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REPRODUCTION-DEVELOPMENT |
Vincent Center for Reproductive Biology, Department of Obstetrics and Gynecology (S.F.C., T.M., M.P.L., J.L.T., B.R.R.), Massachusetts General Hospital/Harvard Medical School, Boston, Massachusetts 02114; Section of Immunobiology and Howard Hughes Medical Institute (R.A.F.), Yale University School of Medicine, New Haven Connecticut 06510; and Departamento De Clínica De Grandes Animais (S.F.C., P.B.D.G.), Universidade Federal De Santa Maria, Faculdade de Medicina Veterinária, Rio Grande do Sul, Brazil
Address all correspondence and requests for reprints to: Bo R. Rueda, Ph.D., Massachusetts General Hospital, VBK137E-GYN, 55 Fruit Street, Boston, Massachusetts 02114. E-mail: . brueda{at}partners.org
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Abstract |
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Introduction |
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Apoptosis is a physiological form of cell death that can be initiated by a number of stimuli, including activation of the so-called death receptors (e.g. Fas, TNF receptor-1) and serum or growth factor withdrawal (7, 8, 9). Once a cell receives a lethal stimulus, the program of apoptosis is then regulated and executed by members of the Bcl-2 and caspase families, respectively. The gene encoding Bcl-2 was discovered at a chromosomal breakpoint that predisposed individuals to development of B-cell lymphoma (10, 11). Since its discovery, numerous Bcl-2-related proteins have been isolated and characterized, with respect to their function in apoptosis. For example, some members inhibit apoptosis (Bcl-2, Bcl-xL, Mcl-1, and Bcl-w), whereas others facilitate or induce apoptosis (Bax, Bak, Bad, Bid, Hrk/DP5, Mtd/Bok, and Bim) (12, 13, 14). Through their interactions at the level of mitochondria, Bcl-2 family members are considered to be the principal determinants of whether or not the execution of apoptosis proceeds via the activation of caspases (13, 15).
Of the 14 identified caspase family members in mammals, caspase-3 is unquestionably the most well-characterized enzyme. Activation of caspase-3, considered by many as a final executioner of the apoptotic cell death program, leads to rapid cleavage of a diverse spectrum of key structural and functional proteins in the cell. These targets for proteolytic attack include cytoskeletal components, signal transduction molecules, DNA repair enzymes, RNA splicing factors, nuclease activity-modulating factors, and nuclear matrix proteins (16, 17). Recent studies have shown that caspase-3 is expressed in the human CL (18), and have provided indirect evidence that caspase-3 is involved in luteal regression in several species (19, 20, 21, 22). However, all of these reports provide correlative data at best, thus raising the question of how functionally important caspase-3 is to the process of luteolysis. Therefore, the present study was designed to determine whether CL of caspase-3-deficient mice exhibit overt defects in luteolysis and whether these defects are, in turn, attributable to the inability of luteal cells to execute apoptosis.
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Materials and Methods |
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Ex vivo culture
Corpora lutea were mechanically isolated, 30 h after induced ovulation, and cleaned of surrounding follicles and stromal tissue. Some CL were immediately frozen (for gel electrophoretic analysis of DNA integrity) or were fixed (for histology and histochemistry) in 10% neutral-buffered formalin, paraffin-embedded, serially sectioned (6 µm), and mounted on glass microscope slides. This group served as time 0 (no culture) data points. The remaining CL were cultured in serum-free conditions as previously described for neonatal mouse ovaries (24). Briefly, after isolation the CL were transferred onto nucleopore polycarbonate membranes (Transwell Membrane, 3.0-µm pore size, 24-mm diameter; Costar, Cambridge, MA) in Waymouth’s MB752/1 culture medium (Life Technologies, Inc., Grand Island, NY) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 1.3 µg/ml Amphotericin, and 0.23 mM pyruvic acid, and cultured for 24, 48, or 72 h, before freezing or fixation as described above. For histochemical assays, the largest diameter sections of the serially sectioned CL were used.
In vivo studies
To validate the induced luteal phase mouse model, ovaries and blood samples were collected from WT mice at 0.5, 1, 2, 3, 4, 5, and 6 d after induced ovulation (n = 3/time point). The ovaries were fixed in 10% neutral-buffered formalin, paraffin-embedded, serially sectioned (6 µm), and mounted, in order, on glass microscope slides. For in situ analysis of DNA fragmentation [terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end-labeling (TUNEL)], the largest diameter sections from each ovary were used. After determining the progesterone levels and incidence of apoptosis in the CL of WT mice, ovaries and blood samples were collected from caspase-3-deficient female mice (n = 3 mice/genotype) on d 2, 4, and 6 after induced ovulation (see above), for comparison.
Immunohistochemistry
Paraffin-embedded sections (6 µm) were analyzed by immunohistochemistry for the presence of cleaved (active) caspase-3, as previously described (21, 25). Briefly, after peroxidase quenching, sections were incubated with a 1:3,000 dilution of a rabbit polyclonal antibody that recognizes cleaved caspase-3 (CM1; IDUN Pharmaceuticals Inc., La Jolla, CA) (25, 26) for 1 h at room temperature. Sections were washed and then incubated with a 1:200 dilution of biotinylated goat antirabbit antibody (Calbiochem-Novabiochem, La Jolla, CA) for 1 h at room temperature. Sections were washed, incubated for 45 min at room temperature with horseradish peroxidase-conjugated streptavidin, washed, and then incubated with ice-cold 0.5-mg/ml 3,3'-diaminobenzidine with 0.03% hydrogen peroxide at room temperature, for antigen detection (brown reaction product). Sections were counterstained with hematoxylin and analyzed by light microscopy. Cells exhibiting the brown cytoplasmic staining from the colorimetric reaction were considered positive for active caspase-3. Negative controls, lacking the labeling enzyme, yielded no reaction product (data not shown). Similarly, ovaries collected from caspase-3-deficient mice had negligible (background) levels of staining. Sections of CL or ovaries from WT and caspase-3-deficient mice were processed in parallel within the same assay. The percent of caspase-3-positive cells was determined by counting CM-1-positive cells/total number of cells per field of view (600x) in sections from three separate CL from 3 different animals.
TUNEL
The occurrence of apoptosis in luteal and ovarian sections was assessed by monitoring the presence of DNA fragmentation in situ, as described previously (27). Slides were analyzed by conventional light microscopy after light counterstaining with hematoxylin. Cells exhibiting dark brown nuclear staining from the colorimetric reaction were considered positive for DNA fragmentation. Negative controls, lacking the labeling enzyme, yielded no reaction product (data not shown). Sections of CL or ovaries from WT and caspase-3-deficient mice were processed for TUNEL in parallel within the same assay. The percent of apoptotic cells was determined by counting TUNEL (brown reaction) positive cells/total number of cells per field of view (600x) in three separate CL from each ovary of three different animals.
DNA isolation, radiolabeling, and gel electrophoretic analysis
Genomic DNA was extracted from freshly isolated or cultured CL, quantitated and 3'-end-labeled with [
32P]-dideoxy-ATP (3,000 Ci/mmol; Amersham Pharmacia Biotech, Inc., Piscataway, NJ) using terminal deoxynucleotidyl transferase (Roche Molecular Biochemicals, Indianapolis, IN) as described (27, 28). Radiolabeled samples were resolved through 2% agarose gels, and the extent of internucleosomal cleavage was qualitatively assessed by autoradiography.
Hormone analysis
Progesterone levels were measured in serum, according to the manufacturer’s instructions, using a direct solid-phase enzyme-immunoassay (DRG Progesterone ELISA kit; ALPCO, Windham, NH). Before the assay, the serum samples were extracted with 6.6 vol ethyl ether; the extracts were dried under nitrogen and reconstituted in zero standard control serum provided by the manufacturer. Sera from WT and caspase-3-deficient mice were assayed in duplicate in the same assay. Progesterone concentration was determined by absorbance, at 450 nm, against a standard curve.
Data presentation and statistical analysis
Each experiment was independently replicated 3 times with different mice in each experiment. Data shown are representative of results obtained in the replicate experiments. For quantitation of immunohistochemical data, the total number of positive cells was determined in at least 3 fields of view per CL or ovary and calculated as a percent of the total number of cells per field of view. An average of 500 cells was counted per field of view. The results are expressed as the percent mean ± SEM. Quantitative data generated by RIA of progesterone levels are expressed as the mean ± SE. Statistical significance was determined by either t test or one-way ANOVA followed by Duncan’s new multiple-range test. A P value less than 0.05 was considered statistically significant.
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Results |
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) or caspase-3-deficient mice (Fig. 1B). However, processing of procaspase-3 to the active form became evident in CL derived from WT mice after 48 h in culture (Fig. 1E) and was widespread after 72 h in culture (Fig. 1G). By comparison, and as anticipated from the genotype, CL isolated from caspase-3-deficient mice had only light cytoplasmic staining of single cells observed on occasion at 48 and 72 h (Fig. 1, F and H). A significant increase (P < 0.0004) in the percent of CM-1-positive cells was observed in the CL of WT mice at 48 h (31.0 ± 1.15%), compared with the percent of CM-1-positive cells in CL of caspase-3-deficient mice at the same time point (4.6 ± 0.08%).
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and 2). The mean number of TUNEL-positive cells increased, over time, in the cultures of CL derived from WT mice (Fig. 2; A, C, E, and G). At 48 h, there was a significant increase (P < 0.005) in the percent of TUNEL-positive cells observed in the CL derived from WT mice (32.6 ± 3.4% TUNEL-positive cells; Fig. 2E), compared with the CL derived from caspase-3-deficient mice (1.04 ± 0.3% TUNEL-positive cells; Fig. 2F). After 72 h in culture without hormonal support, the cells within the CL derived from the WT mice displayed all the expected features of apoptosis, including cellular condensation and detachment, DNA cleavage, and formation of large numbers of apoptotic bodies (Fig. 2G). Precise counts of TUNEL-positive cells were impossible because of the high levels of cellular debris in the CL of the WT mice. By comparison, all of these events were either markedly attenuated or absent in CL isolated from caspase-3-deficient mice cultured in parallel (Fig. 2, B and D, F, H). Because TUNEL alone is not always conclusive of apoptosis, electrophoretic analysis of low-molecular-weight DNA was performed. The attenuated onset of apoptotic cell death in CL collected from caspase-3 null mice was further reflected by gel electrophoretic analysis of DNA integrity (Fig. 3).
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, A and C). In contrast, only a few, if any, TUNEL-positive cells were detected in caspase-3 null CL collected in parallel at d 4 post ovulation (Fig. 4, B and D). Based on counts of TUNEL-positive cells in the CL of WT and caspase-3-deficient mice collected on d 4, the number of TUNEL-positive cells/total number of cells per field of view was significantly higher (P < 0.003) in CL of WT mice (14.75 ± 2.14%), when compared with CL of caspase-3-deficient mice (1.3 ± 0.3%). In keeping with these data that suggest that loss of caspase-3 function delays involution of the CL, 6 d after ovulation, ovaries collected from WT mice contained only remnants of the CL (Fig. 4E). However, caspase-3-deficient ovaries still retained several large CL with a only few TUNEL-positive cells (Fig. 4F).
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). There was no significant difference (P > 0.05) in the levels of progesterone in the caspase-3-deficient [knockout (KO)] mice, when compared with the progesterone levels in WT mice, over time, at either d 4 (WT, 1.99 ± 1.19 ng/ml; KO, 0.85 ± 0.03 ng/ml) or d 6 (WT, 0.9 ± 0.09 ng/ml; KO, 0.82 ± 0.00 ng/ml).
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Discussion |
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and Fas ligand bind distinct membrane receptors, both have the ability to promote caspase-3-mediated apoptosis (29). Moreover, caspase-3 can be activated by more than one signaling pathway coupled to these so-called death receptors. Whether or not apoptosis is induced by an activated death receptor or an alternative apoptotic stimulus, caspase-3 is considered a common mediator of apoptosis (30, 31). Whereas evidence of caspase-3 expression and activity has been shown in regressing CL of many species (18, 19, 20), it remains to be established whether caspase-3 is needed for apoptosis and structural involution associated with luteal regression. Previous studies designed to evaluate the possible role of caspase-3 in the CL have relied heavily on relatively pure or mixed luteal cell populations dispersed in monolayer cultures (21, 22). These studies have demonstrated that caspase-3 is present and activated coincident with apoptosis. Our first series of experiments, using intact CL to maintain the normal three-dimensional structure and cell-to-cell interactions of the CL, revealed a time-dependent onset of apoptosis in CL derived from WT mice, as assessed by both morphological and biochemical parameters. Furthermore, the increase in apoptosis was coordinately associated with an elevation in the level of processed (active) caspase-3 enzyme. These correlative results, which further strengthen the hypothesis that caspase-3 is involved in luteal cell death, were reinforced by our findings derived from comparing the onset of apoptosis in cultured CL collected from WT and caspase-3 mutant female mice. Though freshly isolated CL of WT and caspase-3 null mice were histologically and biochemically similar, the occurrence of apoptosis after in vitro culture was markedly delayed by caspase-3 deficiency.
To confirm and extend these findings, we next tested the importance of caspase-3 to luteal regression in vivo. As a model, prepubertal female mice were treated with exogenous gonadotropins to induce ovulation to ensure that no residual luteal tissue from a previous estrous cycle would be present. Under these experimental conditions, the CL of WT mice maintained their structural integrity for approximately 4 d, at which time there was an easily discernible decrease in overall luteal mass concomitant with the appearance of many TUNEL-positive (apoptotic) cells. By d 6 post ovulation, only remnants of the CL were visible. From a functional standpoint, the levels of progesterone increased after ovulation, reaching peak levels at approximately 30 h after luteal formation. Serum progesterone levels then declined dramatically, well before the onset of structural involution, as determined by TUNEL analysis of apoptosis.
In contrast to these in vivo observations of luteal growth and regression in WT mice, there was little to no evidence of TUNEL-positive cells in the CL of the caspase-3-deficient mice at 4 d post ovulation. Furthermore, luteal mass was clearly maintained by caspase-3 deficiency, as long as 6 d after ovulation, with only a few TUNEL-positive cells becoming detectable. This defect in the temporal onset of apoptosis in caspase-3-deficient mice in vivo was similar to that observed using the ex vivo culture model. Interestingly, the levels of progesterone in caspase-3 null mice did not differ from their WT siblings at any time point examined post ovulation, despite the maintenance of CL mass caused by caspase-3 loss-of-function. Such findings add important insight into mechanisms responsible for luteal regression, because the relationship between the induction of luteal cell death to the loss of steroidogenic capacity has remained unsettled. Indeed, based on our observations herein, it is likely that steroidogenic cells of the CL lose their capacity to produce progesterone via mechanisms independent of those responsible for executing apoptosis during structural involution.
It was previously reported that a reduction in progesterone may accelerate the apoptotic process in bovine luteal cells (32, 33). However, in the present murine model, the onset of apoptosis was delayed by the lack of caspase-3 despite the fact that progesterone had decreased. Because the functional and structural components of luteal regression can be separated temporally and mechanistically, at least in the mouse, the potential may exist to rescue a functionally deficient CL before its structural demise. This could be helpful clinically in cases of luteal insufficiency, which can lead to spontaneous abortions and/or irregular menstrual cycles (34). Interestingly, we have observed a number of reproductive anomalies associated with the caspase-3-deficient female mice, including failure to conceive and/or establish pregnancy. If the female becomes pregnant, the litter sizes are small in number, and it is not uncommon to have birth of nonviable offspring (unpublished data). Whether or not these abnormalities are attributable, entirely or in part, to the disrupted luteal regression that results from caspase-3 deficiency remains to be established. Future experiments will also be needed to characterize whether or not other presumed mediators of apoptosis may influence the loss of steroidogenic function associated with luteolysis. Collectively, these studies may help to further define relationships between steroidogenisis and apoptosis susceptibility in the CL, and whether luteal cells rescued from apoptosis can regain steroidogenic capacity.
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Acknowledgments |
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Footnotes |
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Abbreviations: CL, Corpus luteum (corpora lutea); KO, knockout; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end-labeling; WT, wild-type.
Received October 1, 2001.
Accepted for publication December 6, 2001.
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