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Regular article TA |
a Department of Animal Sciences, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4
ABSTRACT
The present study was designed to 1) investigate whether apoptosis is responsible for the atresia of nonovulatory dominant follicle (DF), 2) to determine if atresia of a nonovulatory DF is associated with alterations in Bcl-2 and Bax expression, 3) to test whether progesterone P4 has a direct effect on apoptosis in bovine follicles, and 4) to study the pattern of expression of Bcl-2 and Bax in follicles at different developmental stages (small, medium, and large). In experiment 1, 16 cycling cows received a norgestomet ear implant at proestrus (Day 1) for 9 days to mimic the subluteal phase. The cows were assigned either to a control (n = 4) or P4-treated groups (n = 12). Injections of P4 (150 mg, i.m.) were given on Day 3 (n = 4); on Days 3 and 4 (n = 4), and on Days 3, 4, and 5 (n = 4) of the implant period. Controls received injections of corn oil on Days 3, 4, and 5. Unilateral ovariectomy was performed on Days 4, 5, and 6 to recover DFs from cows that had been treated with P4 for 24, 48, and 72 h, respectively. DFs in the control group were collected on Day 6. The onset of atresia of DFs was assessed morphologically by ultrasound to determine DF diameters, histologically by light microscopic inspection of tissue sections, and functionally by quantification of follicular fluid steroid hormone levels. Apoptosis was detected by DNA analysis and in situ TUNEL labeling. Expression of Bcl-2 and Bax proteins was examined by Western blot analysis. The earliest signs of atresia were detected 24 h after P4 injection as evidenced by decreased diameter, degeneration and detachment of granulosa cells (GCs) from the basal lamina, and a dramatically reduced ratio of estrogen to P4. Electrophoretic analysis of DNA extracted from DFs of cows treated with P4 for 24 h revealed a distinct ladder pattern of DNA fragments. In contrast, this pattern was not obvious in DFs from control cows. Similar results were also obtained from TUNEL analysis of DFs. Furthermore, both Bcl-2 and Bax were found to be present in all DFs; however, the ratio of Bcl-2 and Bax protein levels was significantly reduced by 24 h of P4 treatment compared with DFs from the control group (P < 0.05). Experiment 2 investigated the direct effect of P4 (4 ng/ml) on apoptosis of cultured GCs using ovaries obtained from a local slaughterhouse. In addition, the pattern of expressions of Bcl-2 and Bax in follicles at different developmental stages (small, medium, and large) was studied. No increase in apoptotic DNA fragments was detected in GCs treated with P4. The ratio of Bcl-2 and Bax protein levels was variable in small follicles; however, Bax protein level was always relatively higher than that of Bcl-2 in medium and large follicles. In conclusion, our study suggests that apoptosis is the mechanism that underlies the atresia of nonovulatory DFs that develops during the luteal phase of bovine estrous cycle.
apoptosis, follicle, granulosa cells, progesterone
INTRODUCTION
The growth of antral ovarian follicles in cows occurs in a wave-like pattern, a wave being characterized by the recruitment of a pool of small antral follicles and the selection and dominance of a single dominant follicle (DF) while the remainder of its cohort regresses. There are two or three waves of follicular growth in each bovine estrous cycle. The DF from the first wave reaches its largest size on Day 7 or 8 of the estrous cycle (day of estrus designated as Day 0). The DF maintains its morphological and functional dominance until around Day 11, then becomes atretic and begins to regress between Days 11–14, to be replaced by a second wave of follicular growth. The number of waves of follicular growth during the estrous cycle is determined by the length of the luteal phase. If progesterone (P4) levels in the circulation begin to decrease because of the spontaneous regression of the corpus luteum (CL) while the second-wave DF is in its growth phase, then the second-wave DF will ovulate. Alternatively, if P4 remains elevated in the presence of an active CL after the second-wave DF has attained its maximum size, the DF undergoes atresia and the third wave of follicular growth will emerge [1–6]. What regulates this pattern and initiates the regression of nonovulatory DFs is not clear. Evidence indicates that P4 may play a major role. It has been suggested that atresia of the nonovulatory DF in bovine estrous cycles is induced by high levels of P4 via regulation of LH [7, 8]; however, a direct action of P4 in the ovary has not been ruled out. Furthermore, although some cellular and biochemical characteristics of atretic DFs induced by P4 have recently been identified, including decreased concentrations of follicular fluid (FF) estradiol-17ß (E2), insulin-like growth factors (IGFs) I and II, reduced aromatase activity, and increased low-molecular-weight IGF binding proteins, these events only provide useful markers for identifying atresia of DFs, which develops during the luteal phase of the estrous cycle in cows [9]. The molecular mechanisms underlying atresia of nonovulatory DFs caused by high P4 levels is still unclear.
Recent studies have suggested that follicle atresia in cows, chickens, pigs, and rodents is associated with apoptosis, which is an active, intrinsic, genetically governed process of selective cell deletion [9–13]. Gonadotropins have been reported to play a critical role in preventing apoptosis in granulosa cells (GCs) of bovine and rat antral follicles [13, 14]. In addition, evidence that ovarian steroids inhibit (estrogens) or induce (androgens) apoptosis in ovarian GCs of estrogen-implanted, hypophysectomized immature rats has been demonstrated [15]. However, the effects of P4 on ovarian follicle apoptosis remain controversial. An in vivo study found treatment with P4 does not affect rat ovarian apoptosis, whereas another in vitro study showed that P4 inhibited cultured rat GC apoptosis [15, 16]. A study of the effects of P4 on apoptotic cell death in bovine ovarian follicles has never been reported. Moreover, nothing is known of the underlying events associated with the initiation of apoptosis during atresia of DFs that develop during the luteal stage of the estrous cycle in cows.
The initiation of apoptosis in various ovarian cell lineages probably depends on cell-specific stimuli via hormonal signals, the absence or presence of which activates or stops repression of gene products that are responsible for the suicidal mechanism [17, 18]. Studies from extragonadal cell systems have shown that among the numerous proteins and genes involved, the Bcl-2 family of proteins constitutes a critical intracellular checkpoint of apoptosis within a distal common cell death pathway. At least 15 mammalian Bcl-2 family members have been identified and categorized into antiapoptotic (Bcl-2, Bcl-w, Bcl-XL, A1, MCL-1) and proapoptotic (Bax, Bak, Bok, Bik, Blk, Hrk, BNIP3, Bim, Bad, Bid, Bcl-XS) subgroups [18]. The Bcl-2 protooncogene was originally identified from a human chromosomal translocation that predisposed affected individuals to malignant transformation of immune cells [19]. It has been found that the Bcl-2 protein prevents apoptosis induced by a variety of stimuli and maintains cell survival by influencing the release of cytochrome c from mitochondria rather than altering proliferation [20]. Bax, identified by coimmunoprecipitation with the Bcl-2 protein, is the first proapoptotic homolog. When Bax is overexpressed in cells, apoptotic death was accelerated in response to death signals. In addition, Bax can heterodimerize with Bcl-2 and thus function to counter the effects of Bcl-2 on cellular survival [21]. Therefore, the ratio of Bcl-2 to Bax expression is important in determining susceptibility to apoptosis [21]. However, the roles of Bcl-2 and Bax in bovine follicular development and atresia remain to be elucidated.
The aims of the present study were 1) to test the hypothesis that apoptosis is the mechanism that underlies the atresia of nonovulatory DFs that develop during the luteal phase of bovine estrous cycle, 2) to determine if atresia of DFs is associated with alterations in Bcl-2 and Bax expression, 3) to test if P4 has a direct effect on apoptosis in bovine follicles, and 4) to study the pattern of expression of Bcl-2 and Bax in follicles at different developmental stages (i.e., small, medium, and large).
MATERIALS AND METHODS
Experimental Protocol
Two experiments were conducted. In experiment 1, an in vivo model of DF maintained by norgestomet (a synthetic progestin, Syncromate-B; Sanofi Inc., Overland Park, KS) was used to determine the mechanism underlying the atresia of nonovulatory DFs that develop during the luteal phase of the bovine estrous cycle, and to determine if atresia of DFs is associated with alterations in Bcl-2 and Bax expression. Our laboratory has demonstrated that with a single norgestomet implant insertion, proestrus DFs remained healthy and functional for 9 days and were capable of ovulation after implant withdrawal. Nine days is long enough to subject a follicle to P4 treatments. In addition, norgestomet has not been reported to cross-react with P4, nor does it alter systemic P4 concentrations; hence, it is very useful for our study in which exogenous P4 was injected to mimic the mid-luteal phase of the bovine estrous cycle [7, 9].
Experiment 2 investigated the direct effect of P4 on bovine follicle apoptosis using ovaries obtained from a local slaughterhouse. In addition, the pattern of expressions of Bcl-2 and Bax was studied in follicles at different developmental stages (small, medium, and large).
Experiment 1
Animals and treatments Sixteen cycling, dry Holstein cows were housed at the University of British Columbia Dairy Teaching and Research Unit and cared for according to the guidelines of the Canadian Council of Animal Care. The cows were chosen for the experiment at the proestrus stage of the estrous cycle. Proestrus phase was confirmed by ultrasonic detection of a large ovulatory follicle and a regressing CL. During proestrus, each cow received a 6-mg norgestomet ear implant (Day 1 = the day of implant insertion), which was removed 9 days later. After implant insertion, the cows were randomly allotted to either the control group (n = 4) or to P4-treated (n = 12) groups. Cows in the P4-treated groups received i.m. injections of 150 mg P4 (Sigma Chemical Company, St. Louis, MO) in corn oil on Day 3 (n = 4), on Days 3 and 4 (n = 4), and on Days 3, 4, and 5 (n = 4) of the implant period. Cows in the control group received injections of corn oil on Days 3, 4, and 5. Unilateral ovariectomy was performed on Days 4, 5, and 6 of the implant period to recover DFs from cows in the groups that had been treated with P4 for 24, 48, and 72 h, respectively. Unilateral ovariectomy was performed on control cows on Day 6. After initial ovariectomy, remaining ovaries were monitored daily by ultrasonography to observe follicular development, and the experiment was repeated. The turnover of follicles in remaining ovaries was similar to that observed for cows with both ovaries and did not influence the emergence and development of the next DF or its maintenance by progestin.
Ovarian ultrasonography The development of a DF in each cow was monitored daily by ultrasound imaging from the day of norgestomet implant insertion until the day of ovariectomy. Ultrasound examination was conducted as described by Rajamahendran and Taylor [22] using a real-time B-mode linear array ultrasound scanner equipped with a 5 MHz rectal transducer (Dynamic Imaging Ltd., Livingston, Scotland, UK). Appropriate images of DFs were arrested on-screen and follicular diameters were measured using a built-in caliper system. Permanent records were created using a video processing unit (Mitsubishi Electronics Co. Ltd., Tokyo, Japan).
Processing of ovaries following ovariectomy Ovariectomy was performed using an ecraser (colpotomy) under epidural anesthesia [23] on designated days. Ovaries were immediately placed in chilled Dulbeccos modified Eagle medium (DMEM) containing glucose, sodium pyruvate, and [sml[nm-glutamine supplemented with 0.1% (w:v) BSA and 100 µg/ml streptomycin (Sigma) on ice and processed in the laboratory within 1 h. DFs from each ovary were individually dissected free from extraneous tissue. FF was aspirated with an 18-gauge needle and a 10-ml syringe, and stored frozen until analysis for P4. Thereafter, DFs were cut into two halves. One half was fixed in 4% neutral buffered formalin for histological study, the other was snap-frozen and stored at -70°C until processed for analysis of DNA integrity or Bcl-2 and Bax expression.
Morphological analysis The follicles (n=8) were fixed in 4% neutral buffered formalin for 48 h. Fixed tissues were washed in PBS solution, dehydrated through a graded series of ethanol (70%–100%), cleared in xylene, embedded in paraffin, and sectioned (5 µm). The sections (n = 48 from 24 follicles) were deparaffinized in xylene, rehydrated through a graded series of ethanol (100%–50%), and then stained with hematoxylin and eosin for morphological analysis.
Progesterone and E2 analysis Concentrations of P4 and E2 in FF were determined by radioimmunoassay (RIA) in samples [22] diluted (1:10 to 1:1000) in assay buffer, with a commercially available solid-phase RIA kit (Coat-A-Count; Diagnostic Products Corp., Los Angeles, CA). The intra-assay coefficients of variation were 9.8% for P4 and 8.4% for E2. The sensitivity of the assay was 0.05 ng/ml and 0.01 ng/ml for P4 and E2, respectively.
In situ 3' end-labeling of DNA fragments DNA fragments in DFs were identified by labeling free 3'-OH DNA ends (TUNEL) using an in situ cell death detection kit (Boehringer-Mannheim, ON, Canada). Briefly, tissue sections (n = 8) were deparaffinized and rehydrated. Sections were then washed in Tris-buffered saline (TBS) and treated with 20 µg/ml proteinase K for 20 min at room temperature. Tissues were further treated with 3% H2O2 for 5 min to inactivate endogenous peroxidase. Finally, the DNA strand breaks were labeled at 3'-ends with fluorescein deoxyuridine 5-triphosphate by incubation with the reaction buffer containing terminal deoxynucleotidyl transferase enzyme for 1.5 h at 37°C in the dark. Samples were immediately analyzed under a fluorescent microscope. Negative control sections were processed identically except that the labeling enzyme (terminal deoxynucleotidyl transferase enzyme) was omitted, whereas positive control sections were treated with DNase I (0.5 mg/ml).
DNA extraction and analysis Genomic DNA was prepared from follicles as previously described [13]. Briefly, follicles were snap-frozen and stored at -70°C to prevent nonspecific activation of DNase. Cells were first disrupted by addition of homogenization buffer and repeated passage through a pipette. Homogenates were then lysed. Genomic DNA was extracted by the phenol/chloroform/isoamyl alcohol (25:24:1, v:v:v) method and spectrophotometrically quantitated at 260 nm.
The DNA was separated (20–25 µg/lane) according to size in a 2% agarose gel by electrophoresis. Gels were stained with ethidium bromide and washed in double distilled H2O. The DNA fluorescence was viewed with a UV transilluminator, and the gels were photographed. The integrated optical density of the stained internucleosomal DNA fragments in each sample lane was measured by densitometry using a gel documentation system and IS-500 Digital Imaging System software version 1.97 (Alpha Innotech Corporation, San Leandro, CA) as described [24].
Analysis of Bcl-2 and Bax expression Samples were thawed and homogenized with ice-cold homogenization solution (pH 7.5) containing 100 mM Tris-HCL, 0.1% SDS, and protease inhibitors (1 mM phenylmethylsulfonyfluoride, 100 µM pepstatin, 50 µM leupeptin, and 50 µM aprotinin) (Sigma, PQ, Canada). Homogenates were centrifuged at 15 000 x g for 1 h at 4°C. The protein content was determined with the standard protein assay (Bio-Rad, Hercules, CA). Proteins were solubilized in Laemmlis sample buffer [18], boiled for 5 min, and separated by electrophoresis on a discontinuous SDS gel system consisting of 6% polyacrylamide stacking and 12% separating components. Electrophoretic proteins were subsequently electrotransferred to nitrocellulose membranes (Hybond-ECL; Amersham Life Science, ON, Canada) in 25 mM Tris-glycine buffer (pH 8.3) containing methanol and 0.1% SDS. The membranes were blocked with Blotto (Santa Cruz Biotechnology, Santa Cruz, CA; 1 mM Tris containing 0.9% NaCl, 0.2% Tween-20, and 5% nonfat dried milk; pH 7.4) for 1 h at room temperature, incubated with Bcl-2 and Bax goat polyclonal antibody (1.4 µg/ml in Blotto) overnight at 4°C, washed, and then incubated with horseradish peroxidase-conjugated secondary antibody in Blotto. Peroxidase activity was visualized with an enhanced chemiluminescence Western blotting immunodetection kit (Amersham Pharmacia Biotech, PQ, Canada). Bcl-2 and Bax protein expression were determined densitometrically. Briefly, images were scanned using a flatbed scanner (Scan-Jet 4C, Hewlett-Packard, BC, Canada) and quantitated by the IS-500 Digital Imaging System software version 1.97 (Alpha Innotech).
Experiment 2
Collection of ovaries and classification of follicles Ovaries were collected and follicles were classified as previously described [25]. Briefly, ovaries were collected at a local slaughterhouse and transported to the laboratory within 2 h of slaughter, on ice, in chilled collection media composed of DMEM/Hams F-12 (1:1) containing 0.1% (w:v) BSA, [SMl[nm-glutamine, and 50 µg/ml gentamicin (Sigma).
The follicles were classified into three groups on the basis of surface diameter: small (
4 mm), medium (5–8 mm), and large (>8 mm). Follicle size categories were selected on the basis of reported gonadotropin dependence and changes in expression of steroidogenic enzyme and LH receptor mRNAs [26]. Small, medium, and large follicles were then classified as healthy or atretic according to previously established morphological criteria with modification [27]. Healthy follicles had vascularized (pink- or red-colored) theca internae, and clear amber FF with no debris.
Granulosa cell culture and P4 treatment GCs were harvested from large healthy follicles (n = 18) and cultured as previously described [13, 25]. Briefly, follicles were punctured with an 18-gauge needle and FF was aspirated. GC collection medium, Ca2+/Mg2+-free buffer (20 mM Tris, 140 mM NaCl, 2 mM EDTA, pH 7.4), was repeatedly flushed in and out of the follicles. The follicles were then cut into hemispheres and the interior walls were gently scraped with an inoculating loop to remove GCs, leaving the basement membrane and theca cells intact. GCs were harvested by centrifugation and washed three times. Cell number and viability were determined using a hemocytometer and trypan blue dye exclusion method.
Viable GCs (2 x 106 cells/ml) were cultured in 6-well plates with 2 ml DMEM/Hams F-12 supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml; Sigma). The cells were incubated in a humidified 5% CO2 atmosphere at 39°C. After 16 h of culture, dead cells were washed off. Only viable GCs remained and were attached tightly to the culture plates. To study the effect of P4 on GC apoptosis, these GCs were cultured for 48 h in serum-free DMEM/Hams F-12 medium with or without P4 (4 ng/ml; n = 3 culture wells of treatment per trial). P4 was dissolved in ethanol and then diluted in DMEM/Hams F-12 to the desired final concentration. The treatment concentration was determined on the basis of the in vivo circulating plasma P4 concentration that can induce atresia of DFs [4, 7]. After culture, GCs were harvested, snap-frozen, and stored at -70°C until processed for DNA analysis as described in experiment 1.
Analysis of Bcl-2 and Bax expression in small, medium, and large follicles Small (n = 12), medium (n = 6), and large (n = 6) follicles were dissected free from extraneous tissue. Protein levels of Bcl-2 and Bax in follicles of different sizes were analyzed by Western blot as described in experiment 1.
Statistical analyses One-way ANOVA was used for comparisons between more than two means, and when a significant difference was found, a Duncans multiple range test was used to determine which means were significantly different. Student's t-tests were used for comparisons between two means. Statistical significance was inferred at P < 0.05.
RESULTS
Experiment 1
Effects of P4 treatment on DF diameter Representative ovarian ultrasound photographs demonstrating the diameter changes of a norgestomet-maintained DF over a 72-h period after P4 treatment are shown in Figure 1a. After 72 h of P4 treatment, DF diameters decreased by 3.28 ± 0.74 mm. In cows treated with P4 for 24 and 48 h, diameters decreased by 0.6 ± 0.17 mm and 2.65 ± 1.29 mm, respectively. On the contrary, diameters of DFs in control cows slightly increased by 0.4 ± 0.28 mm (Fig. 1b).
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Effects of P4 treatment on DF morphology Representative photomicrographs (magnification 200x) demonstrating the morphological changes of norgestomet-maintained DFs after 24, 48, and 72 h of P4 treatment are shown in Figure 2. In control follicles (Fig. 2a), the theca layer was distinct and the granulosa layer appeared thick and well-organized. A few pyknotic cells were observed under higher magnification. Twenty-four h after P4 treatment (Fig. 2b), follicles exhibited features of atresia. The granulosa layer had thinned considerably and had become disorganized. In some cases, the granulosa layer was either partially or completely detached from the underlying basal lamina. With the progression of atresia, by 48 h of P4 treatment (Fig. 2c), few GCs could be observed. The granulosa layer was virtually absent after 72 h of P4 treatment (Fig. 2d).
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Effects of P4 treatment on FF steroid hormone concentrations The effects of P4 treatment on concentrations of P4 and E2 in DFs of treated and control groups are shown in Figure 3. No significant differences in FF P4 concentrations was observed among the groups (P > 0.05); however, there was a great decrease in FF E2 concentrations when control and treated groups were compared (P < 0.01).
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Effects of P4 treatment on apoptosis in DFs (in situ 3' end-labeling, TUNEL) The effects of P4 treatment on apoptosis in DFs are shown in Figure 4. Dominant follicles from the control group exhibited very few detectable levels of DNA fragment labeling (Fig. 4a). In contrast, DFs from the group that had been treated with P4 for 24 h showed heavily fluorescent signals, indicating DNA fragmentation, confined mainly to GCs and in scattered theca cells (Fig. 4b). A lower apoptotic signal was detected in theca cells of DFs in groups treated with P4 for 48 and 72 h because of a lack of GCs (Fig. 4, c and d). Negative control sections did not fluoresce (Fig. 4e), whereas an extreme intense fluorescent signal was observed in positive controls (Fig. 4f), demonstrating that this in situ labeling procedure is specific.
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Effects of P4 treatment on apoptosis in DFs (DNA fragmentation analysis) Results from morphological studies demonstrated that the granulosa layer in DFs of the group that had been treated with P4 for 72 h had entirely disappeared. Therefore, the "DNA ladder" configuration, a characteristic biochemical feature of apoptosis, was examined only in DFs of controls and the groups that had been treated with P4 for 24 and 48 h (Fig. 5). DFs from the control group lacked a signal that indicates low-molecular-weight DNA; however, fragmented DNA extracted from DFs from the group that had been treated with P4 for 24 h, which formed a distinctive ladder pattern, was clearly apparent. The ladder pattern became less distinct for DFs in the group that had been treated with P4 for 48 h, probably because there was an insignificant number of GCs.
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Effects of P4 treatment on Bcl-2 and Bax protein expression The effects of P4 treatment on changes of expression of Bcl-2 and Bax proteins in DFs from the control and groups that were treated with P4 for 24 and 48 h are shown in Figure 6. Both Bcl-2 and Bax were found to be present in DFs. There was no difference in the expression of Bcl-2 with the progression of atresia induced by P4 treatment. In contrast, P4-treated DFs showed a marked increase in Bax expression. Densitometry revealed that the ratio of Bax/Bcl-2 of DFs from cows treated with P4 was significantly increased compared with that of controls (P < 0.05).
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Experiment 2
Effects of P4 on DNA fragmentation in cultured GCs Analysis of DNA fragmentation in GCs cultured for 48 h with or without P4 (4 ng/ml) demonstrated that treatment with P4 had no significant effect on apoptosis (P < 0.05; Fig. 7). In addition, P4 treatment did not affect cell viability (control: 65% ± 4% vs. treatment: 61% ± 5%).
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Bcl-2 and Bax expression in small, medium, and large follicles The ratio of Bcl-2 and Bax protein expression was variable in small follicles (Fig. 8). In contrast, Bax protein expression was relatively higher than that of Bcl-2 in medium and large follicles.
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DISCUSSION
Evidence indicates that a high concentration of P4 may play an important role in initiating the regression of nonovulatory DFs during the bovine estrous cycle [4, 7]. The present study investigated the hypotheses that apoptosis is responsible for the atresia of DFs that develop during luteal phase and that atresia of these DFs is associated with alterations in Bcl-2 and Bax expression. To test this hypothesis, we first employed a model in which proestrus DFs can be maintained healthy and dominant for 9 days and the regression can be induced by P4 injections. Results from our ultrasound study showed that the maintenance and growth of DFs in the control group and regression of DFs in P4-treated groups were similar to those observed in other studies [7, 9]. Histologically, our finding demonstrated that DFs exhibited morphological signs of atresia (e.g., the granulosa layer had thinned considerably and became disorganized just 24 h after P4 injection). These features were further exacerbated with the time of P4 injection. A high ratio of E2:P4 (>1) in FF has been considered to be characteristic of healthy follicles, whereas a low ratio of E2:P4 (<1) is indicative of the atretic status of the follicle [28]. Analysis of steroid hormone concentrations in the present study revealed that significant changes in the ratio of E2:P4 from high (>1) to low (<1) occurred 24 h after P4 injection, which agrees with the morphological changes during atresia. The decrease in E2 production may be the result of a reduction in the number of viable GCs, aromatase activity, or both [29]. These observations demonstrate that P4 treatment could cause atresia of DFs.
Recently, it has been demonstrated that apoptosis is the likely underlying event associated with the initiation and progression of follicular atresia in all vertebrate species studied to date [12, 17, 18, 30]. Examination of the various examples of apoptosis shows that the mediators of apoptosis can be divided into several general classes. The largest group includes those mediators that most likely induce apoptosis by binding to their respective membrane-bound or intracellular receptors and eliciting changes in the intracellular environment. Hormones and growth factors that regulate apoptosis in particular cell types typify this group [31, 32]. Sex steroids are important intraovarian regulators of follicular atresia [33]. In the present study, in situ labeling and electrophoretic analysis of low-molecular-weight DNA of DFs revealed the presence of internucleosomal DNA fragmentation, which is characteristic of apoptosis. Consistent with previous studies [10, 13–15], the identification of much more internucleosomal DNA fragmentation in DFs in the group that had been treated with P4 for 24 h than in those from the control group suggests that apoptosis is the underlying mechanism of follicle degeneration during atresia. The process of apoptosis occurs rapidly, and apoptotic bodies are typically phagocytosed by nearby cells [34]. Combined with the results from our histological study showing decreased numbers of GCs, this may explain why there was less internucleosomal DNA fragmentation in DFs from the group that had been treated with P4 for 48 h. These observations taken together suggest that apoptosis in DFs is regulated in vivo by P4.
How does P4 exert its effects on DFs? Perhaps at two levels; first, via the hypothalamic-pituitary axis. It has long been known that gonadotropins are essential for the growth and development of ovarian follicles and that their secretion can be modulated by negative feedback loops from P4. Previous studies using different models in which DFs are maintained in the absence of a CL using synthetic progestins and low doses of circulating P4 (1–2 ng/ml) have shown that treatments that induce the maintenance of DFs result in a persistent high-frequency and low-amplitude LH pulse pattern. However, high levels of P4 (4–6 ng/ml) reestablish the wave-like growth of antral follicles and result in low frequency LH pulses [3, 7]. Based on these observations, it has been hypothesized that low frequency LH pulses, which are characteristic of the luteal phase, fail to support thecal androgen production, thus impairing GC function and leading to atresia of the DF and a new wave of follicular growth [8]. In addition, recent studies demonstrated that both FSH and LH/hCG inhibited apoptosis and thus are effective survival factors for preovulatory follicles in cows and rats [13, 14].
Alternatively, or coincidentally, P4 may act directly on follicles. The supporting evidence comes from studies showing that P4 suppressed FSH-stimulated E2 production by rat [35] and goat GCs [36]. But this concept remains controversial because some recent studies examining the effects of P4 on FSH-stimulated induction of aromatase mRNA in rat GCs failed to find any suppressive effect [37]. In the present study, P4 had no direct effect on apoptosis in cultured GCs. This is in agreement with the results from an in vivo study in which treatment with P4 had no effect on ovarian apoptosis in hypophysectomized rats [15]. In contrast, a very recent study suggested that P4 can inhibit only large rat GCs from undergoing apoptosis indirectly by stimulating small GCs to synthesize basic fibroblast growth factor [16].
Even though the mechanism by which hormonal factors regulate apoptosis are not well understood, it has been proposed that the absence or presence of cell-specific stimuli via hormonal signals may activate or stop repression of gene products that are responsible for the suicidal mechanism. Because the Bcl-2 family of proteins constitutes a critical intracellular checkpoint of apoptosis within a common cell death pathway [38], we hypothesized that the underlying events involved in high concentration P4-triggered GC apoptosis and atresia of DFs that develop during the luteal phase of the bovine estrous cycle would involve members of this family. Analysis of Bcl-2 and Bax protein expression in cows in the present study indicated that atresia of DFs triggered by in vivo injection of P4 was associated with a marked increase in Bax protein levels. Although Bcl-2 expression was not significantly decreased as expected, the pronounced elevation in Bax expression effectively makes the ratio of Bax to Bcl-2 favor higher death-inducer levels. Therefore, our study supports the concept that proapoptotic proteins such as Bax and Bad accelerate cell death. Conversely, antiapoptotic members such as Bcl-2 and Bcl-XL prevent cell death. Their ratio or balance decides the fate of a cell during development [38]. However, in certain cases, proapoptotic proteins alone are sufficient to cause apoptosis independent of additional signals [39]. In addition, a similar expression pattern of Bcl-2 and Bax has been reported from rat follicles at the mRNA level [40]. In contrast, apoptosis in bovine endothelial cells was associated with altered expression of Bcl-2 protein rather than Bax [41].
Follicles of different sizes (small, medium, and large) were chosen to further study the pattern of Bcl-2 and Bax expression during follicular development. Our data revealed that the ratio of Bcl-2 to Bax was variable in small follicles, whereas the level of Bax protein was higher than that of Bcl-2 protein in medium and large follicles, which suggests that some small follicles may be healthy and most of the medium and large follicles may be undergoing atresia. In a bovine follicular wave, cohorts of antral follicles (2–4 mm) are recruited out of a pool of smaller antral follicles. After 2 to 4 days of recruitment, one follicle is selected. Before selection take place, several medium-size follicles (5–9 mm) can be detected by ultrasonic imaging; however, after selection, only the selected follicle continues to grow, whereas others become atretic [42]. In addition, our results further confirm that an elevated ratio of Bax to Bcl-2 expression occurs in atretic follicles.
In conclusion, our study suggests that apoptosis is the mechanism that underlies atresia of nonovulatory DFs, which develops during the luteal phase of the bovine estrous cycle. P4 does not have a direct effect on apoptosis in bovine GCs; therefore, atresia of nonovulatory bovine DFs is probably via regulation of LH and its inhibitory effect on apoptosis by a P4 negative feedback. Furthermore, our results indicate that atresia in bovine follicles may be linked to a shift in the ratio of antiapoptotic protein (Bcl-2) and proapoptotic protein (Bax) expression and that this shift is mediated primarily through alterations in Bax.
FOOTNOTES
1 Funded by NSERC research grant OGP0036609. 
2 Correspondence: R. Rajamahendran, Faculty of Agricultural Sciences, University of British Columbia, 208-2357 Main Mall, Vancouver, BC, Canada V6T 1Z4. FAX: 604 822 4400; raja{at}unixg.ubc.ca
Accepted: June 6, 2000.
Received: April 11, 2000.
REFERENCES
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L. J. Clark, H. F. Irving-Rodgers, A. M. Dharmarajan, and R. J. Rodgers Theca Interna: The Other Side of Bovine Follicular Atresia Biol Reprod, October 1, 2004; 71(4): 1071 - 1078. [Abstract] [Full Text] [PDF]
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C. Borgeest, K. P. Miller, R. Gupta, C. Greenfeld, K. S. Hruska, P. Hoyer, and J. A. Flaws Methoxychlor-Induced Atresia in the Mouse Involves Bcl-2 Family Members, but Not Gonadotropins or Estradiol Biol Reprod, June 1, 2004; 70(6): 1828 - 1835. [Abstract] [Full Text] [PDF]
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K. Tajima, M. Orisaka, K. Hosokawa, A. Amsterdam, and F. Kotsuji Effects of Ovarian Theca Cells on Apoptosis and Proliferation of Granulosa Cells: Changes During Bovine Follicular Maturation Biol Reprod, June 1, 2002; 66(6): 1635 - 1639. [Abstract] [Full Text] [PDF]
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