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BOR - Papers in Press, published online ahead of print June 11, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.017897
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BIOLOGY OF REPRODUCTION 69, 1379–1387 (2003)
DOI: 10.1095/biolreprod.103.017897
© 2003 by the Society for the Study of Reproduction, Inc.

Ovary

Determination of Onset of Apoptosis in Granulosa Cells of the Preovulatory Follicles in the Bonnet Monkey (Macaca radiata): Correlation with Mitogen-Activated Protein Kinase Activities1

J. Uma3, P. Muraly3, Shalu Verma-Kumar3, and R. Medhamurthy2,3,4

Department of Molecular Reproduction, Development and Genetics3 Primate Research Laboratory,4 Indian Institute of Science, Bangalore 560012, India


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During reproductive life, only a selected few ovarian follicles mature and ovulate, while the vast majority of follicles undergo a degenerative process called atresia. Recent studies have indicated that follicular atresia is mediated through apoptosis of follicular granulosa cells. The objectives of the present study were to determine the time of onset of apoptosis in granulosa cells of preovulatory follicles and to evaluate the consequences of gonadotropin withdrawal on mitogen-activated protein (MAP) kinase activities. Bonnet monkeys (Macaca radiata) undergoing controlled ovarian stimulation cycles were utilized for stimulation of multiple follicles, and granulosa cells were retrieved from preovulatory follicles at 24, 48, 72, and 96 h after stopping gonadotropin treatment. Serum and follicular fluid estradiol concentrations were highest at 24 h but declined precipitously (P < 0.05) to reach the lowest concentrations at 96 h; however, progesterone concentrations during this period did not increase, indicating the absence of luteinization. Quantitative analysis of genomic DNA by 3'-end labeling revealed the presence of low-molecular-weight fragments from 48 h onward, but by agarose gel electrophoresis, DNA laddering could be visualized only after 72 h. Messenger RNA expression for Bax, caspase-2, and caspase-3 increased with the onset of apoptosis. Immunoblot analysis of MAP kinases in lysates of granulosa cells (48–72 h) indicated increased (P < 0.05) levels of phosphorylated extracellular response kinase-1 and -2, Jun N-terminal kinase (JNK)-1 and -2, and p38. However, in vitro kinase assay data indicated that only phospho-JNK and -p38 activities were higher at 72 h compared to 24 h. These results demonstrate that granulosa cells of preovulatory follicles undergo apoptosis and that increased activities of phospho-JNK and -p38 are correlated with apoptosis in the primate.

apoptosis, follicular development, granulosa cells, ovary, signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue homeostasis is dependent on the proper relationships among cell proliferation, differentiation, and cell death. Apoptosis (also referred to as programmed cell death) is a highly regulated process by which an organism eliminates unwanted cells. Apoptosis plays an important role in the regulation of ovarian function in mammals (for reviews, see [1–3]). During the course of reproductive life span, only a selected few follicles mature and ovulate, while the vast majority of follicles (up to 99%) do not grow to ovulate but degenerate during various stages of follicular development because of the process of initiation of apoptosis of granulosa cells [4–7]. The mechanism(s) by which only a few follicles (the number of which is more or less specific to each species) mature during each reproductive cycle, while many of the follicles undergo atresia, remains to be delineated. Although gonadotropins, FSH and LH, are primary regulators of ovarian follicular growth, the involvement of paracrine/autocrine factors originating within the ovary during the process of folliculogenesis has become increasingly apparent during the last few years [8]. For instance, several studies indicate that rapidly growing follicles produce higher levels of autocrine and paracrine growth factors that stimulate increases in vasculature and FSH responsiveness, which may result in escape of these follicles from undergoing atresia [9, 10].

Although extensive data exist regarding characterization of apoptosis on the basis of morphological and biochemical changes that occur in the dying granulosa cells (for review, see [1, 22]), the sequence of signaling events that commit the granulosa cells to death remains to be characterized. Recent studies demonstrate the critical roles played by gonadotropins, steroids, and growth factors in attenuation of follicular (granulosa) apoptosis. One of the mechanisms by which these so-called "survival factors" attenuate apoptosis in granulosa cells is by altering the expression of cell death-related genes [6, 8] (for review, see [11]). Moreover, mechanisms such as increased expression of survival factors and phosphorylation of critical phosphoproteins also play a important role in mediating the action of trophic growth factors on survival of cells (for review, see [3]).

Among the major types of signal transduction pathways in eukaryotic cells are protein kinase cascades that culminate in activation of protein kinases known as mitogen-activated protein (MAP) kinases. In mammals, four major groups have been identified, and each of these groups of MAP kinase is activated by a protein kinase cascade (reviewed in [12]). They are extracellular response kinase (ERK), Jun N-terminal kinase (JNK), p38MAPK (p38), and the big MAP kinase (ERK-5). The hallmark of MAP kinase signaling is the stimulation-dependent nuclear translocation of the involved kinases, which regulate gene expression and the cytoplasmic acute response to mitogenic, stress-related, apoptotic, and survival stimuli. It has been shown that in Rat1 and PC12 cells, apoptosis induced by the withdrawal of trophic growth factors involves a rapid increase in p38 and JNK and an inhibition of ERK [13, 14]. However, FSH has been shown to activate ERK and p38 in granulosa cells in vitro [15–17]. These findings suggest that determination of survival as well as apoptosis is also critically regulated by MAP kinases.

The present study was undertaken to examine whether deprivation of trophic support to the preovulatory follicle would lead to apoptosis as well as when the apoptotic process is initiated and whether this could be used as a model for delineating the apoptotic signal transduction pathways in the nonhuman primate ovary. Because apoptosis induced by withdrawal of trophic growth factors involves activation of MAP kinases, we sought to examine the consequences of withdrawal of gonadotropin treatment on MAP kinase activities in granulosa cells under in vivo conditions. The preovulatory follicle model was chosen, because the collection of granulosa cells after cessation of gonadotropin could be precisely timed. In addition, granulosa cells could be obtained from a class of follicles known to be functional without removing the ovaries, thus precluding the potential for obtaining cells from smaller-sized follicles that could have been atretic at the time of collection.


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

The polyclonal antibodies specific to phospho-p38 MAP kinase (no. 9211), phospho-SAPK/JNK (no. 9251), phospho-p42/44 MAP kinase (no. 9100), p38 MAP kinase (no. 9212), ERK-1 (no. sc-19), ERK-2 (no. sc-154), and JNK-1 (no. sc-571) were purchased from Cell Signaling Technology (Beverly, MA; no. 9100, no. 9211, no. 9212, and no. 9251) and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA; no. sc-19, no. sc-154, no. sc-571, and no. sc-572). In vitro kinase activity assay kit for p38 MAP kinase (no. 9820) and protein kinase substrates for ERK (Elk-1 fusion protein, no. 9184) and JNK (c-jun fusion protein, no. 6093) were purchased from Cell Signaling Technology.

Omniscript reverse transcriptase was obtained from Qiagen (Valencia, CA). Taq DNA Polymerase, random hexamers, RNasin, and dNTPs were from Promega (Madison, WI). Oligonucleotide primers were synthesized by Sigma-Genosys (Cambridgeshire, U.K.). All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO), Gibco BRL (Gaithersburg, MD), or local sources.

Animal Protocols

All procedures involving monkeys in the present study were cleared by the Institutional Animal Ethics Committee of the Indian Institute of Science. Adult female bonnet monkeys (Macaca radiata) weighing 3.1–5 kg with a history of regular menstrual cycles of 27–29 days were utilized for the study. The general care and housing of monkeys at the Primate Research Laboratory, Indian Institute of Science, have been described elsewhere [18]. During the study period, the temperature in the animal rooms, which were supplied with fresh, filtered air (filter size, 5 µm), ranged from 22 to 28°C (dry-bulb temperature) and from 17 to 22°C (wet-bulb temperature) maximum and minimum, respectively. Studies were conducted during January to February and during July to December, because female bonnet monkeys exhibit summer amenorrhea during March through June [18]. Monkeys were monitored daily for onset of menses. Starting from Day 1 of menses, monkeys were treated with human (h) FSH (25 IU of Metrodin twice daily i.m., 0900 and 1700 h; Ares Serono, Aubonne, Switzerland) for 6 days, followed by hFSH plus hLH (25 IU each; Pergonal, twice daily i.m.; Ares Serono) for 2.5 days to promote multiple follicular growth and development. Blood samples were collected daily or on alternate days to estimate serum estradiol (E2) and progesterone (P4) for monitoring follicular growth during and following withdrawal of gonadotropin treatment. A variety of methods have been used to stimulate the growth and maturation of multiple follicles in a number of nonhuman primate species [19] (for review, see [20]). The protocol in the present study was similar (except for the lower dose and shortened duration of treatment) to that reported previously [19]. To rule out that spontaneous endogenous LH surges did not occur during the course of gonadotropin treatment and the withdrawal period, selected serum samples were analyzed for monkey LH concentration using a specific RIA [21], and the results confirmed the absence of LH elevations during or after stopping gonadotropin treatment. Also, serum and follicular fluid levels of P4 were low, which is indicative of the absence of luteinization.

Collection and Processing of Granulosa Cells

Transabdominal ultrasonography (Aloka SSD500V equipped with a 7.5-MHz linear transducer; Aloka Co. Ltd., Tokyo, Japan) was performed for visual inspection of the follicle number and diameter on the 10th day after initiation of the treatment. It was consistently observed that five to six follicles greater than 5 mm could be identified per ovary. Granulosa cells were harvested from follicles after accessing ovaries by performing laparotomy on ketamine hydrochloride (15 mg/kg body wt)-anesthetized monkeys. Granulosa cells (n = 3/time point) were collected at different periods 1 day after stopping gonadotropin treatment—that is, on Day 10 (24 h), on Day 11 (48 h), on Day 12 (72 h), and on Day 13 (96 h) after initiation of treatment. The follicular fluid was aspirated with the help of a 25-gauze, 1.5-inch needle attached to a 1-ml syringe. As far as possible, care was taken to aspirate the follicular contents from follicles larger than 5 mm, and whenever possible, efforts were made to flush the follicle after aspiration to maximize the yield of granulosa cells. For RNA isolation, follicular fluid was collected separately from one or two follicles and centrifuged at 290 x g, and the cell pellet was stored at -70°C until processed for RNA isolation. Immediately after collection, follicular fluid was centrifuged at 290 x g for 7 min at 4°C. The supernatant was discarded, and the cell pellet was suspended in PBS solution. After counting the cells, the cell suspension was aliquoted and centrifuged again to obtain the cell pellet. One or two of the aliquots were snap-frozen in liquid nitrogen and stored at -70°C until analysis, and the remaining aliquot was subjected to cell lysate preparation as described below. When the follicular fluid was mixed with blood, it was collected separately into a separate test tube for further processing. The blood-mixed follicular fluid was centrifuged at 290 x g for 7 min, and the cell pellet was suspended in 1 ml of Percoll buffer (1x Hanks balanced salt solution containing 0.252% [w/v] Hepes and 0.1% [w/v] BSA). The cell suspension was then carefully layered over a 40% Percoll gradient and centrifuged for 30 min at 480 x g. Granulosa cells were recovered from the interface, resuspended in 5-fold their volume with Percoll buffer, and centrifuged at 130 x g for 10 min. The cell pellet was dissolved in PBS and centrifuged at 290 x g for 7 min, and the cell pellet was then used for cell lysate preparation (see below) or stored at -70°C until analysis. The cell number was determined using a hemocytometer, and cell viability was judged by 0.4% (w/v) Trypan blue exclusion. The total number of cells recovered per retrieval ranged from 3.6 to 10.5 x 106 cells (per monkey/time at 24, 48, 72, or 96 h after stopping gonadotropin treatment), with cell viability of 51–89%.

Isolation of Genomic DNA and Analysis

Genomic DNA was extracted from granulosa cells, precipitated, dissolved in distilled water, and spectrophotometrically quantitated as described previously for DNA fragmentation analysis [22, 23]. Genomic DNA (10–15 µg for agarose electrophoresis or 500 ng for quantitative analysis) was either subjected to agarose gel electrophoresis, stained with ethidium bromide and DNA visualized by ultraviolet (UV) transillumination, or analyzed for quantitation of low-molecular-weight (LMW) DNA fragments as described previously [22, 23].

RNA isolation

Total RNA was extracted from granulosa cells using Trizol reagent according to the manufacturer's recommendations and quantitated spectrophotometrically. Analysis of RNA at optical density A260 versus A280 consistently yielded a ratio above 1.8.

Reverse Transcription-Polymerase Chain Reaction

Reverse transcription-polymerase chain reaction (RT-PCR) was carried out using a Peltier Thermal Cycler PTC-200 MiniCycler Instrument (MJ Research, Waltham, MA). Computer searches and sequence alignments were performed at http://www.ncbi.nlm.nih.gov and http://searchlauncher.bcm.tmc.edu/. The identity of the PCR products was confirmed by sequence analysis. Total RNA (1 µg) was reverse transcribed using the following RT mixture: 200 µM of dNTPs, 10 U of RNasin, 10x RT buffer (250 mM Tris-HCl [pH 8.3 at 25°C], 250 mM KCl, 50 mM MgCl2, 2.5 mM Spermidine, and 50 mM dithiothreitol [DTT]), 10 µM of oligo dT, and 4 U of Omniscript reverse transcriptase in a total reaction volume of 20 µl. The RNA was allowed to stand at 65°C in a water bath for 5 min before chilling on ice for 5 min. After addition of the RT mixture, RT was carried out for 1 h at 37°C. For PCR, cDNA equivalent to 500 ng of total RNA was used. The PCR mix was made up of 200 µM dNTPs, 1x Taq buffer (50 mM KCl, 10 mM Tris-HCl [pH 9.0 at 25°C], 1.5 mM MgCl2, and 0.1% [v/v] Triton X-100), 10 µM of each gene-specific primer, and 2 U of Taq DNA Polymerase in a total reaction volume of 50 µl. Semiquantitative, multiplex RT-PCR was carried out according to the method of Wong et al. [24] with a few modifications and using the following cycling parameters: For caspase-2 and -3, an initial denaturation was performed at 95°C for 2 min, followed by five cycles of denaturation at 94°C for 45 sec, annealing at 65°C for 45 sec, and extension at 72°C for 1 min and then 30 cycles of touchdown PCR with denaturation at 94°C for 45 sec, annealing from 65 to 58°C for 45 sec, and extension at 72°C for 1.5 min. The caspase-2 primers were added to allow 30 cycles of amplification, and ribosomal phosphoprotein (RPLO) primers were added to allow 18 cycles of amplification at an annealing temperature of 58°C. This was followed by a final extension at 72°C for 10 min. Similarly, for Bax, touchdown PCR was carried out with an initial denaturation at 95°C for 2 min; five cycles of denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C for 45 sec; followed by 30 cycles with denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, and extension at 72°C for 1 min. The RPLO primers were added to allow 18 cycles of amplification for RPLO at an annealing temperature of 58°C. The PCR products were separated on a 2% (w/v) agarose gel containing ethidium bromide and photographed under UV light using the Alpha Imager 1200 Documentation and Analysis System (Alpha Innotech Corp., San Leandro, CA). Oligonucleotides used for PCR are listed in Table 1.


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  		TABLE 1. Primer sequences used for RT-PCR analysis

Preparation of Cell Lysates for Immunoblotting

Granulosa cell lysate was prepared following a previously published procedure [23]. In brief, an aliquot of washed cell pellet (~2 x 106 cells) was transferred to 150 µl of RIPA lysis buffer, sonicated using a Branson Cell Disrupter at 20% power for 10 sec, and incubated for 30 min on ice with intermittent vortexing before centrifugation at 15 000 x g for 10 min at 4°C. The clarified supernatant was recovered, aliquoted, and stored at -70°C until analyzed for various MAP kinases. The protein was estimated using the Bradford method [27].

Western Blot Analysis

Whole-cell lysate (25–40 µg) was resolved by 10% SDS-PAGE and electroblotted onto polyvinyl fluoride (PVDF) membrane using a semidry transfer unit (Bio-Rad Laboratories, Richmond, CA) as described previously [23].

In Vitro MAP Kinase Assays

Assays were carried out with few modifications of published procedures (ERK and JNK [28, 29]) or according to the manufacturer's protocol (phospho-p38 MAP kinase). In brief, 100–200 µg of granulosa cell lysate protein were incubated with either 20 µl of immobilized phospho-p38 MAP kinase antibody or phospho-JNK (1:200)/phospho-ERK (1:100) MAP kinase antibodies overnight, followed by additional incubation with 20 µl of Protein-A-Agarose for 3 h at 4°C. The resultant immune complexes were collected by centrifugation at 15 000 x g for 30 sec, and after washing, the immunoprecipitates were used directly in the assay. The MAP kinase activities were assayed in the immune complexes using respective glutathione-S-transferase (GST) fusion proteins as substrates. For phospho-p38 MAP kinase activity assay, the immune complexes were washed in kinase buffer (25 mM Tris [pH 7.5], 5 mM ß-glycerophosphate, 10 mM MgCl2, 2 mM DTT, and 0.1 mM Na3VO4), and the pellet was resuspended in 50 µl of kinase buffer supplemented with 200 mM ATP and 2 µg of ATF-2 fusion protein and then incubated for 30 min at 30°C. The reaction was terminated by adding 25 µl of 3x SDS sample buffer. Samples were separated on a 10% acrylamide gel, transferred to PVDF membrane, and probed with phospho-ATF-2 antibody (1:1000). For phospho-JNK and ERK MAP kinase activity assays, the immune complexes were washed in kinase buffer, and the pellet was resuspended in 25 µl of kinase buffer supplemented with 20 µM ATP, 2.5 µCi [{gamma}-32P]ATP, and 2 µg GST-c-jun (JNK assay)/or Elk-1 (ERK assay) fusion proteins and then incubated for 30 min at 30°C. The reaction was terminated by adding 12.5 µl of 3x SDS sample buffer, and samples were separated on a 12% (v/v) acrylamide gel, followed by gel drying and autoradiography.

Steroid Assays

Estradiol and P4 concentrations in serum were determined by specific RIA as reported previously [30]. The E2 (GDN no. 244) and P4 (GDN no. 337) antisera were kindly provided by Professor G.D. Niswender (University of Colorado, Fort Collins, CO). Follicular fluid was diluted with 0.1% (w/v) gelatin-PBS before ether extraction and assay. The sensitivities of the assays for E2 and P4 were 39 pg/ml and 0.1 ng/ml, respectively. The inter- and intraassay coefficients of variation for both the hormones were less than 10%.

Statistical Analyses

Wherever applicable, data are expressed as the mean ± SEM. The arbitrary densitometric units were represented as the percentage relative to the 24-h time point, which was set at 100%. The data were analyzed by one-way ANOVA, followed by the Newman-Keuls multiple-comparison test (PRISM Graph Pad, version 2; Graph Pad Software, Inc., San Diego, CA). A P value of less than 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Steroid Concentrations in Monkeys During and after Gonadotropin Treatment

Mean serum E2 concentrations in monkeys receiving exogenous human gonadotropin treatments to promote multiple follicular growth are presented in Figure 1. Also shown in Figure 1 are serum E2 concentrations at different time intervals after stopping gonadotropin treatment. Serum E2 concentrations increased slowly during the first 7 days of the treatment period, but they increased briskly after initiation of combination hFSH and LH treatment to reach peak concentrations of 2920 ± 233.2 pg/ml at 24 h after stopping gonadotropin treatment. Thereafter, serum E2 concentrations declined precipitously (P < 0.05) and were lowest at 96 h after stopping gonadotropin treatment (Fig. 1).



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  		FIG. 1. Serum estradiol levels before and during exogenous gonadotropin treatment (6 days of hFSH preparation; 25 IU twice daily) followed by 2.5 days of a combination of hFSH and hLH preparation (25 IU twice daily) and after stopping gonadotropin treatment in bonnet monkeys. The open and solid bars represent duration of the hFSH and the hFSH-plus-hLH treatment, respectively. Data are represented as the mean ± SEM for n = 15 monkeys on Days 1–9 of gonadotropin treatment and, at different time intervals after stopping treatment, n = 15 (24 h), n = 9 (48 h), n = 6 (72 h), and n = 3 (96 h) monkeys. (For analysis details, see Fig. 3)

The follicular fluid concentrations of E2 and P4 on different days after stopping gonadotropin treatment are shown in Figure 2. The pattern of E2 concentrations in the follicular fluid paralleled the serum E2 patterns, with high concentrations at 24 h but significantly lower (P < 0.05) concentrations at 48, 72, and 96 h after stopping gonadotropin treatment. Follicular fluid P4 concentrations were not significantly different after stopping gonadotropin treatment (Fig. 2).



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  		FIG. 2. Follicular fluid estradiol (white bars) and progesterone (shaded bars) concentrations at different time intervals after stopping gonadotropin treatment. Bars with different letters are significantly different (P < 0.05) from each other

Based on the E2 (and P4) secretory pattern, the duration of experimental period was arbitrarily divided into two phases, gonadotropin treatment and gonadotropin withdrawal, and represented with a log scale y-axis, as shown in Figure 3. Also shown in Figure 3 is the pattern of P4 concentrations on different days after stopping gonadotropin treatment. Serum P4 concentration was lowest (P < 0.05) at 96 h compared to 24 h. It is evident that E2 concentrations decreased following withdrawal of gonadotropin treatment and that P4 concentrations did not increase, suggesting the absence of differentiation of granulosa cells in the absence of a midcycle gonadotropin surge.



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  		FIG. 3. Serum estradiol and progesterone concentrations after stopping gonadotropin treatment. Serum estradiol concentrations before and during gonadotropin treatment period (proliferative phase of granulosa cells) and serum estradiol and progesterone concentrations after stopping gonadotropin treatment period (nonproliferative phase of granulosa cells) in bonnet monkeys are shown. The time points with different letters are significantly different (P < 0.05) from each other

Biochemical Analyses of Apoptosis

Agarose gel electrophoresis and ethidium bromide staining of genomic DNA isolated from granulosa cells retrieved at different time intervals after stopping gonadotropin treatment showed a characteristic pattern of DNA laddering, which was indicative of the presence of apoptotic granulosa cells at 72 and 96 h (Fig. 4A). At 24 h, no evidence was observed for DNA laddering, and at 48 h, although no appearance of DNA laddering was found, evidence was seen for smearing of DNA (Fig. 4A). When genomic DNA was analyzed for LMW fragments by 3'-end labeling, the results showed the presence of LMW DNA fragments, which could be visualized in granulosa cells retrieved at 48, 72, and 96 h after stopping gonadotropin treatment (Fig. 4B). Quantitation of DNA fragments indicated that the LMW DNA fragments increased (P < 0.05) 300–700% during the period from 48 to 96 h compared to 24 h (Fig. 4C).



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  		FIG. 4. Analysis of apoptotic DNA fragmentation in granulosa cells. Genomic DNA isolated from monkey granulosa cells retrieved from preovulatory follicles at different time intervals after stopping exogenous gonadotropin treatment was subjected to either qualitative (A) or quantitative (B) analysis. Bar graph (C) represents the quantitative measurement of low-molecular-weight DNA labeling as a percentage of change from the 24-h time point. Bars with different letters above them are significantly different (P < 0.05). M, 100-base pair ladder

Bax mRNA was detectable in granulosa cells retrieved at 24 h after stopping gonadotropin treatment (Fig. 5A). The Bax mRNA expression tended to be higher (P > 0.05) at 48 and 72 h, but the expression was significantly higher (P < 0.05) at 96 h compared to the 24- and 48-h time points. Figure 5B depicts the expression patterns of caspase-2 and capase-3 at different time intervals after stopping gonadotropin treatment.



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  		FIG. 5. Bax (A) as well as caspase-2 (B; shaded bars) and caspase-3 (B; white bars) mRNA expression in the monkey granulosa cells retrieved from follicles at different time intervals after stopping gonadotropin treatment. Messenger RNA of Bax and of caspase-2 and caspase-3 were coamplified with the internal standard RPLO, a constitutive housekeeping gene, and intensities of the amplified Bax, caspase-2, and caspase-3 products were normalized against the internal standard. A representative gel is shown for Bax (A) and for caspase-2 and caspase-3 (B) mRNAs. Because of the inavailability of sufficient RNA in a few of the monkeys, the densitometric data represented for caspase-2 and -3 are an average of two monkeys/time point, with no statistical analysis performed on the data (B). Bars with different letters are significantly different (P < 0.05). M, 100-base pair ladder.

Caspase-2 and -3 mRNAs were detectable in granulosa cells retrieved at 24 h and progressively increased, but the caspase-3 expression increased only 50% at 96 h compared to the 24-h time point. On the other hand, during the corresponding period, caspase-2 expression increased nearly 2-fold (Fig. 5B).

Correlation of MAP Kinase Activities During Granulosa Cell Apoptosis

Figure 6 shows representative immunoblots and integrated arbitrary densitometric unit data of phospho- and total-ERK-1 and -2 levels (expressed as a percentage of results obtained at 24 h) from granulosa cells retrieved during 24–96 h after stopping gonadotropin treatment. Phospho-ERK-1 and -2 levels were higher at 48 h but were significantly difference (P < 0.05) at 72 h. Total ERK-2 levels, although slightly higher at 48 and 72 h, were not statistically significantly different from the 24-h time point. On the other hand, total ERK-1 levels showed an increase that mirrored its phospho-levels. Granulosa cell lysate from 24 and 72 h were subjected to in vitro ERK assay, and the result did not confirm the increased phospho-levels observed by immunoblot analysis (Fig. 6).



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  		FIG. 6. Autoradiographic analysis of phospho-GST-Elk-1 and immunoblot analyses of phospho-ERK-1 and -2 (white bars) and total ERK-1 and -2 (shaded bars) in protein lysates of granulosa cells retrieved from follicles at different intervals after stopping gonadotropin treatment. The blots shown are from one of the three independent experiments (granulosa cells from one monkey from each time point were used per experiment). The arbitrary densitometric units from the 24-h time point were set as 100%, and results at other time points were expressed in relation to the 24-h value. In vitro ERK kinase (phospho-Elk-1) was performed on only two time points. Bars with different letters are significantly different

Representative immunoblots and integrated arbitrary densitometric unit data of phospho-JNK-1 and -2 and of total JNK-1 are shown in Figure 7A. Phospho-JNK-1 levels increased significantly (P < 0.05) at 48 and 72 h after stopping gonadotropin treatment. At 96 h, although the levels were higher, the levels were not significantly (P > 0.05) different from the 24-h time point. Total JNK-1 levels were not different from the 24-h time point. Total JNK-2 could not be detected in the granulosa cells using the conditions described in Materials and Methods, whereas it can be detected in the monkey corpus luteum tissue [21]. Phospho-JNK-2 levels increased similar to phospho-JNK-1 levels at all time points compared to the 24-h time point (data not shown). In vitro kinase assay performed on granulosa cells retrieved at 24 and 72 h after stopping gonadotropin treatment showed increased phospho-JNK activity at 72 h compared to 24 h, confirming the immunoblot analysis data.



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  		FIG. 7. Autoradiographic analysis of phospho-GST-c-jun and immunoblot analyses of (A) phospho-JNK-1 (white bars) and -2 and total JNK-1 (black bars) and of (B) phospho-ATF2, phospho-p38 MAP kinase (pp38; white bars), and total p38 (shaded bars) in protein lysates of granulosa cells retrieved from follicles at different intervals after stopping gonadotropin treatment. The blots shown are from one of three independent experiments (granulosa cells from a single monkey from each time point was used per experiment). Bars with different letters are significantly different

Figure 7B shows representative immunoblots and integrated arbitrary densitometric unit data of phospho-p38 and total p38 levels. Phospho-p38 levels were significantly increased (P < 0.05) in granulosa cell lysates collected at 48 and 72 h after stopping gonadotropin treatment. At 96 h, the level was not increased compared to the 24-h time point. Total p38 levels did not change (P = 0.07) during 48 to 96 h compared to the 24-h time point levels. In vitro kinase assay performed for granulosa cell lysates at 24 and 72 h revealed increased activity at 72 h compared to the 24-h time point.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although rhesus monkeys undergoing controlled ovarian stimulation cycles comparable to those in clinical in vitro fertilization (IVF)/assisted reproduction treatment protocols for induction of multiple preovulatory follicles for studying preovulatory, periovulatory, or oocyte retrieval for IVF and related research have been reported [31–33], to our knowledge this is the first report that describes standardization of protocol for successful induction of multiple follicular growth in the bonnet monkey. In the present study, a GnRH-antagonist treatment was not included in the protocol as reported by others (e.g., [31, 33]) to rule out the possibility of direct effect on the ovary. It has been shown that GnRH may have a direct effect on the ovary and may even increase the incidence of apoptosis in rat granulosa cells [34]. Interestingly, however, GnRH receptors have not been demonstrated in granulosa cells of human preovulatory follicles, but luteinized granulosa cells appear to have specific binding sites for GnRH [35].

Studies carried out in rodent, primate, and a number of domestic animal species have established that unwanted ovarian somatic and germ cells from nondominant follicles are eliminated by the process of apoptosis (for review, see [1, 2, 36]). Ovarian follicular atresia in all vertebrates that have been studied to date is mediated via apoptosis that involves well-organized morphological and biochemical processes characterized by membrane blebbing, cell shrinkage, oligonucleosome formation, and apoptotic bodies (for review, see [1, 3, 37]). In the present study, we investigated the feasibility of studying apoptosis in monkey granulosa cells to delineate intracellular signaling events associated with initiation of apoptosis.

Before the present study, reports concerning granulosa cell apoptosis in human and nonhuman primates were scarce [25, 36]. After having established a procedure for induction of multiple preovulatory follicles in the monkey, we then determined the time course of the appearance of DNA oligonucleosomal formation in granulosa cells following withdrawal of exogenous gonadotropins, because formation of the typical "DNA ladder" is considered to be a biochemical hallmark of apoptosis. Several studies have suggested that LMW DNA fragmentation precedes or occurs coincident with morphological changes of apoptosis [38–40]. However, it has been observed that a small percentage of histologically early atretic ovarian follicles were not accompanied by DNA laddering [40]. On the other hand, it has been suggested that the apoptotic process of cells characterized by chromatin condensation, reduced cell volume, and formation of apoptotic cell bodies can proceed in the absence of oligonucleosomal formation, and in other situations, the formation of LMW fragments occur only subsequent to the cleavage of genomic DNA into at least two different pools of high-molecular-weight fragments (50 and 300 kilobases) [41–43]. We assessed the time of onset of apoptosis on the basis of biochemical DNA analysis, because morphological assessment of apoptosis in a previous study correlated well with the appearance of LMW DNA fragments [23]. Moreover, the biochemical analysis appears to be a reliable index for determining the time of onset of apoptosis. In the present study, because the granulosa cells did not show evidence of differentiation and their steroid biosynthetic machinery was severely compromised, the cells began to show evidence of DNA fragmentation following clearance of exogenous gonadotropins due to metabolism. In an elegant study by Nahum et al. [39], DNA degradation in rat granulosa cells of preovulatory follicles was observed within 8 h after hypophysectomy, but in this study, DNA degradation was not observed until 48 h after stopping gonadotropin treatment. This is not surprising, because exogenously administered gonadotropin preparations used in this study have a circulating biological half-life in excess of 20 h [44], which is perhaps in contrast to a rapid clearance of endogenous gonadotropins after hypophysectomy in rats. Also, it should be pointed out that E2 secretion continued to increase and was highest at 24 h after stopping gonadotropin treatment, indicating that the effect of exogenously administered gonadotropins was still present after stopping treatment.

Great strides have been made in recent years toward a better understanding of events that occur in ovarian follicles after initiation of apoptosis, but the exact sequence of events responsible for triggering apoptosis in granulosa cells is still poorly understood. One key group of intracellular factors regulating apoptosis is the Bcl-2 family of proteins (for review, see [11, 45]). Members of the ever-increasing mammalian Bcl-2 family currently number in excess of 20, and they encode proteins that are classified as either antiapoptotic (e.g., Bcl-2 and Bcl-XL) or proapoptotic (e.g., Bax and Bad), which are important in the decision step of apoptosis [2, 11]. We unsuccessfully tried to examine the expression of Bcl-XL by RT-PCR (data not shown) with the published primers, but the reason(s) for lack of expression was not clear. We next examined the expression of Bax in granulosa cells at different time intervals after stopping gonadotropin treatment, and we observed an increased expression coincident with the onset of apoptosis, an observation that corroborates findings in the human ovary [25]. Interestingly, our attempts to determine the Bax protein expression were not successful, and Kugu et al. [25] also could not detect the protein in the atretic follicles. It could be that the Bax antibody used may not be suitable for detecting the protein in monkey tissues. Nevertheless, that Bax plays an important role in granulosa cell apoptosis is further reinforced by the reported observation of decreased Bax expression associated with the inhibition of eCG-mediated granulosa cell apoptosis [6].

Caspases represent a family of intracellular cysteine proteases, the actions of which are linked to the initiation and execution phase of apoptosis (for review, see [37]). We examined the expression pattern of two of the caspases, caspase-2 and caspase-3, both of which are involved in the penultimate stage of apoptosis. It is well established that caspase-3 is involved in virtually every type of vertebrate cell apoptosis (for review, see [46]). Surprisingly, only marginal increase in caspase-3 mRNA levels was observed in granulosa cells undergoing apoptosis. It could be that it is the activity levels, more than the expression, of caspase-3 (i.e., cleaved caspase-3 protein levels) that are important for apoptosis. We also examined the expression of caspase-2 with a view to correlate its activity with LMW DNA fragmentation. Unlike the caspase-3 expression, a clear-cut increase in the caspase-2 expression was associated with the increase in LMW DNA fragments, suggesting its involvement in granulosa cell apoptosis. It has been reported that oocytes lacking caspase-2 are resistant to apoptosis in caspase-2-deficient mice [47]. Kugu et al. [25] also found increased expression of caspase-2 in apoptotic human granulosa cells. The extent of caspase-2 involvement and delineation of its intracellular apoptotic cascade need to be investigated.

The role of both FSH and LH/hCG in suppression of ovarian follicular atresia both in vivo and in vitro is well established (for review, see [3, 48]). Because follicles are also exposed to a large number of substances, such as growth factors, cytokines, hormones, etc., in vivo it is possible that the presence (or withdrawal) of these factors, either alone or in combination, may activate the apoptotic signaling cascades [3, 49]. In this regard, it has been reported that elements of extrinsic (receptor-mediated) apoptotic signaling cascade, such as Fas and Fas ligand, are expressed in granulosa cells following gonadotropin withdrawal (for review, see [3]). In a recent study [50], it was conclusively demonstrated that under in vitro follicle culture conditions, FSH stimulated X-linked inhibitor of apoptosis (X-IAP) and that adenoviral X-IAP sense cDNA expression in granulosa cells reduced apoptosis, suggesting that at least one of the consequences of FSH withdrawal is the reduced expression of X-IAP from theca cells, leading to apoptosis. However, it is possible that withdrawal of gonadotropin support to follicles leads to activation of multiple signaling apoptotic cascades.

Many extracellular stimuli are converted into specific cellular responses through activation of MAP kinase signaling pathways [12]. It has been observed that serum withdrawal stress often involves activation of JNK and p38 [14]. In the present study, phosphorylated levels of JNK and p38 and ERK-2 levels were higher following withdrawal of gonadotropin treatment, coinciding with the onset of LMW DNA fragments. However, in eCG-induced granulosa cell apoptosis in rats, it was reported that the ERK signaling pathway was attenuated [51]. The reason for this discrepancy between the monkey (although in vitro kinase assay results did not confirm the increase in the ERK activity) and the rat results is not clear, but in the rat study [51], the phospho-ERK levels were lower at 48 h in the absence of LMW DNA fragments and continued to be lower at 96–120 h, when LMW DNA fragments were present. We have observed increased phospho-ERK levels in rat granulosa cells 48 h after eCG treatment, which decreased at 96 h, but the levels increased again at 120 h after treatment [52]. Recently, apoptosis induced by hydrogen peroxide in porcine granulosa cells in vitro showed increased phospho-ERK levels [17]. More studies are required to further determine the exact role of the ERK signaling pathway. The findings that phosphorylated JNK and p38 levels were elevated suggest that deprivation of gonadotrophic support to granulosa cells resulted in stress-induced activation of MAP kinase activity, which is indicative of their critical role during apoptosis. Although immunoblot analysis data of total p38 and JNK appeared to be higher, especially at the 48- and 72-h time points, the increase was not statistically significant, and moreover, the increase in phosphorylated levels during the corresponding period was clearly much larger. More detailed in vitro kinase assays are needed to precisely establish the temporal changes in the MAP kinase activities.

In summary, the present study demonstrates that monkey granulosa cells of preovulatory follicles undergo apoptosis following withdrawal of gonadotropin support. Serum and follicular fluid E2 levels appear to be inversely correlated with the various biochemical changes associated with the onset of apoptosis. Moreover, MAP kinases appear to be activated during granulosa cell apoptosis. Future studies will be aimed at delineation of MAP kinase-activated pathways associated with initiation of apoptosis.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. S.G. Ramachandra of the Primate Research Laboratory for help with surgeries and Mr. Vijay Kumar Yadav for help with in vitro MAP kinase assays. Dr. A.F. Parlow kindly supplied the monkey LH RIA kit.


   FOOTNOTES
 
1 Supported by Life Sciences Research Board, Indian Council of Medical Research and the DBT postdoctoral training program (S.V.-K.). Presented in part at the 12th National Conference on Recent Advances in Reproductive Health, Jaipur, India, 2003. Back

2 Correspondence: R. Medhamurthy, Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India. FAX: 91 80 3600 999; rmm{at}mrdg.iisc.ernet.in Back

Received: 9 April 2003.

First decision: 2 May 2003.

Accepted: 28 May 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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