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a Department of Anatomy and Human Biology, The University of Western Australia, Nedlands, Perth, Western Australia, Australia
b School of Biomedical and Sports Science, Edith Cowan University, Joondalup, Western Australia, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One of the physiological processes in which apoptosis has been implicated is regression of the corpus luteum (CL) [7], an organ within the ovary that differentiates from the ruptured follicle after ovulation. The main function of the CL is secretion of progesterone to maintain the lining of the uterus for implantation and support of a fertilized ovum [8]. Regression of the CL occurs at the end of each ovarian cycle or when it is no longer required for the maintenance of pregnancy [8]. Functional regression of the CL, evidenced by a significant decrease in progesterone secretion, begins 4 days prior to parturition in the rat [9–11]. Although previous studies have shown that the number of luteal cells in the rat CL remains constant [12, 13], recent studies have indicated that some cell death is occurring at Day 20 of pregnancy [14]. Structural regression of luteal cells becomes apparent at Day 1 postpartum, and the weight of the CL of pregnancy continues to decline over numerous estrous cycles following parturition [15]. Little is known about the mechanisms involved in structural regression; however, apoptotic cell death has been found to occur during luteolysis in cattle [16], pseudopregnant and pregnant rabbits [17, 18], marmoset monkeys [19], and humans [20]. Apoptosis can be induced in CL of pregnant rats by treatment with a gonadotropin-releasing hormone agonist [21], and DNA fragmentation increases in luteal cells of rat CL after parturition [14]. Also, apoptotic cell death has been identified in CL of cycling rats treated with prolactin and during proestrus of the normal rat cycle [22].
Apoptosis may be triggered by a range of stimuli, and many molecular pathways have been identified that facilitate apoptotic cell death; thus it is necessary to identify the mediator(s) of apoptosis in regressing luteal cells. One possibility is the Fas (or APO-1 or CD95) receptor, a transmembrane protein that induces apoptosis in the cell when bound to its ligand. Although the mechanisms by which Fas induces apoptosis are not well characterized, cysteine proteases, or caspases, have been identified as downstream messengers in this process [23].
Research on Fas and Fas ligand (FasL) has been primarily concerned with the immune system [24–27]; however, roles within the reproductive system are also indicated. Expression of mRNA for the Fas receptor has been detected in the mouse ovary [24], and Fas appears to be involved in the regression of vaginal epithelium following ovariectomy and during the estrous cycle in the mouse [28]. Fas receptor mRNA is also expressed by human granulosa and luteal cells, and a Fas monoclonal antibody (mAb) can induce apoptosis in these cells when they are pretreated with interferon gamma in culture [29]. In addition, abundant expression of the Fas receptor in the regressing CL of the normal adult human ovary has been reported [30].
These findings indicate that Fas-mediated apoptosis may have a role in the mechanisms of regression of the CL. The present study was an examination of the expression of Fas receptor and FasL in the rat CL during pregnancy and postpartum. An in vitro CL culture model, in which CL undergo spontaneous apoptosis when cultured in serum-free medium [17, 21], was used to determine the effect of treatment with an anti-rat Fas mAb.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The species used were sexually mature (10–12 wk old) female Wistar rats. They were housed at 21°C with 55% humidity in a 12L:12D cycle. Their feed was autoclaved normal cubes and acidified water, both provided ad libidum. Rats were mated overnight. Day 1 of gestation was designated the morning on which spermatozoa were found in a vaginal smear. Litters were born on Day 23 of pregnancy. All protocols were reviewed and approved by the University of Western Australia Animal Ethics Committee.
Immunohistochemistry
Localization of Fas receptor and FasL within the rat CL during pregnancy and postpartum was established using immunohistochemistry. Four rats from each stage of pregnancy (Days 1, 8, 16, and 22 of gestation) and postpartum (Days 1 and 3) were used. One ovary from each rat was used for this study. Portions of spleen were excised from rats for use as positive controls. After excision, the ovaries and portions of spleen were fixed in 4% paraformaldehyde at 4°C for 24 h. Tissue was dehydrated and embedded in paraffin wax. Sections of 5 µm were cut.
Paraffin wax was removed from sections using toluene (BDH, Kilsyth, Victoria, Australia); then slides were rehydrated through alcohol solutions of decreasing concentrations. Sections were incubated in 3% hydrogen peroxide in methanol for 10 min. After this and each subsequent step in the immunohistochemistry protocol, sections were washed in fresh PBS (137 mM NaCl, 2.7 mM KCl, 13 mM NaH2PO4, 1.5 mM KH2PO4, 1 mM MgCl2, and 0.9 mM CaCl2, pH 7.4) for 5 min three times.
For Fas receptor staining, sections were first incubated in 1% (w:v) SDS for 5 min and then incubated with primary antibody (mouse anti-rat Fas monoclonal IgG, 250 µg/ml; Transduction Laboratories, Lexington, KY), diluted 1:100, for 2 h. The secondary antibody (sheep anti-mouse immunoglobulins, 0.5 mg/ml; Amersham Corporation, Arlington Heights, IL), which was diluted 1:100, was incubated on sections for 45 min.
For FasL staining, sections were incubated with the primary antibody (goat anti-rat FasL polyclonal IgG, 200 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA), diluted 1:50, for 2 h. Sections were incubated with the secondary antibody (biotinylated rabbit anti-goat IgG, 1.5 mg/ml; Vector Laboratories, Burlingame, CA) diluted 1:50 for 45 min. Streptavidin-horseradish peroxidase conjugate (Amersham Corporation) was diluted 1:50 and incubated on sections for 45 min.
All sections were then incubated for 10 min with 3,3'-diaminobenzidine tetrahydrochloride (DAB; 1.2 mg/ml) and hydrogen peroxide (5 µg/ml) solution. Sections were counterstained by being placed in hematoxylin for 6 sec, then dehydrated through alcohol solutions of increasing concentrations and toluene. Slides were coverslipped using DPX mountant (Merck Pty. Ltd., Kilsyth, Victoria, Australia), then viewed by light microscopy.
Negative controls for each section were treated in the same manner except that they received diluent only in place of primary antibody. Sections of rat spleen were placed in each run as a positive control.
Relative Quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Expression of Fas receptor and FasL mRNA in the rat ovary during pregnancy and postpartum was initially confirmed using RT-PCR. A relative quantitative method of RT-PCR was then developed to analyze expression of FasL mRNA at the stages of pregnancy and postpartum examined in the rat ovary. The right ovary from each rat was dissected (Days 1, 8, and 16 of pregnancy, n = 4; Day 22 of pregnancy, n = 5; Day 1 postpartum, n = 8; Day 3 postpartum, n = 4). Immediately after excision, each ovary was snap frozen in liquid nitrogen, then stored at -80°C until further analysis.
The RNAzol B method (Bresatec, Thebarton, SA, Australia) was used to extract total RNA from snap-frozen rat ovaries according to manufacturer's instructions. Briefly, the tissue was homogenized with RNAzol B (2 ml/100 mg tissue). Chloroform was then added (one tenth the volume of the homogenate); then samples were mixed well and left on ice (4°C) for 15 min. The suspension was then centrifuged at 12 000 x g (4°C) for 15 min. The aqueous phase was transferred to a fresh tube, and an equal volume of isopropanol was added to allow precipitation of RNA. Tubes were incubated at 4°C for 1 h. Samples were then centrifuged at 12 000 x g for 15 min. RNA pellets were then washed with 75% ethanol by vortexing and subsequent centrifugation for 8 min at 7500 x g. Pellets were air dried briefly, then dissolved in dimethyldicarbonate-treated RNase-free double-distilled H2O, and stored at -80°C. RNA integrity was later confirmed by running 3 µg RNA on a 1% agarose denaturing gel containing formaldehyde, 3-[N-morpholino]propanesulfonic acid, and ethidium bromide.
Complementary DNA was synthesized from 2 µg RNA using oligo-dT (Promega, Annandale, NSW, Australia) and avian myeloblastosis virus (AMV) reverse transcriptase (Promega). Fas primers (upstream sequence: 5' CTG CAG ATA TGC TGT GGA TCA 3'; downstream sequence: 5' TTT GGT GTT GCT GGT TGG T 3'; synthesized by Bresatec on the basis of rat Fas sequence published by Kimura et al. [31]) and Taq polymerase (Gibco BRL, Gaithersburg, MD) were used to amplify a portion of the cDNA. Reaction mixes were heated to 94°C for 4 min 30 sec. Amplification steps comprised 94°C for 30 sec, 55°C for 45 sec, and 72°C for 1 min repeated 38 times. Samples were incubated at 72°C for 5 min. PCR products were run on a 1.5% agarose gel containing ethidium bromide and viewed under UV light. The expected size of the PCR product was 491 bp.
Complementary DNA was synthesized from 1 µg RNA using 3'-end primers for FasL (sequence: 5' AGT CTC TAG CTT ATC CAT GA 3'; synthesized by Bresatec on the basis of rat FasL sequence published by Suda et al. [32]) and AMV reverse transcriptase (Promega). FasL primers (upstream sequence: 5' AAA GAC CAC AAG GTC CAA CA 3', downstream sequence: 5' AGT CTC TAG CTT ATC CAT GA 3'; synthesized by Bresatec on the basis of rat FasL sequence published by Suda et al. [32]) and PLATINUM Taq polymerase (Gibco BRL) were used to amplify a portion of the cDNA. Reaction mixes were heated to 94°C for 4 min 30 sec. Amplification steps comprised 94°C for 30 sec, 58°C for 1 min, and 72°C for 1 min repeated 35 times. Samples were incubated at 72°C for 5 min. PCR products were run on a 1.5% agarose gel containing ethidium bromide and viewed under UV light. The expected size of the PCR product was 341 bp.
For relative quantitative RT-PCR, cDNA was synthesized from 1 µg RNA using the 3'-end primer for FasL as described above and AMV reverse transcriptase (Promega). All samples were reverse transcribed in one run. FasL primers as described above and PLATINUM Taq polymerase (Gibco BRL) were used to amplify a portion of the cDNA, which was labeled with the addition of [
-32P]dCTP (DuPont, Sydney, NSW, Australia). Reaction mixes were heated to 94°C for 4 min 30 sec. Amplification steps comprised 94°C for 30 sec, 58°C for 1 min, and 72°C for 1 min repeated 32 times. Samples were incubated at 72°C for 5 min. Isopropanol extraction, as previously described [33], was used to remove unincorporated nucleotides from PCR products, which were then quantitated with a beta counter. Initially 4-fold serial dilutions (i.e., neat, 1/4, 1/16, 1/64, 1/128) of one sample from each time point were amplified by PCR and quantitated, and the results were plotted on a logarithmic graph. A dilution in the exponential phase of the PCR reaction was chosen for each time point as previously described [34]. One PCR run with cDNA samples from each time point at the specified dilution was completed and quantitated as described above. Data from different time points were compared after allowing for the dilution factor used in the PCR reaction.
In Vitro CL Culture Model
Four rats from Day 16 of pregnancy were used. Ovaries were excised and new CL dissected from ovarian stroma. Two CL were placed in each sterile culture vial. Vials contained either 2 ml serum-free medium only (Gibco BRL); or 2 ml serum-free medium plus 40 µl antibody storage buffer, consisting of 50% glycerol, 20 mM NaH2PO4, pH 7.5, 1.5 mM NaN3, and 1 mg/ml BSA; or 2 ml serum-free medium plus 40 µl anti-rat Fas mAb, 250 µg/ml (Transduction Laboratories). Four vials were used for CL placed in medium with anti-Fas mAb. Five vials per treatment were used for CL placed in either medium with antibody storage buffer or medium only.
Sterile culture vials were gassed with a 95% O2:5% CO2 mixture and their lids sealed with vacuum grease. Vials were incubated at 37°C in 95% O2:5% CO2 for 4 h. CL were then snap frozen in liquid nitrogen.
Genomic DNA was prepared from CL as originally described by Gross-Bellard et al. [35] and modified by Dharmarajan et al. [17]. After extraction, purification, and quantitation of DNA, samples were labeled on the 3' ends with [-32P]dideoxy-ATP (3000 Ci/mmol; Amersham Australia, Sydney, Australia) using the terminal transferase reaction as described previously [17, 36]. Radiolabeled DNA samples were resolved by electrophoresis through a 2% agarose gel at 50 volts (6.5 volts/cm) for 3–3.5 h. The gel was dried for 2 h without heat in a slab gel drier, sealed in plastic wrap, and exposed to Kodak X-Omat film (Eastman Kodak, Rochester, NY) at -80°C for autoradiographic analysis. After autoradiography, low molecular weight DNA fractions (< 15 kilobases [kb]) were excised from the gel, mixed with 3 ml scintillation fluid, and counted in a beta counter to provide a quantitative estimate of the degree of internucleosomal DNA cleavage among samples.
Statistical Analyses
Statistical differences between the mean values of FasL mRNA expression were analyzed by one-way ANOVA followed by F-test. A representative autoradiograph is presented where appropriate for quantitative analysis, whereas quantitative results obtained from beta-counting of radiolabeled low molecular weight DNA fragments represent mean ± SEM of combined data from replicate samples.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunohistochemical staining indicated that the Fas receptor was present in the rat CL at Day 1 of pregnancy (Fig. 1a). No staining was observed in CL of rats at Days 8 (data not shown) and 16 of pregnancy (Fig. 1b). The stain was most intense in the CL at Day 22 of pregnancy and Day 1 postpartum (Fig. 1, c and d). Staining was present, but less intense, at Day 3 postpartum (Fig. 1e). Within the CL, staining was concentrated in the cytoplasm of luteal cells. Endothelial cells within the CL displayed no positive staining for Fas receptor. Negative control sections showed no nonspecific staining (Fig. 1f). The staining pattern and intensity in the spleen (positive control; Fig. 1g) were consistent between immunohistochemical runs.
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FasL was present in the rat CL at all stages of pregnancy and postpartum examined. At Day 1 of pregnancy FasL was localized in most CL in each rat ovary (Fig. 2a), staining all CL observed by Day 16 of pregnancy (Fig. 2b). Expression of FasL increased in CL at Day 22 of pregnancy (Fig. 2c). Staining of FasL was intense at Day 1 postpartum (Fig. 2d) and had decreased by Day 3 postpartum (Fig. 2e). Endothelial cells within the CL did not stain positively for FasL. Nonspecific staining was not apparent in negative control sections (Fig. 2f). The staining pattern and intensity in the spleen (positive control; Fig. 2g) were consistent between immunohistochemical runs.
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Relative Quantitative RT-PCR
Fas and FasL mRNA were expressed at each of the stages of pregnancy and postpartum examined (Fig. 3, a and b). Relative quantitative RT-PCR indicated that the expression of FasL mRNA in the rat ovary decreased from Day 1 to Day 16 of pregnancy. A significant increase in expression was observed from Day 16 to Day 22 of pregnancy (p < 0.001). Expression of FasL decreased at Day 1 postpartum; however, the results were not significantly different from those at Day 22 of pregnancy. The decrease in expression from Day 22 of pregnancy to Day 3 postpartum was significant (p < 0.05, Fig. 4).
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In Vitro CL Culture Model
Treatment with the antibody storage buffer demonstrated a trend in reduction in occurrence of spontaneous apoptosis in cultured CL. A further reduction in apoptosis was apparent when CL were treated with an anti-rat Fas mAb in culture (Fig. 5, a and b).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When Fas is expressed on the cell surface, it can be bound to FasL and will then transduce an apoptotic signal to the cell. Thus, it is expected that if the Fas/FasL system is involved in cell death occurring during CL regression, the presence of both proteins would correlate with luteolysis. Fas receptor was localized in the rat CL at Day 1 of pregnancy, which may relate to natural regression of CL during the normal estrous cycle of the rat, and at Day 22 of pregnancy and Days 1 and 3 postpartum. FasL was present in the rat CL throughout pregnancy and postpartum, with immunohistochemical staining intensity increasing around the time of parturition. Thus, localization of both proteins within the rat CL during luteolysis was demonstrated.
After formation and growth, rat CL maintain their maximum size through metestrus of the following cycle and then regress slowly, so that three or more generations of CL may be seen in the ovary [37]. Throughout pregnancy, rat CL of previous cycles grow and display characteristics of steroidogenic tissue [38]. This indicates that even following the onset of luteolysis, regression can be halted and the CL may be functional. Our immunohistochemistry results show that Fas receptor is not present in the CL of previous estrous cycles at times of peak function in the CL of pregnancy, suggesting that factor(s) that maintain the newly formed CL in the rat also positively affect the functioning of aged CL.
While progesterone production in the rat declines by Day 22 of pregnancy (functional regression of the CL) [39], the weight of the CL of pregnancy does not fall until after parturition (structural regression of the CL) [15, 38]. The staining intensity for Fas receptor was highest in CL at Day 22 of pregnancy and Day 1 postpartum. Thus, it is not clear at this stage whether the Fas/FasL system is involved in functional regression of the CL, but a role in structural regression is implicated. Studies localizing Fas and FasL at time points between Days 16 and 22 of pregnancy will be conducted to further confirm changes in expression during functional regression. The weight of the CL of pregnancy continues to decline over numerous estrous cycles following parturition [15]. Staining intensity for both Fas receptor and FasL decreased by Day 3 postpartum, although both proteins were still present. Further research will be required to determine whether Fas receptor and FasL are present in the rat CL throughout lactation.
Previous investigators found no evidence of vascular degeneration during natural regression of the pregnant rat CL [38]. In agreement with this report, both Fas and FasL were localized in the cytoplasm of luteal cells at the time of luteolysis, but staining was not observed in endothelial cells at any stage of pregnancy or postpartum.
The presence of FasL in rat CL throughout pregnancy and postpartum indicates that luteal regression may not be its only function. FasL contributes to immune privilege displayed by the Sertoli cells of the mouse testis [40] and by the mouse eye [41]. The ovary has also been identified as an immune-privileged site [42]. Further, as luteal cells are not present at the time that the immune system is adapting to "self" [43], they might be susceptible to immune attack. Thus, maintenance of CL may involve mechanisms of immune privilege. Research using gld mice [44], which express nonfunctional FasL, is required to establish a role for FasL-mediated immune privilege in the ovary.
A significant increase in expression of FasL mRNA was observed from Day 16 to Day 22 of pregnancy, just prior to parturition. This increase is consistent with the immunohistochemical findings and coincides with luteolysis. The decrease in expression of FasL mRNA from Day 22 of pregnancy to Day 3 postpartum was significant. In summary, FasL mRNA expression increases significantly at the time of luteolysis, and regulation appears to follow a pattern similar to that seen in localization of the protein.
The buffer in which the anti-rat Fas mAb was stored reduced the occurrence of spontaneous apoptosis observed in the in vitro CL culture model. A further reduction in spontaneous apoptosis was demonstrated by treatment with the anti-rat Fas mAb, although neither result was significant. Our finding with regard to the action of the anti-rat Fas mAb supplied by Transduction Laboratories differs from that of Foghi et al. [45], who used this product to induce apoptosis in cultured thecal/interstitial cells placed in serum-free medium. Research indicates that cross-linking of Fas receptor on the cell surface is required for transduction of an apoptotic signal and that pentameric IgM anti-Fas mAb or aggregated IgG3 anti-Fas mAb is able to trigger the Fas pathway through receptor cross-linking [46–48]. Monomeric IgG1 mAb may not produce efficient cross-linking of Fas receptor. As stated previously, luteal cells undergo spontaneous apoptosis when placed in culture in serum-free medium [17, 21]. The rationale for placing thecal/interstitial cells in serum-free medium was not stated in the study cited above, nor is it clear whether the serum-free culture medium contributes to apoptosis in this model. Nonetheless, as well as using different ovarian cell types, we used an organ culture model while Foghi et al. [45] used a cell culture model. These factors may have contributed to the conflicting results obtained.
This study suggests that the Fas/FasL system has a role in CL function in the rat. Expression of both proteins increased during natural regression in rat pregnancy, implicating a role in luteolysis. Future research is required in order to further define this role and to relate the function of the Fas/FasL system to the many factors that influence the CL.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 61 8 9380 1051; dharma{at}anhb.uwa.edu.au
Accepted: November 2, 1998.
Received: July 6, 1998.
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