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Department of Biological Regulation (K.Y., A.T., A.G.), Weizmann Institute of Science, Rehovot 76100, Israel; and Department of Animal Physiology (A.W.), Institute of Zoology, Jagiellonian University, Krakow 30-060, Poland
Address all correspondence and requests for reprints to: Atan Gross, Ph.D, Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: atan.gross{at}weizmann.ac.il.
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Abstract |
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Introduction |
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More than 99% of the follicles in the mammalian ovary do not complete their development and die rather than ovulate, in a process termed atresia. In early stages of ovarian and preantral follicle development, apoptotic death of oocytes is the major mechanism for germ cell elimination, whereas at later stages of follicular development atresia is initiated by the death of GC (2, 3). The theca-interstitial cells (TIC), which comprise only a minor fraction of the somatic compartment of the mature follicle, were described as undergoing hypertrophy in atretic follicles in rodents and primates (reviewed in Ref. 4) and are considered to play a minor role during atresia. In follicles undergoing atresia, the GC display morphological and biochemical changes associated with apoptosis [such as chromatin condensation, membrane blebbing, cell shrinkage, and DNA fragmentation (2, 5, 6)].
Multiple factors that control the development of the follicle also serve as survival factors, preventing follicular atresia. These factors include the gonadotropins FSH and LH, the growth factors IGF-I and epidermal growth factor, the cytokine IL-1ß, and the steroid hormones progesterone and estrogens. Apoptosis promoting factors include TNF
, Fas ligand, GnRH, androgens, and IL-6 (7, 8, 9). Follicles at different stages of development are dependent on the regulation of different survival factors (9).
BCL-2 family members are major regulators of apoptosis (10, 11), whereas caspases are the major executioners of this process (12). Two major pathways leading to apoptosis exist in cells: the extrinsic pathway, which involves the activation of the TNF/Fas death receptor family and the intrinsic pathway, which involves the mitochondria. In both pathways, an apoptotic death stimulus results in the activation of caspases, either directly or via activation of the mitochondrial death program. The caspase family includes initiator caspases (i.e. caspase-8 and -9), which activate effector caspases (i.e. caspase-3, -6, and -7). All caspases are expressed as inactive enzymes (zymogens) and their activation involves two cleavage events. These cleavages result in the generation of a large and small subunit, which associate to form an active heterotetrameric enzyme. All caspases cleave after an aspartic acid and have a signature cleavage site of XXXD. In the case of caspase-3 and -7, this signature is DEXD.
Caspase-3 is the most characterized effector caspase, and its activation leads to the final stages of cellular death by proteolytic dismantling of a large variety of cellular components on the one hand, and activation of proapoptotic factors on the other. Caspase-3 substrates include structural cytoskeletal proteins, signal transduction proteins, and DNA repair proteins (12). Deletion of the caspase-3 gene in mice leads to major defects in brain development (13, 14). In the ovary, such mice display aberrant GC death (15) and aberrant luteal regression (16). In addition, caspase-3 levels were reported to increase in GC of preovulatory follicles undergoing atresia (17). Thus, caspase-3 seems to be involved in several cell death processes in the ovary but is probably not essential for GC death due to the fact that caspase-7 can compensate for its loss (15).
Caspase-7 is an effector caspase that is very similar to caspase-3 in terms of substrate specificity (18). Caspase- 7 -/- mice are not viable and die early during embryogenesis (19). Caspase-7 is expressed in the oocyte and in GC (20) and was suggested to play a role together with caspase-3 in follicular atresia (15).
To further explore the regulation of caspase-3 and -7 in atresia of preovulatory follicles, we have used a well-established in vitro model system in which apoptosis is induced by serum starvation and suppressed by LH, FSH, or IGF-I (21). Using this model, we have demonstrated that apoptosis is induced in the absence as well as in the presence of serum and that under both culture conditions caspase-3 and -7 are activated. Surprisingly, the addition of either LH or FSH further increased caspase-3/-7 cleavage/activity. We have found that active caspase-3 localizes to TIC, and we demonstrated that its increase in activity was accompanied by an increase in apoptosis. LH treatment also increased the activity of caspase-3/-7 in GC, but this increase was accompanied by a decrease in apoptosis. Thus, gonadotropins have a dual action on cultured rat preovulatory follicles: they increase apoptosis in TIC and decrease apoptosis in GC.
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Materials and Methods |
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Follicle cultures
All animal experiments were approved by the Institutional Animal Care and Use Committee. Follicle cultures were performed as previously described (22). Briefly, immature female Wistar rats at the age of 23–25 d were injected with 10 IU eCG in PBS (Life Technologies, Inc., Grand Island, NY) to induce follicular growth. After 48 h, rats were killed by CO2, the ovaries were placed in Leibovitz medium (L-15; Life Technologies, Inc.) containing 2 mM L-glutamine, 0.1% BSA (Fraction V, Sigma, St. Louis, MO) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin; Life Technologies, Inc.), and immediately dissected. The largest preovulatory follicles (>800 µm in diameter) were isolated from the ovaries and cleaned from adherent tissue using fine forceps. Five to 15 intact follicles of similar size, were transferred into an organ culture dish (Falcon, NJ) and cultured on stainless steel grids. Cultures were carried at 37 C under 50% O2/50% air and sufficient CO2 to give a pH value of 7.2. Follicles were cultured for 24 h in medium containing 5% fetal calf serum or in serum-free medium in the presence or absence of LH (100 ng/ml), FSH (2 IU/ml), IGF-I (100 ng/ml), or zVAD-fmk (50 µM). In the experiments in which GC were separated from TIC, cultured follicles were transferred to serum-free medium and punctured with a 27.5-gauge needle. GC were first gently squeezed out of the follicle to the medium. Subsequently, the follicle was opened and gentle scraping of the follicular wall was performed to remove adherent GC. GC and the remaining tissue (TIC) were transferred to separate 1.5-ml tubes (Axygen Scientific, Inc., Union City, CA), centrifuged at 1000 x g, washed once in PBS, centrifuged again, and the cell pellets were snap frozen. The resulting GC were a clean population of cells and did not contain TIC, based on the absence of the TC enzyme 17-hydroxylase cytochrome P450 from Western blots of GC extracts (data not shown). On the other hand, the residual tissue that contained mainly TIC was not a pure population of cells and may have contained some GC.
Western blotting
Proteins were size-fractionated by SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Immobilon-P, Bio-Rad, Hercules, CA). Antibodies included polyclonal anticaspase-3 (H-277; Santa Cruz Biotechnology, Inc., Santa Cruz, CA; dilution of 1:1000; this antibody recognizes full-length caspase-3 and weakly recognizes the p20 and p17 cleavage products; this antibody does not cross-react with caspase-7 because we did not obtain any bands in the 30- to-40-kDa range on Western blots using lysates of MCF-7 cells, which are breast carcinoma cells that do not express caspase-3 (Ref. 23 and data not shown); polyclonal anticleaved caspase-3 (Cell Signaling Technology, Inc., Beverly, MA; catalog no. 9661; dilution of 1:300; this antibody recognizes the p20 and p17 cleavage products of caspase-3 but not full-length caspase-3), monoclonal anticaspase-7 [a gift from Dr. Y. Lazebnik, Cold Spring Harbor, NY; dilution of 1:1000; this antibody recognizes the full-length and p20 cleavage product of caspase-7 (24)], monoclonal anti--fodrin (Chemicon, Temecula, CA; dilution of 1:1000) and monoclonal anti-ß-actin (Sigma dilution of 1:5000). Western blots were developed by use of the enhanced chemiluminescence reagent (Amersham, Buckinghamshire, UK). Individual bands were quantified from films by densitometry using the Quantity One software program (Bio-Rad Laboratories, Inc.).
Caspase-3/-7 activity assays
Follicles (10 per sample) were homogenized twice in lysis buffer [20 mM HEPES (pH 7.3), 5 mM EGTA, 5 mM EDTA, 10 µM digitonin, 2 mM dithiothreitol]. The lysates were clarified by centrifugation and the supernatants were used for the assays. Protein concentrations were determined by Bradford (Bio-Rad). Enzymatic reactions were carried out in lysis buffer containing 20 µg of protein and 50 µM DEVD-AMC to assess caspase-3/-7 activity. Each sample was divided into three parts: one of them included in addition to the extract and substrate, 50 µM zVAD-fmk to inhibit caspase activity and two replicates that included extract and substrate, without inhibitor. The reaction mixtures were incubated at 37 C for 1 h, and fluorescent AMC formation was measured at excitation 380 nm and emission 460 nm using a microplate spectrofluorometer (SPECTRAmax, Molecular Devices Corp., Sunnyvale, CA). Specific activity was calculated for each sample as the mean of the duplicate sample minus the value obtained for the sample containing zVAD-fmk.
Immunohistochemistry
Follicles (five to six per treatment in duplicates) were paraffin embedded, and 6-µm sections were analyzed by immunohistochemistry using the Histostain-SP Kit (Zymed Laboratories, South San Francisco, CA), according to the manufacturer’s instructions. Briefly, after deparaffinization, dehydration and blocking of endogenous peroxidases with 3% of H2O2 in methanol, sections were incubated with 10% goat serum for 1 h, followed by incubation with the primary antibody. Anticleaved caspase-3 antibodies (Cell Signaling; dilution of 1:5) were used to detect the cleaved/large subunit of caspase-3 and anticaspase-3 antibodies (H-277; Santa Cruz Biotechnology, Inc.; dilution of 1:25) were used to detect procaspase-3. To demonstrate the specificity of these antibodies, anticleaved caspase-3 antibodies were preincubated with the peptide used to generate the antibody (Cell Signaling; antibody/peptide 1:1) and anticaspase-3 antibodies were preincubated with recombinant caspase-3 (Calbiochem, San Diego, CA; antibody/recombinant protein 1:5). The sections were then washed and incubated with the biotinylated secondary antibody for 10 min at room temperature, followed by another 10 min with horseradish streptavidin-peroxidase conjugated. After washing, sections were incubated with the chromogen substrate for 10 min and counterstained with hematoxylin. Slides were analyzed under a light microscope (Nikon, Tokyo, Japan) at a magnification of x100/x400. Pictures were taken with a 1310 digital camera (DVC, Imaging Research, Ontario, Canada).
Fluorescence-activated cell sorting (FACS) analysis of sub-G1 DNA content
GC were isolated from cultured follicles, washed once in PBS, and fixed with methanol at -20 C (overnight). The cells were recovered by centrifugation at 1000 x g for 5 min, washed once in PBS, and incubated in PBS containing 25 µg/ml propidium iodide and 50 µg/ml ribonuclease A. The percentage of cells displaying a sub-G1 DNA content was determined by FACScan (Becton Dickinson, Franklin Lakes, NJ).
Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay
Apoptosis was assessed by in situ 3'-end labeling of DNA fragments using the TUNEL assay. TUNEL was performed using the ApopTag in situ apoptosis detection kit (Intergen, Norcross, GA), according to the manufacturers’ instructions. Briefly, follicles were fixed in Carnoy solution (ethanol/chloroform/acetic acid 6:3:1), dehydrated, embedded in paraffin, and serially cut at 6 µm. After deparaffinization, hydrated sections were incubated in a humidified chamber with 10 µg/ml proteinase K (Sigma) at room temperature for 5 min and endogenous peroxidases were neutralized by incubation with 3% H2O2. The sections were then incubated with terminal deoxynucleotidyl transferase for 1 h at 37 C, followed by incubation with antidigoxigenin-peroxidase antibodies. Sections were stained with diaminobenzidine solution and counterstained with hematoxylin. Slides were analyzed and photographed as described above.
DNA fragmentation analysis
DNA was extracted by digestion of the tissue in a buffer containing 0.5% sodium dodecyl sulfate, 0.1 M NaCl, 0.05 M Tris (pH 8.0), 4 mM EDTA, and 100 µg/ml of proteinase K (Sigma) in a water bath (57.5 C) for 5 h. After digestion, the DNA was incubated with 1.5 M potassium acetate/chloroform (1:1) on ice for 1 h followed by centrifugation (8', 7000 x g, 4 C). The aqueous phase of the samples was collected and precipitated in ethanol (first in 100% and later in 70%) and then resuspended in double distilled water. The DNA concentration was determined and samples were treated with ribonuclease (10 µg/ml, deoxyribonuclease free; Roche, Basel, Switzerland) at 37 C for 1 h. One microgram of DNA from each sample was 3' end labeled with [32P]dideoxy-ATP (1000 µCi/100 µl; Amersham) using terminal transferase (Roche). Labeled samples were fractionated on 2% agarose gels, dried and exposed to film (Fuji, Tokyo, Japan). After autoradiography, low molecular weight DNA fragments (<2 kb) from each sample were excised from the gels and analyzed in a ß-counter (Beckman Coulter, Inc., Fullerton, CA).
Statistical analysis
Data regarding sub-G1 DNA analysis, caspase activity assays, low molecular DNA laddering assays, and densitometric analysis were expressed as the mean ± SEM of pooled results obtained from at least three independent experiments. Data regarding caspase activity assays needed a root square variance-stabilizing transformation. Statistical analysis was performed by one-way ANOVA test followed by Fisher’s protected least significant difference posttest for multiple comparisons using the StatView Program (Abacus Concepts, Berkeley, CA). Significance level was considered as P < 0.05.
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Results |
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). Surprisingly, incubation of the follicles in serum-containing medium for 24 h resulted in a similar level of sub-G1 DNA content as in follicles incubated in the absence of serum, suggesting that serum components are insufficient in suppressing apoptosis of follicles in culture. Because there was no difference in the level of apoptosis between follicles cultured in the presence or absence of serum, most cultures from this point on were performed only in the absence of serum.
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, middle panel). Unexpectedly, addition of either LH or FSH, but not of IGF-I, to the culture medium resulted in an increase in the intensity of the p17 cleavage product. The approximately p20 band that appears in this panel represents a partial cleavage product of caspase-3 (27), which can also be detected using the anticaspase-3 antibody (data not shown). Of note, each of the different treatments did not seem to significantly change the level of procaspase-3 (Fig. 1B, top panel). A dose-response analysis of the LH-induced cleavage of caspase-3 revealed that a dose of 1 ng/ml resulted in a slight elevation in p17, whereas 10 ng/ml resulted in a substantial elevation, which was not further enhanced by higher doses of this gonadotropin (Fig. 1C).
To assess whether there were additional effector caspases involved in this process, we examined caspase-7 by Western blot analysis using an anticaspase-7 antibody, which recognizes procaspase-7 as well as its p20 large/active subunit (24). As in the case of caspase-3, the p20 cleavage product of caspase-7 appeared in lysates of cultured follicles and the addition of either LH or FSH, but not of IGF-I, resulted in a significant increase in its intensity (Fig. 1D).
To assess whether the increase in the cleavage of caspase-3 and -7 correlated with an increase in their activity, the cleavage of DEVD-AMC, a fluorogenic peptide substrate, specific for both caspase-3 and -7, was measured. As shown in Fig. 1E, DEVDase activity was induced in follicles cultured in the presence or absence of serum and the addition of either LH or FSH further increased this activity. Addition of IGF-I, however, reduced DEVDase activity. The activity measured in the follicle lysates was specific for caspases because addition of the broad caspase inhibitor zVAD-fmk to the culture medium completely abolished this activity (Fig. 1E; LH + Z).
To determine whether the activity of caspase-3 correlated with the cleavage of a known caspase-3 substrate, we analyzed the cleavage of p240 -fodrin. This protein is a major component of the cortical cytoskeleton, which is cleaved by caspase-3 to form a p120 product (28). In cultured follicles as well as in follicles treated with IGF-I, but not in freshly isolated follicles, -fodrin was cleaved to form the p120-truncated product (Fig. 1F). As expected, a significant elevation in the intensity of the p120 product was observed in follicles cultured with either LH or FSH. Cleavage of -fodrin was due to caspases because it was inhibited in follicles cultured in the presence of zVAD-fmk (Fig. 1F; LH + Z/FSH + Z).
Gonadotropins induce the appearance of active caspase-3 in TIC of cultured preovulatory follicles
To determine the location of active caspase-3, sections of rat follicles were immunostained using the anticleaved caspase-3 antibodies. As expected, positive staining was not detected in freshly isolated follicles (Fig. 2; time 0). In cultured follicles, very few cells were positively stained, which surprisingly were mainly TIC (nontreated; N/T). Most strikingly, in follicles cultured in the presence of either LH or FSH, prominent staining of many TIC was apparent. In follicles cultured in the presence of IGF-I, positive staining was lower than in follicles cultured in the absence of hormones. The positive staining that appeared in all slides indicated the presence of activated caspase-3 because follicles cultured in the presence of both LH and zVAD-fmk showed no positive staining (LH + Z). Moreover, preincubation of the antibody with the peptide that was used to generate the antibody completely eliminated the positive staining (data not shown).
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; N/T). Strikingly, addition of either LH or FSH to the culture medium resulted in a marked increase in positive TUNEL staining in TIC and a decrease in TUNEL staining in GC. Addition of LH in the presence of zVAD-fmk largely inhibited the positive TUNEL staining, indicating that the positive staining was due to active caspases. Finally, the IGF-I treatment did not seem to have an apparent effect compared with N/T follicles.
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).
Gonadotropins increase caspase-3 and -7 activity in both TIC and GC
Next we assessed the expression of both pro-forms and cleaved products of caspase-3 and -7 in GC and TIC, separated from freshly isolated follicles or from follicles cultured in the presence or absence of LH. After mechanical separation, both cell populations were lysed and analyzed by Western blot. Interestingly, in freshly isolated follicles there was much less expression of procaspase-3 in GC than in TIC, whereas procaspase-7 was similarly expressed in both cell population (Fig. 4A, top panel, and 4B). Culture of follicles induced the appearance of the p17 caspase-3 cleavage product only in TIC, whereas the p20 caspase-7 cleavage product appeared in both cell populations [Fig. 4, A (middle panel) and B]. The addition of LH to the culture medium further increased the levels of p17 and p20 in both cell populations. Similar results were obtained with FSH (data not shown). We have also measured the DEVDase activity in both cell populations and found that it correlated with the Western blot results (Fig. 4C). Thus, placing follicles in culture induces the activation of caspase-3 and -7 in TIC (and caspase-7 in GC) and the addition of gonadotropins further augments this effect.
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suggested that procaspase-3 is predominantly expressed in TIC of preovulatory follicles. To further confirm these observations, we immunostained follicle sections with the anticaspase-3 antibody. Using this antibody, we obtained positive staining in TIC of freshly isolated preovulatory follicles (Fig. 5A, left), which was inhibited by preincubation of the antibody with recombinant caspase-3 (Fig. 5A, right). To determine the expression pattern of procaspase-3 in intact ovaries, we used the same antibody to stain ovarian sections taken from eCG-treated rats. These studies clearly indicated that procaspase-3 was predominantly expressed in TIC of antral follicles (Fig. 5B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Unexpectedly, LH and FSH, which are considered to be survival factors in this model system, further increased caspase activity. Most of the increase in caspase-3 and -7 activity was localized to TIC and was accompanied by an increase in apoptosis (Figs. 2–4). This effect was restricted to gonadotropins because IGF-I treatment resulted in a reduction in caspase-3 and -7 activity, which correlated with its antiapoptotic effect.
The increase in caspase-3 and -7 activity in response to gonadotropins was not restricted to the TIC because we also detected an increase in GC (Fig. 4). Paradoxically, this increase was accompanied by a decrease in apoptosis. The fact that gonadotropins inhibit apoptosis in GC is in agreement with their antiapoptotic effect on whole follicles (Fig. 1 and Ref. 21). Several studies using GC have demonstrated the involvement of multiple survival signaling pathways triggered by gonadotropins (29). Progesterone is one of the factors that is induced by LH and that was reported to act as an antiapoptotic factor in luteinized rat and human GC (30, 31). Gonadotropins were also demonstrated to affect the apoptotic machinery by suppressing the expression of proapo-ptotic proteins (32, 33) as well as inducing the expression of antiapoptotic proteins (34, 35). Because GCs outnumber any other follicle cells, the response of the whole follicle to LH is a decrease in apoptosis. Thus, gonadotropin administration to cultured preovulatory follicles induces an antiapoptotic response in GC, which overrides the proapoptotic response due to caspase-3 and -7 activation.
Our studies demonstrate that both LH and FSH induce apoptosis of TIC in cultured intact rat follicles (Fig. 3). In the preovulatory follicle, LH can act directly on both GC and TC via LH receptors. On the other hand, the action of FSH is restricted to the GC because TCs do not express FSH receptors. Thus, the proapoptotic effect of FSH on TIC is probably mediated via GC. The finding that both gonadotropins act similarly suggests that this effect is controlled by a common downstream mechanism. Several intraovarian factors that induce atresia and are produced by GC include TNF, Fas ligand, and IL-6. Whether these factors participate in gonadotropin-induced apoptosis of TIC remains to be determined.
Atretic degeneration of antral follicles in the ovary is mainly due to apoptosis of GC. On the other hand, thecal tissue hypertrophy and apoptosis occurs at later stages of follicular degeneration and therefore is considered to play a minor role in the atretic process (2, 4). Therefore we have expected that the expression levels of procaspase-3, a major executioner of apoptosis, would be high in GC and low in TIC. Unexpectedly, the levels of procaspase-3 were found to be high in TIC and very low in GC (Figs. 4 and 5). Similar results were obtained by Boone and Tsang (17), who noticed that procaspase-3 is predominantly expressed in TIC of healthy rat follicles. Unlike caspase-3, caspase-7 is similarly expressed in both cell populations (Fig. 4). Taken together, these data suggest that caspase-7 is playing a role in apoptosis of both GC and TIC, whereas caspase-3 is mostly playing a role in apoptosis of TIC.
One may question whether the results we have obtained in vitro reflect changes occurring in vivo. One possible answer is that LH and FSH have both pro- as well as antiapoptotic effects in vivo. If this is indeed the case, then follicle cells that do not respond to the gonadotropins surge by producing sufficient levels of survival factors will be more susceptible to their proapoptotic effects. Another possibility is that elevation in caspase-3 and -7 activity in TIC is related to the process of ovulation. LH induces the activation of several proteolytic enzymes that degrade extracellular matrix and thus facilitate follicle rupture and ovulation (36). Elevation in caspase-3 and -7 activity leading to apoptosis of a limited number of TIC might be an additional mechanism to facilitate formation of the stigma and the rupture of the follicle, enabling the release of the oocyte. In support of this idea, Murdoch and his colleagues (37, 38) have detected apoptotic cells in the apical follicular wall and surrounding ovarian tissues in preovulatory dominant ovine follicles after the endogenous LH surge triggering ovulation. Another possibility is that low levels of active caspase-3 and -7 do not result in apoptosis but are rather necessary for the differentiation of follicular cells into luteal cells. This idea is based on the fact that several reports have described the involvement of active caspase-3 in the process of terminal differentiation (39, 40, 41, 42).
In summary, we have demonstrated that gonadotropins induce an increase in caspase-3 and -7 activity in intact rat preovulatory follicles. This increase mainly occurs in TIC and is accompanied by an increase in apoptosis in these cells. The fact that high levels of procaspase-3 are expressed in TIC of antral follicles in intact ovaries strongly suggests that caspase-3 is playing a functional role in these cells in vivo.
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Footnotes |
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Abbreviations: DEVD-AMC, Acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin; eCG, equine chorionic gonadotropin; FACS, fluorescence-activated cell sorting; GC, granulosa cells; N/T, nontreated; TIC, theca-interstitial cells; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling; zVAD-fmk, Z-Val-Ala-Asp-fluoromethylketone.
Received October 17, 2003.
Accepted for publication January 7, 2004.
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References |
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-fodrin cleavage but dispensable for cleavage of other death substrates in apoptosis. J Biol Chem 273:15540–15545
B-mediated X-linked inhibitor of apoptosis protein expression prevents rat granulosa cells from tumor necrosis factor -induced apoptosis. Endocrinology 142:557–563This article has been cited by other articles:
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