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Endocrinology Vol. 145, No. 11 5033-5043
Copyright © 2004 by The Endocrine Society

Progesterone Receptor and the Cell Cycle Modulate Apoptosis in Granulosa Cells

Susan M. Quirk, Robert G. Cowan and Rebecca M. Harman

Department of Animal Science, Cornell University, Ithaca, New York 14853

Address all correspondence and requests for reprints to: Susan M. Quirk, Department of Animal Science, Morrison Hall, Cornell University, Ithaca, New York 14853. E-mail: smq1{at}cornell.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Our previous studies showed that exposure of bovine preovulatory follicles to the LH surge-induced resistance of granulosa cells, but not theca cells, to apoptosis. Here, the temporal development of resistance to apoptosis and potential roles of progesterone receptor (PR) and alterations in the cell cycle in mediating this effect were examined. Injection of cows with GnRH induced an LH surge within 2 h. Granulosa cells isolated 0, 6, and 10 h after GnRH were sensitive to Fas ligand-induced apoptosis, but cells isolated at 14 h were resistant. PR was first detectable in granulosa cells at 10 and 14 h and was not detectable in theca. Treatment of granulosa cells isolated 14 h after GnRH with the PR antagonist, RU486, induced susceptibility to apoptosis, an effect mediated by PR and not glucocorticoid receptor. After GnRH treatment, granulosa cells, but not theca cells, exited the cell cycle, expression of cyclin D2 was reduced, and p27Kip1 was elevated. Treatment of granulosa cells isolated from small antral follicles with the G1 phase blocker, mimosine, reduced Fas ligand-induced killing, suggesting that nonproliferating cells are resistant to apoptosis. Treatment of granulosa cells isolated 14 h after GnRH with RU486 induced reentry of some cells into the cell cycle and reversed resistance to apoptosis, suggesting that cycling cells became susceptible to apoptosis. Treatment with mimosine prevented the ability of RU486 to promote susceptibility to apoptosis. In summary, the LH surge induces expression of PR by granulosa cells and withdrawal from the cell cycle, and these events promote resistance to apoptosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MOST GROWING FOLLICLES in the mammalian ovary undergo atresia, a process that occurs by programmed cell death, or apoptosis, of somatic and germ cells. Somatic cells of healthy follicles are supported by survival pathways, activated by gonadotropins and a number of growth factors, which prevent apoptosis (reviewed in Ref. 1). The Fas pathway is a well-studied proapoptotic pathway that is present in most cells, including granulosa and theca cells (2, 3). Fas is a cell surface receptor belonging to the TNF receptor family that, in sensitive cells, triggers apoptosis upon binding Fas ligand (FasL) (reviewed in Ref. 4). Expression of Fas and FasL, and susceptibility to FasL-induced apoptosis in vitro, are elevated in granulosa cells from atretic relative to healthy follicles (2, 5). Survival pathways can block Fas-mediated apoptosis in ovarian and other cell types (6, 7, 8). Despite the fact that healthy preovulatory follicles escape atresia in vivo, granulosa cells from these follicles may be susceptible to induction of apoptosis by various treatments in vitro. When granulosa cells from bovine preovulatory follicles, isolated before exposure to the LH surge, were cultured in serum-free, defined medium, approximately 20–30% died by apoptosis in response to treatment with recombinant FasL (9). In the same study, addition of serum to the culture medium protected granulosa cells from undergoing apoptosis in response to treatment with FasL. These findings suggest that survival factors in serum-containing culture medium mimic the protective environment of the healthy follicle in vivo. Our study also showed that granulosa cells from bovine preovulatory follicles differed in their susceptibility to FasL-induced apoptosis depending on whether they were isolated before or after exposure to an LH surge in vivo (9). Granulosa cells isolated from preovulatory follicles after the LH surge and cultured in serum-free, defined medium, were completely resistant to FasL-induced apoptosis. In addition, cells isolated after the LH surge were unaffected by abrupt removal of serum from the culture medium, a treatment that induced apoptosis of approximately 50% of granulosa cells isolated from follicles before the LH surge. In contrast to results with granulosa cells, theca cells from bovine preovulatory follicles isolated before or after the LH surge were equally susceptible to FasL-induced apoptosis (9).

The preovulatory surge of LH induces profound changes in follicular somatic cells that promote ovulation and luteinization (reviewed in Ref. 10). The LH surge induces the expression of progesterone receptor (PR) by granulosa cells (11, 12, 13, 14, 15, 16, 17, 18). PR modulates the final stages of development of the preovulatory follicle and is required for ovulation (19). Progesterone is reported to act as a survival factor in rat and human granulosa cells (20, 21) and in bovine luteal cells (22). It was therefore of interest to determine the role of PR in the development of granulosa cell resistance to apoptosis after the LH surge. The LH surge also signals cessation of granulosa cell proliferation as part of the process of terminal differentiation into cells of the corpus luteum (23, 24, 25). In a number of cell types, the process of terminal differentiation, and accompanying withdrawal from the cell cycle, is associated with resistance to apoptosis (26, 27, 28). In the present study, we examined the relationships among induction of PR expression, changes in cell proliferation after the LH surge, and the development of granulosa cell resistance to apoptosis. In addition, experiments using cultured granulosa cells from preovulatory follicles were performed to test the hypothesis that expression of PR in response to the LH surge mediates withdrawal of granulosa cells from the cell cycle and their resulting resistance to apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Prostaglandin F2{alpha} (PGF2{alpha}) was obtained from Pharmacia & Upjohn Corp. (Lutalyse, Kalamazoo, MI). GnRH (Cystorelin) was obtained from Merial Ltd. (Iselin, NJ). Culture media, fetal bovine serum (FBS), BSA, penicillin, streptomycin, and fungizone were obtained from Life Technologies, Inc. (Grand Island, NY). Dexamethasone, RU486, and medroxyprogesterone acetate (MPA) were from Sigma Chemical Co. (St. Louis, MO). Mimosine was from Calbiochem (La Jolla, CA). Tissue culture plates were obtained from Corning-Costar (Cambridge, MA), and slide wells were obtained from Nalge Nunc International (Naperville, IL). Soluble recombinant human FasL was obtained from Upstate Biotechnology (Lake Placid, NY). Frozen tissue embedding media (Histo Prep), and microscope slides (Superfrost/Plus) were from Fisher Scientific (Pittsburgh, PA). Mouse anti-ß-actin (clone AC-15) was obtained from Sigma Chemical Co. Rabbit antihuman cyclin D2 (sc-181) and rabbit antihuman p27Kip1 (sc-528) were from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse antihuman Ki67 was from Novocastra Laboratories (New Castle upon Tyne, UK). Mouse antihuman PR (clone PR-AT 4.14) was from Affinity BioReagents (Golden, CO). Mouse anti-BrdU (bromodeoxyuridine) (clone 3D4) was from BD PharMingen (San Diego, CA). Alexa 488-conjugated goat antimouse IgG and propidium iodide (PI) were from Molecular Probes (Eugene, OR), and horseradish peroxidase-conjugated goat antimouse IgG and goat antirabbit IgG were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).

Animals
All procedures were approved by the Cornell University Institutional Animal Care and Use Committee and conducted in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. Preovulatory follicles were obtained from cycling, nonlactating Holstein cattle at precise times relative to the preovulatory LH surge using an established protocol (9, 29). After an observed estrus, cows were monitored daily by transrectal ultrasonography to determine the onset of the first wave of follicular growth and to identify the dominant follicle in the wave. On d 6 or 7 of the estrous cycle, cows were injected with PGF2{alpha} (25 mg Lutalyse im) to induce luteolysis and initiate a follicular phase. Luteolysis was confirmed by RIA of progesterone in plasma samples (30): mean plasma progesterone concentration (n = 31 surgeries) decreased from 2.38 ± 0.19 and 2.86 ± 0.18 ng/ml at 12 h and 0 h before PGF2{alpha}, to 0.69 ± 0.07 and 0.53 ± 0.06 ng/ml at 12 and 24 h after PGF2{alpha}, indicating that luteolysis had occurred. At 36 h after PGF2{alpha} treatment, cows were injected with a GnRH analog (Cystorelin) to induce a LH surge. Previous studies showed that this protocol resulted in induction of a LH surge within 2 h after GnRH, ovulation between 22 and 31 h, and development of a normal corpus luteum (9, 29). Induction of a LH surge was confirmed by RIA of LH in plasma samples (31): mean plasma LH concentrations in cows receiving GnRH (n = 21 post-GnRH surgeries) were 1.05 ± 0.14 and 1.23 ± 0.35 ng/ml at 1 h and 0 h before injection of GnRH, and 5.85 ± 0.70 and 11.18 ± 0.82 ng/ml at 1 and 2 h after GnRH. The ovary bearing the preovulatory follicle was removed by colpotomy (32) at 36 h after PGF2{alpha} (0 h, before GnRH injection) or at 6, 10, and 14 h after GnRH. Ovaries were placed in PBS, and transported to the laboratory within 15 min.

Tissue preparation
The preovulatory follicle was dissected from the ovary as previously described (9). Follicular fluid was removed by aspiration using an 18-gauge needle. The follicle was bisected in DMEM-F12, and the follicle wall, composed of theca interna and attached granulosa cells, was isolated using fine forceps. Several pieces of follicle wall were dissected, placed in embedding media, and frozen in liquid nitrogen for subsequent immunohistochemistry. Granulosa cells were scraped from the remaining theca interna using a finely drawn Pasteur pipette, collected by centrifugation, and counted. Granulosa cells were either cultured, fixed for subsequent cell cycle analysis, or frozen in liquid nitrogen for subsequent preparation of lysates (described below). Theca interna were carefully scraped to remove adherent granulosa cells and frozen in liquid nitrogen for subsequent preparation of lysates. Potential contamination of theca samples with granulosa cells has been shown to be insignificant using this dissection protocol (2). In one experiment, granulosa cells were isolated by aspiration of multiple 2- to 4-mm follicles on ovaries obtained at an abattoir and pooled as described (8).

Granulosa cells from the preovulatory follicle were plated in DMEM-F12 (supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml fungizone, 1 mM pyruvate, and 2 mM glutamine) containing 10% FBS with or without 0.5 µM RU486, a PR antagonist. In one experiment, MPA or dexamethasone (0.2, 1, or 5 µM) was added in addition to RU486. Cells were plated at a density of 5 x 104 cells/well in 96-well culture plates, 1 x 106 cells/well in six-well culture plates, or 2 x 105 cells/well in eight-chambered slide-well chambers. At 24 h, media were changed to serum-free defined media:DMEM-F12 supplemented as above and containing 100 ng/ml insulin, 5 µg/ml transferrin, 20 nM Na-selenite, and 0.1% BSA. Cultures received RU486 as before, and treatments were applied at this time (described below). Media from some granulosa cell cultures were collected at 24 and 48 h for measurement of progesterone production by RIA. Granulosa cells from ovaries obtained from an abattoir were plated as described above and maintained in media containing 10% FBS for 48 h. Treatments were then applied in defined media.

Assay of granulosa cell susceptibility to apoptosis
To determine the susceptibility of granulosa cells to FasL-induced apoptosis, cells were treated at 24 h with 0 or 100 ng/ml FasL in DMEM-F12-ITS in the presence or absence of RU486, MPA, and dexamethasone. At 48 h, the number of viable cells attached to each well was determined. In some experiments, cells were pretreated with 0 or 1000 µM mimosine for 4 h before treatment with FasL. Preliminary dose-response experiments determined the minimum dose of mimosine effective in blocking progression from G0/G1 to S phase but without deleterious effects on cell viability (data not shown). Cells were trypsinized, collected and stained with trypan blue, and live cells were counted in a hemacytometer. The percent of granulosa cells killed by FasL was calculated by comparing the number of live cells present in FasL-treated cultures vs. the number of cells in control cultures receiving no FasL but otherwise treated identically. Each experiment was performed using granulosa cells from a single preovulatory follicle. Treatments within each experiment were done in quadruplicate wells. Experiments were repeated three or four times (as indicated in figure legends). The cell viability assay employed provides a quantitative measure of the percentage of granulosa cells susceptible to FasL-induced apoptosis. Our previous studies have verified that FasL-induced killing of bovine granulosa cells occurs by apoptosis by measuring externalization of phosphatidylserine residues on the plasma membrane (3, 7, 33) and by measuring the percent of cells with sub-diploid DNA content (8). Because dying granulosa cells become detached from the culture dish and undergo degeneration as apoptosis progresses, specific assays for apoptosis are useful for detection of early stages of apoptosis but cannot be used to quantify the percentage of cells within a culture that are susceptible to apoptosis over a 24-h culture period.

Assays of proliferation in cultured granulosa cells
The effects of various treatments on the percentage of proliferating granulosa cells was examined by staining for the cell proliferation marker, Ki67, and by determining the percent of cells incorporating the thymidine analog, BrdU, into DNA. Granulosa cells plated in eight-well slide-well chambers were treated at 24 h with or without FasL in the presence or absence of 0.5 µM RU486, or in the presence or absence of 1000 µM mimosine. In some experiments, 10 µM BrdU was also added to cultures at 24 h. At 48 h, cells were fixed in 80% ethanol. Cells were stained for Ki67 using the same protocol as was used for tissue sections (described below). Detection of BrdU incorporation was performed as previously described with minor modifications (8). Briefly, cells were fixed in acetone and incubated for 1 h with 1 µg/ml mouse anti-BrdU in PBS-0.1% BSA-0.1% Tween containing 200 ng/ml deoxyribonuclease. Cells were rinsed, and Alexa 488-conjugated goat antimouse IgG was used to detect the BrdU antibody. For both Ki67 and BrdU staining, nuclei were counterstained with PI to facilitate cell counts. The proliferative index (percentage of cells expressing Ki67) and proportion of cells passing through S phase (percentage of cells incorporating BrdU) were quantified as follows: cells were examined under epifluorescent illumination using the following filters: excitation 460–500 nm, emission 500–540 nm for Alexa-488; and excitation 536–556 nm and emission greater than 590 nm for PI. For each treatment in each experiment, images were obtained of four randomly chosen fields using a Spot II Digital Camera (Diagnostic Instruments, Sterling Heights, MI), and the number of Ki67-positive cells and total cells (PI-stained nuclei), or BrdU-positive and total cells, were counted. For Ki67, fields contained 89 ± 39 cells/field (mean ± SD), and for BrdU, fields contained 104 ± 49 cells/field. Experiments were repeated using granulosa cells from three separate preovulatory follicles.

Immunohistochemistry
Expression of PR and Ki67 in sections of follicle wall were examined by immunohistochemistry. Ten-micrometer-thick sections of follicle wall were fixed in acetone, hydrated in PBS, and incubated with antibody to PR (2 µg/ml) or Ki67 (2 µg/ml) in PBS-0.1% BSA. Cells were rinsed in PBS, and Alexa 488-conjugated goat antimouse IgG was used to detect the antibodies. Cell nuclei were counterstained with PI. Cells were examined under epifluorescence and images obtained as above. For Ki67, the proliferative index for granulosa and theca cells was determined as described above. The granulosa and thecal layers of follicle wall were easily differentiated by morphology, evident in both phase contrast and PI-stained images. Granulosa cells were small, evenly spaced, round to cuboid cells with distinct nuclei. The theca cell layer consisted of a linear band of cells with fibroblastic appearance in a connective matrix separated from the granulosa cell layer by a basement membrane. For Ki67 staining, 272 ± 94 cells/field and 74 ± 35 cells/field (mean ± SD) were counted for granulosa and theca cells, respectively. For both PR and Ki67, follicle walls obtained from three separate preovulatory follicles at 0, 6, 10, and 14 h post GnRH were examined.

Cell cycle analysis
Cellular DNA content was determined by flow cytometric measurement of PI binding as validated previously for bovine granulosa cells (8). Freshly isolated granulosa cells, or granulosa cells cultured in six-well culture plates and collected by trypsinization, were fixed in 80% ethanol and stored at 4 C until staining for flow cytometry. Cells (5 x 105) were stained with 5 µg/ml PI in 0.01 M PBS containing 0.01% Triton X-100 and 30 µg/ml deoxyribonuclease-free ribonuclease A. Cells (10,000 per sample) were analyzed on a FACScan flow cytometer (Becton Dickinson and Co., Franklin Lakes, NJ). Data were gated for single cells and DNA content assigned to G0/G1, S or G2/M phases based on the method of Ormerod (34) using WinMDI software (The Scripps Research Institute, La Jolla, CA).

Detection of cyclin D2 and p27Kip1 by immunoblotting
Freshly isolated granulosa cells (1 x 106 cells/sample) or pieces of theca interna (~3 x 5 mm) were suspended in RIPA buffer (1.0% Nonidet P-40, 0.05% Na-deoxycholate, 0.1% SDS in PBS) containing freshly added proteinase inhibitors (100 µg/ml phenylmethylsulfonyl fluoride and 3 mg/ml aprotinin). Samples were sonicated for 10 sec and lysates frozen until further analysis. Protein content of lysates was determined using the DC Protein assay kit (Bio-Rad, Hercules, CA). Lysates (20–30 µg) were separated by SDS-7.5% PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked in Tris-buffered saline [TBST: 20 mM Tris (pH 8.0), 150 mM NaCl, 0.05% Tween 20] containing 5% nonfat milk for 30 min at room temperature. Membranes were incubated at 4 C overnight in TBST-5% BSA containing antibodies to cyclin D2 (0.4 µg/ml) or p27Kip1 (0.8 µg/ml). Membranes were washed, incubated with horseradish peroxidase-conjugated secondary antibodies in TBST-5% nonfat milk for 30 min at room temperature, and washed. A chemiluminescent signal was generated using Western blot chemiluminescence reagent (NEN Life Science Products, Boston, MA) and membranes were exposed to x-ray film (Kodak, Rochester, NY). After detection of cyclin D2 or p27Kip1, membranes were stripped and reprobed with an antibody to ß-actin (dilution 1:1000). Signals were quantified by densitometry of digitized images using AlphaImager 2200 software (Alpha-Innotech Corp., San Leandro, CA) and ratios of cyclin D2 or p27Kip1 to ß-actin were calculated. Lysates were obtained from three separate follicles each at 0, 6, 10, and 14 h after GnRH.

Statisitcal analysis
Data were analyzed by one-way ANOVA using a completely randomized design except as noted below. Duncan’s new multiple range test was used for comparison of means when overall significance was observed. The experiments examining the effect of mimosine were analyzed by paired t tests, and experiments examining the combined effects of mimosine and RU486, or of MPA, dexamethasone and RU486, were analyzed by ANOVA using a randomized complete block design with experiment replicates as blocks, and Duncan’s new multiple range test was used for comparison of means.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Resistance to FasL-induced killing is mediated by PR
Studies were performed to determine the time course of development of granulosa cell resistance to apoptosis after the LH surge and to test whether resistance to apoptosis is mediated by PR. Granulosa cells were obtained from preovulatory follicles isolated at 0, 6, 10, and 14 h after injection of cows with GnRH (equivalent to ~0, 4, 8, and 12 h after the LH surge). The susceptibility of cells to FasL-induced apoptosis was tested in the presence and absence of the PR antagonist, RU486. Granulosa cells were susceptible to FasL-induced apoptosis at 0, 6, and 10 h after GnRH (18–21% killing) but were resistant to apoptosis at 14 h (<1% killing; Fig. 1Go). Treatment with RU486 had no effect on FasL-induced apoptosis in cells isolated at 0, 6, and 10 h after GnRH but induced susceptibility to FasL in cells isolated 14 h after GnRH (Fig. 1Go). Treatment with 0.5 µM RU486 in the absence of FasL did not alter the number of viable granulosa cells (data not shown). These results suggest that granulosa cells become resistant to apoptosis between 10 and 14 h after GnRH and that this effect is dependent on PR.



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  		FIG. 1. Resistance to FasL-induced apoptosis in granulosa cells isolated before and after the LH surge is mediated by PR. Granulosa cells were isolated from preovulatory follicles obtained 0, 6, 10, and 14 h after injection of GnRH. Cells were cultured in media containing 10% FBS and 0 or 0.5 µM RU486, a PR antagonist. At 24 h, cells were treated with 0 or 100 ng/ml FasL in defined media with 0 or 0.5 µM RU486. The cells were counted 24 h later using trypan blue exclusion. The percent of cells killed by FasL was calculated by comparing the number of viable cells in wells treated with or without FasL. Bars represent the mean ± SEM of results obtained with granulosa cells isolated from four individual follicles at each time point. *, P < 0.05 vs. other values.

 
To determine whether the effect of RU486 was mediated by antagonism of the PR or the glucocorticoid receptor, cells isolated 14 h after GnRH were cotreated with RU486 and increasing doses of MPA or dexamethasone. MPA has high affinity for both PR and the glucocorticoid receptor (35) and, at equivalent or higher doses than RU486, should inhibit the action of RU486 mediated by either receptor. Dexamethasone has very little affinity for PR but high affinity for the glucocorticoid receptor (35). Therefore, dexamethasone should have no effect on actions of RU486 mediated by PR but should block actions of RU486 mediated by glucocorticoid receptor. Cotreatment with 1 or 5 µM MPA blocked the effect of RU486 (0.5 µM) to increase susceptibility to FasL-induced killing, whereas cotreatment with increasing doses of dexamethasone from 0.2–5 µM had no effect (Fig. 2Go). These results support a role of PR, and not the glucocorticoid receptor, in mediating resistance to apoptosis.



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  		FIG. 2. The effect of RU486 to promote susceptibility of granulosa cells to FasL-induced killing is mediated through PR and not the glucocorticoid receptor. Granulosa cells were isolated from preovulatory follicles obtained 14 h after injection of GnRH. Cells were cultured in media containing 10% FBS and 0 or 0.5 µM RU486 alone, or 0.5 µM RU486 plus increasing doses of either MPA or dexamethasone (Dex). At 24 h, cells were treated with 0 or 100 ng/ml FasL in defined media containing 0 or 0.5 µM RU486 alone, or RU486 plus increasing doses of either MPA or Dex. The cells were counted 24 h later using trypan blue exclusion. The percent of cells killed by FasL was calculated by comparing the number of viable cells in wells treated with or without FasL. Bars represent the mean ± SEM of results obtained with granulosa cells isolated from four individual follicles at each time point. *, P < 0.05 vs. unlabeled bars.

 
Expression of PR protein was examined by immunohistochemistry in sections of follicle wall (Fig. 3Go). Staining for PR in the granulosa cell layer was absent at 0 h after GnRH and present in only a few cells at 6 h after GnRH. However, at 10 and 14 h, staining was clearly present, predominantly in the nuclei, of most granulosa cells. In the theca cell layer, expression of PR was absent from cells at all times examined. The results show that bovine granulosa cells begin to express PR protein after exposure to the LH surge and that expression of PR is prominent in cells before development of resistance to apoptosis.



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  		FIG. 3. Top and middle, PR expression in preovulatory follicles obtained before and after an LH surge. Sections of follicle wall were obtained 0, 6, 10, and 14 h after injection of GnRH and PR detected by fluorescent immunohistochemistry. Follicle walls were examined from three follicles at each time point, and representative sections are shown. Magnification, x220. Bottom left, Background fluorescence obtained using nonspecific IgG in place of PR antibody. Magnification, x220. Bottom center, bottom right, High magnification (x800) coincident images of PR staining and PI staining of DNA showing predominantly nuclear localization of PR. The follicle was obtained 14 h after GnRH. G, Granulosa cell layer; T, theca cell layer; I, interstitium.

 
Granulosa cells developed increased capacity to secrete progesterone after the LH surge. Levels of progesterone in follicular fluid increased from 88 ± 14 nM (mean ± SEM, n = 6 follicles) at 0 h after GnRH to 177 ± 11 nM (n = 14 follicles) at 14 h after GnRH (P < 0.05). Granulosa cells isolated 14 h after GnRH were analyzed for the capacity to secrete progesterone in vitro. Cumulative progesterone production over 48 h (mean ± SEM) was 198 ± 78 nmol/well in control cultures and 184 ± 47 nmol/well in cultures treated with RU486 (P > 0.05; n = 4 cultures prepared from different preovulatory follicles). These results show that after the LH surge granulosa cells express PR and have the capacity to secrete progesterone.

Bovine granulosa cells from preovulatory follicles exit the cell cycle after the LH surge
A number of studies have suggested that cells are resistant to apoptosis when in G0 or G1 stages of the cell cycle (see Discussion). Cell proliferation was examined in bovine preovulatory follicles to assess whether resistance to apoptosis after the LH surge was associated with withdrawal from the cell cycle. Ki67 is a nuclear protein that is expressed in proliferating cells during S, G2, and M phases and, in some cells, during G1 phase (36, 37). Expression of Ki67 in sections of follicle wall was determined by immunohistochemistry (Fig. 4Go, B and C). The percent of granulosa cells expressing Ki67 was similar in follicles isolated at 0, 6 and 10 h after GnRH and decreased by 50% at 14 h. Expression of Ki67 in theca was relatively low and did not change after the LH surge. The percent of granulosa cells in various stages of the cell cycle was determined by staining granulosa cells with PI and determining DNA content by flow cytometry (Fig. 4Go, A and C). Within 6 h after GnRH, the percent of granulosa cells in G0/G1 phases increased and the percent of cells in S phase decreased. These changes appeared more pronounced at 14 h after GnRH.



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  		FIG. 4. Granulosa cells of preovulatory follicles withdraw from the cell cycle after exposure to a LH surge. A, Cell cycle phase distribution of granulosa cells isolated from follicles obtained at 0, 6, 10, and 14 h after injection of GnRH. Cell cycle phase was determined by flow cytometric analysis of PI binding to DNA. Cells were analyzed from three follicles at each time point. *, P < 0.05 vs. other times. B, Percent of cells expressing Ki67 in sections of follicle wall obtained at 0, 6, 10, and 14 h after injection of GnRH. Ki67 expression was determined by immunohistochemistry and total cells by nuclear PI staining. Three follicles were analyzed at each time point. *, P < 0.05 vs. other times. C, Representative cell cycle histograms (left column), follicle wall sections stained for Ki67 (center column) and follicle wall sections stained with PI (right column) in samples obtained at the indicated times after GnRH. Magnification, x150.

 
Levels of expression of the cell cycle proteins, cyclin D2 and P27Kip1, were examined in lysates of granulosa and theca cells by immunoblotting. D-Type cyclins are associated with entry into G1 phase and passage through G1 phase. Cyclin D2 is the crucial D-type cyclin in granulosa cells (24, 38). Expression of cyclin D2 was elevated in lysates of granulosa cells prepared from follicles isolated 0 and 6 h after GnRH, and decreased in lysates of granulosa cells from follicles isolated at 10 and 14 h (Fig. 5Go). Expression of cyclin D2 in lysates of theca cells was not detectable by immunoblotting (data not shown), consistent with reports that theca cells express cyclin D3 and not cyclin D2 (24). P27Kip1 inhibits passage of cells through G1 phase and into S phase (39). P27Kip1 expression was low in lysates of granulosa cells isolated at 0, 6, and 10 h after GnRH but was elevated in lysates of granulosa cells isolated at 14 h (Fig. 5Go). Expression of P27Kip1 did not differ in lysates of theca cells isolated from follicles at various times after GnRH (Fig. 5Go). Results of Ki67 staining, cell cycle analysis, and immunoblotting for cyclin D2 and P27Kip1 proteins are consistent with withdrawal of granulosa cells but not theca cells from the cell cycle beginning as early as 6 h and continuing progressively through 14 h after GnRH. The termination of granulosa cell proliferation is coincident with development of resistance to apoptosis.



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  		FIG. 5. Expression of cell cycle proteins in granulosa cells obtained after GnRH injection is consistent with withdrawal from the cell cycle. Cyclin D2 expression in granulosa cells (left column), and P27Kip1 expression in granulosa cells (center column) and in theca cells (right column), were analyzed by immunoblotting. Immunoblots were standardized by reprobing blots for ß-actin expression to obtain relative intensity. Representative blots are shown at top. Mean relative intensities are shown at bottom. Cyclin D2 was undetectable in theca and is not shown. Bars represent the mean ± SEM of results obtained with lysates from three follicles at each time point. *, P < 0.05 vs. unlabeled bars.

 
Granulosa cells in G0 and G1 phases of the cell cycle are resistant to apoptosis
The effect of withdrawal from the cell cycle on the susceptibility of granulosa cells to FasL-induced apoptosis was tested. Granulosa cells were pooled after aspiration of 2- to 4-mm follicles on ovaries obtained from an abattoir. Cells were treated with mimosine, a drug that blocks cells in G1 phase of the cell cycle before the G1/S interface (40). Mimosine treatment reduced killing by FasL by 62% (Fig. 6AGo). Cell cycle analysis confirmed that mimosine blocked cells in G0/G1 phases: the percent of cells in G0/G1 was increased and the percent cells in S and G2/M phases were reduced (Fig. 6BGo).



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  		FIG. 6. Susceptibility to apoptosis requires passage through G1 phase. Granulosa cells isolated from 2- to 4-mm follicles on ovaries obtained at an abattoir were treated in defined media with 1000 µM mimosine, which blocks cells in G1, and 4 h later with 0 or 100 ng/ml FasL. The cells were counted 24 h later using trypan blue exclusion or fixed for flow cytometric analysis of DNA content. A, Percent killing by FasL, calculated by comparing the number of viable cells in wells treated with or without FasL. B, Cell cycle stages in cells treated with or without mimosine. Bars represent the mean ± SEM of results obtained with three separate granulosa cell preparations. *, P < 0.05 vs. control.

 
RU486 induces reentry of granulosa cells into the cell cycle and susceptibility to apoptosis
The results indicate that PR mediates the development of granulosa cell resistance to apoptosis after the LH surge, that resistance to apoptosis occurs at a time after the LH surge when cells have withdrawn from the cell cycle, and that granulosa cells arrested in G0/G1 are relatively resistant to apoptosis. We therefore tested whether PR mediates withdrawal of granulosa cells from the cell cycle after the LH surge and whether this affects susceptibility to FasL-induced apoptosis. Granulosa cells isolated 14 h after GnRH were cultured for 24 h and then treated with or without the PR antagonist, RU486, and with or without FasL. The percent of proliferating cells was determined 24 h later using two methods: measurement of BrdU incorporation into DNA and immunohistochemical staining for Ki67. In the absence of RU486, BrdU incorporation and Ki67 staining were low and FasL treatment did not alter the percent of positively stained cells (Fig. 7Go). Treatment with RU486 increased the percent of cells staining positively for Ki67 and BrdU. Treatment with FasL in the presence of RU486 reduced the percent of cells staining positively for Ki67 and BrdU. These results suggest that antagonizing the PR with RU486 induces proliferation of a subset of cells and increases susceptibility of those cells to FasL-induced apoptosis.



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  		FIG. 7. Treatment with RU486 induces granulosa cells to enter the cell cycle. A, Incorporation of BrdU. Cells isolated from follicles obtained 14 h after GnRH were cultured in media containing 10% FBS and 0 or 0.5 µM RU486. At 24 h, cells were treated with 10 µM BrdU in defined media with 0 or 0.5 µM RU486, and 4 h later cells were treated with 0 or 100 ng/ml FasL. Cells were fixed at 48 h, and BrdU was detected by immunohistochemistry. * and {dagger}, P < 0.05 vs. other treatments. B, Expression of Ki67. Cells isolated from follicles obtained 14 h after GnRH were cultured in media containing 10% FBS and 0 or 0.5 µM RU486. At 24 h, cells were treated with 0 or 100 ng/ml FasL in defined media with 0 or 0.5 µM RU486. Cells were fixed at 48 h. Ki67 was detected by immunocytochemistry. Bars represent the mean ± SEM of results obtained with granulosa cells from three individual follicles. *, P < 0.05 vs. other treatments.

 
Cell cycle withdrawal is necessary for the protective effect of PR against apoptosis
We tested whether reentry of granulosa cells into the cell cycle in response to RU486 is necessary to promote susceptibility to apoptosis. Granulosa cells isolated 14 h after GnRH were cultured for 24 h and then treated with or without mimosine. After 4 h, cells were treated with or without RU486 and FasL in the presence or absence of mimosine. The number of viable cells was determined 24 h later, and the percentage of cells killed by FasL was calculated. As observed previously, little killing in response to FasL occurred in control cultures, whereas significant killing was observed in cultures treated with RU486 (Fig. 8Go). Treatment with mimosine prevented the effect of RU486 to promote susceptibility to FasL-induced killing. The results show that the effect of RU486 to induce susceptibility of granulosa cells to apoptosis requires entry into the cell cycle.



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  		FIG. 8. The ability of RU486 to induce susceptibility to apoptosis is dependent on passage through G1 phase. Granulosa cells obtained from follicles isolated 14 h after GnRH were cultured with or without 0.5 µM RU486. At 24 h, cells were treated with 1000 µM mimosine in defined media and 4 h later with 0 or 100 ng/ml FasL. The cells were counted 24 h later using trypan blue exclusion and the percent of cells killed by FasL was calculated by comparing the number of viable cells in wells treated with or without FasL. Bars represent the mean ± SEM of results obtained with granulosa cells from three individual follicles at each time point. *, P < 0.05 vs. other treatments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Granulosa cells obtained from bovine preovulatory follicles before and after the LH surge provide a model to study the effect of differentiation, and associated withdrawal from the cell cycle, on susceptibility to apoptosis. In cows and humans, the interval from the LH surge to ovulation is prolonged compared with the interval in rodents (about 28–30 h in cows, 36 h in women, and 14 h in rats; reviewed in Ref. 41). Granulosa cells of bovine preovulatory follicles became resistant to FasL-induced apoptosis after development of PR expression and withdrawal from the cell cycle. Treatment of granulosa cells isolated at 14 h after GnRH with a PR antagonist, RU486, increased cell proliferation and reversed the resistance to FasL-induced apoptosis. Cotreatment with the G1 phase blocker, mimosine, prevented the effect of RU486 to induce susceptibility to apoptosis. These data suggest that the acquisition of granulosa cell resistance to apoptosis after the LH surge is mediated by PR and requires withdrawal from the cell cycle.

Our previous study showed that granulosa cells from bovine preovulatory follicles isolated before the LH surge were susceptible to induction of apoptosis by treatment with FasL or withdrawal of serum from the culture medium, whereas granulosa cells isolated 14 h after GnRH were resistant to apoptosis (9). The results of the present study show that resistance of granulosa cells to FasL-induced apoptosis develops between 10 and 14 h after GnRH (8–12 h after the LH surge). The time course of events is consistent with a role for PR in promoting resistance of differentiating granulosa cells to apoptosis. RU486 was effective in promoting FasL-induced apoptosis in cells isolated at 14 h after GnRH, a time point when granulosa cells have become resistant to apoptosis and when development of prominent expression of PR protein in granulosa cells occurs. At other time intervals tested, RU486 had no significant effect. At 6 h after GnRH, expression of PR protein by immunohistochemistry was apparent in some granulosa cells, and at 10 and 14 h, PR was expressed in most granulosa cells. Previous studies showed that PR mRNA was elevated in bovine preovulatory follicles at 6 h after GnRH, which was the earliest time point studied (17), or from 3.5–6 h after GnRH (18). Both studies indicated that PR mRNA levels declined to baseline from 12–18 h after GnRH. In one study, a secondary increase in PR mRNA occurred at 24 h (18), whereas in the other study, levels remained basal at 24 h (17). A comparison of our findings to other studies (17, 18) suggests that detectable expression of PR protein after GnRH treatment (10–14 h) is delayed relative to increases in PR mRNA (3.5–6 h). The possibility that expression of PR protein may be prominent at time points when PR mRNA is decreased requires further study. The results presented here support and extend previous studies: at 12 h after hCG [human chorionic gonadotropin (CG)] treatment to equine (e) CG-primed rats, granulosa cells from preovulatory follicles were relatively resistant to apoptosis induced by culture in serum-free medium, and treatment with a PR antagonist increased apoptosis (20). Treatment with a PR antagonist also increased spontaneous apoptosis of luteinizing granulosa cells obtained from women undergoing oocyte retrieval for in vitro fertilization (cells isolated at 34–36 h after administration of hCG) (21).

Our previous study showed that theca cells differed from granulosa cells in that they were equally sensitive to FasL-induced apoptosis before and after the LH surge (9). In the current study, theca cells differed from granulosa cells in that they did not express levels of PR protein detectable by immunohistochemistry, and they did not exit the cell cycle after the LH surge. A previous study, which used in situ hybridization, demonstrated expression of PR mRNA in granulosa cells but not theca cells in sections of bovine preovulatory follicles (17), whereas another study, which used a ribonuclease protection assay, showed induction of PR mRNA in cultured theca tissue from preovulatory follicles in response to treatment with LH (18). The apparent lack of, or lower levels of, PR protein expression in bovine theca cells compared with the prominent expression in granulosa cells after the LH surge is consistent with findings in rodents. In situ hybridization of ovaries from eCG-primed immature rats showed an increase in PR mRNA in granulosa cells, but not theca cells, of preovulatory follicles within 3–4 h after injection of hCG (14, 42). PR protein was readily detectable by immunohistochemistry in granulosa cells, but not theca cells, of preovulatory follicles within 4 h after injection of hCG (43). In the nonhuman primate and human ovary, however, PR protein was detected by immunohistochemistry in theca cells of all follicular classes but only in granulosa cells of preovulatory follicles that had been exposed to an LH surge (11, 12, 16). Comparison of data obtained in a variety of species, therefore, indicate a common induction of PR expression in granulosa cells of preovulatory follicles by the LH surge and suggest possible differences among species in the regulation of thecal expression of PR.

The present study demonstrates an association between the development of granulosa cell resistance to apoptosis and their withdrawal from the cell cycle after the LH surge. Analysis of the cell cycle by flow cytometry indicated a progressive withdrawal of granulosa cells from the cell cycle beginning 6 h after GnRH. Immunohistochemistry of follicle wall for the cell proliferation marker Ki67 showed that staining in the granulosa cell layer decreased significantly between 10 and 14 h after GnRH. Ki67 is expressed in all cells progressing through the cell cycle, including cells in G1, S, G2, and M phases. Staining for Ki67 is therefore less informative than flow cytometry in detecting early changes in the cell cycle. Staining for Ki67, however, provided a means to compare proliferation in nondispersed granulosa and theca cell layers of the follicle. Changes in the expression of cell cycle regulatory proteins were consistent with measures of cell proliferation. Cyclin D2 protein, which is required for entry into and progression through G1 phase, decreased in granulosa cells between 6 and 10 h after GnRH and remained low at 14 h. The cdk inhibitor P27Kip1, which inhibits progression from G1 to S phase, was low between 0 and 10 h after GnRH and increased at 14 h. In contrast, in the thecal cell layer there was no difference in the percentage of cells staining positively for Ki67 and no change in expression of P27Kip1 protein before and after the LH surge. Thus, after the LH surge, granulosa cells exit the cell cycle beginning by 6 h and continuing through 14 h and become resistant to apoptosis during this time. Proliferation of theca cells and their susceptibility to apoptosis does not change. The earliest time point at which FasL-induced killing of granulosa cells was significantly reduced was 14 h after GnRH. At 6 h and 10 h, however, the percentage of cells killed by FasL was reduced relative to 0 h, but these effects were not statistically significant. In addition, whereas effects of RU486 to increase FasL-induced killing were significant at the 14-h time point alone, small effects were apparent at 6 and 10 h that were not statistically significant. These results suggest the possibility that changes in cell proliferation, effects of PR, and resistance to apoptosis begin by 6 h and intensify through 14 h after GnRH. Changes in cell proliferation in the bovine follicle are consistent with findings in other species. Proliferation of granulosa cells in preovulatory follicles of hormonally primed rats decreased within 4 h after an ovulatory dose of hCG (23). Expression of cyclin D2 mRNA in granulosa cells decreased within 4 h after hCG, whereas expression of the cdk inhibitor p21cip1 increased at 4 h and P27Kip1 increased between 12 and 24 h (24). Potential changes in the theca cell layer were not detailed in these studies (23, 24). In nonhuman primates, about 50% of granulosa cells from preovulatory follicles stained positively for Ki67 before the LH surge, whereas relatively few theca cells were positive (25). Within 12 h after the LH surge (the first time point studied), Ki67 staining decreased in the granulosa cell layer and remained low at 36 h (detailed information on the theca was not provided; Ref. 25).

Although cell proliferation can continue as cells differentiate, the final stage of differentiation, often referred to as "terminal differentiation," is frequently associated with exit from the cell cycle (reviewed in Ref. 44). Granulosa cells in growing follicles proliferate while they progressively develop differentiated characteristics, such as acquisition of LH receptors, steroidogenic capacity, and ability to synthesize inhibin. The terminal differentiation of a granulosa cell is considered its final development into a luteal cell and is associated with exit from the cell cycle (reviewed in Ref. 23). Terminal differentiation of cells and their withdrawal from the cell cycle is associated with resistance to apoptosis (reviewed in Ref. 45). For example, induction of p21cip1 during differentiation of neuroblastoma cells is required for their survival (26). Differentiation of myoblasts into myotubes is associated with induction of p21cip1, cell cycle withdrawal, and resistance to apoptosis (27). Granulosa cells appear to be relatively resistant to apoptosis in early G1 phase of the cell cycle because treatment of proliferating granulosa cells with the G1 phase blocker, mimosine, increased the percent of cells in G0/G1 and decreased FasL-induced apoptosis. Other evidence supports the resistance of quiescent cells to apoptosis. Treatment of T cells with agents that block the cell cycle in early G1 induces resistance to apoptosis, whereas blocking them at the G1 to S phase transition increases susceptibility to apoptosis (reviewed in Ref. 46). Members of the bcl-2 family of proteins, some of which are potent inhibitors of apoptosis, also prevent quiescent cells from entering the cell cycle (reviewed in Ref. 45). In contrast to quiescent cells, proliferating granulosa cells are dependent on growth factors to stimulate mitosis as well as to protect against apoptosis (8). Interestingly, in the current study, treatment of proliferating granulosa cells with mimosine, which blocked cells in G0/G1 phase, protected against apoptosis. Our previous studies with proliferating granulosa cells from 2- to 4-mm follicles showed that the protective effect of IGF-I against FasL-induced apoptosis was prevented by blocking cells later in the cell cycle, at the G1 to S phase transition (8). Taken together, our studies suggest that proliferating granulosa cells are susceptible to apoptosis during transit through the cell cycle and require growth factors for protection, whereas granulosa cells residing in G0 or early G1 phases of the cell cycle are resistant to apoptosis.

We hypothesized that the effect of RU486 to promote susceptibility of granulosa cells to FasL-induced apoptosis was mediated by reentry of cells into the cell cycle. Treatment with RU486 increased the number of proliferating cells, as measured by staining for Ki67 and BrdU incorporation, and the number of proliferating cells was reduced by simultaneous treatment with FasL. Therefore, induction of cell cycle reentry by RU486 appeared to promote susceptibility to FasL-induced apoptosis. In a subsequent experiment, treatment with mimosine prevented the effect of RU486, demonstrating that cell cycle reentry was required to induce susceptibility to apoptosis. Effects of progesterone on cell proliferation have been demonstrated previously: treatment with progesterone inhibited proliferation of human granulosa cells isolated after an ovulatory dose of hCG, and treatment with aminoglutethamide blocked progesterone synthesis and increased proliferation (47). In addition, a progesterone-binding protein has been implicated in antiapoptotic and antiproliferative effects of progesterone in nonluteinized rat granulosa cells (reviewed in Ref. 48). This binding protein, which is distinct from the classical nuclear PR isoforms, has been identified in rat granulosa cells and its presence is independent of the LH surge (49). It is likely that antiapoptotic and antiproliferative effects mediated through PR in the present study are mediated by classical nuclear PR, which is expressed in bovine granulosa cells only after exposure to the LH surge.

In summary, the LH surge induces expression of PR by granulosa cells of the preovulatory follicle and their exit from the cell cycle, and these events promote resistance to apoptosis. These changes do not occur in the theca cells. Our experiments suggest that the effect of PR to promote resistance of granulosa cells to apoptosis requires withdrawal from the cell cycle. Increased resistance to apoptosis during differentiation may play a role in promoting the long-term survival of granulosa-derived cells of the corpus luteum.


   Acknowledgments
 
The authors thank Dr. Dale Porter (Novartis Institute for Biomedical Research, Cambridge, MA) for initiating these studies.


   Footnotes
 
This work was supported by National Institutes of Health Grant HD 32535.

Abbreviations: BrdU, Bromodeoxyuridine; CG, chorionic gonadotropin; eCG, equine CG; FasL, Fas ligand; FBS, fetal bovine serum; hCG, human CG; MPA, medroxyprogesterone acetate; PGF2{alpha}, prostaglandin F2{alpha}; PI, propidium iodide; PR, progesterone receptor.

Received February 5, 2004.

Accepted for publication July 12, 2004.


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