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Ovary |
Laboratory of Reproductive Endocrinology,3Department of Animal Science, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan
Division of Reproductive Endocrinology and Pathophysiology,4 Institute of Animal Reproduction and Food Research, PAS, Olsztyn 10-747, Poland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, bcl-2 gene family, corpus luteum, Fas-Fas ligand, progesterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, which initiates a complex cascade of events that finally interrupt steroidogenesis and induces structural regression of the corpus luteum (CL) [1]. The cells of the CL undergo apoptosis during structural luteolysis [2, 3]. In the bovine CL, the appearance of internucleosomal fragmentation of DNA characteristic of apoptosis is associated with the action of cytokines, especially of tumor necrosis factor- (TNF) and interferon-
(IFN) [4–6]. TNF and its specific type-I receptors (TNF-R; responsible for the transduction of cell death signaling) are present in the bovine CL during luteolysis [4, 7]. TNF belongs to the TNF superfamily (TNF-SF), which consists of 18 members. Fas ligand (Fas L), another member of the TNF-SF, has also been shown to be a potent trigger for cell death during structural luteolysis [6, 8–10]. Fas antigen (Fas) is a cell surface receptor that triggers apoptosis in sensitive cells when bound to Fas L or agonistic anti-Fas antibody (Fas mAb) [11, 12]. We recently demonstrated that Fas mRNA is expressed in the bovine CL throughout the estrous cycle [6]. However, Fas L alone did not induce the death of cultured bovine luteal cells [6]. Therefore, it is possible that bovine luteal cells are refractory to Fas-mediated apoptosis. In mouse luteal cells, although Fas mAb alone also did not induce apoptosis, it was cytotoxic when the cells were simultaneously treated with an inhibitor of protein synthesis, cycloheximide [9]. In addition, the onset of apoptosis in the bovine CL is not observed until progesterone (P4) production has declined [2, 13]. Thus, some survival substances, including P4, within the CL may prevent Fas-mediated apoptosis via the synthesis of intracellular protein inhibitors of the Fas pathway. Moreover, it is not known whether the decline in P4 production and its autoparacrine actions within the bovine CL are sufficient to initiate expression of Fas receptors on the surface of luteal cells.
In addition to being controlled by Fas, apoptosis is controlled by the expression of a number of regulatory genes, for example bcl-2 and bax, which belong to the bcl-2 gene/ protein family [14, 15] and caspases [16]. Bcl-2 is known to protect cells from apoptosis, whereas an increased level of Bax expression accelerates the cell death [14, 15, 17]. It has been shown that bcl-2 mRNA levels in the human CL during the menstrual cycle are highest in the midluteal phase and lowest in the regressing CL, whereas bax mRNA levels are highest in the regressing CL [17]. Moreover, Rueda et al. [3] showed that the increase of apoptosis in the regressed bovine CL was associated with a significant increase of bax mRNA expression as compared with that in the functional CL. Caspases, a family of aspartic acid-specific cysteine proteases, are pivotal mediators of apoptosis during regression of the CL [16, 18]. Of the 14 identified caspase family members, caspase-3 is the best-characterized enzyme [18]. Rueda et al. [19] showed that the levels of caspase-3 mRNA were 3-fold higher in the CL at 12 and 24 h after induction of luteolysis by PGF2 treatment in comparison to the levels measured in matched CL from untreated ewes. Thus, some intraluteal regulatory factors, including P4, may be involved in the regulation of bcl-2 family gene expression during the estrous cycle in cattle. Moreover, changes of caspase-3 mRNA expression may coincide with the initiation of luteolysis and with the cessation of P4 production/action in the bovine CL.
The current study was undertaken to determine whether intraluteal P4 is involved in Fas L-mediated luteal cell death in the bovine CL in vitro. Moreover, we studied whether an antagonist of P4 influences gene expression of the bcl-2 family and caspase-3 in the bovine CL.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovaries with CL from Holstein cows were collected at a local abattoir within 10–20 min after exsanguination. The luteal stages were classified as early, mid, late, or regressed by macroscopic observation of the ovary as described previously [20].
Cell Isolation
Only those CLs classified in the midluteal stage (Days 8–12 after ovulation) were collected for the cell culture. The ovaries with CL were submerged in ice-cold physiological saline and transported to the laboratory. Dissociation of the luteal tissue and culture of luteal cells were performed as described previously [21, 22]. Briefly, CL were perfused for 15 min with EGTA-buffer (0.1 mM EGTA [E-4378; Sigma-Aldrich, Inc., St. Louis, MO], 10 mM Hepes [H-9136; Sigma], 140 mM NaCl, 7.1 mM KCl; pH 7.4) to remove vascular blood and to loosen the connections between the vascular endothelial cells. CL were then perfused for 15 min with wash buffer (10 mM Hepes, 140 mM NaCl, 7.1 mM KCl, 5.0 mM CaCl2; pH 7.4). These perfusion buffers were bubbled with 5% CO2 in 95% O2 during perfusion. The dissociation of the cells was achieved by perfusing the tissue for 30 min with wash buffer containing 0.05% (w/v) collagenase (C-0130; Sigma) and 0.1% (w/v) BSA (735078; Roche Diagnostics GmbH, Mannheim, Germany). The cells were dispersed from the CL matrix with steel combs. Finally, the dissociated luteal cells were pooled and stirred for 30 min in Dulbecco Modified Eagle medium (DMEM; D-1152; Sigma) containing 0.05% collagenase, 0.005% DNase I (D-5025; Sigma), and 0.1% BSA in a water bath at 37°C. After stirring, cells were filtered through metal wire meshes (150 µm and then 80 µm) to remove undissociated tissue fragments. The filtrate was washed three times by centrifugation for 5 min at 50 x g with DMEM, supplemented with 60 µg/ml penicillin, 100 µg/ml streptomycin, and 0.1% BSA. After the three washes, the cells were resuspended in a culture medium, DMEM and Ham F-12 medium (D/F medium; 1:1 [v/v], D-8900; Sigma) containing 5% calf serum (CS; C-6278; Sigma) and 20 µg/ml gentamicin (15750-060; Invitrogen Co., Carlsbad, CA). Cell viability was higher than 85% as assessed by trypan blue exclusion. The cell suspension contained only about 5% endothelial cells or fibrocytes and no erythrocytes, and it consisted of about 25% large luteal cells and 70% small luteal cells [21, 22].
Experiment 1. Effect of a Specific P4 Antagonist on Fas L-mediated Killing of Bovine Luteal Cells
Dispersed luteal cells (2.0 x 105/ml) were cultured in 100 µl of basal medium (BM; D/F) containing 5% CS in 96-well culture dishes (3860-096; Iwaki, Chiba, Japan). After 18 h of culture, the medium was replaced with D/F medium containing 0.1% BSA, 5 ng/ml sodium selenite, and 5 µg/ml transferrin (BM-BSA). The cells were then exposed to a highly specific P4 antagonist (onapristone [OP], ZK98.299; Schering AG, Berlin, Germany; 10–4 M) for 24 h. The dose of OP, dissolving vehicle (DMSO), and manner of cell stimulation were chosen and tested in our previous study [22]. After 24 h of culture, the medium was replaced with the same medium. The cells were then exposed to OP in the presence or absence of 100 ng/ml soluble recombinant human Fas L (05-351; Upstate Biotechnology, Lake Placid, NY) or were stimulated with human recombinant TNF (Dainippon Pharmaceutical Co., Ltd., Osaka, Japan; 50 ng/ml) together with recombinant bovine IFN (kindly donated by Dr. S. Inumaru, NIAH, Ibaraki, Japan; 50 ng/ml) as a control for 24 h [4–6]. The viability of the cells was determined using a Dojindo Cell Counting Kit including WST-1 (345-06463, Dojindo, Kumamoto, Japan) as described previously [6]. WST-1, a derivative of MTT (3-[4, 5-dimethyl-2 thiazolyl]-2,5-diphenyl– 2H-tetrazolium/Br), is a yellow tetrazolium salt that is reduced to formazan by live cells containing active mitochondria. For the viability assay, the culture medium was replaced with 100 µl of D/F medium without phenol red, and 10 µl of assay solution (0.3% WST-1, 0.2 mM 1-methoxy PMS in PBS, pH 7.4) was added to each well. The cells were then incubated for 4 h at 37°C. The absorbance (A) was read at 450 nm using a microplate reader (Model 450; BIO-RAD, Hercules, CA). The cell viability was determined by dividing the mean A of OP- and/or Fas L-treated wells (Atest) by the mean A of nontreated wells (Acontrol). The mean A of wells in the absence of the cells was subtracted from the mean A of all experimental wells. The cell viability (%) was calculated as follows:
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Experiment 2. Effect of a Specific P4 Antagonist on the Fas L-mediated Apoptosis in Bovine Luteal Cells (TUNEL Assay)
Dispersed luteal cells were seeded at 5.0 x 104 viable cells/1 ml on glass slides in 6-well cluster dishes (MS-80060; Sumitomo Bakelite, Tokyo, Japan). After 18 h of culture in BM containing 5% CS, the medium was replaced with fresh BM-BSA. The cells were then exposed to a specific P4 antagonist (OP, 10–4 M) for 24 h. After 24 h of culture, the medium was replaced with the same medium, and the cells were then exposed to OP in the presence of Fas L (100 ng/ml) for 24 h. After the final 24 h of culture, the cells were washed twice with 1 ml of phosphate buffered saline (PBS, 05193; Seikagaku Corporation, Tokyo, Japan). The cells were fixed for 1 h at room temperature in PBS containing 4% paraformaldehyde, followed by two washes in PBS before permeabilization with 0.5% Triton X-100 (BIO-RAD) in PBS for 20 min. The cells were then briefly washed twice in PBS. The cells were incubated in 30 µl of fluorescein-conjugated dUTP and TdT (TUNEL reagents, 8445; MBL, Nagoya, Japan) for 1 h at 37°C in a dark, moist chamber. Following TUNEL, the cells were washed twice in PBS and once in PBS containing 0.0002% propidium iodide (PI, P-4170; Sigma). Then the cells were washed three times in PBS and stored in the dark at 4°C. Cells were observed under fluorescent illumination using a 470-nm excitation filter and a 515-nm absorption filter for FITC, and a 545-nm excitation filter and a 610-nm absorption filter for PI. The number of nuclei (=cell number) was at first counted by PI staining, and then the number of cells with fragmented DNA was counted by TUNEL. A proportion of DNA fragmented cells from 500 cells (nuclei) was analyzed.
Experiment 3. Effect of a Specific P4 Antagonist on Fas, bcl-2 Family, and Caspase-3 mRNA Expression in Cultured Luteal Cells
Dispersed luteal cells were seeded at 2.0 x 105 viable cells in 1 ml of medium in 24-well culture dishes (3524; Costar, Cambridge, MA). After 18 h of culture in BM containing 5% CS, the medium was replaced with BM containing 0.1% BSA with or without OP (10–4 M), or replaced with 5% CS-supplemented medium (BM-CS). After 24 h of culture, the cells were disrupted with TRIZOL reagent (15596; Invitrogen) and frozen at –80°C until they were processed for RNA isolation and RT-PCR.
Experiment 4. Effect of a Specific P4 Antagonist and Fas L on P4 Secretion and Caspase-3 Activity in Cultured Luteal Cells
Dispersed luteal cells were seeded at 2.0 x 105 viable cells/ml in culture medium in 24-well culture dishes (Costar). After 18 h of culture, the medium was replaced with D/F medium containing 0.1% BSA, 5 ng/ ml sodium selenite, and 5 µg/ml transferrin. The cells were then exposed to OP in the presence or absence of 100 ng/ml soluble recombinant human Fas L (05-351; Upstate) or were stimulated with human recombinant TNF (Dainippon; 50 ng/ml) in combination with recombinant bovine IFN (Dr. S Inumara; 50 ng/ml) as a control for 24 h [4–6]. After 24 h of culture, culture media were collected and stored at –30°C until P4 could be determined. Then, the cells were washed 3 times in PBS, and caspase-3 activity was measured using a commercially available caspase-3 calorimetric assay kit (CASP3; Sigma) according to the instructions of the manufacturer (www.sigmaaldrich.com/sigma/bulletin/casp3cbul.pdf).
Semiquantitative RT-PCR
Total RNA was prepared from the cultured luteal cells using TRIZOL reagent according to the manufacturer's directions. One microgram of each sample of total RNA was reverse transcribed using a SuperScript First-Strand Synthesis System for RT-PCR (11904-018; Invitrogen), and the reaction mixture was used in each PCR together with appropriate oligonucleotide primer pairs. The PCR amplification was calibrated to determine the optimal number of cycles that would allow detection of the appropriate mRNA transcripts while still keeping amplification of these genes in the log phase. Semiquantitative RT-PCR was carried out using the housekeeping gene G3PDH as an internal standard. G3PDH primer was added at the appropriate cycle number by the "primer-dropping method" as described by Wong et al. [23] with our own modification [24]. The primers for G3PDH, which were designed as described by Tsai et al. [25], were 5'-TGT TCC AGT ATG ATT CCA CCC-3' (5' primer, 21 mer) and 5'-TCC ACC ACC CTG TTG CTG TA-3' (3' primer, 20 mer). These primers generated a specific 850-base pair (bp) product. The primers for bcl-2, which were designed as described by Long et al. [26], were 5'-GAT GAC TTC TCT CGG CGC TAC-3' (5' primer, 21 mer) and 5'-AGT GCC TTC AGA GAC AGC CAG-3' (3' primer, 21 mer). These primers generated a specific 356-bp product. The sequences of the bax primers, which were based on a report by Rueda et al. [3], were 5'-GGT TTC ATC CAG GAT CGA GC-3' (5' primer, 20 mer) and 5'-ACA AAG ATG GTC ACT GTC TGC C-3' (3' primer, 22 mer). These primers generated a specific 446-bp product. The sequences of the Fas primers, which were based on a report by Vickers et al. [27], were 5'-ATG GGC TAG AAG TGG AAC AAA AC-3' (5' primer, 23 mer) and 5'-CAG GAG GGC CCA TAA ACT GTT TGC-3' (3' primer, 24 mer). These primers generated a specific 206-bp product. The PCRs were carried out using TaKaRa Taq (R001A; TaKaRa; Takara Bio Inc., Ohtsu, Shiga, Japan) and a thermal cycler (iCycler; Bio-Rad). The conditions for the PCRs were as follows: 30 (bcl-2), 27 (bax and Fas), or 18 (G3PDH) cycles of reactions including denaturation for 30 sec at 95°C, annealing for 1 min at 60°C, and extension for 1 min at 70°C were performed, followed by an additional extension for 5 min at 72°C. The primers for caspase-3 were based on the ovine caspase-3 (AF068837) sequence [19]. The primers were chosen using the Primer3 online software (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) and were as follows: caspase-3 forward 5'-AGC AAA CCT CAG GGA AAC CT-3' and reverse 5'-GGC AGG CCT GAA TAA TGA AA-3' (278 bp). The conditions for the PCRs were as follows: 27 (caspase-3) or 18 (G3PDH) cycles of reactions including denaturation for 30 sec at 95°C, annealing for 1 min at 60°C, and extension for 1 min at 70°C were performed, followed by an additional extension for 5 min at 72°C.
Aliquots of PCR reaction products were electrophoresed on a 1.5% agarose gel containing ethidium bromide with a known standard (100 bp Ladder; N3231S; New England BioLabs Inc., Beverly, MA) and photographed under ultraviolet illumination. The band intensities were analyzed by computerized densitometry using the National Institutes of Health (NIH; Bethesday, MD) image software. This method allowed only relative quantification. The amplified cDNA fragments were sequenced directly after being subcloned into pGEM3Zf (+). The nucleotide sequence was determined by the dideoxy chain termination method with an ABI310 sequencer (ABI310; Applied Biosystems, Foster City, CA). Sequence analysis was carried out using GENETYX software and the Blast program (available at: http://www.ncbi.nlm.nih.gov/).
Progesterone Determination
Progesterone concentrations in the culture medium were assayed using a direct enzyme immunoassay (EIA). P4 labeled by horseradish peroxidase (P4-HRP) was used as a tracer. Cross-reactivities of the anti-P4 serum (donated by Dr. S. Okrasa, University of Warmia and Mazury in Olsztyn, Poland) were determined by comparing the inhibition of binding of P4-HRP to antiserum. Results were as follows: 100% with P4; 38.9% with pregnenolone; 11.1% with 17-hydroxy-progesterone; 9.8% with 17ß-estradiol; 1.2% with dihydrotestosterone and testosterone; and less than 0.5% with 11-deoxycortisol, estrone, cortisol, and 4-androsten-3,17-dione. In brief, 20-ml aliquots of standards or medium samples (1:200 dilution factor) were incubated in the dark at room temperature for 18–24 h with 100 µl P4 antiserum (1:100 000 final dilution) and with 100 µl P4-HRP (1: 150 000 final dilution) in duplicates in 96-well ELISA plates (Corning Inc., Corning, NY) coated with ovine anti-rabbit secondary antibody. After discarding reagents, the plates were washed three times with 300 µl of Tween 80 (0.05%), and 150 µl substrate buffer with 3,3' 5,5' tetramethylbenzidine (Sigma) was added to each well. The plates were further incubated at 36°C for 40 min in the dark. The reaction was stopped by addition of 50 µl of 2 M H2SO4 to each well. The absorbance was measured at 450 nm with a plate reader (LABSYSTEM). The P4 standard curve ranged from 0.008 to 2 ng per well, and the effective dose of 50% inhibition (ED 50) of the assay was 0.09 ng. The intraassay coefficients of variation (CV) were averaged 5.3% and 7.9%, respectively.
Statistical Analysis
All experimental data are shown as the mean ± SEM. For the statistical analysis of differences in the viability of the cells, the percentages relative to the control were used. Statistical significance of differences of the viability of the cells, P4 concentrations, and caspase-3 activity between control and treated groups were analyzed using one-way analysis of variance followed by Bonferroni multiple comparison test (ANOVA; GraphPAD PRISM version 4.00, GraphPad Software, San Diego, CA). Statistical significance of differences in Fas, bcl-2, bax, and caspase-3 mRNA expression between control and OP-treated cells was assessed by Student t-test (GraphPAD PRISM).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Treatment of cells with OP alone or OP in combination with Fas L killed 30% and 45% of the cells (P < 0.05; Fig. 1), respectively, whereas Fas L alone did not show a cytotoxic effect on the cells (P > 0.05). As expected, TNF/ IFN treatment significantly reduced the viability of the cultured cells (P < 0.001; Fig. 1).
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Experiment 2. Effect of a Specific P4 Antagonist (OP) on Fas L-mediated Apoptosis in Bovine Luteal Cells
As shown by staining with PI, the cells treated with Fas L in the presence of OP underwent a change of morphology, and we found several cells with already condensed chromatin in their nuclei (Fig. 2; asterisks). TUNEL staining indicated that DNA fragmentation occurred in the cells treated with Fas L in the presence of OP in BM-BSA (Fig. 2d; arrows). The percentages of the control cells and the cells treated with OP in combination with Fas L that had fragmented DNA were 3.1% ± 0.2% and 37.1% ± 12.1% (mean ± SEM, n = 4), respectively.
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Experiment 3. Effect of a Specific P4 Antagonist (OP) on the Fas, bcl-2 Family, and Caspase-3 mRNA Expression in Cultured Bovine Luteal Cells (Semiquantitative RT-PCR)
Representative samples of the Fas- and G3PDH-specific RT-PCR products (206 and 850 bp) are shown in the upper panel of Fig. 3. The relative signal intensities for PCR products specific for Fas were assessed after correction based on the G3PDH signal intensities (lower panel of Fig. 3). The expression of Fas mRNA increased significantly in the cells treated with an antagonist of P4 (OP; P < 0.01).
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The relative signal intensities for PCR products specific for bcl-2 and bax were assessed after correction based on the G3PDH signal intensities (data not shown). OP did not affect the expression of bcl-2 (P > 0.05) or bax mRNA (P > 0.05). Moreover, the ratio of bcl-2 to bax mRNA was not changed in the OP-treated compared with the control cells (P > 0.05).
Representative samples of the caspase-3–and G3PDH-specific RT-PCR products (278 and 850 bp, respectively) are shown in the upper panel of Fig. 4. The relative signal intensities of PCR products specific for caspase-3 were assessed after correction based on the G3PDH signal intensities (lower panel of Fig. 4). The expression of caspase-3 mRNA was significantly increased in the cells treated with an antagonist of P4 (OP; P < 0.01).
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Experiment 4. Effect of a Specific P4 Antagonist (OP) and Fas L on P4 Synthesis and Caspase-3 Activity in Cultured Luteal Cells
The concentration of the P4 in the medium after 24-h culture was lower in the cells treated with OP (65% of the control) with OP together with Fas L (40% of the control) and in the cells treated with TNF together with IFN (62% of the control) compared with that in the medium of control cells (P < 0.05; data not shown). The data on P4 secretion and caspase-3 activity (Fig. 5) were corrected for viable cells (as shown on Fig. 1). Fas L alone did not affect P4 secretion in viable cultured luteal cells (P > 0.05; Fig. 5a). When Fas L and OP were added concomitantly, OP treatment revealed the inhibitory effect of Fas L on P4 secretion (66.8% of the control, P < 0.05; Fig. 5a).
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TNF/IFN increased caspase-3 activity (511.2% of the control, P < 0.001) in viable cultured luteal cells after 24 h of incubation, confirming the reactivity of the cells (Fig. 5b). Treatment with Fas L or OP alone increased caspase-3 activity (164.9% and 248.7% of the control, respectively; P < 0.05). Treatment with both Fas L and OP simultaneously increased caspase-3 activity by 412.4% (P < 0.001; Fig. 5b).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has recently been shown that Fas mRNA was expressed throughout the estrous cycle in bovine CL tissue, with the highest level in the regressed bovine CL [6]. Moreover, although cultured bovine luteal cells expressed Fas mRNA, Fas L alone did not induce cell death [6]. In the present study, bovine luteal cells became sensitive to Fas L–induced cell death in response to the inhibition of intraluteal P4 action using a highly specific P4 antagonist (OP). In addition, considerable DNA fragmentation was observed in the cells treated with Fas L in the presence of OP, as shown by TUNEL assays. Taken together, these findings strongly suggest that intraluteal P4 blocks the Fas-mediated apoptosis pathway in bovine luteal cells. Three major intracellular apoptosis signaling cascades have been characterized, namely the death receptors, the mitochondrial pathway including Bcl-2 protein, and the caspases cascade. Therefore, we studied whether intraluteal P4 is involved in the regulation of Fas receptor or bcl-2 family gene expression changes of caspase-3 mRNA expression and activity.
T lymphocytes, which are known to be a major source of Fas L, were observed not only at the time of luteolysis but also at early stages of the estrous cycle in bovine CL [34]. Therefore, we were interested whether intraluteal P4 reduces Fas-mRNA expression in the functional CL, resulting in blockage of Fas L–induced apoptosis during the luteal phase. Since P4 down-regulated Fas mRNA expression in rat luteal cells [35], we expected that the blockage of intraluteal P4 action could induce Fas mRNA expression. In fact, OP treatment significantly increased Fas mRNA expression, suggesting that the antiapoptotic action of P4 in the bovine CL involves mechanisms including the regulation of Fas protein expression on the surface of bovine luteal cells.
We hypothesized that the antiapoptotic action of P4 includes modification of the expression of apoptosis-specific modulating proteins (e.g., proteins of the bcl-2 family). It has been reported that the ratio of Bcl-2 to Bax expression is the critical determinant of cell fate, such that elevated Bcl-2 favors extended survival of cells, whereas increasing levels of Bax expression accelerate cell death [14, 15, 17]. Thus, we expected that the blockage of intraluteal P4 action using OP would reduce bcl-2 mRNA expression and induce bax mRNA expression in the bovine luteal cells. However, no changes of bcl-2 and bax mRNA expression were observed upon treating bovine luteal cells with OP. Consequently, the ratio of bcl-2 to bax mRNA was not altered in the cells treated with OP either. Thus, the antiapoptotic mechanisms during maintenance of bovine CL, including the inhibition of apoptosis-promoting genes/proteins (e.g., Bax), are regulated via P4-independent pathways in vitro. Recently, we found that some intraluteal growth factors could directly modulate the expression of bcl-2 family proteins and inhibit apoptosis of CL cells during the luteal phase (Skarzynski et al., unpublished data). The implications of this finding are now under further examination.
It has been shown that induction of caspase-3 is a step of PGF2-mediated luteolysis in the ewe [19]. Interestingly, those authors noted that a marked increase in the levels of caspase-3 mRNA in luteal tissue from PGF2-treated ewes was associated with a decrease in circulating levels of P4 and the appearance of internucleosomal DNA cleavage in the CL [19]. Moreover, there were no differences in the levels of caspase-3 mRNA in fully functional CL between Days 12 and 14 of the estrous cycle and pregnancy [19]. These data suggest that the cessation in P4 production is one of the important stimuli increasing the expression/activity of caspase-3 in the CL. In fact, the inhibition of auto/ paracrine P4 action (OP-treatment) significantly increased caspase-3 mRNA expression and activity in the cultured bovine CL cells in the present study. This provides the first reported indication that P4 may act on caspase-3 mRNA expression and activity. Moreover, inhibition of P4 action in the cells (OP treatment) augmented Fas L stimulatory effect on caspase-3 activity and concomitantly inhibited P4 secretion by the live cells in the culture. These findings support the general conclusion that the inhibition of intraluteal P4 action by a specific antagonist (OP) amplifies Fas L–mediated apoptosis. On the other hand, other cytokines (TNF and IFN) without additional support from OP action also strongly induced apoptosis of the bovine luteal cells and strongly stimulated activity of caspase-3. TNF given together with IFN killed over 50% of the cells in the culture. This treatment also inhibited strongly the accumulation of P4 in the medium. However, when the P4 secretion was counted on the viable cells, the inhibitory effect of cytokines on P4 accumulation was covered. Thus, it may be suggested that such inhibition of total accumulation/concentration of P4 may also amplify apoptosis induced by other cytokines.
The mechanisms or signaling pathways by which P4 exerts its action on the bovine CL are unknown. Although a synthetic antagonist of P4 (onapristone) has been shown to be specific for intracellular P4 receptors [28], P4 seems to act on the secretory function of bovine CL [36] and endometrium nongenomically [37] (i.e., through membrane binding sites [32], rather than through nuclear mediation). In support of this idea, Cannon et al. [38] has recently shown that P4 suppresses luteal cells–stimulated T lymphocyte proliferation. However, classical–genomic P4 receptors were not expressed in T lymphocytes, showing that P4 may act nongenomically on the cells [38]. On the other hand, it has been also suggested that P4 plays an active role in the inhibition of luteal regression by a direct effect on the classical P4 genomic binding sites in bovine luteal cells [29]. Although classical genomic P4 receptors are present in luteal cells during the midluteal phase, not all steroidogenic luteal cells express P4 receptors [29]. Therefore, it could be proposed that the increase of cell death by the treatment with a specific P4 antagonist in the present study is limited to those bovine luteal cells that express P4 receptors. Moreover, Rueda et al. [29] suggested that P4 promotes survival of the bovine luteal cells by inhibiting apoptosis via the glucocorticoid receptors (GR). In support of this idea, it has been previously shown that, despite the absence of the P4 receptors in the rat CL, P4 can act through the GR to down-regulate the expression of 20-HSD, an enzyme that catabolizes P4 and reduces P4 secretion by the CL [39]. Thus, further studies are needed to clarify the receptor and intracellular mechanisms of the P4 autocrine and/or paracrine action on steroidogenic bovine CL cells.
In conclusion, the overall results of the present study showed that the inhibition of intraluteal P4 action by a specific antagonist (OP) amplifies Fas L–mediated apoptosis via the increase of Fas and caspase-3 expression and caspase-3 activity in cultured bovine luteal cells. Thus, it could be assumed that intraluteal P4 is implicated in a survival pathway in the CL by the inhibition of Fas and caspase-3 mRNA expression without changing the gene expression of bcl-2 and bax, and consequently prevents luteolysis in cattle.
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ACKNOWLEDGMENTS |
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(HF-13). |
FOOTNOTES |
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2 Correspondence: FAX: +81 86 251 8388; kokuda{at}cc.okayama-u.ac.jp
Received: 31 January 2004.
First decision: 23 February 2004.
Accepted: 12 August 2004.
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J. Komiyama, R. Nishimura, H.-Y. Lee, R. Sakumoto, M. Tetsuka, T. J. Acosta, D. J. Skarzynski, and K. Okuda Cortisol Is a Suppressor of Apoptosis in Bovine Corpus Luteum Biol Reprod, May 1, 2008; 78(5): 888 - 895. [Abstract] [Full Text] [PDF]
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R. Nishimura, J. Komiyama, Y. Tasaki, T. J. Acosta, and K. Okuda Hypoxia Promotes Luteal Cell Death in Bovine Corpus Luteum Biol Reprod, March 1, 2008; 78(3): 529 - 536. [Abstract] [Full Text] [PDF]
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 X. Hou, E. W. Arvisais, C. Jiang, D.-b. Chen, S. K. Roy, J. L. Pate, T. R. Hansen, B. R. Rueda, and J. S. Davis Prostaglandin F2{alpha} Stimulates the Expression and Secretion of Transforming Growth Factor B1 Via Induction of the Early Growth Response 1 Gene (EGR1) in the Bovine Corpus Luteum Mol. Endocrinol., February 1, 2008; 22(2): 403 - 414. [Abstract] [Full Text] [PDF]
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 M. C. Peluffo, R. L. Stouffer, and M. Tesone Activity and expression of different members of the caspase family in the rat corpus luteum during pregnancy and postpartum Am J Physiol Endocrinol Metab, November 1, 2007; 293(5): E1215 - E1223. [Abstract] [Full Text] [PDF]
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 M. C Peluffo, L. Bussmann, R. L Stouffer, and M. Tesone Expression of caspase-2, -3, -8 and -9 proteins and enzyme activity in the corpus luteum of the rat at different stages during the natural estrous cycle. Reproduction, September 1, 2006; 132(3): 465 - 475. [Abstract] [Full Text] [PDF]
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 R. Nishimura, R. Sakumoto, Y. Tatsukawa, T. J. Acosta, and K. Okuda Oxygen Concentration Is an Important Factor for Modulating Progesterone Synthesis in Bovine Corpus Luteum Endocrinology, September 1, 2006; 147(9): 4273 - 4280. [Abstract] [Full Text] [PDF]
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J. J. Peluso Multiplicity of Progesterone's Actions and Receptors in the Mammalian Ovary Biol Reprod, July 1, 2006; 75(1): 2 - 8. [Abstract] [Full Text] [PDF]
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 K. A Slot, M. Voorendt, M. de Boer-Brouwer, H. H van Vugt, and K. J Teerds Estrous cycle dependent changes in expression and distribution of Fas, Fas ligand, Bcl-2, Bax, and pro- and active caspase-3 in the rat ovary J. Endocrinol., February 1, 2006; 188(2): 179 - 192. [Abstract] [Full Text] [PDF]
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 M. C. Peluffo, K. A. Young, and R. L. Stouffer Dynamic Expression of Caspase-2, -3, -8, and -9 Proteins and Enzyme Activity, But Not Messenger Ribonucleic Acid, in the Monkey Corpus Luteum during the Menstrual Cycle J. Clin. Endocrinol. Metab., April 1, 2005; 90(4): 2327 - 2335. [Abstract] [Full Text] [PDF]
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