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Endocrinology Vol. 140, No. 5 2307-2317
Copyright © 1999 by The Endocrine Society

ARTICLES

Involvement of the Fas/Fas Ligand System in p53-Mediated Granulosa Cell Apoptosis during Follicular Development and Atresia1

Jong-Min Kim2, Yong-Dal Yoon and Benjamin K. Tsang

Reproductive Biology Unit (J.-M.K., B.K.T.), Departments of Obstetrics & Gynecology and Cellular & Molecular Medicine, University of Ottawa; and the Hormones, Growth and Development Unit, Loeb Health Research Institute (J.-M.K., B.K.T.), Ottawa, Ontario, Canada K1Y 4E9; and the Department of Biology (Y.-D.Y.), College of Natural Sciences, Hanyang University, Seoul 133–791, Korea

Address all correspondence and requests for reprints to: Dr. Benjamin K. Tsang, Reproductive Biology Unit, Department of Obstetrics and Gynecology, The Ottawa Hospital (Civic Site), 1053 Carling Avenue, Ottawa, Ontario, Canada K1Y 4E9. E-mail: btsang{at}lri.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study we have examined the presence of Fas, Fas ligand (FasL), and p53 in rat granulosa cells during follicular development and atresia, especially in relation to the granulosa cell cycle progression and the onset of granulosa cell apoptosis. Fas, FasL, and p53 proteins were immunolocalized, and their contents were determined by Western blotting. Granulosa cell apoptosis was assessed by DNA fragmentation analyses (DNA ladder) and in situ terminal deoxynucleotidyl transferase mediated deoxy-UTP-biotin nick end labeling (TUNEL) as well as by flow cytometry. Ovaries not exposed to gonadotropins (control) consisted predominantly of preantral and early (small) antral follicles, the latter of which were mostly atretic and demonstrated intense TUNEL staining in granulosa cells exhibiting positive immunoreactivities for FasL and Fas. Granulosa cells isolated from these follicles were apoptotic, as evident by clear ladder pattern of DNA fragmentation upon electrophoretic analysis and the high percentage (>10%) of the cell population in the A0 phase of the cell cycle. After gonadotropin treatment, these features completely disappeared during each of the 3 days of follicular growth to the medium to large antral stages. Cell cycle analysis showed significantly higher proportion of the cells in S and G2/M phases compared with controls, which was accompanied by marked decrease in immunoreactivities for Fas, FasL, and p53. By days 4 and 5, widespread atresia and extensive granulosa cell apoptosis were noted in large antral and preovulatory follicles and were coincidental to increased expression of p53 and Fas, but not of FasL, as well as an apparent arrest of granulosa cell G1/S progression, as evident by an increased cell population in G0/G1 and a decrease in the S and G2/M. Granulosa cells from equine CG-primed ovaries exhibited marked increases in p53 and Fas protein contents and apoptosis after adenoviral p53-sense complementary DNA infection in vitro and were more responsive to Fas activation by an agonistic Fas monoclonal antibody challenge. Taken together, these findings are consistent with the well accepted concept that gonadotropin plays a central role as a survival factor in the regulation of granulosa cell Fas/FasL and p53 expression during ovarian follicular development. In addition, the control of granulosa cell apoptosis may involve two consecutive cellular/molecular events: cell cycle arrest at G1/S and exit from G0 into A0 phase, via regulation of the p53 and Fas/FasL death pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FOLLICULAR atresia is an important, negatively selective degenerative process during mammalian ovarian follicular growth and development (1, 2) and is believed to involve granulosa cell death by apoptosis (3, 4, 5). Although several intracellular molecules, including Bcl-2 (6, 7), Bax (6), caspases (8, 9), inhibitor of apoptosis proteins (10), and deoxyribonuclease I (DNase I)-like endonuclease (11), have been implicated to be directly involved in the regulation of this process, recent studies on the cellular and molecular events leading to granulosa cell apoptosis have suggested that Fas antigen (Fas)- and/or p53-mediated death pathways may be central in the induction of follicular atresia (12, 13, 14, 15).

Fas belongs to the family of cell membrane integral glycoproteins that includes nerve growth factor receptor and tumor necrosis factor receptor (16). Its ligand [Fas ligand (FasL)] is a type II integral membrane protein homologous to tumor necrosis factor and is known to be a potent cell death factor (17). Binding of FasL to Fas or cross-linking Fas with agonistic antibody induces apoptosis in Fas-bearing cells (18, 19). Although transcripts for both Fas and FasL are detectable in a variety of tissues, including thymus, liver, heart, lung, small intestine, kidney, and testis (20, 21), only the presence of the Fas protein and message has been demonstrated in granulosa cells (14, 22, 23). A recent report by Hakuno et al. (14) suggested a possible involvement of Fas as a mediator in rat granulosa cell apoptosis during follicular atresia. However, these studies did not examine the expression of Fas during antral follicular development and failed to detect FasL in granulosa cells. Thus, information on dynamic changes in Fas and FasL protein expression during follicular development and atresia remains to be determined.

The p53 protein is a antiproliferative transcription factor that enhances the rate of gene transcription important for p53-dependent functions (24). p53 is believed to be involved in cell cycle arrest during the G1 to S phase transition, DNA repair, control of genome integrity, and apoptosis (25, 26). It is known that mutation of the p53 gene is an important etiological factor for tumorigenesis in a variety of tissues, including human ovarian surface epithelium (27, 28). DNA damage (e.g. induced by UV irradiation) and growth factor withdrawal lead to p53-dependent apoptosis (29, 30). However, the mechanism(s) by which p53 controls apoptosis has not been well established. In this context, Tilly et al. (12) demonstrated that gonadotropin suppresses p53 messenger RNA expression in the immature rat ovary, and that p53 protein is present in apoptotic granulosa cell nuclei of atretic follicles. In addition, involvement of p53 in cAMP-mediated apoptosis has been emphasized in immortalized granulosa cells (13). However, the dynamic changes in p53 protein content during follicular development and atresia, particularly in relation to those of the Fas/FasL system, remains to be elucidated.

The purpose of the present study was to examine the expression of ovarian Fas, FasL, and p53 as well as the possible interaction/relationship between p53 and the Fas/FasL system in granulosa cell apoptosis during follicular development and atresia, particularly in the context of granulosa cell cycle progression and apoptosis. Using an eCG-primed immature rat model and an adenoviral p53-sense complementary DNA (cDNA) expression system to induce granulosa cell apoptosis in vitro, we have demonstrated for the first time that the induction of granulosa cell apoptosis and follicular atresia may involve Fas activation in a p53-mediated mechanism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
[{alpha}-32P]Dideoxy-ATP and the enhanced chemiluminescence Western blotting detection kit were obtained from Amersham (Arlington Heights, IL). Acetic acid, ethanol, formalin, hydrogen peroxide, and xylene were purchased from BDH (Toronto, Canada). The Bio-Rad DC protein assay kit, dithiothreitol, horseradish peroxidase (HRP)-conjugated goat antirabbit secondary antibody, protein mol wt standards (low range), and nitrocellulose membrane were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). Biotinylated 16-deoxy (d)-UTP, DNase-free ribonuclease (RNase), HRP-conjugated streptavidin, proteinase K, terminal deoxynucleotidyl transferase (TdT), and yeast transfer RNA were purchased from Boehringer Mannheim (Indianapolis, IN). X-Ray films were obtained from Eastman Kodak Co. (Rochester, NY). HBSS, MEM, nonessential amino acids, penicillin, streptomycin, and X-galactosidase were obtained from Life Technologies (Burlington, Canada). Hamster antimouse Fas monoclonal antibody (clone Jo2) and hamster IgG were obtained from PharMingen (San Diego, CA). Rabbit IgG, goat IgG, rabbit and goat peroxidase kits, rabbit polyclonal antimouse Fas (sc-716) and antirat FasL (sc-834) antibodies, goat polyclonal antirat p53 antibody (sc-1313), and neutralization peptides (Fas, sc-716P; FasL, sc-834P; p53, sc-1313P) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Acrylamide, agarose, ammonium acetate, ammonium persulfate, aprotinin, BSA (fraction V), chloroform, cobalt chloride, diaminobenzidine tetrahydrochloride (DAB), EDTA, equine CG (eCG), Giemsa, glycine, hematoxylin, Hoechst 33258, imidazole, isoamyl alcohol, leupeptin, methyl green, N,N'-methylenebis-acrylamide, Nonidet P-40, normal goat serum, paraformaldehyde, phenol, phenylmethylsulfonylfluoride, PBS, potassium acetate, propidium iodide, sodium acetate, sodium azide, sodium carcodylate, sodium chloride, sodium deoxycholate, SDS, sucrose, tetramethylene diamine, Trizma base, Triton X-100, Tween-20, and veronal acetate were purchased from Sigma Chemical Co. (St. Louis, MO).

Animals and tissue/cell preparations
Immature female Sprague Dawley rats (21 days old) from Charles River Canada (Montréal, Québec, Canada) were injected ip with eCG (15 IU in 200 µl saline) at the age of 22–23 days for the induction of ovarian follicular development and atresia (4, 31). This treatment induces atresia (histologically and biochemically) in large antral and preovulatory follicles resembling those in animals after gonadotropin deprivation, hypophysectomy, and pentobarbitone administration. In addition, ovulation (on day 3) and the presence of corpus lutea (on days 4 and 5) were rarely evident (<5%) in these experimental animals. The animals were fed Pro Lab RMH 4018 (Agway, Inc., Syracuse, NY) and water ad libitum. A 14-h light, 10-h dark photo-cycle was maintained, with lights on at 0600 h. Rats were killed by cervical dislocation 0, 1, 2, 3, 4, and 5 days after gonadotropin treatment, and ovaries were excised. After removal of connective tissues, the ovaries were briefly washed in PBS (pH 7.4) to remove excess blood and either immediately fixed in 10% neutral buffered formalin (pH 7.4) for histological processing or used for granulosa cell isolation by follicle puncture (32).

DNA extraction for fragmentation analysis
Total DNA was extracted according to the modified procedure of Gross-Bellard et al. (33). Briefly, granulosa cells were homogenized in a buffer (pH 8.0) containing 300 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA, and 200 mM sucrose. The homogenates were incubated in 0.6% SDS (65 C; 30 min) and 35 mM potassium acetate (0 C; 60 min), and centrifuged (5,000 x g, 4 C, 10 min). The supernatants were extracted with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1, vol/vol/vol), followed by chloroform-isoamyl alcohol (24:1, vol/vol). The nucleic acid in the aqueous phase was precipitated with 2.5 vol absolute ethanol (-70 C, 60 min) and collected by centrifugation (14,000 x g, 4 C, 30 min). The pellets were resuspended in TE buffer (10 mM Tris-HCl and 1 mM EDTA; pH 8.0), incubated with DNase-free RNase (500 µg/ml, 37 C, 60 min), and reextracted with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1, vol/vol/vol) followed by an equal volume of chloroform-isoamyl alcohol (24:1, vol/vol). DNA in the resulting supernatants was precipitated [with 0.1 vol 3 M sodium acetate and 2.5 vol absolute ethanol (-70 C, 60 min)], collected by centrifugation (14,000 x g, 4 C, 30 min), washed with ethanol (80%, 0 C), and dried in a Speed-Vac concentrator (Savant Instruments, Farmingdale, NY). DNA content was determined spectrophotometrically at 260 nm, using a Spectronic 1201 (Milton Roy, NY).

For biochemical assessment of DNA fragmentation, DNA was radiolabeled at the 3'-ends according to an established method (34). One microgram of DNA was incubated (37 C, 60 min) with 50 µl of the labeling buffer (25 mM Tris-HCl, 200 mM sodium cacodylate, 5 mM cobalt chloride, and 250 µg/ml BSA, pH 6.6) containing TdT (25 U) and [{alpha}-32P]dideoxy-ATP (5 mCi, 3000 Ci/mmol), and the reaction was terminated with the addition of 5 µl 250 mM EDTA (pH 8.0). Unincorporated radionucleotide was removed by the addition of 0.2 vol 10 M ammonium acetate and 3 vol absolute ethanol (-70 C, 60 min), using yeast transfer RNA (50 µg). The nucleic acid was collected by centrifugation (14,000 x g, 4 C, 20 min), precipitated with ethanol, and air-dried. The labeled DNA were resolved on 1.8% agarose gel for 3.5 h at 60 V in 40 mM Tris-acetate and 1 mM EDTA. The gel was dried without heating and exposed to a Bio-Rad Laboratories, Inc., phosphorimager screen to quantify low mol wt DNA (<4 kb) densitometrically and were subsequently exposed to x-ray film at -70 C.

Nuclear morphological assessment of granulosa cell apoptosis
Granulosa cells were stained with either Giemsa or Hoechst 33258 in the assessment of apoptotic nuclei. The cell suspension (attached and floating cells) was centrifuged (8000 x g, 4 C, 10 min), and the pellet was resuspended in paraformaldehyde solution (4% in PBS, 10 min), smeared on the glass microscope slide, left to air-dry, and stained for 10 min with either Hoechst 33258 (10 µg/ml; in the dark) or Giemsa solution (0.4% in buffered methanol, pH 6.8). At least 400 cells were assessed in each treatment group for apoptotic morphology. Apoptotic cells exhibited distinct characteristics (smaller size, condensed chromatin, and fragmented nuclei) compared with healthy and nonapoptotic ones.

Flow cytometric analysis of granulosa cells
Granulosa cells were fixed in 80% ethanol (1–2 x 106 cells/ml, 4 C, 2–3 h) and resuspended in 1 ml 50 µg/ml propidium iodide (room temperature) in modified HBSS (pH 7.4) containing 0.1% Triton X-100, 0.1 mM EDTA, and 50 µg/ml RNase. Samples were filtered through a 35-µm pore size nylon mesh to remove cell clumps, and the percentage of cells with degraded DNA and cell cycle distribution were determined at an excitation wave length of 488 nm, using a Coulter EPICS Profile II (Hialeah, FL) flow cytometer. Cell cycle histograms were obtained from 10 determinations, each with a total of 10,000 cells/group. G0/G1 doublets were gated out by pulse-processing methods, using peak and integrated DNA fluorescence. The Profile II histogram analysis option was used to set up analysis cursors for data acquisition in the A0 (subpopulation of cells with degraded DNA and lower DNA fluorescence than G0/G1 cells), G0/G1, S, and G2/M regions of the DNA histogram.

Histological assessment of stages of follicular atresia
Based on the number of granulosa cells exhibiting pyknotic nuclei (pyknosis) and the morphological condition of the granulosa cell layers (as observed on hematoxylin-eosin-stained sections), follicles were classified into three stages of atresia: weakly atretic, one or two pyknotic cells and intact granulosa cell layer; moderately atretic, three to six pyknotic cells and granulosa cell layer loose attached to basement membrane; and extremely atretic, more than seven pyknotic cells and fragments of granulosa cell layer scattered in follicular fluid.

In situ localization of apoptotic cells: TdT-mediated dUTP-biotin nick end labeling (TUNEL)
The TUNEL method of Gavrieli et al. (35) was used to localize apoptotic cells in paraffin whole ovarian sections (4–5 µm) mounted on positively charged slides (ProbeOn Plus, Fisher Scientific, Pittsburgh, PA). The sections were deparaffinized, hydrated, treated with 10 µg/ml proteinase K (37 C, 30 min), washed in Tris buffer (100 mM Tris-HCl and 150 mM NaCl, pH 7.5) for 15 min, and dipped in methanol containing 0.3% H2O2 (room temperature, 20 min). The sections were then rinsed in distilled water (15 min), soaked in the TdT buffer (25 mM Tris-HCl, 200 mM sodium cacodylate, 5 mM cobalt chloride, and 250 µg/ml BSA, pH 6.6; 15 min) and incubated in 50 µl TdT buffer (10 U TdT and 1 nmol biotinylated 16-dUTP) at 37 C for 60 min. The biotinylated dUTP molecules incorporated into nuclear DNA were incubated with HRP-conjugated streptavidin (diluted 1:100, room temperature, 30 min), and the peroxidase coloring reaction was performed with 0.3 mg/ml DAB in 0.05 M Tris-HCl, 0.65 mg/ml sodium azide, 10 mM imidazole, and 0.003% H2O2 for 1–5 min. The nuclei were counterstained with 5% methyl green buffered with 0.1 M veronal acetate at pH 4.0. In negative control slides, TdT enzyme or biotinylated 16-dUTP were omitted in the labeling reaction.

Immunohistochemistry
For Fas, FasL, and p53 immunohistochemistry, paraffin-embedded whole ovarian sections were incubated in 0.3% H2O2 for 20 min and rinsed thoroughly with PBS for 15 min. The sections were blocked with 1.5% normal goat/horse serum in PBS (room temperature 1 h), and then incubated with rabbit polyclonal antimouse Fas (0.3 µg/ml), rabbit polyclonal antirat FasL antibodies (0.3 µg/ml), and goat polyclonal antirat p53 (0.3 µg/ml) in 1.5% normal goat/horse serum (room temperture, 45 min) in PBS. The sections were incubated at room temperature with biotin-conjugated secondary antibody (1:200, 1 h), avidin-biotin-peroxidase complex (Santa Cruz Biotechnology, Inc., rabbit and goat peroxidase kit; 1 h) and DAB solution (1–5 min). The nuclei were counterstained with hematoxylin. For negative controls, rabbit or goat IgG (1 µg/ml) instead of the primary antibodies was added to the reaction.

Overexpression of p53 following adenoviral p53-sense cDNA infection in vitro
Granulosa cells (2.5 x 106), isolated 48 h after eCG injection (15 IU, ip), were plated for 6 h (5% CO2; 37 C) in 60-mm plastic culture dishes in medium [MEM supplemented with 0.1% BSA, 1 x nonessential amino acids, penicillin (100 U/ml), and streptomycin (100 µg/ml)], and subsequently incubated for 1 h in 500 µl of serum-free MEM containing adenoviral p53 (AdCMV.p53) and LacZ (AdCMV.LacZ;control) sense cDNA at different multiplicities of infection (MOI) (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25). Additional adenovirus-free medium (2 ml) was then added, and incubation was allowed to continue for 24 h. The efficiency of infection, as assessed by the X-galactosidase test on granulosa cells infected for 24 h with adenoviral LacZ, was more than 85%. AdCMVp53 was provided by Dr. F. L. Graham (McMaster University, Hamilton, Canada). It is a replication-defective adenovirus containing the cDNA for human wild-type p53 driven by the cytomegalovirus promoter (36). The plasmid contains two segments of adenovirus type 5 DNA that were separated from each other by an expression cassette composed of the immediate early promoter of human cytomegalovirus followed by a multiple cloning region and simian virus 40 late polyadenylation signal. The 1.8-kb fragment of p53 was inserted into the BamHI site of the multiple cloning region. The virus was amplified in 293 cells using standard procedure (37). AdCMV.LacZ is a replication-deficient adenovirus encoding bacterial galactosidase. To assess the functionality of the Fas pathway following p53 overexpression, LacZ- and p53-infected cells were incubated for 6 h with either agonistic Fas monoclonal antibody (hamster antimouse Fas mAb, IgG; 1 µg/ml) or hamster IgG (control; 1 µg/ml), and apoptosis in the infected cells was assessed as described.

Protein extraction and Western blot analysis for Fas, FasL, and p53
Freshly isolated and cultured granulosa cells were lysed with ice-cold PBS (pH 7.4) containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors (1 mM phenylmethylsulfonylfluoride, 10 mg/ml aprotinin, and 10 mg/ml leupeptin). To facilitate the complete solubilization of cellular proteins, the cell lysates were sonicated (5 sec/cycle, three cycles, 0 C), incubated on ice for 30 min, and centrifuged (13,000 x g, 4 C, 30 min). The protein content of the supernatant was determined with the DC protein assay from Bio-Rad Laboratories, Inc. Equal amounts of proteins (20 µg) in the cell extracts were resolved by SDS-PAGE (10% or 12%) and electrotransferred to nitrocellulose membranes. The membranes were then blocked (room temperature, 1 h) with Blotto (Tris-buffered saline, pH 8.0, with 0.05% Tween-20 and 5% dried nonfat milk) and incubated (room temperature, 1 h) with 0.1 µg/ml rabbit polyclonal antimouse Fas, rabbit polyclonal antirat FasL, or goat polyclonal antirat p53 antibody. The membranes were washed in Tris-buffered saline, pH 8.0, with 0.05% Tween-20 (twice, 7 min each time) and incubated (room temperature, 30 min) in HRP-conjugated secondary antibody (1:3,000) in Blotto. Peroxidase activity was visualized with the Western enhanced chemiluminescence system (Amersham) according to the manufacturer’s instruction, and Fas, FasL, or p53 protein contents were determined densitometrically. Antibody specificity was confirmed by antibody preabsorption test, using 0.1–0.3 µg Fas, FasL, and p53 neutralizing peptides in the respective primary antibody reaction.

Statistical analysis
Data were expressed as the mean ± SEM of three experiments. Statistical analysis was performed by one- or two-way ANOVA. Significant differences between treatment groups were determined by the Tukey test. Statistical significance was inferred at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DNA fragmentation and protein levels of Fas, FasL, and p53 in granulosa cells during follicular development and atresia
The DNA degradation pattern and the abundance of low mol wt DNA fragments (<4 kb) in granulosa cells on each of 5 days after eCG treatment are shown in Fig. 1Go. DNA isolated from granulosa cells of immature animals not exposed to exogenous gonadotropin (day 0) or injected with PBS (vehicle; data not shown) exhibited the typical apoptotic DNA degradation pattern (presence of internucleosomal fragments of 185-bp multiples), which completely disappeared on the days 1, 2, and 3 post-eCG injection. This DNA ladder pattern, however, reappeared on day 4 and was sustained until day 5 (Fig. 1Go, A and B). Although the 185- and 370-bp fragments appeared in greater abundance on day 0 than on days 4 and 5 (Fig. 1AGo), no significant overall quantitative differences were observed in low mol wt DNA degradation on days 0, 4, and 5 (Fig. 1BGo). Fas and FasL protein contents decreased progressively after gonadotropin treatment, and a significant difference in FasL levels was noted on days 2 and 3 (P < 0.001, compared with day 0; Fig. 1Go, C and D). Although the Fas level on day 4 significantly increased compared with those on days 2 (P < 0.05) and 3 (P < 0.01), the FasL protein content remained low and was not significantly different from that on day 3 (P > 0.05; Fig. 1DGo). p53 in granulosa cell extracts migrated on SDS-PAGE as a doublet; its level also decreased immediately after eCG treatment and was significantly elevated on days 3, 4, and 5 (P < 0.01; Fig. 1DGo). Although the intensity of the fast migrating band of the protein (indicated by the arrowhead) remained relatively high throughout days 3–5, coincident to the increase in apoptotic DNA degradation (Fig. 1Go, A and B) was the increased immunoreactivity of the slow migrating band on days 4 and 5 as well as the decreased immunosignal of the fast migrating protein (Fig. 1CGo).



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  		Figure 1. Fas, FasL, and p53 protein contents and apoptotic DNA degradation in granulosa cells from immature rats at different times (0–5 days) after eCG injection (15 IU, ip). A, Representative autoradiogram; extracted DNA was 3'-end labeled with [{alpha}-32P]dideoxy-ATP and resolved on 1.8% agarose gel electrophoresis. Mol wt are indicated on the left. B, Densitometric quantification of low mol wt (<4 kbp) DNA content (mean ± SEM of four experiments). C, Representative immunoblot of Fas, FasL, and p53 protein contents in granulosa cell protein extracts. D, Densitometric quantification of Fas, FasL, and p53 protein contents (mean ± SEM of four experiments). Compared with day 0: *, P < 0.05; ***, P < 0.001. Compared with day 2: +, P < 0.05; ++, P < 0.01; +++, P < 0.001.

 
In situ colocalization of apoptosis and Fas/FasL system in various stages of follicles
The relationship between apoptosis and Fas and FasL expression during follicular development was examined in situ (Fig. 2Go). Primordial follicle cells showed weak TUNEL activity (Fig. 2Go-Ia, shown in circle) but very intense immunoreactivities for Fas and FasL (Fig. 2Go-I, b and c). Similarly, granulosa cells in the preantral (primary/secondary) follicle also exhibited positive immunoreactivity for Fas (Fig. 2Go-IIa) and FasL (Fig. 2Go-IIb), but were TUNEL negative (Fig. 2Go-IIc). In early and, to a lesser extent, medium antral stages of follicular development, apoptosis was widespread throughout the granulosa layer of atretic follicles (Fig. 2Go, IIId and IVd), and the TUNEL signal was localized in cells showing an intense and aggregated pattern of immunoreactivities for Fas (Fig. 2Go, IIIe and IVe) and FasL (Fig. 2Go, IIIf and IVf). On the contrary, the TUNEL-negative granulosa cells (Fig. 2Go-IIIa) in healthy follicles showed Fas-positive immunoreactivity (Fig. 2Go, IIIb and IVb), but very weak FasL signal (Fig. 2Go, IIIc and IVc). The granulosa cells in healthy, large antral follicles (including preovulatory follicles) did not exhibit TUNEL positivity (Fig. 2Go-Va) or Fas/FasL immunoreactivities (Fig. 2Go, Vb and Vc), whereas granulosa cells mainly lining the antrum of the large atretic follicle were intensely TUNEL positive (Fig. 2Go-Vd), Fas positive (Fig. 2Go-Ve), and FasL positive (Fig. 2Go-Vf). The relative TUNEL positivity and Fas/FasL immunoreactivities in granulosa cells during follicular development are summarized in Table 1Go.



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  		Figure 2. In situ detection of apoptotic cells (TUNEL) and immunolocalization of Fas and FasL proteins on adjacent sections of healthy (a, b, and c, respectively) and/or atretic (d, e, and f, respectively) primordial (I), preantral (II), early antral (III), medium antral (IV), and large antral (V) follicles from immature rats 0–5 days after eCG injection (15 IU, ip). GC, TC, and O represent granulosa cell, thecal cell, and oocyte, respectively. Arrows indicate Fas- and FasL-immunoreactive cells. Magnification, x400 (I, II, and III) and x200 (IV and V).

 

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  		Table 1. Summary of TUNEL and immunohistochemistry of Fas and FasL in granulosa cells during follicular development

 
Cell cycle analysis of granulosa cells after eCG treatment in vivo
The results of flow cytometric cell cycle analysis of granulosa cells recovered from immature rat ovaries during the 5 days after eCG injection (15 IU) are shown in Fig. 3Go. A flow cytogram of granulosa cell DNA distribution revealed accumulation of cells in G0/G1, S, and G2/M phases of the cell cycle and the presence of a subpopulation of granulosa cells (A0) with less DNA fluorescence than the G0/G1 peak (Fig. 3AGo). Seven percent of granulosa cells from animals not exposed to exogenous gonadotropin (day 0) were found in the A0 phase, with the remaining cells distributed among G0/G1 (76.0%), S (7.4%), and G2/M (8.8%; Fig. 3BGo). A marked decrease (~70%) in the population of A0 granulosa cells was observed on days 1, 2, and 3 after gonadotropin treatment, whereas a significant increase in S (P < 0.001) and a decrease in G0/G1 (P < 0.05) phases were detected on day 1. On day 2 and thereafter, the populations of granulosa cells in S and G2/M phases gradually decreased, and by day 5, they reached only 25% and 50% of their respective day 1 levels, whereas that in G0/G1 phase increased markedly (P < 0.001) on days 3, 4, and 5. The A0 cell populations on days 4 and 5 were significantly larger than that on day 1 (P < 0.01 and P < 0.001, respectively). Granulosa cell preparations with a high percentage of the population in the A0 phase (days 0, 4, and 5) also showed higher incidences of apoptotic fragmented nuclei (indicated by arrowheads), whereas normal nuclear morphology (presence of intact and relatively large size nuclei) was observed on days 1, 2, and 3 (Fig. 3AGo, insets).



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  		Figure 3. Cell cycle analysis by DNA fluorescence flow cytometry of granulosa cells from immature rats during 5 days after eCG injection (15 IU, ip). A, Representative DNA histograms with insets showing apoptotic nuclear fragmentation (arrowheads; magnification, x1,000); the percentage of granulosa cells containing subdiploid amounts of DNA (A0, region 1) and at different stages of the cell cycle (G0/G1, region 2; S, region 3; G2/M, region 4). A total of 10,000 propidium iodide-stained cells were counted at least 10 times/experimental group (top left corner of each panel, days after eCG injection). The square box in each histogram in A shows typical nuclear morphology of the cells (stained with Giemsa) in each group. B, Integrative summary of cell cycle changes in granulosa cell populations in ovaries after eCG treatment (mean ± SEM of four experiments). Compared with day 0: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Compared with day 2: ++, P < 0.01; +++, P < 0.001.

 
In situ evaluation of relationship between p53 accumulation and Fas/FasL system in granulosa cell apoptosis during follicular atresia
The relative abundance of p53, Fas, and FasL in relation to granulosa cell death at different stages of follicular atresia is shown in Fig. 4Go and Table 2Go. As shown in Fig. 4Go-I, intense p53 immunoreactivity was observed in the nuclei of granulosa cells of very early antral follicle (Fig. 4Go-Ib), in which these cells also showed positive immunoreactivities for Fas (Fig. 4Go-Ib) and FasL (Fig. 4Go-Ic), and TUNEL-positive signals were restricted to cells around the oocyte and antral region of the follicle (Fig. 4Go-Ib). Intense immunoreactivity for p53 was also detected in thecal cells in this follicle type (Fig. 4Go-Ib). In the small and large antral follicles, intense p53 immunoreactivities and nuclear accumulation were evident in the granulosa cells of moderately atretic follicles (Fig. 4Go, I-b and II-b), as determined by the integrity of the granulosa cell layer and TUNEL positivity. Consistent with the observation in very early antral follicles, Fas (Fig. 4Go, IIc and IIIc) and FasL (Fig. 4Go, IId and IIId) were also colocalized in p53-positive cells in the small and large antral follicled. However, p53 was not detectable in granulosa cell nuclei of extremely atretic follicles (Fig. 4Go, IIf and IIIf), whereas Fas (Fig. 4Go, IIg and IIIg) and FasL (Fig. 4Go, IIh and IIIh) immunoreactivities were evident.



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  		Figure 4. Histological localization of apoptosis, p53, Fas, and FasL in weakly and moderately (a, b, c, and d, respectively) and extremely (e, f, g, and h, respectively) atretic follicles of different stages of development [very early antral (I), early/small antral (II), and large antral (III)] in eCG-treated rat ovaries. GC, TC, and O represent granulosa cell, thecal cell, and oocyte, respectively. Arrows indicate p53-immunoreactive cells. Magnification, x200.

 

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  		Table 2. Summary of TUNEL and immunohistochemistry of p53, Fas, and FasL in granulosa cells at different stages of atretic follicles

 
Overexpression of p53, up-regulation of the Fas/FasL system, and apoptosis
Results from the present studies have shown that Fas, FasL, and p53 levels are high in apoptotic granulosa cells in atretic follicles from ovaries of immature rats not exposed to exogenous gonadotropin and that eCG treatment in vivo decreases the cellular content of these death gene products and apoptotic cell death (Figs. 1Go and 4Go). Whether the changes in Fas, FasL, and p53 levels under these experimental conditions are coincidental or causally related is not known. To determine whether the expression of Fas and/or FasL is directly related to that of p53, granulosa cells isolated 2 days after eCG treatment (at a stage when granulosa cell p53, Fas, and FasL contents were low; Fig. 1DGo) were infected with adenoviral p53 sense cDNA or vector control (LacZ), and p53, Fas, and FasL expressions in vitro were assessed (Fig. 5Go). As our preliminary experiments have demonstrated that infection of granulosa cells for 1 h with viral densities of MOI of 5 or 25 was insufficient to elicit significant changes in Fas and FasL contents compared with the controls (data not shown), granulosa cells were infected for a longer period in the present investigation. Infection of granulosa cells over a 24-h culture period with the p53 sense cDNA at a MOI of 5 markedly increase p53 and Fas, but not FasL, levels (Fig. 5AGo, compared with vector control). To confirm whether newly expressed Fas in response to p53 overexpression is functional, the influence of an agonistic Fas monoclonal antibody (Fas mAb) on cell morphology and apoptosis was examined. Analysis of overall cell and nuclear morphology of granulosa cells infected with the vector control (LacZ) indicates the cells were healthy, well attached to the growth surface, and exhibited round and intact nuclei (Fig. 5BGo). Overexpression of p53 resulted in increased detachment of the cells from the growth surface, the presence of floating cells (~55%), and a number of cells with fragmented nuclei (Fig. 5Go, B, arrows, and C; ~35%). Although treatment of the infected control cells (LacZ) with Fas mAb (1 µg/ml) slightly increased cell floating (to ~25%) and the number of cells exhibiting apoptotic nuclear fragmentation (to ~15%), p53 overexpression resulted in a further enhancement of these responses to the agonistic Fas antibody (Fig. 5CGo). Two-way ANOVA indicates that there were highly significant effects of Fas mAb and p53 overexpression on both cell attachment (P < 0.002 and P < 0.001, respectively) and apoptosis (P < 0.001 and P < 0.001, respectively). A significant interaction between these factors was, however, only observed in the number of cells exhibiting nuclear fragmentation (P < 0.02; Fig. 5CGo).



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  		Figure 5. Influence of adenoviral infection (MOI = 5) of granulosa cells with p53 sense cDNA or LacZ (control) for 24 h on p53, Fas, and FasL protein contents (A), and general cellular and nuclear morphology (B) and cell detachment from growth surface and apoptosis (C) with or without (IgG control) subsequent challenge with an agonistic monoclonal Fas antibody (Fas mAb) for 24 h. A and B are representative panels from three experiments. In B, black and white arrowheads indicate floating cells and fragmented apoptotic cells, respectively (magnification, x400). Results in C are the mean ± SEM of three experiments. *, P < 0.05 (compared with IgG-treated LacZ control). ***, P < 0.001 (compared with IgG-treated LacZ control). +++, P < 0.001 (compared with Fas mAb-treated LacZ control). #, P < 0.05 (compared with IgG-treated p53 sense group). ###, P < 0.001 (compared with IgG-treated p53 sense group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although Fas, FasL, and p53 has been implicated in the control of granulosa cell apoptosis during follicular atresia in several mammalian species (12, 13, 14, 22), the regulation of these cell death proteins and their dependence on follicular maturation have not been addressed. Although anti-eCG antiserum-induced gonadotropin withdrawal in eCG-primed immature rats increased Fas and FasL protein content and induced apoptosis in granulosa cells in the early antral stage of follicular development (15), using an alternate eCG-primed rat model that induces atresia in follicles at a wide range of development, the present study has demonstrated that the Fas/FasL system may also be involved in the regulation of granulosa cell apoptosis during the late stage of follicular maturation. Whereas metabolic clearance of gonadotropin (days 4 and 5) resulted in a marked increased in Fas protein levels, FasL content was only slightly, but nonsignificantly, elevated in granulosa cells of large antral follicles. These observations do not exclude the possibilities that FasL expression may be very low or transient and undetectable (by Western blot) in highly differentiated granulosa cells undergoing apoptosis, but sufficient to activate the Fas pathway and induce follicular atresia. Alternately, FasL was already present in sufficiently high level, and apoptosis was initiated merely by an increase in Fas content.

Consistent with our previous findings on the granulosa cell Fas and Fas ligand expression at the penultimate state of follicular development (15), the present histochemical studies indicate that TUNEL-positive granulosa cells in atretic early and medium antral follicles also exhibited intense and aggregated Fas and FasL immunoreactivities, whereas healthy (TUNEL-negative) early antral and medium-sized follicles also showed Fas and FasL positivity, but immunoreactivities were much weaker and more evenly distributed. These results suggested that intense and aggregated immunoreactivities may be required to observe sufficient intensity of TUNEL-positive signals that could be only detectable at the end stage of the apoptotic event. Although the biological significance of the aggregated appearance of Fas and FasL immunostaining remains to be determined, it is possible that it might reflect oligomerization of the Fas/FasL system required for the induction of apoptosis (38, 39). In contrast, Fas, but not FasL, was expressed in significant levels in the apoptotic antral granulosa cells of large atretic ones, suggesting that this stage of follicular development is potentially less susceptible to apoptotic insults.

Although primordial and preantral follicles exhibited intense immunoreactivities for Fas/FasL, minimal or no TUNEL signals were observed in follicle cells at these stages of development. Although the reason(s) for the apparent resistance of the granulosa cells to this death signal is unknown, it is possible that components of this pathway downstream from the Fas receptor may be either absent or not operational. In this context, we have recently demonstrated that DNase I, an endonuclease believed to be involved in apoptotic DNA degradation in rat granulosa and luteal cells, is present but inactive in nuclei of healthy granulosa cells of antral, but not preantral, follicles, suggesting that its expression may be developmentally regulated and dependent on cell differentiation (11, 40). Moreover, our recent immunolocalization of caspase-3, a protease involved in a number of cell death pathways, in apoptotic but not healthy cells of rat follicles suggests that the expression of this death factor is under the influence of gonadotropin and may be up-regulated as part of the apoptotic process in granulosa cells (9).

The present studies demonstrated that immature rat ovaries contain a significant number of atretic early antral follicles with granulosa cells that were TUNEL positive, exhibited considerable nuclear fragmentation, and were highly apoptotic, as determined by their high abundance in the A0 phase of the cell cycle. Exogenous gonadotropin suppressed nuclear fragmentation and decreased the proportion of cells in the A0 phase on days 1–3. Presumably as a consequence of gonadotropin metabolism and clearance on days 4 and 5, the population of granulosa cells in the A0 phase was significantly increased. These changes appeared to be coupled to a concomitant increase in cells in G0/G1 and a decrease in cells in S, suggesting that this induction of granulosa cell apoptosis in large antral follicles (days 4 and 5) may be a result of G1/S arrest. In this context, p53 levels were markedly elevated during metabolic clearance of gonadotropin on days 3, 4, and 5, suggesting that the induction of follicular atresia in large antral and preovulatory follicles may be p53 dependent. Although the cellular mechanism(s) involved in control of this latter event remains to be determined, it is of interest to note that this inhibition of cell cycle progression is accompanied by marked increases in granulosa cell p53 content. These findings are consistent with the well established role of p53 as an important check point or gatekeeper in cell cycle regulation in a variety of normal and neoplastic cells (26, 41) and its involvement in the induction of apoptosis (42).

It has been reported that p53 up-regulates several apoptosis-related proteins, such as Bax (43), cyclin G (44), p21 (45), IGFBP-3 (46), Gadd45 (47), mdm-2 (48), and Fas (49). In the present study, we have demonstrated for the first time that changes in granulosa cell p53 protein content during follicular development are closely coupled to the expression of Fas. Intense p53 immunoreactivities in the granulosa cell nuclei were consistently colocalized with Fas and FasL in the weakly and moderately atretic (TUNEL-positive) follicles, whereas p53 was found in low abundance in the extremely atretic (TUNEL-positive) follicles, where Fas and FasL were present at a similar or higher level than those in weakly or moderately atretic ones. These immunohistochemical observations for p53, Fas, and FasL raise the interesting possibility that nuclear p53 accumulation may be required for the up-regulation of Fas and/or FasL in granulosa cells in vivo and that p53 could be degraded in cells undergoing the end stage of apoptosis after Fas activation. To further examine whether Fas expression was causally related to p53 accumulation, the influence of adenoviral p53 sense cDNA expression on Fas and FasL contents in granulosa cells isolated 2 days after eCG treatment was investigated in vitro. Overexpression of p53 resulted in an increase in Fas protein content and extensive apoptosis. This increase in Fas content appeared to be functional, as a significantly greater increase in apoptosis was observed if the death pathway was activated with agonistic Fas mAb after p53 sense expression. These findings raise the interesting possibility of an involvement of Fas activation in the p53-mediated granulosa cell apoptosis.

In the present studies, we have consistently observed a p53 protein doublet in granulosa cell extracts after SDS-PAGE, and the relative intensities of these bands (53 and 55–57 kDa) appeared to be dependent on gonadotropic support. Although the nature and function of this slow migrating immunopositive band are unknown, it is possible that it may represent an active, phosphorylated form of the p53 protein and may be central to the induction of granulosa cell apoptosis in large antral follicles. Recently, the importance of p53 phosphorylation, transactivation, and translocation has been emphasized in several different cell types (50, 51). Although the 53-kDa protein in the granulosa cells was highly expressed, apoptotic granulosa cell death on day 3 was not evident, as judged by the minimal DNA degradation, the low proportion of cells in the A0 phase of the cell cycle, and the minimal nuclear fragmentation. However, the switch in the relative abundance of the immunoactive 53- and 55- to 57-kDa protein observed on days 4 and 5 appears coincidental to the marked increase in Fas expression and cell death at this stage of follicular development. The high Fas and FasL protein contents and the much lower level of p53 observed in immature rat granulosa cells not exposed to eCG are consistent with the observation that the granulosa cell death in these atretic early antral follicles had already been reached at the end stage of apoptosis, as confirmed by flow cytometric and DNA fragmentation analyses.

It is of interest that thecal cells in healthy, but not atretic, follicles exhibited marked FasL immunoreactivity throughout follicular development. Although the physiological significance of such observation is not known, it is possible that this cell death factor may serve to confer immune privilege to the follicle, as has been demonstrated in several organs, including the testes and eye (52, 53). As the ovary has been known to be immune privileged (54, 55), which excludes cells of the immune system, the high FasL content of the theca in healthy follicles may function as a barrier by inducing apoptosis in invading Fas-containing immune cells. The role and regulation of FasL in the theca in the induction of ovarian follicular atresia remain to be investigated.

In summary, the present studies demonstrate a central role of gonadotropin as a survival factor in the regulation of granulosa cell Fas/FasL and p53 expression during ovarian follicular development. Our findings suggest that the gonadotropic control of granulosa cell apoptosis involves two consecutive cellular/molecular events, cell cycle arrest at G1/S and exit from G0 into A0 phase, via regulation of p53 and Fas/FasL death pathways. We have also shown that accumulation of the gatekeeper protein p53 at the cell cycle check-point (G1 phase) may be important for the up-regulation of Fas and FasL proteins needed for G0 to A0 exit, granulosa cell apoptosis, and, ultimately, follicular atresia.


   Acknowledgments
 
The authors express their gratitude to Mr. Sang-Ho Ahn (Asan Medical Center, Seoul, Korea) for his excellent assistance with the flow cytometric cell cycle analysis and thank Dr. F. L. Graham (McMaster University, Hamilton, Ontario, Canada) and Dr. R. Korneluk (University of Ottawa, Ottawa, Ontario, Canada) for providing the adenoviral p53-sense cDNA and LacZ, respectively.


   Footnotes
 
1 This work was supported by Research Grant MT-10369 from the Medical Research Council of Canada (to B.K.T.) and Research Grant BSRI 4437/KOSEF HRC 0401 (to Y.-D.Y.) and was presented in parts at the 28th and 29th Annual Meetings of the Society for the Study of Reproduction (July 9–12, 1995, Davis, CA, and July 27–30, 1996, London, Ontario, Canada, respectively). Back

2 Present address: Division of Reproductive Biology, Department of Biochemistry, The Johns Hopkins University School of Hygiene and Public Health, 615 North Wolfe Street, Baltimore, Maryland 21205. Back

Received October 14, 1998.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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