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Regular Article |
a Instituto de Biología y Medicina Experimental, CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, 1428 Buenos Aires, Argentina
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, follicle, gonadotropin-releasing hormone, growth factors, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Apoptosis is the biological process by which follicular cells are eliminated in atretic follicles [12–18]. FSH and LH are the primary survival factors for ovarian follicles; the antiapoptotic effects of these gonadotropins are probably mediated by the production of ovarian growth factors. Various growth factors and cytokines (insulin-like growth factor I, epidermal growth factor [EGF], transforming growth factor 
[TGF], basic fibroblast growth factor, keratinocyte growth factor, interleukin Iß) prevent apoptosis in antral follicles [15, 19–21]. Many researchers have used preovulatory follicles obtained from eCG-treated prepubertal rats and have observed a significant degree of apoptosis within 24 h of incubation in serum-free medium and prevention of apoptosis in the presence of FSH or growth factors. EGF plays an important role in the modulation of apoptotic cell death within ovarian follicles [15]. The presence of these growth factors and their receptors in the ovary has been demonstrated at both the protein and mRNA levels. Apoptotic cell death in granulosa cells of those follicles selected for ovulation may be prevented by the paracrine actions of EGF and TGF (a homologue of EGF that has similar cell membrane receptors), which are produced by theca-interstitial cells [15].
Protooncogene and tumor suppressor gene products of the Bcl-2 family may function as intracellular mediators of cell survival. The protein products of the bcl-2 and bax genes were described as anti- or proapoptotic factors, respectively [22–24]. The bcl-x gene generates several alternative spliced products. One of them encodes the long isoform (Bcl-xL), which has been described as an antiapoptotic protein, and another encodes the short isoform (Bcl-xS), which counters the death repressor effects of Bcl-2 and Bcl-xL [24, 25].
In hypophysectomized estrogen-treated immature rats, Billig et al. [26] showed that the in vivo administration of a GnRH-a increased apoptotic cell death in ovarian tissue and granulosa cells, and Papadopoulos et al. [27] demonstrated that administration of a GnRH-a enhances the rate of DNA degradation in the corpora lutea of pregnant rats. No studies, however, have been performed on the in vivo effects of GnRH-a on follicular development induced by gonadotropins in relation to the apoptotic cell death regulation and their interactions with growth factors and members of the Bcl-2 gene family. The hypothesis to be addressed in this study is that GnRH interferes with gonadotropin follicular stimulation by alterations in some apoptosis-related process.
The aim of the present study was to examine the in vivo effect of the GnRH-a leuprolide acetate (LA) on apoptosis in preovulatory ovarian follicles obtained from prepubertal eCG-treated rats, the interaction of LA with EGF, and the control of protein expression of bcl-2-related genes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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LA (Lupron; Abbott Laboratories, Buenos Aires, Argentina) in the original ampoule (2.8 mg/5 ml) was dissolved in saline solution to obtain the appropriate concentration. The eCG (Novormon) was provided by Syntex S.A. (Buenos Aires, Argentina). Steroid hormones, Hepes, EGF, and SDS were purchased from Sigma Chemical Co. (St. Louis, MO). 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 (Grand Island, NY). All other chemicals were of reagent grade and were obtained from standard commercial sources. Rabbit polyclonal anti-Bax (N-20), anti-Bcl-2 (N-19), and anti-Bcl-x (S-18) were from Santa Cruz Biotechnology (Santa Cruz, CA). Extravidin-peroxidase, anti-rabbit IgG biotin conjugate, and diaminobenzidine were from Sigma.
In Vivo LA Treatment and Superovulation
Female Sprague-Dawley rats 23–25 days old were kept at 21–23°C and allowed food and water ad libitum. Animals were injected subcutaneously with eCG (25 IU/rat, control group) to induce growth of multiple follicles. LA alone was injected (0.5 µg/rat) at Time 0 and then at 12-h intervals. The last LA injection was administered 3 h before rats were killed. Control animals were injected with vehicle only. Animals were killed by cervical dislocation 48 h after eCG injection. The ovaries were removed and cleaned of adhering tissue in culture medium for subsequent assays. The experimental protocols were approved by the Animal Experimentation Committee of the Instituto de Biología y Medicina Experimental.
Ovarian Morphology
To evaluate changes in general structure, representative ovaries from control and treated rats were immediately fixed in 4% neutral buffered formalin for 12 h and then embedded in paraffin. Three-micrometer 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 count the number of follicles per ovary section, and the others were immunostained with the TUNEL technique. Atresia was defined as the presence of >10 pyknotic granulosa cells; in the smallest follicles, the criterium for atresia was a degenerate oocyte and/or precocious antrum formation [1, 28]. Follicles were classified as preantral or antral according to the presence or absence of an antrum. The numbers of preantral, antral, and atretic follicles were determined in ovarian sections obtained from animals (n = 8) after 48 h of vehicle or LA treatment. To study ovarian morphology or the number of apoptotic cells by TUNEL, five randomly selected fields were analyzed from each ovarian section (six sections/ovary, six to eight ovaries).
TUNEL
For immunohystochemical quantification of apoptosis, formalin-fixed tissue sections were processed for in situ localization of nuclei exhibiting DNA fragmentation by the TUNEL technique [29] using an apoptosis detection kit (Oncor, Gaithersburg, MD) as previously described [1]. The 4-µm-thick tissue sections were deparaffinized and digested for 15 min at room temperature with 20 µg/ml of proteinase K (Gibco). Endogenous peroxidase was quenched with 2% hydrogen peroxide in PBS. The labeling reaction was carried out by incubating tissue sections with buffer containing digoxygenin-dUTP 30 min prior to incubation with TdT for 1 h at 37°C. Tissues were then incubated 30 min with a peroxidase-conjugated anti-digoxygenin monoclonal antibody, and apoptotic cells were visualized as positively immunostained structures after reaction with diaminobenzidine (DAB). Negative controls included TdT omission. Sections were counterstained with hematoxylin. The number of apoptotic cells was determined by counting labeled cells from preantral and antral follicles in 400x randomly selected microscopic fields as described above. The apoptotic index was calculated as a percentage of the total number of cells.
Follicle Isolation and Incubation
Healthy preovulatory follicles (>400 µm in diameter) from 12 ovaries were dissected microscopically using fine needles. Culture was initiated within 1 h of ovary removal.
For DNA or protein analysis, four follicles or 100 follicles/glass vial, respectively, were incubated under serum-free conditions at 37°C in 500 µl DMEM:F12 (1:1 vol/vol) containing 10 mM Hepes supplemented with fungizone (250 µg/ml) and gentamicin (10 mg/ml). Follicles were gassed with 95% O2/5% CO2 at the start of culture.
DNA Isolation and Fragmentation Analysis
Cellular DNA was extracted from follicles incubated for 24 h in the presence or absence of EGF (200 ng/ml) as previously described [2, 30]. The 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) at 55°C for 4 h to facilitate membrane and protein disruption. After incubation, samples were cooled for 30 min on ice in 1 M potassium acetate and 50% chloroform to initiate protein precipitation and were centrifuged at 9000 x g for 8 min at 4°C. Supernatants were then precipitated for 30 min in 2.5 volumes of ethanol at -70°C, centrifuged for 20 min at 5000 x g at 4°C, extracted in 70% ethanol, and resuspended in water. DNA was quantitated by reading the absorbance at 260 nm, and the samples were then incubated for 1 h with RNase (10 µg/ml) at 37°C.
Agarose Gel Electrophoresis and Quantitation of DNA Fragmentation
DNA samples (4 µg) were electrophoretically separated on 1.9% agarose gels containing ethidium bromide (0.4 µg/ml) in Tris-borate-EDTA buffer. Within each agarose gel, equal amounts of DNA were loaded into each well. To enhance sensitivity, gels were further stained with ethidium bromide for 15 min. DNA was visualized in an ultraviolet (302 nm) transilluminator and photographed with a Polaroid camera system. Densitometric analysis of low-molecular-weight (<15 kilobases) DNA was performed with an Image Scanner (Genius, KYE Systems Corp., Taiwan, China) using the software program Image Quant (Molecular Dynamics, NIH, Washington, DC). Quantitative results obtained by densitometric analysis of the low molecular weight DNA fragments represent the mean ± SEM of three or four independent gel runs.
Western Blots
To examine the correlation of the LA-induced apoptosis effect with the stability of pro- or anti-apoptotic proteins, a number of Bcl-2 family members were studied. After incubations at 0, 1, 2, and 5 h, the preovulatory follicles obtained from the control and LA treatment groups were lysed during 20 min at 4°C in five volumes of lysis buffer (20 mM Tris-HCl, pH 8, 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 lysate was centrifuged at 4°C for 10 min at 10 000 x g, and the resulting pellet was discarded. Protein concentration in the supernatant was measured by the Bradford assay (Bio-Rad Laboratories, Richmond, CA). After boiling for 5 min, 100 µg of protein was applied to a 15% SDS-polyacrylamide gel, and electrophoresis was performed at 25 mA for 1.5 h. The resolved proteins were transferred for 2 h onto nitrocellulose membranes in transfer buffer containing 20% methanol (vol/vol), 0.19 M glycine, 0.025 M Tris base (pH 8.3) at 4°C. Blots were then blocked for 1 h in Tris-buffered saline (4 mM Tris-HCl, pH 7.5, 100 mM NaCl) containing low-fat powdered milk (2%) and Tween 20 (0.2%) at room temperature. Rabbit polyclonal anti-Bax, anti-Bcl-2, and anti-Bcl-x (1:200, overnight) were used as primary antibodies. Protein bands were visualized by incubating with a biotin-conjugated secondary anti-rabbit IgG (1:500, 1 h) followed by extravidin-peroxidase complex and DAB solution. Negative controls were obtained in the absence of the primary antibody. The levels of protein were compared in extracts from the control and LA treatment groups and analyzed by densitometry. Optical density data are expressed as arbitrary units ± SEM (n = 3). The proper loading was evaluated by staining the membranes with Ponceau-S.
Other Methods
Serum progesterone and estradiol (E2) levels were measured by RIA following ether extraction. RIAs were performed as described previously [31] using specific antibodies supplied by Dr. G.D. Niswender (Fort Collins, CO). Under our conditions, the within-assay and between-assay variations were 8.0% and 14.2%, respectively, for progesterone and 7.2% and 12.5%, respectively, for E2.
All experiments were repeated at least three times with six to eight animals per group. Incubations were done in triplicate. A representative gel is shown in the figures. Statistical comparisons were performed using a one-way ANOVA followed by Scheffé multiple range test. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A significant decrease in circulating estradiol levels was observed in the LA treatment group (control: 531 ± 52.2 pg/ml, LA: 141.3 ± 27.3 pg/ml, n = 6; P < 0.05), whereas serum progesterone levels remained unchanged (control: 13.08 ± 2.04 ng/ml, LA: 13.48 ± 1.09 ng/ml, n = 6). These results suggest that LA treatment could interfere with the mechanism of follicular recruitment induced by eCG and as a consequence decrease serum estradiol levels. To test this possibility, ovarian morphological studies were performed.
Morphological and Immunohistochemistry Studies
Ovarian histology revealed an inhibitory effect of LA treatment on the follicular development induced by eCG (Fig. 1, A and B). After 48 h of LA treatment, the numbers of atretic and preantral follicles (ATF and PF, respectively) were increased compared with controls (ATF = control: 2.95 ± 0.17, LA: 5.49 ± 0.23; PF = control: 2.06 ± 0.13, LA: 7.37 ± 0.43; P < 0.05), whereas the number of antral follicles (AF) had decreased (AF = control: 48.26 ± 1.03, LA: 37.39 ± 1.06, P < 0.05) (Fig. 2A). In addition, LA treatment caused an increase in the percentages of apoptotic cells measured by TUNEL in preantral and antral follicles (PF = control: 0.28 ± 0.07, LA: 0.91 ± 0.11; AF = control: 0.26 ± 0.03, LA: 1.34 ± 0.26, P < 0.05). As controls, the colorimetric reaction did not occur in cells of sections processed without either the terminal transferase enzyme (data not shown). Ovarian photomicrographs from the LA treatment group are shown in Figure 1, C and D, and Figure 2B. In all follicles, apoptosis was confined to the granulosa cell layers.
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Agarose Gel Electrophoresis and Quantitation of DNA Fragmentation
Preovulatory antral follicles cultured in serum-free medium showed spontaneous onset of apoptotic DNA fragmentation (Fig. 3, A and B, lane 1). Follicles obtained from LA-treated rats showed a significant increase in the spontaneous onset of apoptotic DNA fragmentation (lane 3; 24 h follicle culture: 253% ± 55%, P < 0.05 vs. controls). Quantitative estimation of DNA cleavage from ovarian follicles revealed a significant suppression in DNA fragmentation after in vitro EGF treatment (200 ng/ml) when compared with untreated cultures. This effect was observed both in control and LA-treated follicles (lane 2, 38% ± 3.3%, and lane 4, 57% ± 2.3%, respectively; P < 0.05 vs. controls). DNA fragmentation was minimal in freshly isolated antral preovulatory follicles, showing an increase in those follicles obtained from LA-treated rats (data not shown).
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Effects of LA on Protein Expression of Bcl-2-Related Genes
Follicular contents of Bcl-2, Bax, and Bcl-x (short and long) proteins were measured by Western blotting at different follicle incubation times (Fig. 4). No changes in the levels of Bcl-2 were observed in fresh follicles (Fig. 4A, Time 0); this protein was not detected after different incubation times in control or LA-treated follicles (Fig. 4A; 1, 2, and 5 h). The levels of Bax detected in extracts obtained from control and LA-treated follicles decreased similarly during the incubations (Fig. 4B). The ratio of Bcl-xL:Bcl-xS was significantly diminished in follicles obtained from LA-treated rats (control = 0 h: 2.52 ± 0.27, 1 h: 2.47 ± 0.23, 2 h: 1.4 ± 0.1, 5 h: 1.29 ± 0.05; LA = 0 h: 3 ± 0.1, 1 h: 1.73 ± 0.19, 2 h: 0.83 ± 0.04, 5 h: 0.62 ± 0.08; P < 0.05) (Fig. 4C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The decrease in the circulating levels of estradiol suggests that follicles from the LA treatment group may be less developed after gonadotropin treatment. In studies performed on histological ovarian sections, LA injections produced lower numbers of antral follicles and greater numbers of atretic and preantral follicles. The present observations corroborate previous findings suggesting that GnRH-a treatment of cycling rats reduces the total number of growing follicles present in ovaries, with no change in the number of corpora lutea [33]. In our experimental model, high doses of eCG were administered, and we postulate that the agonist acts directly at the ovarian level by interfering with the follicular stimulation produced by gonadotropin treatment. However, the possibility of an indirect action of the analogue via changes in pituitary gonadotropin release cannot be discarded. Taking into account that follicular atresia is mediated by apoptosis, the TUNEL assays have shown that in vivo GnRH-a administration to eCG-treated rats produces an increase in the number of apoptotic cells in growing follicles. We also investigated the effect of in vivo GnRH-a treatment on apoptotic DNA fragmentation in preovulatory follicles cultured in serum-free medium. This system is a model currently used to investigate the hormonal factors and the pathways that control apoptosis and follicular atresia [13–15, 19, 20, 26, 32]. It has the advantage of conserving the integrity of the follicle to be studied. DNA isolated after follicle incubation exhibited the typical apoptotic DNA degradation pattern, and an increase in the spontaneous apoptotic DNA fragmentation in preovulatory follicles obtained from LA-treated rats was observed. These results demonstrate that in vivo LA treatment sensitizes granulosa cells to undergo apoptosis.
Previous reports [15] have indicated that spontaneous apoptosis observed in preovulatory follicles placed in serum-free culture could be inhibited by treatment with EGF/TGF and that this effect is completely blocked by an inhibitor of tyrosine kinase activity. We found that in vitro treatment of follicles with EGF suppressed the spontaneous onset of DNA fragmentation, and a similar effect was observed in LA-treated follicles. Thus, these findings reinforce the importance of EGF as an important growth factor in the ovary. However, EGF did not reverse the apoptotic effect of LA completely, suggesting that there are other factors responsible for follicular survival. In this regard, the mechanism by which EGF is able to reverse the apparently irreversible process of apoptosis remains unclear. A possible explanation would be that follicles obtained from the LA-treated rats were at an early stage of atresia (as was demonstrated by measuring DNA fragmentation by TUNEL and by agarose gel electrophoresis of fresh follicles), and this condition is exacerbated in serum-free medium culture but protected by EGF.
Members of the bcl-2 gene family have been described as main participants in the cascade of events that activate or inhibit apoptosis [24, 25]. The Bcl-2-related proteins can be separated into anti- and proapoptotic members, and the balance between these counteracting proteins presumably determines cell fate [34]. Tilly and Tilly [20] demonstrated that the gonadotropin inhibition of granulosa cell apoptosis and follicular atresia is related to the ability of gonadotropins to reduce the expression of Bax in granulosa cells by producing a change in the ratio between Bax and the constitutive levels of Bcl-2 and Bcl-xL. In this regard, Papadopoulos et al. [27] examined the correlation between apoptosis produced by a GnRH-a in corpora lutea from pregnant rats and changes in the expression of some bcl-2 gene family members. In our experimental model, preovulatory follicles 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 reduction of Bcl-xL than of Bcl-xS during incubation, suggesting that the stability of this Bcl-xL isoform is lower in LA-treated rats than in the control group.
In the present study, in vivo GnRH-a treatment produced an increase in the apoptosis process in preovulatory follicles from eCG-treated rats, and this effect was mostly reversed by EGF. The apoptotic action of GnRH-a was correlated with an imbalance in the ratio of anti-apoptotic:proapoptotic proteins, as observed for the Bcl-xL/Bcl-xS pair. This GnRH analogue reduces the stability of the Bcl-xL protein, interfering with follicular development by an as yet undetermined mechanism.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by the ANPCYT (PICT 6384 BID 1201 OC-AR), UBA (01/TW05, University of Buenos Aires, Argentina), Roemmers Foundation, and PLACIRH. 
2 Correspondence: Marta Tesone, Instituto de Biología y Medicina Experimental, Obligado 2490, 1428 Buenos Aires, Argentina. FAX: 54 011 4786 2564; mtesone{at}dna.uba.ar
Accepted: February 13, 2002.
Received: September 21, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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