. Moreover, bcl-xlong message levels were significantly reduced in follicles incubated for 24 h to 69 +/- 4% of those levels detected in freshly isolated, healthy follicles. However, no significant change in the level of bcl-2 mRNA was detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Folliculogenesis in hypophysectomized rats after treatment with recombinant human follicle-stimulating hormone.

To examine the role of FSH and LH in follicular growth and atresia, immature hypophysectomized (hypox) rats were treated twice daily for four days with a total dose either of 2.5 to 40 IU recombinant human FSH (recFSH; Org 32489) or of 8 IU recFSH supplemented with 0.2 to 5 IU hCG. RecFSH alone caused dose-dependent increases in ovarian weight and intraovarian estradiol (E2) but was unable to elevate circulating E2 levels. The number of antral follicles was also increased in a recFSH dose-dependent manner, and a gradual shift of small antral follicles to large preovulatory follicles was noted. The latter ovulated after a single bolus injection of 10 IU hCG. In comparison with follicles from hypox vehicle-treated animals, these follicles showed a diminished incidence of atresia, especially in the smallest size class of antral follicles. A total dose of > or = 10 IU recFSH increased uterine weight accompanied by endometrium proliferation. When 8 IU recFSH was supplemented with 0.2 to 5 IU hCG, ovarian weight was augmented in an hCG dose-dependent fashion, but no further increases in total number of antral follicles were noted except with the highest hCG dose given. Nevertheless, addition of relatively low doses of hCG caused considerable shifts of small follicles to large, preovulatory follicles. Furthermore, supplementation with hCG, especially low dosages of hCG (0.2 and 0.5 IU), reduced the incidence of atresia in antral follicles of all size classes. These data suggest that in the complete absence of LH activity, recFSH induces follicular growth up to the stage of mature preovulatory follicles and induces ovarian estradiol production and endometrium proliferation. The addition of small amounts of LH activity increases the percentage of healthy follicles.

Synergistic effects of steroids with FSH on folliculogenesis, steroidogenesis and FSH- and hCG-receptors in hypophysectomized mice

Injection of ovine FSH (4 micrograms day-1) for 4 days into hypophysectomized mice does not restore folliculogenesis to normal cyclic values. This may be due to insufficient production of oestradiol. The present study was designed to determine whether FSH- and LH-induced oestradiol was critical for growth and differentiation of follicles at all stages. Twelve days after hypophysectomy, mice were injected s.c. with 10, 50 or 250 micrograms oestradiol cyclopentylpropionate daily with or without ovine FSH (4 micrograms day-1) for 1-4 days. One ovary from each animal was used for histology. From the second ovary, follicles were isolated at different stages and incubated with [3H]thymidine for 3 h to determine the rate of DNA synthesis. Incubation medium and serum were used for steroid determinations. After oestradiol treatment alone, there was a dose-dependent response in serum oestradiol, but ovarian and uterine weights did not increase further with the increasing doses of oestradiol administered. This finding was consistent with an increase in the number of preantral follicles and small antral follicles but excluding the development of preovulatory follicles. Treatment with 10 and 50 micrograms of oestradiol cyclopentylpropionate did not prevent antral follicles from undergoing atresia. The higher dose (250 micrograms day-1) of oestradiol cyclopentylpropionate delayed atresia of antral follicles and maintained more large healthy antral follicles. After concurrent injection of oestradiol and FSH, ovarian weight was 2-3 times greater than with either FSH or oestradiol alone; the number of follicles and follicular DNA synthesis at all stages of development increased without any signs of atresia; the in vitro accumulation of oestradiol also increased. Oestradiol alone induced FSH receptors in granulosa cells, but did not induce hCG receptors in any ovarian compartment; FSH alone induced FSH and hCG receptors in granulosa cells but not in thecal-interstitial tissues, whereas, oestradiol plus FSH enhanced FSH receptors in granulosa cells and LH/hCG receptors in granulosa and thecal-interstitial tissues. The synergistic effect of oestradiol with FSH was mimicked by the same dose of diethylstilboestrol, testosterone or dihydrotestosterone, but the latter steroids increased only the number of antral follicles, presumably because of their shorter half-lives. These results indicate that in mice oestradiol stimulates the growth of preantral and antral follicles and delays follicular atresia; oestrogens and androgens act synergistically with FSH to enhance follicular proliferation and differentiation, and prevent follicles from undergoing atresia.

Relative levels of thecal blood flow in atretic and non-atretic ovarian follicles of the conscious sheep.

The relative level of blood flow through thecal capillaries (RTF) in individual atretic and non-atretic follicles greater than 1 mm was measured in conscious ewes by injecting 15 micron radioactive microspheres into the aorta near the origin of the ovarian artery, and determining the ratio of radioactivity per mg protein in theca of individual follicles to that in the non-luteal portion (stroma plus follicles less than 1 mm diameter) of the corresponding ovary. In follicles greater than 1 mm diameter, RTF in those showing advanced stages of atresia (stages IV and V) was significantly less than that in non-atretic (stage I) follicles. RTF values in non-atretic (stage I), early atretic (stage II) and moderately atretic (stage III) follicles did not differ significantly. There was no effect of follicular diameter (P greater than 0.05) on the relative level of blood flow within individual thecae or of atresia on thecal protein content. These data indicate that reduced thecal blood flow is associated with advanced atresia of antral follicles and are in accordance with the findings of previous histological studies. Whether thecal ischaemia is a factor involved in the onset of atresia, or occurs as a consequence of the degenerative process, remains uncertain.

Ovarian follicular population changes with the advance of the breeding season in intact and unilaterally ovariectomized ewes.

In ewes at the 1st, 2nd or 4th oestrous cycle after unilateral ovariectomy, the ovulation rate remained constant at 1.5 in the control (sham-operated) ewes, but increased from 1.3 to 2.0 in unilaterally ovariectomized ewes. In control ewes, the proportion of preantral follicles declined significantly (P less than 0.05) with each oestrous cycle while the antral follicles increased as the breeding season progressed (P less than 0.05). In contrast, after unilateral ovariectomy, the proportion of preantral and antral follicles remained constant throughout the cycles studied. The rate of atresia of antral follicles, especially those from small size classes, decreased significantly after one cycle of unilateral ovariectomy (P less than 0.05). Larger antral follicles had a different rate of atresia as the breeding season advanced. It is concluded that unilateral ovariectomy acutely decreased the rate of atresia and maintained the within-ovary equilibrium between preantral and antral follicles which otherwise would have decreased due to the depletion of preantral follicles with the advance of the breeding season.

Preovulatory follicular development in sheep treated with PMSG and/or prostaglandin.

Summary. The patterns of growth and atresia of antral follicles including that of the presumptive preovulatory follicle were examined in sheep ovaries for a 24–48-h period after the induction of luteolysis with a prostaglandin analogue, cloprostenol or cloprostenol + PMSG. Ewes were ovariectomized at various times after the initiation of the treatments. All follicles ≥1 mm in diameter were dissected from the excised ovaries and the antral fluid and granulosa cells recovered. Individual follicles were classified as healthy or atretic on the basis of the number of granulosa cells recovered and then subclassified as to whether they contained intrafollicular levels of oestradiol that were ≥ or < than 100 ng/ml. In another series of similarly treated ewes, the ovarian secretion rates of oestradiol and the intrafollicular concentrations of oestradiol in all large antral follicles (≥5 mm diameter) as well as the levels of progesterone in peripheral plasma were measured at different times after induction of luteolysis. The results showed that a large 'oestrogenic' follicle (≥5 mm diameter and secreting ≥1 ng oestradiol/min) appears around 10 h after the cloprostenol injection and that this presumptive preovulatory follicle emerges before the corpus luteum has ceased to function. Moreover, the presumptive preovulatory ('oestrogenic') follicle appears to develop from the pool of small 'oestrogenic' follicles (1–3 mm diameter) after the onset of luteolysis. The emergence of a large 'oestrogenic' follicle is accompanied by a widespread increase in atresia (> 80%) in all other classes of antral follicles (≥1 mm in diameter). During the first 10 h of cloprostenol-induced luteolysis, PMSG (a) prevented the normal occurrence of atresia in the large follicle population; (b) enhanced oestrogen secretion in a greater proportion of large antral follicles compared to that in control animals; (c) temporarily 'rescued' and/or prevented small antral follicles (1–4 mm diameter) from undergoing atresia; but (d) had little, or no, effect on the overall population of antral follicles (≥1 mm diameter). After 24 h, the atresia-preventing effects of PMSG were no longer discernible and the only obvious difference noted, compared to the controls, was the number of large oestrogen-secreting follicles.