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BOR - Papers in Press, published online ahead of print October 4, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.003673
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Biology of Reproduction 67, 1989-1995 (2002)
DOI: 10.1095/biolreprod.102.003673 © 2002 Society for the Study of Reproduction, Inc.

Ovary

Inhibin A Increases Apoptosis in Early Ovarian Antral Follicles of Diethylstilbestrol-Treated Rats1

Alejandra M. Vitalea, Olga M. Gonzalezb, Fernanda Parborella, Griselda Irustaa, Stella Campoc, and Marta Tesone2,a

a Instituto de Biología y Medicina Experimental CONICET, b Facultad de Ciencias Exactas y Naturales, Facultad de Agronomía, c Departamento de Producción Animal, Universidad de Buenos Aires, and Centro de Investigaciones Endocrinológicas, Buenos Aires 1428, Argentina


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The purpose of this study was to evaluate the role of inhibin A in follicular development and apoptosis-related mechanisms in preantral and early antral follicles from prepubertal diethylstilbestrol (DES)-treated rats. Granulosa cells isolated from the ovaries of 23- to 25-day-old rats were cultured in serum-free medium containing FSH (20 ng/ml), transforming growth factor ß (5 ng/ml), and estradiol (50 ng/ml) in the presence or absence of different concentrations of recombinant human inhibin A. 3H-Thymidine incorporation was decreased in the presence of Inh, but no significant changes were observed in progesterone and estradiol levels in culture medium. An increase in low molecular weight DNA fragmentation indicative of apoptosis and an increase in the levels of Bax protein with no changes in Bcl-2 protein levels were evident in early antral follicles incubated for 24 h with Inh. For each animal, Inh (0.5 µg/ovary) was injected intrabursally in one ovary, and the contralateral ovary served as a control. Ovarian histology revealed an inhibitory effect of Inh treatment on the follicular development induced by DES. At 24 h after Inh injection, the number of preantral follicles was increased compared with controls, whereas the number of early antral follicles was decreased. In addition, in vivo Inh treatment caused an increase in the percentage of apoptotic cells in preantral and early antral follicles. These results suggest that inhibin produced by the dominant follicle may act as a paracrine factor inhibiting the growth of neighboring follicles, thus participating in the mechanism of follicular selection.

apoptosis, follicle, granulosa cells, inhibin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhibin and activin, members of the transforming growth factor ß (TGFß) superfamily, have been proposed as autocrine/paracrine factors that modulate follicular growth, atresia, gonadotropin responsiveness, and steroidogenesis [1–4]. Inhibins are peptides secreted mainly by the gonads, and the role of inhibins is to inhibit FSH synthesis and secretion [5, 6]. These peptides are heterodimers formed from two subunits ({alpha} and ß) linked by disulfide bonds. Inhibins A and B, the dimeric forms of inhibin, are formed by a common {alpha} subunit linked to either a ßA or a ßB subunit, respectively. The {alpha} and ß subunits and inhibin binding sites have been identified in the ovary [3, 7, 8]. In several studies performed in rats, inhibin mRNA levels increased as the cycle of follicle maturation progressed. High levels of inhibin mRNA have been observed in the dominant follicle, and low or undetectable levels have been found in atretic follicles [9]. Similar findings have been described in human follicular fluid, suggesting that in the follicular phase the dominant follicle produces more inhibin A than do the cohort follicles [10, 11]. These observations and the effect of inhibin on ovarian steroidogenesis described by Hillier et al. [12] support the hypothesis that inhibin may be an autocrine/paracrine ovarian hormone.

Endocrine and ovarian factors are involved in the emergence of a dominant follicle, which in turn indirectly limits the growth of subordinate follicles by releasing increasing concentrations of estradiol and inhibin in circulation. These ovarian products produce a decrease in FSH concentration, which induces atresia of subordinate follicles [13–15]. Several studies have demonstrated that apoptotic cell death is the molecular mechanism underlying follicular atresia [16, 17]. Protooncogene and tumor suppressor gene products of the bcl-2 family have been postulated as intracellular mediators of cell survival, whereas the protein products of the bcl-2 and bax genes have been described as anti- and proapoptotic factors, respectively [18–20].

Atretogenic factors produced by the dominant follicle have been postulated as responsible for the inhibition of the development of subordinate neighboring follicles [21, 22]. Although a number of putative dominance factors have been reported [23, 24], the possibility that the inhibin secreted by the dominant follicle may be involved in the regulation of the growth of subordinate follicles has not been explored. To test this hypothesis, we examined the in vitro and in vivo effects of inhibin A on proliferation of granulosa cells and apoptosis in early antral follicles (EAF) from prepubertal diethylstilbestrol (DES)-treated rats.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Recombinant human inhibin A (Inh) [25] was provided by the National Institute for Biological Standards and Controls (Hertfordshire, U.K.). Ovine FSH (o FSH S17) was kindly donated by the National Hormone and Pituitary Agency, (NIADKK-NIH, Bethesda, MD). Estradiol, androstenedione, DES, and Hepes were from Sigma (St. Louis, MO). TGFß, Dulbecco modified Eagle medium (DMEM, 4.5 g glucose/L), Ham F-12 nutrient mixture (F12), fungizone (250 µg/ml), and gentamicin (10 mg/ml) were from Gibco Laboratories (Buenos Aires, Argentina). All other chemicals were of reagent grade from standard commercial sources. Rabbit polyclonal anti-Bax (N-20) and goat polyclonal anti-Bcl-2 (N-19) were from Santa Cruz Biotechnology (Santa Cruz, CA). Extravidin-peroxidase, anti-rabbit IgG biotin conjugate, anti-goat IgG peroxidase conjugate and diaminobenzidine (DAB) were from Sigma. The enhanced chemiluminescence (ECL) kit was from Amersham (Buckinghamshire, U.K.).

Animals

Female Sprague-Dawley rats, 23–25 days old, were allowed food and water ad libitum and kept at room temperature at 21–23°C. Rats were injected s.c. with 1 mg DES (dissolved in corn oil) daily for 3 days to stimulate the development of EAF. Animals were killed by cervical dislocation, and the ovaries were removed for granulosa cell isolation or follicle dissection. Experimental protocols were approved by the Animal Experimentation Committee of the Instituto de Biología y Medicina Experimental.

Granulosa Cell Isolation and Culture

Granulosa cells from DES-treated rats were isolated by follicular puncture, as described previously [26]. Cells were seeded onto plastic 96-well plates (Nunc, Roskilde, Denmark) precoated with rat tail collagen. Initial plating density was 3 x 105 viable cells/well, and cells were maintained at 37°C with 5% CO2. After 3 h, medium was changed to remove nonattached cells and replaced with fresh medium containing the different factors to be tested.

Proliferation Assays

Tritiated thymidine (0.4 µCi/well, final specific activity 1.2 Ci/mmol; Dupont, Boston, MA) was added to the culture 24 h after stimulus. After an additional 24 h, cells were harvested in hollow glass fibers using a multiwell harvester [26]. Excess 3H-thymidine was removed by washing with 6 volumes of distilled water followed by 1 volume of ethanol. Filters were allowed to dry and then transferred to vials, and radioactivity was counted in a scintillation counter (efficiency 50%).

The activity of the dehydrogenase enzymes found in metabolically active cells was measured by the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega, Buenos Aires, Argentina) according to the manufacturer's instructions (MTS technique).

Dimeric Inhibin ELISA

Inhibin A concentration was determined using a specific two-site ELISA as previously described [10]. Conditioned medium was diluted in fetal calf serum (FCS) according to the amount of inhibin present. Before assaying, a 0.5 volume of 5% aqueous SDS was added to all samples, and standards were preheated for 3 min at 100°C. Samples were then treated for 30 min with freshly prepared 1% hydrogen peroxide solution at room temperature. A sensitive enzyme-amplified assay (Ampak; DAKO Corp., Carpinteria, CA) was used to amplify alkaline phosphatase activity. Inh was used as the standard. Activin A, activin B, and follistatin showed <0.1% cross-reaction for both assays. Cross-reaction for inhibin B in the inhibin A assay was <0.1%, and the assay sensitivity was 7 pg/ml. Intra- and interassay coefficients of variation were <10%.

Radioimmunoassay

Progesterone and estradiol production by granulosa cells was evaluated in the culture medium by RIA 48 h after the addition of the factors to be tested. For estradiol measurement, 0.25 µM of 4-androstene-3,17-dione was added. RIAs were performed as described previously [27] using specific antibodies supplied by Dr. G.D. Niswender (Animal Reproduction and Biotechnology Lab, Department of Physiology, Colorado State University, Fort Collins, CO). Under our conditions, within-assay and between-assay variations were 8.0% and 14.2%, respectively, for progesterone and 7.2% and 12.5%, respectively, for estradiol.

Follicle Culture

EAF (~350 µm in diameter) were dissected from ovaries collected following DES treatment and cleansed of adhering tissue in culture medium. Ovarian follicles from six animals were pooled, and cultures were initiated at 37°C within 1 h of ovary removal. Twenty follicles per culture plate well were incubated under serum-free conditions in 300 µl DMEM:F12 (1:1 vol/vol) supplemented with streptomycin and gentamicin in the absence or presence of 4 or 40 ng/ml Inh. Follicles were maintained at 37°C in an incubator with 95% O2 and 5% CO2. Following incubation for 24 h, follicles were stored at -70°C until DNA extraction. Other follicles were incubated for 24 h in the absence or presence of 0.4, 4, 40, or 100 ng/ml Inh with an additional inhibin stimulus added at 8 h of culture and were then stored at -70°C until protein extraction.

DNA Isolation, Agarose Gel Electrophoresis, and Quantitation of DNA Fragmentation

Cellular DNA was extracted from follicles as previously described [28]. Follicles from each culture were homogenized in a buffer containing 100 mM NaCl, 4 mM EDTA, 50 mM Tris-HCl, 0.5% SDS, pH 8, and proteinase K (100 µg/ml) and incubated for 3 h at 55°C to facilitate membrane and protein disruption. Following incubation, samples were cooled 30 min on ice in 1 M potassium acetate and 50% chloroform to initiate protein precipitation and then centrifuged for 8 min at 5000 x g at 4°C. Supernatants were precipitated for 30 min in 2.5 volumes of ethanol at -70°C and then centrifuged for 20 min at 5000 x g at 4°C. Samples were then extracted in 70% ethanol and resuspended in water. DNA content was measured by reading the absorbance at 260 nm, and samples were incubated 1 h with RNase (10 µg/ml) at 37°C. RNase treatment did not affect the DNA content.

DNA samples (3–4 µg) were electrophoretically separated on 1.7% agarose gels containing ethidium bromide (0.4 µg/ml) in TBE buffer (89 mM Tris-borate, 2 mM EDTA). Within each agarose gel, equivalent amounts of DNA were loaded into each well. DNA was visualized in an ultraviolet (302 nm) transilluminator (UVP GDS-8000; Ultraviolet Products Ltd., Cambridge, U.K.), and densitometric analysis of low molecular mass (<15 kilobases) DNA was performed using the software program Image Quant (Molecular Dynamics, Sunnyvale, CA).

Western Blot Analysis of Bax and Bcl-2 Proteins

Protein extracts were prepared by lysing EAF for 20 min at 4°C in 5 volumes of lysis buffer (20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 1% Nonidet P-40, and 10% glycerol) supplemented with protease inhibitors (0.5 mM PMSF, 0.025 mM N-CBZ-L-phenylalanine chloromethyl ketone, 0.025 mM N'-p-tosyl-lysine chloromethyl ketone, and 0.025 mM L-1-tosylamide-2-phenyl-ethylchloromethyl ketone). The resulting lysate was then centrifuged for 10 min at 4°C at 10 000 x g, and the pellet was discarded. Protein concentration in the supernatant was measured by the Bradford assay (Bio-Rad, Hercules, CA). After adjusting to sample buffer and boiling for 5 min, 60 µg of protein was applied on a 15% SDS-polyacrylamide gel, and electrophoresis was performed at 25 mA for 1.5 h. The resolved proteins were transferred onto nitrocellulose membranes in transfer buffer containing 20% methanol (vol/vol), 0.19 M glycine, and 0.025 M Tris-base (pH 8.3) for 2 h at 4°C. Blots were then blocked for 1 h in TBS (4 mM Tris-HCl, pH 7.5, 100 mM NaCl) containing low-fat powdered milk (2%) and Tween 20 (0.2%) at room temperature. Polyclonal anti-Bax and anti-Bcl-2 (1:200, overnight) were used as primary antibodies. Bcl-2 bands were visualized by incubating with a biotin-conjugated secondary anti-goat IgG (1:500, 1 h), extravidin-peroxidase complex and diaminobenzidine solution. Bax bands were visualized by incubating with a peroxidase-conjugated secondary anti-rabbit IgG (1:1000, 1 h) and by using the ECL kit (NEN Life Science Products Inc., Boston, MA), according to the manufacturer's instructions. Negative controls were obtained by omitting the primary antibody. Protein levels were analyzed by densitometric studies using Image Quant. The proper loading was evaluated by staining the membranes with Ponceau-S.

TUNEL and Ovarian Morphology

Rats were injected s.c. with 1 mg DES (dissolved in corn oil) daily for 3 days. On the third day, rats were anesthetized with ketamine HCl (80 mg/kg), a dorsal incision (1 cm) was made and the left ovary was exteriorized for injection of 10 µl of Inh (0.5 µg/ovary) intrabursally using a 10-µl Hamilton syringe. The contralateral ovary was exposed and injected with saline as a control. Ovaries were replaced, the incision was sealed, and the animals were returned to their cages. Twenty-four hours later, the animals were killed and the ovaries were collected for histological analysis in vials containing 10% formalin. Ovaries were fixed for 12 h and then embedded in paraffin. According to the method of Woodruff et al. [3], 3-µm step sections were mounted at 50-µm intervals onto microscope slides to prevent counting the same follicle twice. One set of slides was stained with hematoxylin and eosin to determine the number of follicles per ovarian section, and the other was immunostained using the TUNEL technique [29]. The number of healthy preantral and EAF was determined in ovarian sections obtained from animals following DES (n = 4) or DES + Inh (n = 4) treatment. To study ovarian morphology and the percentage of apoptotic cells by TUNEL, five randomly selected fields were analyzed from each ovarian section (six sections/ovary, four ovaries). Follicles were classified as preantral or early antral according to size and the presence or absence of an antrum.

For detection of apoptotic cells, the ApopTag Plus Peroxidade In Situ Apoptosis Detection Kit (Intergen, Purchase, NY) was employed, and procedures were followed as indicated in the manufacturer's instructions. Negative controls included omission of the terminal deoxynucleotidyl transferase. Sections were counterstained with methyl green. The number of apoptotic cells was determined by counting labeled cells from preantral and antral Gfollicles in 400x randomly selected microscopic fields as described above. The apoptotic index was calculated as a percentage of total cell number.

Data Analysis

Data are expressed as the mean ± SEM of three experiments. Incubations were made in triplicate. Statistical comparisons were performed using a one-way ANOVA followed by the Tukey test for multiple comparisons. For the Western blot, a Kruskal-Wallis test followed by the Dunn test was used. Differences were considered significant at P < 0.05. Fitting of the dose-response curve and calculation of the ID50 was performed using a computer program based on a three-parameter exponential decay equation (Sigma Plot 4.01).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In Vitro Effect of Inhibin on Granulosa Cell Proliferation

3H-Thymidine incorporation was chosen as a measure of DNA synthesis instead of the usual approach of measuring cell numbers. This technique is more sensitive because of the low in vitro replicating potential of rat granulosa cells; only a small fraction of these cells are able to enter into the S phase [26].

We had previously found that a combination of FSH (20 ng/ml), TGFß (5 ng/ml), and estradiol (50 ng/ml) produces a significant increase in thymidine incorporation in this system [30]. 3H-Thymidine incorporation induced by this means was considered control proliferation (100%). Addition of Inh at a concentration of 4 ng/ml produced a 16% proliferation inhibition (P < 0.05), and a higher inhibin concentration (40 ng/ml) resulted in 35% inhibition (P < 0.05) (Fig. 1). A dose-response effect for Inh was observed with an estimated IC50 of 4.8 ng/ml.



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  		FIG. 1. Effects of different concentrations of inhibin A on DNA synthesis inhibition measured as 3H-Thymidine incorporation. Granulosa cells (105 cells/well) were cultured in the presence of FSH (20 ng/ml), TGFß (5 ng/ml), and estradiol (500 ng/ml). Proliferation was considered at 100% in cultures containing FSH + TGFß + estradiol. 3H-Thymidine was added 24 h after plating, and cultures were harvested 24 h later. Values are expressed as the mean ± SEM of triplicate incubations of four independent experiments. *P < 0.05 vs. the respective control by ANOVA and Tukey test

The effect of Inh on granulosa cell proliferation was also measured by the MTS technique, and the results confirmed those obtained for 3H-thymidine incorporation. Thus, a similar pattern of inhibition was observed: 17.5% inhibition with 4 ng/ml Inh (P < 0.05) and 60% inhibition with 40 ng/ml Inh (P < 0.05).

Progesterone, Estradiol, and Inhibin Production by Cultured Granulosa Cells

Progesterone and estradiol accumulations by granulosa cells were determined after 48 h of culture (Table 1). Addition of FSH and TGFß stimulated the production of both steroids, but Inh failed to modify steroidogenic response of the cells to FSH and TGFß.


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  		TABLE 1. Effect of FSH, TGFß, and inhibin A on progesterone and estradiol production by cultured granulosa cells

FSH and TGFß are known to stimulate inhibin production in rat granulosa cells [31]. To test Inh levels in this system, its secretion was measured in the conditioned medium of stimulated granulosa cell cultures (FSH, TGFß, and estradiol) at different times (2, 4, 8, 16, 24, and 48 h), and the values obtained were 0.46 ± 0.004, 0.22 ± 0.01, 0.77 ± 0.01, 1.55 ± 0.04, 3.26 ± 0.25, and 5.3 ± 1.6 ng/ml, respectively. Maximal inhibin production was observed at 48 h.

In Vitro Effect of Inhibin on Apoptosis in EAF

To test the possibility that the decrement observed in cellular growth was related to apoptosis, in vitro studies were performed using incubations of EAF isolated by ovarian microdissection from DES-treated rats. EAF were cultured for 24 h in serum-free medium with different Inh concentrations. Serum-free follicle culture is currently used to investigate the hormonal factors and pathways that control apoptosis and follicular atresia. Furthermore, this system has the advantage of conserving the integrity of the follicle to be studied. After incubation, cellular DNA was extracted from EAF and electrophoretically separated. Follicles cultured for 24 h in serum-free medium showed spontaneous onset of apoptotic DNA fragmentation (Fig. 2). In contrast, DNA fragmentation was minimal in freshly isolated EAF (no culture, Time 0). Quantitative estimation of DNA cleavage from ovarian follicles revealed a significant increase in DNA fragmentation after in vitro Inh treatment when compared with untreated cultures. Maximal DNA fragmentation was observed using 40 ng/ml Inh (P < 0.05). To evaluate a possible correlation between the inhibin-induced apoptosis effect and the stability of pro- or antiapoptotic proteins, two Bcl-2 family members were studied.



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  		FIG. 2. Apoptotic DNA fragmentation in EAF incubated 24 h in serum-free medium in the presence or absence of inhibin A (4 or 40 ng/ml). A) Representative agarose gel. DNA extracted from follicles in each culture was visualized by ethidium bromide staining. B) Quantitative estimation of low molecular mass DNA content (<15 kilobases). Results obtained by densitometric analysis of the gel are expressed as percentage change compared with a 24-h culture in serum-free medium, arbitrarily designated as 100%. Values are expressed as the mean ± SEM of DNA from three different pools of follicles run on three independent gels. Those without the same letter are significantly different by ANOVA and Tukey test (P < 0.05)

Effects of Inhibin on Protein Expression of Bcl-2-Related Genes

EAF obtained from follicles isolated by microdissection were incubated with 0.4, 4, 40, and 100 ng/ml of Inh. Bax and Bcl-2 follicular protein content was measured by Western blotting. The dose-response curve shows that Bax protein levels increased in the presence of inhibin, and this increase was significant at a dose of 40 ng/ml (P < 0.05) (Fig. 3, A and B). No significant changes were observed in the Bcl-2 protein content (Fig. 3, C and D).



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  		FIG. 3. Concentration-response curve of Bax and Bcl-2 protein content in EAF incubated 24 h with inhibin A (0.4, 4, 40, or 100 ng/ml). A and C) Representative immunoblot of Bax and Bcl-2, respectively. B and D) Densitometric quantification of the immunoblot of Bax and Bcl-2, respectively. Data are expressed as the mean ± SEM of three independent blots. *P < 0.05 vs. the respective control by Kruskal-Wallis test followed by Dunn test

In Vivo Effect of Inhibin on Apoptosis: Morphological and Immunohistochemical Studies

To test whether the inhibin effect on follicular growth would be observed after in vivo treatment, morphological and follicular apoptosis changes were evaluated after ovarian intrabursal injection of Inh. Ovarian histology revealed an inhibitory effect of Inh on the follicular development induced by DES (Fig. 4A). After 24 h of in vivo Inh treatment, the number of preantral follicles (PF) was increased (control = 4.1 ± 0.4; Inh = 7.5 ± 1.1; P < 0.05), whereas the number of EAF was decreased (control = 48.3 ± 1.9; Inh = 39.3 ± 2.3; P < 0.05), indicating a delay in follicular development. The number of healthy follicles (PF + EAF) was similar in control (97.6%) and Inh (96.9%) groups.



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  		FIG. 4. Follicular content and apoptosis evaluated after ovarian intrabursal injection of inhibin A. A) Values represent the number of healthy follicles (PF, EAF) counted in inhibin-treated or control ovaries in sections stained with hematoxylin and eosin. B) In situ DNA fragmentation analysis (TUNEL technique). The number of apoptotic cells was determined by counting labeled cells in randomly selected 400x microscopic fields of healthy PF and EAF. Data are expressed as the mean ± SEM. *P < 0.05 vs. the respective control by ANOVA and Tukey test

To investigate whether these changes were mediated by cellular death induced by the Inh treatment, the degree of apoptosis was measured. Cells undergoing DNA fragmentation were quantified by TUNEL on ovarian sections (Figs. 4B and 5). In vivo Inh treatment caused a 3-fold and a 4-fold increase in apoptotic cell number per follicle in PF and EAF, respectively, compared with saline-treated ovaries. In all follicles, apoptosis was confined to the granulosa cell layers.



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  		FIG. 5. Representative ovarian section from DES-treated rats following ovarian intrabursal injection of inhibin A (0.5 µg/rat) or saline. Apoptosis was determined by the TUNEL technique. Note immunostained apoptotic cells (arrowheads) in EAF. Cells of sections processed without the terminal transferase enzyme, in which no colorimetric reaction was observed, were considered controls (data not shown). Oo, Oocyte; Gc, granulosa cells; Tc, theca cells. A) x100. B) x200


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Much information has accumulated in recent years on the local autocrine/paracrine actions of activin, inhibin, and follistatin on ovarian folliculogenesis and steroidogenesis [3, 32–35]. The purpose of the present study was to evaluate the in vitro and in vivo effect of human recombinant inhibin A on granulosa cell proliferation, follicular development, and apoptosis-related mechanisms in immature granulosa cells and EAF obtained from prepubertal DES-treated rats. Several researchers have demonstrated that follicular atresia is mediated by apoptosis and that FSH and LH are the primary survival factors, with effects probably mediated by the production of unidentified local ovarian factors [36–40]. However, this study is the first to demonstrate that the paracrine action of inhibin A involves a decrease in granulosa cell proliferation and an increase in the apoptotic process in EAF from DES-treated rats. In addition, the increased expression of follicular Bax protein observed after incubation with inhibin is novel.

These results indicate that inhibin A is produced in vitro by granulosa cells and is stimulated by FSH, TGFß, and estradiol. In addition, physiological concentrations of inhibin A interfere with granulosa cell proliferation in cultures stimulated with FSH, TGFß, and estradiol. This effect could be produced by interference in the mechanism of action of these stimulating factors or by an enhancement of the apoptotic process. However, a decrease in DNA repair in response to inhibin could not be excluded, because the cleavage of the DNA repair enzyme poly ADP-ribose polymerase by caspase-3 following induction of apoptosis has been demonstrated in the ovary [41]. Regarding the possible interference in the mechanism of action of TGFß, Bernard et al. [42] determined that inhibin can block activin/TGFß binding and subsequent signal transduction. However, we observed that 3 h of preincubation with TGFß did not modify the inhibin effect (data not shown). In addition to the antiproliferative effect, in vitro inhibin treatment revealed a significant increase in DNA fragmentation in EAF cultured in serum-free medium. The physiological importance of these findings resides in the fact that although atresia occurs at all stages of follicular development, the majority of follicles undergoing degeneration, presumably by apoptosis, are at the EAF stage [36, 43].

Members of the bcl-2 gene family have been described as main participants in the cascade of events that activate or inhibit apoptosis [20, 44, 45]. One of these proteins, termed Bax, counters the effects of Bcl-2 on cell survival. Tilly and Tilly [46] demonstrated that the inhibition of granulosa cell apoptosis and follicular atresia, mediated by gonadotropin treatment, is related to the ability of gonadotropins to reduce the expression of Bax in granulosa cells, producing a change in the ratio of Bax to the constitutive levels of Bcl-2 and Bcl-x long. In our experimental model, EAF incubated with inhibin A showed no changes in Bcl-2 protein levels but showed an increase in Bax content after 24 h of follicular incubation.

Woodruff et al. [3] reported that intraovarian inhibin treatment of virgin prepubertal rats increased the total number of growing follicles present in ovaries and increased follicular thymidine incorporation. According to this report, activin administration blocked the follicular development and caused follicular atresia both in untreated and in eCG-treated rats. Our observations disagree with these results, and the difference we found in the inhibin effect may be due to the experimental model used in the present study. It is difficult to evaluate a paracrine/autocrine ovarian effect in such different hormonal environments such as virgin prepubertal and eCG- or DES-treated rats. Administration of eCG to prepubertal rats induces an advanced stage of follicular development, whereas DES treatment induces follicular development mainly to the EAF stage.

The present results show that inhibin A did not modify progesterone or estradiol production. This finding is in agreement with previous findings demonstrating that neither inhibin nor follistatin directly modulate steroidogenesis in cultured unluteinized granulosa cells [34].

The emergence of dominant and subordinate follicles is a result of the complex interplay between FSH stimulation and the action of local factors [21]. In this context, the relevance of interfollicular regulation in this process is well recognized. Factors from granulosa cells or in follicular fluid, presumably produced by the dominant follicle, that appear to inhibit the development of its subordinate neighbors have been described in various species [23, 24, 30, 47–49]. Our results suggest that inhibin produced by the dominant follicle may be an additional factor participating in this process.

In the present study, in vitro and in vivo inhibin A treatment produced an increase in the apoptotic process in EAF from DES-treated rats. The apoptotic action of inhibin may be correlated with an imbalance in the ratio of antiapoptotic to proapoptotic proteins as observed for the Bcl2/Bax pair, and it may interfere with follicular development by an as yet unknown mechanism. These results demonstrate that inhibin produced by the dominant follicle may act as a paracrine factor inhibiting the growth of neighboring follicles, thus participating in the mechanism of follicular selection.


   FOOTNOTES
 
1 This study was supported by the ANPCYT (PICT 6384 BID 1201 OC-AR), UBA (01/TW05, University of Buenos Aires, Argentina), and the Roemmers Foundation. Back

2 Correspondence: Marta Tesone, Instituto de Biología y Medicina Experimental, Obligado 2490, Buenos Aires 1428, Argentina. FAX: 54 011 4786 2564; mtesone{at}dna.uba.ar Back

Received: 17 January 2002.

First decision: 6 February 2002.

Accepted: 8 July 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Knight PG, Glister C. Potential local regulatory functions of inhibins, activins and follistatin in the ovary. Reproduction 2001 121:501-512
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  6. Burguer HG. Evidence for a negative feedback role of inhibin in follicle stimulating hormone regulation in women. Hum Reprod 1993 8:suppl 2129-132
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