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Ovary |
a Department of Physiology and Pharmacology, Göteborg University, SE-40530 Göteborg, Sweden
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, granulosa cells, progesterone receptor
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Progesterone receptors consist of two different molecular isoforms, an A and a B form [1, 9–11]. In mice, two proteins of the PR isoforms, with a molecular mass of 83 and 115 kDa, designated A and B forms, respectively, have been identified [11, 12]. The A form lacks 164 amino acids at the N-terminus. The two isoforms arise from the use of two distinct promoters in a single gene [13]. They have similar hormone and DNA binding affinities [14] and can either homodimerize or heterodimerize with each other [15].
The biological effects of progesterone are dependent on the activation of both PR isoforms [9, 10]. In the absence of progesterone, PR is maintained in an inactive complex that contains heat shock proteins in the nuclei of target cells. After progesterone binding, PR starts to change distinctly, including dissociation of the heat shock proteins, phosphorylation, and dimerization. Binding of PR to progesterone response elements (PREs) promotes the formation of a stable initiation complex, resulting in gene transcription [2]. Several studies have shown that the A and B forms have different transcriptional effects on progestin-responsive promoters depending on the cell and promoter context to activate target genes. The A form is transcriptionally less active than the B form and can function as a strong dominant inhibitor of the B form and other steroid receptors [16–19]. Given the functional transcriptional difference between the two isoforms, an unequal distribution of these isoforms may provide a biologically important regulatory mechanism in the ovary. Indeed, PR-A null mice, which express PR-B only, have decreased ovulation [20], and those that lack only the B form are not affected [21], whereas ablation of both isoforms inhibits ovulation [7, 8].
The cellular responses to the progesterone-PR complex are determined by the total level of PR in diverse target cells. It is interesting that the two isoforms exist at different relative levels in different tissues in vivo [1, 12, 22, 23]. Moreover, overexpression of the B form is related to ovarian cancers [24], which suggests that differing relative PR isoforms and their regulation may be as important as the total PR level in determining the ovarian response to the action of progesterone. However, the regulation of the relative expression pattern of PR isoforms in the ovary has not been described so far.
Follicular atresia, a degenerative process in the mammalian ovary that is mediated via cell apoptosis [25] and regulated by hormones [26], is characterized by internucleosomal DNA fragmentation and the appearance of cell death intermediate molecules, such as active caspase-3 in humans, rats, and mice [27–30]. The marked increase in internucleosomal DNA fragmentation and caspase-3 activity in human and rat periovulatory granulosa cells treated with PR antagonists in vitro [31–33] indicates that expression of functional PR mediates inhibition of apoptosis.
The present study was undertaken to examine the regulation of the relative expression of the PR A and B isoforms in granulosa cells during gonadotropin-induced follicular development and ovulation, and to determine the cellular localization of PR in relation to granulosa cell apoptosis and proliferation in the mouse ovary in vivo and in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Equine gonadotropin (eCG), monoclonal anti-ß-actin (catalog number A-5441) and alkaline phosphatase-conjugated goat-anti-mouse immunoglobulin (Ig; A-1682) were obtained from Sigma Chemical Company (St. Louis, MO). Human chorionic gonadotropin (hCG) and the PR antagonist Org 31710 were obtained from N.V. Organon (Oss, Holland). The PR antagonist RU 486 was obtained from Exelgyn (Paris, France). Rabbit polyclonal anti-PR (sc-538 and sc-539) and their respective blocking peptides (sc-538p and sc-539p), as well as normal mouse IgG (sc-2025), were obtained from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA). Rabbit polyclonal antiactivated caspase-3 (557035) was obtained from BD PharMingen (San Diego, CA). Mouse monoclonal antiproliferating cell nuclear antigen (PCNA) (PC-10; NCL-PCNA) was obtained from Nobacastra Laboratories Ltd. (United Kingdom). PCNA is a nuclear protein required for DNA synthesis and is used as a marker protein for identification of follicular growth [34, 35]. Normal rabbit serum (x-0902) was obtained from DAKO Corp. (Carpinteria, CA). Alkaline phosphatase-conjugated goat-anti-rabbit Ig (AC31RL) was obtained from Tropix (Bedford, MA). All other chemicals were purchased from Sigma unless otherwise specified.
Animals and Preparation of Tissues
Immature female mice (C57BL/6) were obtained from M&B (Denmark). All mice were housed in individual cages in an air-conditioned, controlled environment with a 12L:12D cycle and were fed standard rodent chow and tap water ad libitum during a minimum of 5 days before the start of the experiments. All animal studies described in this study were reviewed and approved by the ethics committee at Göteborg University, Sweden.
Mice (26 days old, body weight 14.05 ± 0.44 g, mean ± SD) were killed at 6, 24, or 48 h after treatment with eCG (5 IU i.p.) to stimulate the development of multiple follicles. In addition, some mice were randomly assigned to receive hCG (5 IU i.p.) at 48 h after administration of eCG to achieve synchronized follicular development and ovulation [36]. From these animals, tissues were collected at 3, 6, 12, 24, and 48 h after hCG treatment. Untreated littermate mice served as controls. After the mice were killed at various time intervals, the ovaries were quickly dissected and trimmed. Isolated granulosa cells were washed three times in PBS and immediately frozen in liquid nitrogen and stored at -135°C until further processing. The ovaries from two mice from each group at various time intervals were fixed in 4% formaldehyde with neutral-buffered solution (Sigma, St. Louis, MO) for 24 h at 4°C, embedded in paraffin, and used for immunohistochemical evaluation of PR protein expression.
Immunoblot Analysis
Granulosa cells or tissues (ovary, uterus, small intestine and liver) were homogenized in a pellet pestle mixer (Merck KGaA, Darmstadt, Germany) using the following ice-cold lysis buffer: 50 mM Tris-HCl, 0.5 M NaCl, 0.5% Nonidet-P40, 50 mM NaF, 0.5 mM Na3VO4, 20 mM Na4P2O7 · 10 H2O pH 7.4, and a cocktail of protease inhibitors (2 mM EDTA, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM dithiothreitol). Lysates were kept on ice for 30 min and centrifuged at 12 000 x g for 30 min at 4°C to remove solid material. The supernatants were collected and their protein concentrations were determined via a bicinchoninic acid protein assay (Pierce, Rockford, IL) with BSA as the standard.
Protein aliquots (30 µg) were pretreated with 4x SDS before loading and separated on SDS-polyacrylamide gels (6% gels for detection of PR, 10% gels for detection of caspase-3 and PCNA; Novex) with a Tris-glycine or Bis-Tris-Mes buffer system under reducing conditions. The separated samples were electrophoretically transferred to polyvinylidene difluoride membranes (Amersham International, Buckinghamshire, U.K.) and treated with blocking buffer (0.2% I-Block, 0.2% BSA, 5 mM MgCl2, 3 mM NaN3, and 0.3% Tween-20 in PBS pH 7.4) for 4 h. The membranes were incubated with primary antibody at 1:1000 dilutions in blocking buffer overnight at 4°C. After washing in blocking buffer for 2 h, the membranes were incubated with alkaline phosphatase-linked secondary antibody (polyclonal secondary antibody at a dilution of 1:40 000 or monoclonal secondary antibody at a dilution of 1:80 000, respectively) in blocking buffer for 4 h with gentle shaking, and detected using the CDP-Star substrate for alkaline phosphatase (Tropix). The second antibody contributed no nonspecific bands at the concentrations employed. Immunoblotted signals were exposed and developed using enhanced chemiluminescence film (Amersham) and subsequently scanned into a computer. Individual bands were quantified directly from membranes by densitometry using the ImageQuant (version 5.0) software program (Molecular Dynamics, Inc., Sunnyvale, CA). No electronic modifications of the images, such as contrast or brightness adjustment, were performed prior to quantitation. All steps were carried out at room temperature unless otherwise stated. Equal loading of proteins was determined by the expression of ß-actin, and were confirmed by staining the gels with colloidal blue after transfer (Novex). In addition, two PR antibodies were preabsorbed with 10-fold excess of neutralizing synthetic PR peptides for 4 h at room temperature before use to demonstrate antigen specificity.
Immunohistochemical Studies
Paraffin sections of 5 µm were mounted onto poly-L-lysine coated slides, deparaffined in xylene, rehydrated through a graded series of ethanol, digested with 20 µg/ml of proteinase K (Boehringer-Mannheim, Mannheim, Germany), treated with 3% H2O2 to remove endogenous peroxidase activity, and blocked for nonspecific binding. The primary antibody was diluted 1:100 in Tris-buffered saline (TBS) containing 1% BSA and incubated overnight at 4°C in a humidified chamber. After washing with TBS, sections were stained using the avidin-biotinylated-peroxidase complex detection system (ABC kit, Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer's instructions. Immunostaining was then visualized using 3,3-diaminobenzidine tetrahydrochloride (0.5 mg/ml in PBS and 0.01% H2O2 pH 7.6) for 10 min. Sections were consecutively counterstained with Richardson modified methylene blue for 1 min, dehydrated, and coverslipped using mounting medium (Mountex, Histolab, Sweden). Slides were viewed on a Nikon E-1000 microscope (Japan) under brightfield optics, and photomicrographed using Easy Image 1 (Bergström Instrument AB, Sweden). Negative control slides were prepared in an identical manner and processed with either TBS containing 1% BSA, normal mouse IgG, or the normal rabbit serum with equivalent concentration in place of primary antibodies. In addition, the specificity of two PR antibodies was monitored during preincubation with a 10-fold excess of neutralizing synthetic PR peptides for 4 h at room temperature and substituted with the same volume for omission of PR antibodies. All controls gave negative results.
In Vivo Experiments
Fifteen mice chosen at random received eCG (5 IU i.p.). The animals were randomly divided into three groups of five mice each. Mice in the first group were given 2 mg of RU 486 in 100 µl of sesame oil by i.p. injection after treatment with eCG for 44 h. Mice in the second and third groups were injected with either 100 µl of sesame oil or 2 mg of RU 486 dissolved in 100 µl of sesame oil after treatment with eCG for 48 h. All mice in the three groups received hCG (5 IU, i.p.) at 48 h after administration of eCG. After 6 h, the mice were killed and the ovaries were rapidly removed and trimmed. Granulosa cells isolated from the left ovary in each mouse were frozen in liquid nitrogen and stored at -135°C, whereas the right ovary from each mouse was fixed in 4% formaldehyde with 10% neutral-buffered AFIP formulation for 24 h at 4°C and embedded in paraffin and prepared for either immunoblot or immunohistochemical studies. In order to evaluate RU-486 inhibition of ovulation, mice were handled and treated as described above and mice ova were flushed from the oviducts at 24 h after hCG treatment and were counted in another experiment.
In Vitro Experiments
Isolation, culture, and treatment of granulosa cells In selected experiments, the granulosa cells were isolated by puncturing either all follicles from immature mice or only big follicles from mice at various time intervals after treatment with eCG, hCG, or both in vivo, and by allowing expulsion of the cells into Earles modified Eagle media (MEM with glutamax-I; Life Technologies, Carlsbad, CA) supplemented with 0.1% BSA fraction V (Sigma), 100 U/ml of penicillin, and 100 µg/ml of streptomycin and pelleted at 200 x g for 5 min at 4°C. Cell numbers were quantified in a hemocytometer with trypan blue dye exclusion (Life Technologies) to estimate viability (between 52% and 75%). The granulosa cells were placed in tubes (Falcon 12 x 75 mm, Becton-Dickinson, Franklin Lakes, NJ) at about 5 x 105/0.5 ml and incubated at 37°C in a humidified atmosphere with 95% air and 5% CO2 for 24 h. At the end of the incubation period, tubes were centrifuged at 200 x g for 5 min at 4°C, and media were removed. Cells were collected for measurement of DNA fragmentation. In selected experiments, granulosa cells isolated from the ovaries in mice treated with either eCG for 48 h or additional hCG for 12 h, were incubated in the absence or presence of RU 486 or Org 31710 for 24 h under the same conditions as described above. RU 486 and Org 31710 were solubilized in absolute ethanol or dimethyl sulfoxide (DMSO). Aliquots from each stock solution were added to fresh medium and each preparation contained less than 0.1% ethanol or DMSO (v/v, final concentration), and served as the control treatment in all experiments.
DNA fragmentation DNA fragmentation was detected as previously described [33]. In brief, 50 µl of cold lysis buffer (5 mM Tris-HCl, 20 mM EDTA, 0.5% Triton pH 8) was added to pelleted granulosa cells and placed on ice for a further 20 min. After adding 250 µl of buffer (5 mM Tris-HCl, 20 mM EDTA pH 8), samples were centrifuged at 11x000 x g for 20 min at 4°C in order to separate fragmented DNA in the supernatant from the intact DNA in the pellets. The pellets were incubated with 300 µl of buffer (10 mM Tris-HCl, 1 mM EDTA pH 7.5) and 30 µg of proteinase-K (Merck, Darmstadt, Germany) overnight at 45°C. Hoechst dye H33258 (0.2 µg/ml in 2 M NaCl, 1 mM EDTA, and 10 mM Tris-HCl pH 7.4) was added to the samples. The DNA content in the supernatant and pellet was measured using a fluorescence spectrophotometer (356 nm excitation and 458 nm emission; F-2000, Hitachi, Kebo, Sweden). Apoptosis reflects the ratio between low molecular weight DNA isolated from the supernatant and total DNA including the supernatant and the pellet. The value for the control group was set to 100%.
DNA synthesis Granulosa cells were plated at a density of approximately 500 000 cells/tube (Falcon 12 x 75 mm) with 0.5 ml Eagle MEM supplemented as above either with or without the progesterone antagonists (RU-486 or Org 31710) for 20 h at 37°C in 95% air and 5% CO2. [methyl-3H]thymidine was added to the culture medium to a final concentration of 1 µCi/ml for a further 4 h, and each group consisted of six tubes. After approximately 24 h, the labeled cells were washed three times with ice-cold PBS, twice with 10% (w/v) trichloroacetic acid for 10 min at 4°C, and once with 95% ethanol. Cells were solubilized in 0.5 ml of 0.1 M NaOH and 0.2% SDS followed by neutralization with 50 µl of 10 N HCl at room temperature, and the incorporated radioactivity was measured by scintillation counting.
Data Analysis and Statistics
In the protein expression studies, the results presented were derived from at least three densitometric values in three independent experiments and from separate mice unless otherwise stated. All in vitro cell culture experiments were performed a minimum of three times (run in triplicate at least in each group) unless otherwise stated. Each experiment represents a separate pool of dispersed granulosa cells. The data are presented as means ± SEM. All statistical analyses were performed using one-way ANOVA followed by Analyse-It multiple comparison tests (Analyse-It Software, Ltd., U.K.) in which differences were indicated to be significant among groups, P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The developmental changes of PR isoform protein levels in the granulosa cells were detected by quantitative immunoblot analysis. Two isoforms of PR protein were detected in mouse granulosa cells (Fig. 1A). When the two PR antibodies were neutralized by incubation with their blocking peptide or replaced with normal rabbit IgG, no PR bands were detected in samples from granulosa cells or uterus, which suggests that the signals are specific for PR. Both the A and B forms remained at low levels after eCG treatment. However, treatment of eCG-primed mice with hCG caused a rapid and significant increase of the expression of both isoforms. The maximum level of both isoforms was found at 6 h after hCG treatment as compared to no eCG treatment or no hCG treatment (time 0 h). The levels of both isoform proteins gradually decreased and declined by 24 h after hCG treatment (Fig. 1B). The average ratio of A form to B form protein levels during hCG treatment was 2.09 ± 0.22 (mean ± SD).
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Immunolocalization of PR Expression in Mouse Ovary
We further explored the expression of PR protein by immunohistochemical analysis to determine the cell type that expresses PR in the ovary. In accordance with the data obtained by immunoblot analysis, expression of PR was absent in all stages of follicles in ovaries during eCG treatment (Fig. 2, A and B), whereas immunoreactivity was apparent in granulosa cells of healthy and large antral follicles in ovaries isolated from mice treated with hCG for 6 and 12 h after 48 h of eCG stimulation (Fig. 2, C and D). There was no detectable immunoreactivity in theca and interstitial cells in any stage of follicles. Furthermore, histological examination of atretic follicles as determined by positive TUNEL staining failed to detect any PR immunoreactivity (data not shown). The PR expression was transient, and no positive immunostaining for PR was observed in granulosa cells at 24 h after hCG treatment or in corpora lutea at 48 h or 1 wk after hCG treatment (data not shown). In addition, immunostaining for PR was negative in corpora lutea in adult, nonpregnant mouse ovaries (data not shown). Sections from human breast carcinoma tissues, which highly expressed PR, were always run in parallel as experimental controls (Fig. 2, E and F).
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Effect of RU 486 on Ovulation and Expression of PR Isoforms, Caspase-3, and PCNA In Vivo
The stage-dependent expression of PR protein in granulosa cells prompted us to investigate PR-mediated functions in vivo using eCG/hCG-primed mice treated with the PR antagonist RU 486.
Quantitative assessment of ovulation showed that RU 486 suppressed ovulation and a reduction in the number of ovulated ova was detected (Table 1).
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RU 486 down-regulated the A and B isoforms of the PR only when mice were treated with RU 486 4 h before they were treated with the bolus of hCG (Fig. 3A). In addition, there was no significant change in the ratio of the A and B forms during RU 486 treatment. Similarly, PCNA protein expression was significantly down-regulated by treatment with RU 486 only when given 4 h before hCG treatment (Fig. 3C). In contrast, treatment with RU 486 activated caspase-3, whether it was administered with hCG or 4 h before hCG (Fig. 3B).
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Ovaries isolated from eCG/hCG-primed mice treated without and with RU 486 were morphologically different. The former resulted in the presence of a large number of antral follicles at 6 h that expressed PR (Fig. 4, A and C), but they showed no positive staining for active caspase-3 (Fig. 4E); the latter resulted in a reduction of the number of large antral follicles, which failed to express PR (Fig. 4, B and D). These large antral follicles were positively stained for active caspase-3 protein expression (Fig. 4F), in agreement with immunoblot analysis studies.
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Gonadotropin Regulation of the Susceptibility of Differentiated Granulosa Cells to Undergo Apoptosis In Vitro
After treatment with eCG, hCG, or both in vivo, the differentiated granulosa cells had significantly decreased DNA fragmentation compared with cells that had been isolated from immature mice before gonadotropin stimulation (Fig. 5). The results were in agreement with histological examination of atretic follicles using a TUNEL assay and associated with a time-dependent decrease of active caspase-3 protein levels using immunoblot analysis (data not shown). In addition, granulosa cells isolated from mice treated with eCG, hCG, or both, incubated under serum-free conditions for 24 h showed a significant decrease of DNA fragmentation compared with cells that had been isolated before gonadotropin stimulation (Fig. 5).
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Effect of RU 486 and Org 31710 on Granulosa Cell DNA Fragmentation and DNA Synthesis In Vitro
Isolated granulosa cells from mice treated with either eCG for 48 h or hCG for 12 h following by 48 h eCG stimulation were cultured in vitro with either 10 µM RU 486 or 10 µM Org 31710 (a specific progesterone antagonist). Addition of RU 486 or Org 31710 to the incubation medium to granulosa cells isolated from ovaries of eCG-treated mice, which lack PR expression, had no effects on DNA fragmentation or [3H]thymidine incorporation as an index of DNA synthesis and cell proliferation. In contrast, a significant increase in DNA fragmentation and a significant decrease in DNA synthesis were observed in granulosa cells isolated from ovaries of eCG-treated mice with an additional 12 h of hCG treatment. These granulosa cells indeed expressed PR (Fig. 6, A and B). Although RU 486 can interact with the glucocorticoid receptor [37], DNA fragmentation in granulosa cells was not affected by treatment with dexamethasone (1–50 µM) during 24 h of incubation (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our results have shown that there was no expression of the total PR in immature mice or during administration of eCG, but it was up-regulated by treatment with an ovulatory dose of hCG. The expression of PR was specific for the granulosa cells of healthy periovulatory follicles and was significantly down-regulated when the granulosa cells were undergoing luteinization. Moreover, a complete absence of detectable PR protein was observed after ovulation and formation of the corpora lutea. These observations confirm that induction of PR expression in the ovary is dependent on stimulation of the LH receptor for induction of progesterone formation in granulosa cells during follicular development. Previous studies on the regulation of this gene's expression have shown that the PR expression is rapidly increased in periovulatory rat granulosa cells and is dramatically decreased after the LH surge that occurs at the time of ovulation in vivo [38–40] and is absent in the corpora lutea in the rat ovary [41]. In contrast, the expression of PR persists in the corpus luteum of the human ovary [42, 43]. Taken together, our results suggest that the expression pattern of ovarian PR in mice is similar to that for rats but differs from that for humans.
Ovulation is one of the major female reproductive events, and appears to involve the expression of a variety of specific genes [10]. Although the functional role of PR and its isoforms associated with ovulation has been well delineated with PR gene-deficient mice [7, 8, 20, 21], the possible consequences of the regulation of the relative expression of PR isoforms in the ovary is unknown. Previous studies have suggested that the cellular ratio of PR isoforms is tissue specific and their relative levels are under developmental control depending on the species [1, 12, 22, 23]. In addition, failure of ovulation in mice that lack both PR isoforms seems unlikely to be due to a deficiency of pituitary gonadotropins or ovarian steroid hormones because the ovaries from these knockout mice exhibit normal follicular development [7, 8]. Therefore, it could be assumed that the gonadotropin-induced ovulation may require the coordinated action of both PR isoforms because the expression of either isoform may cause distinct phenotypes [9] (i.e., PR A-null mice are anovulatory, whereas PR B-null mice ovulate) [20, 21]. We have shown that both PR isoform proteins increased approximately in equal proportion in response to gonadotropin stimulation, peaking at 6 h and maintaining significant high levels at 12 h after hCG treatment. In our experimental models, ovulation occurs approximately 12 to 14 h after hCG treatment [7, 36]. The ratio of the A to B forms was constant during hCG treatment until a decline of the total PR expression. However, the levels of PR-A protein were always approximately 2-fold higher than the levels of PR-B protein. Moreover, treatment with the PR antagonist RU 486 in vivo suppressed the expression of both PR A and B isoforms in the mouse ovary. This finding is consistent with an in vivo study of rat ovary showing down-regulation of PR mRNA by administration of RU 486 [44], indicating that the regulation of PR protein is dependent on a change in its mRNA in the ovary. In our experiments, RU 486 was able to reduce both A and B form protein levels as well as the rate of ovulation in vivo, suggesting that both PR isoforms may be involved in RU 486 inhibitory mechanism and is essential for successful ovulation.
During follicular development in the mammalian ovary, only a few follicles reach ovulation, whereas the majority of follicles are depleted by apoptosis [25, 26, 45]. Granulosa cell apoptosis has been recognized as the cellular mechanism responsible for follicular atresia and is developmentally regulated by gonadotropins [25, 26, 45]. The in vitro system for spontaneous apoptosis development in mouse granulosa cells regulated by gonadotropin stimulation in vivo presented in this paper is consistent with gonadotropin-dependent granulosa cell survival during follicular development [26, 45]. In addition, our data show that in vitro, mice ovarian granulosa cells became less susceptible to apoptosis in response to in vivo treatment with eCG and hCG in a time-dependent manner, which indicates that apoptosis of mouse granulosa cells is stage-dependently regulated, as is apoptosis of human and rat granulosa cells [45].
In general, the regulation of follicular cell death shares the intracellular apoptotic pathways with other cell types [45]. Caspase-3 is a member of the aspartate-specific protease family that plays a central role in apoptosis because they are activated in response to apoptotic stimuli [46, 47]. It has been shown that activation of caspase-3 is involved in follicular apoptosis in rats in vivo [29] and in murine and human granulosa cells in vitro [27, 28, 30]. Furthermore, granulosa cells in atretic ovarian follicles of caspase-3 knockout mice fail to undergo apoptosis [48], which suggests that caspase-3 plays an important role in the programmed death of granulosa cells. An important aim of this study was to evaluate whether the timing of induction of the functional PR protein is of physiological significance in the regulation of granulosa cell fate. We have demonstrated that the induction of PR protein paralleled with decreased activation of caspase-3 in vivo after hCG treatment in periovulatory granulosa cells (data not shown). In addition, treatment with the PR antagonist RU 486 in vivo increased the proportion of periovulatory follicles undergoing apoptosis, which resulted from the activation of caspase-3 and a decreased level of PCNA in PR-expressed granulosa cells. These findings are in agreement with in vitro data. The low level of apoptosis observed in PR-expressed granulosa cells isolated 12 h after hCG treatment in vivo was reversed by the addition of the PR antagonists in vitro. DNA synthesis was also decreased after PR antagonist treatment in vitro. Thus, stage-dependent expression of PR is not only necessary for ovulation, but it also mediates the inhibition of granulosa cell apoptosis through the suppression of the caspase-3 activation pathway, which is in agreement with previous studies of rat and human granulosa cells in vitro by us and others [32, 33, 49]. In addition to gonadotropins, PR as an intraovarian regulator may act in an autocrine manner to influence granulosa cell survival. It has recently been shown that the B form of PR but not the A form can up-regulate PCNA in human breast cancer cells [50] and elicit proliferation of uterine and mammary glands [20]. However, whether the combined expression of both isoforms is necessary for involvement in apoptosis and cell proliferation or whether one isoform is more important than the other requires further investigation.
In summary, we have demonstrated that the developmental and relative expression of PR isoforms in mouse granulosa cells in response to gonadotropin stimulation is associated with the ovulation process. The effect of the PR antagonists on PR-expressed granulosa cells in the mouse ovary suggests that stage-dependent PR acts as a positive regulator for preventing granulosa cells from undergoing apoptosis and shifts the balance toward cell proliferation both in vivo and in vitro.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Håkan Billig, Department of Physiology, Göteborg University, P.O. Box 434, SE-40530 Göteborg, Sweden. FAX: 46 31 7733531; e-mail: hakan.billig{at}fysiologi.gu.se
Received: 3 July 2002.
First decision: 4 August 2002.
Accepted: 20 September 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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