Departments of 1Community and Environmental Medicine, 2Medicine, and 3Developmental and Cell Biology, University of California at Irvine, Irvine, California
Submitted 4 November 2004 ; accepted in final form 26 March 2005
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ABSTRACT |
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glutathione; -glutamylcysteine synthetase; preovulatory gonadotropin surge
The ovary depends on de novo synthesis to maintain intracellular GSH. GSH is synthesized in two sequential, ATP-dependent enzymatic reactions that are catalyzed by glutamate cysteine ligase (GCL, also called -glutamylcysteine synthetase) and glutathione synthetase, respectively. The GCL holoenzyme is composed of a catalytic (GCLc) subunit and a modifier (GCLm) subunit (11, 45). GCLc has all substrate binding sites and possesses the catalytic activity of the GCL enzyme. GCLm does not have any catalytic activity but modulates the affinity of GCLc for substrates and inhibitors. De novo synthesis of GSH can be regulated by transcriptional and posttranscriptional regulation of GCL, by the availability of cysteine, and by negative feedback of GSH on GCL, resulting in decreased enzymatic activity (11, 45).
Although uncontrolled production of ROS can result in oxidative stress and damage to cells, ROS may also function as signaling molecules. Within the rat ovary, levels of superoxide anion increase and enzymatic activity of superoxide dismutase falls on the day of estrus (23). Moreover, administration of exogenous superoxide dismutase blocks ovulation (41). Leukocytes appear to be the major source of superoxide anion within the ovary during the periovulatory period (22), and infusion of leukocytes into perfused rat ovaries enhances the LH-stimulated ovulation rate (14). ROS in the ovary may also arise as byproducts of cellular respiration and of steroid synthesis, as they do in other steroidogenic tissues (15, 36). Adverse effects of ROS have been demonstrated in ovarian cells. Hydrogen peroxide interferes with normal granulosa cell function, decreasing the cAMP and progesterone responses to FSH stimulation (29). ROS may also play a role in follicular apoptosis. Follicles in serum-free culture spontaneously undergo apoptosis within hours (49). This can be prevented by FSH or by antioxidants, including the GSH precursor N-acetylcysteine (49). Gonadotropin administration to prepubertal rats in vivo also inhibits granulosa cell apoptosis (48), upregulates ovarian expression of the antioxidant genes manganese superoxide dismutase and secreted superoxide dismutase (28, 49), and increases ovarian levels of GSH and GCL subunit protein (3, 28). Together, these data suggest that ovarian antioxidants, and particularly GSH, must be tightly regulated to maintain ROS-dependent signaling pathways without allowing oxidative stress to occur.
We (28) previously reported that ovarian GSH concentrations and GCL subunit protein levels increase during the first 24 h of follicular growth induced by pregnant mare's serum gonadotropin (PMSG), before any increase in ovarian GCL mRNA levels. The goals of the present studies were 1) to further delineate the time course of changes in ovarian GCL subunit expression after PMSG, 2) to localize GCL subunit protein and mRNA within the ovary after PMSG administration, 3) to investigate the effects of an ovulatory gonadotropin stimulus on ovarian GCL protein and mRNA expression, and 4) to investigate the effects of PMSG and an ovulatory gonadotropin stimulus on GCL enzymatic activity.
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METHODS |
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Female Sprague-Dawley rats [Crl:CD(SD)IGS BR] were obtained from Charles River Laboratories (Wilmington, MA). Upon arrival, the animals were housed in an American Association for the Accreditation of Laboratory Animal Care-accredited facility, 3 or 4 to a cage, with free access to deionized water and standard laboratory chow, on a 14:10-h light-dark cycle. The experimental protocols were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (33) and were approved by the Institutional Animal Care and Use Committee at the University of California, Irvine.
Experimental Protocols
Effect of PMSG priming on ovarian GCL subunit protein and mRNA expression levels and intraovarian localization. Twenty-five-day-old female rats were injected subcutaneously with 10 IU of PMSG (obtained from Dr. A. F. Parlow, Director, National Institute of Diabetes and Digestive and Kidney Diseases National Hormone and Peptide Program) in sterile 0.9% sodium chloride to stimulate ovarian follicular development or saline alone. PMSG possesses both FSH and LH activity in the rat (25). At 4, 8, 24, and 48 h after injection, animals were euthanized by decapitation for collection of trunk blood, and both ovaries were dissected out. Body weights at the time the animals were killed ranged from 45.5 to 87 g. These time points were chosen because we had previously observed statistically significant increases in whole ovary GCLm protein levels in this time frame (28). In the present study, the combined whole ovary GCL subunit protein, GCL subunit mRNA, and GSH data from the present experiment and the previous study (Ref. 28; with permission of the publisher) are presented together so that the time course of changes in these parameters can best be appreciated. Ovaries were processed for one of several assays as follows. For GSH assay, one ovary was immediately homogenized on ice in 1:6 (wt/vol) 5% sulfosalicylic acid and centrifuged at 15,800 g, and supernatants were stored at 70°C (n = 414 ovaries/group). For protein extraction and Western analysis (n = 69 ovaries/group) or for RNA extraction and Northern analysis (n = 512 ovaries/group), ovaries were immediately frozen on dry ice and stored at 70°C. For immunohistochemistry (n = 6 ovaries/group) or in situ hybridization (n = 6 ovaries/group), the ovary was pre-fixed in 4% paraformaldehyde in PBS for 1 h at 4°C, dehydrated in 15% sucrose in PBS for 34 h at 4°C, embedded in Tissue Tek OCT (Sakura Finetek, Torrance, CA), and stored at 70°C until sectioning at 10-µm thickness using a cryostat. Ovaries were serially sectioned, and every fourth serial section was processed for GCLc immunohistochemistry. Adjacent sections were processed for in situ hybridization for gclc or gclm.
Effect of ovulatory dose of human chorionic gonadotropin on whole ovary GCL protein and mRNA expression and intraovarian localization of GCL mRNA in PMSG-primed rats. Female rats (2324 days old) weighing 41.558 g were injected subcutaneously with 10 IU of PMSG. Forty-eight hours later, some of the rats were killed by carbon dioxide asphyxiation followed by decapitation for collection of trunk blood. The remaining rats were injected subcutaneously with 10 IU of human chorionic gonadotropin (hCG; CG-10, Sigma-Aldrich, St. Louis, MO) in PBS and were killed 4, 6, or 12 h later. Unlike PMSG, hCG possesses exclusively LH activity in the rat (34). Ovaries were processed for GSH assay (n = 511 ovaries/group), protein extraction for Western analysis (n = 510 ovaries/group), RNA extraction for quantification by real-time PCR (n = 38 ovaries/group), or in situ hybridization (n = 23 ovaries/group) as above. gclc and gclm in situ hybridization was performed in three separate experiments, each including two or three slides probed with antisense riboprobe and one slide probed with sense riboprobe for each subunit from each ovary, as well as "positive control" kidney sections.
Effect of endogenous gonadotropin surge on GCL expression in the ovary.
Nine- to ten-week-old adult female rats weighing 202269 g that had displayed at least two 4-day estrous cycles, as determined by daily vaginal cytology, were killed by carbon dioxide anesthesia followed by decapitation for collection of trunk blood 1) at 0900 on proestrus; 2) at 2200 on proestrus evening, 4 h after the preovulatory surge; 3) at 0000 on estrus,
6 h after the surge; and 4) at 0900 on estrus morning,
15 h after the surge (n = 4 at each time point). Carbon dioxide asphyxiation was carried out in the dark, with only safelight illumination for the 2200 and 0000 hour time points to avoid disrupting the animals' light-dark cycles. One ovary was processed for GSH assay, and one ovary was processed for RNA extraction as described above.
Effect of PMSG and hCG on ovarian GCL enzymatic activity. Female rats (2324 days old) weighing 48.5 to 63 g were injected subcutaneously with 10 IU of PMSG at 0800 or 1200 or with saline vehicle at 1200 (n = 8/group). The animals treated with PMSG at 0800 were injected subcutaneously with 10 IU of hCG 48 h after PMSG. All animals were killed by carbon dioxide asphyxiation followed by decapitation for collection of trunk blood at 1200 on the second day after injection (48 h after PMSG or saline or 4 h after hCG). The timing was chosen so that GCL activity assays could be done immediately upon removal of the ovaries. Ovaries were homogenized on ice in 1:6 (wt/vol) TES-SB buffer (20 mM Tris, 1 mM EDTA, 250 mM sucrose, 20 mM boric acid, 2 mM L-serine). An aliquot of homogenate was snap-frozen for subsequent protein assay. The remaining homogenate was immediately used for GCL enzymatic activity assay.
Total GSH and Oxidized GSH Assays
Total GSH and oxidized GSH (GSSG) were measured using the enzymatic recycling method (12). 2-Vinylpyridine (Sigma-Aldrich) was used to remove reduced GSH from aliquots of sample, permitting the measurement of GSSG (12). GSSG assay was carried out on separate aliquots of the supernatants from 5% sulfosalicylic acid tissue homogenates used for the total GSH assay. Supernatants (25 µl) were diluted 1:1 with GSH assay buffer (143 mM Na2HPO4, 6.3 mM EDTA, pH 7.5). Standards of GSSG (Sigma-Aldrich) were also prepared in 1:1 GSH assay buffer-5% sulfosalicylic acid. The resulting 50 µl of sample or standard were mixed with 7 µl of triethanolamine (diluted 1:10 with deionized water) to a pH of 5.57.0. Four microliters of 2-vinylpyridine were added per tube, and the tubes were vortexed for 1 h at room temperature. Sixty microliters of chloroform were added per tube. The tubes were vortexed again for 15 min and were then centrifuged at 15,800 g for 5 min at 4°C. Five microliters of the aqueous phase from samples or standards were pipetted in triplicate into a 96-well microplate. Thirty-three microliters of water were added per well, and the plate was incubated at 30°C for 10 min. Next, a 162-µl reaction mixture containing 0.26 mM NADPH, 0.74 mM 5,5'-dithiobis(2-nitrobenzoic acid), and 0.62 U/ml glutathione reductase (all from Sigma-Aldrich) were added to each well, and the absorbance was monitored at 412 nm every 10 s for 30 min using a VersaMax Tunable Microplate Reader (Molecular Devices, Sunnyvale, CA). The assay protocol for total GSH was identical to that of the GSSG assay from the step at which 5 µl of sample or standard were pipetted in triplicate into a 96-well microplate, except that the absorbance was monitored for only 90 s. The interassay coefficient of variation (CV) for total GSH in a rat liver pool was 12.2%; the intra-assay CVs ranged from 4.0 to 5.3%. For GSSG, the interassay CV was 6.8%, and the intra-assay CVs for the two assays for this experiment were 3.9 and 2.3%. The sensitivity of these assays in our hands is 0.015 nmol.
Northern Analysis
RNA extraction, RNA gel electrophoresis, and Northern Blotting for gclc, gclm, and gapdh were carried out as described (28). Total ovarian RNA was prepared using the TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. Samples of RNA were analyzed by separation in 1% agarose-formaldehyde-containing gels, followed by capillary transfer to nylon membranes and hybridization with 32P-labeled nucleic acid probes. 32P-labeled random-primed probes were prepared using template full-length cDNAs of the mouse gclc, gclm (38, 39), and gapdh (Ambion, Austin, TX) genes with the DECAprime II DNA Labeling Kit (Ambion). Visualization was by autoradiography. Semiquantitative analysis of autoradiographs was performed using a Stratagene molecular documentation and image analysis system with EagleSight software. Ovarian gclc absorbance readings and the mean of the absorbance readings for the two gclm transcripts were normalized to control mRNA (gapdh) and relative differences among treatment groups were calculated.
Fluorogenic 5'-Nuclease mRNA Quantitation (Real-Time PCR)
RNA was extracted using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. Two micrograms of RNA from each sample were incubated for 30 min at 37°C with DNAse I (0.25 U), dithiothreitol (10 mM), and RNAse inhibitor (10 U) and then inactivated at 70°C. Oligo(dT)15 primer (0.13 µg, Invitrogen) was added and the mixture incubated at 70°C for 5 min. Finally, reverse transcription was carried out by adding RT Master Mix containing Superscript II RNase H-Reverse Transcriptase (100 U), dNTPs (1.43 mM), first-strand buffer, and dithiothreitol (14.3 mM; all from Invitrogen) at 45°C for 1 h. RT reactions were shipped on dry ice to the University of Washington, where fluorogenic 5'-nuclease assays (TaqMan) were carried out using an ABI Prism 7700 sequence detection system (PerkinElmer Applied Biosystems). The thermal cycling conditions comprised an initial denaturation step at 95°C for 10 min, followed by 40 cycles at 95°C for 20 s and 62°C for 60 s. The gene-specific sequences of all primer pairs and probes used in the TaqMan assays are listed in Table 1. The PCR primers and the dual-labeled probes were designed using the primer design software Primer Express (PerkinElmer Applied Biosystems). The specific PCR primers and/or the corresponding dual-labeled probes were designed to span intron/exon boundaries, thus eliminating spurious results derived from DNA-contaminated RNA samples. Synthesis of probes was performed by IDT (Coralville, IA). Probes were labeled at the 5' end with the fluorescent molecule 6-carboxyfluorescein (6-FAM) and at the 3' end with the quenching molecule 6-carboxytetramethylrhodamine (TAMRA). A standard curve derived from serial dilutions of rat kidney RNA was used to determine concentrations of the gclc and gclm mRNAs normalized to gapdh.
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Protein extraction, gel electrophoresis, and Western blotting for GCLc, GCLm, and -actin were carried out as described (28). Briefly, 40 µg of protein extract from each ovary were loaded onto polyacrylamide gels, separated by electrophoresis, transferred to PVDF membranes, blocked, and incubated with GCLm and GCLc antisera (47). Blots were subsequently reprobed with
-actin antiserum (Sigma-Aldrich) as a loading control. The second antibody was horseradish peroxidase-conjugated goat antirabbit (for GCLc and GCLm) or antimouse (for
-actin) immunoglobulin G (Amersham Pharmacia Biotech, Piscataway, NJ). Visualization was accomplished using enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech) followed by exposure to Hyperfilm ECL (Amersham). Semiquantitative analysis of films was performed using a Stratagene molecular documentation and image analysis system with EagleSight software.
In Situ Hybridization
In situ hybridization for gclc and gclm was adapted from Wilcox et al. (51) as previously described (27). Briefly, 35S-labeled antisense and sense riboprobes were transcribed from full-length 0.82-kb mouse gclm cDNA in pCR II (39) or a 0.6-kb mouse gclc cDNA fragment in pBluescript II (20). Sections were postfixed in 4% paraformaldehyde, treated with proteinase K, and prehybridized before hybridization overnight at 55°C with 3 x 106 cpm/slide of the appropriate riboprobe. Sections of kidney were used as "positive controls" because of the known high levels of GCL subunit mRNA in this organ (24). Negative control slides were incubated with sense gclc or gclm riboprobes.
Serial sections of ovaries were examined under bright- and dark-field microscopy. At least eight slides per ovary, each with eight sections, were evaluated in a blinded manner, and the evaluator's impressions of the intensity of hybridization in the granulosa cells, theca cells, and oocytes of each follicle class [primary (Pederson stages 13), secondary (Pederson stages 45), or antral (Pederson stages 68) (35, 37)] were recorded. The reported changes were consistently observed between three or more in situ hybridization runs.
Immunohistochemistry
Immunohistochemistry was performed using a polyclonal rabbit anti-GCLc and the Vectastain ABC Kit (Vector Laboratories, Burlingame, CA). This antibody has previously been shown to be specific for GCLc (6). The GCLm antibody used for Western blotting was not useful for immunohistochemistry. For antigen retrieval, slides were heated at high power in a conventional microwave oven in 1 mM sodium citrate and 1 mM citric acid buffer for 3 min. The slides were blocked with 3% hydrogen peroxide in methanol for 10 min, 1.5% goat serum in PBS for 20 min, and avidin D-biotin blocking solution (Vector Laboratories) for 15 min each. The slides were incubated with primary antibody (1:1,000) in PBS for 90 min, washed, incubated with biotinylated goat anti-rabbit IgG in PBS for 30 min, washed, incubated with ABC reagents for 30 min, washed, and incubated with diaminobenzidene substrate for 8 min. Finally, the slides were counterstained with hematoxylin, dehydrated in graded ethanol washes, washed in xylene, and mounted with Permount (Fisher Scientific, Pittsburgh, PA). Kidney sections were used as positive controls because GCLc is highly expressed in the paracortical region of the kidney. Negative controls were incubated with nonimmune rabbit serum without primary antibody.
Serial sections of ovaries were examined in a blinded manner under light microscopy. Follicles were classified and impressions of the intensity of immunostaining were recorded as described for in situ hybridization.
Estradiol and Progesterone Assays
Estradiol and progesterone radioimmunoassays were performed as previously described (26). All samples were assayed in duplicate. The progesterone assay utilized the Progesterone CL Radioimmunoassay Kit (Diagnostic Systems, Webster, TX). The interassay CV for a high-progesterone rat serum pool was 9.6%; the intra-assay CV was 4.3%. The sensitivity of the assay is 7.5 pg. For the estradiol assay, a standard curve was prepared in charcoal-stripped, ovariectomized rat serum by use of a stock solution of 17-estradiol (Sigma) dissolved in 100% ethanol at a concentration of 100 ng/ml that was serially diluted to concentrations of 7.8, 15.6, 62.5, 125, 250, 500, and 1,000 pg/ml. The other reagents were from the Estradiol Double Antibody Radioimmunoassay Kit (Diagnostic Products, Los Angeles, CA). All of the estradiol measurements for the present studies were done in one assay. The intra-assay CV was 4.9%. The sensitivity of this assay is 1.6 pg.
GCL Enzymatic Activity Assay
GCL activity was measured essentially as described by White et al. (50). Ovaries homogenized in TES-SB buffer (20 mM Tris, 1 mM EDTA, 250 mM sucrose, 2 mM L-serine, 20 mM boric acid) were centrifuged, and the supernatant was diluted again in TES-SB for a final dilution of 1:24 (wt/vol). Fifty microliters of sample (in duplicate) or GSH standards in TES-SB were added to prewarmed 1.5-ml microcentrifuge tubes containing GCL reaction cocktail (in mM: 400 Tris, 40 ATP, 20 L-glutamic acid, 2 EDTA, 20 boric acid, 2 L-serine, 40 MgCl2) in a 37°C water bath. After 11 min of preincubation, the reaction was initiated by the addition of 50 µl of 2 mM L-cysteine to one of each of the two sample tubes. After exactly 20 min of incubation, the reaction was stopped by the addition of 50 µl of 200 mM sulfosalicylic acid to all tubes. Then, 50 µl of cysteine were added to the second tube for each sample (the baseline GSH tube) and to the standard tubes. Samples were vortexed, incubated on ice for 20 min, and centrifuged at 1,800 g at 4°C for 5 min. Twenty microliters of supernatant (in triplicate) were pipetted into the wells of a 96-well microplate. One hundred eighty microliters of NDA solution (1.4 parts 50 mM Tris base, pH 10, 0.2 parts 0.5 N NaOH; 0.2 parts 10 mM 2,3-naphthalenedicarboxaldehyde in DMSO) were then added to each well. The plate was incubated at room temperature for 30 min and read at an excitation wavelength of 485 nm and emission wavelength of 530 nm in a Biotek FL600 spectrofluorometer microplate reader (Biotek Instruments, Winooski, VT). Results were expressed as nanomoles of GSH synthesized above the baseline GSH level per minute per milligram ovary or per milligram protein [determined using the Pierce BCA Protein Assay Kit (Pierce, Rockford, IL)]. The intra-assay CVs for the two assays for this experiment were 3.3 and 3.3%. The sensitivity of the assay in our hands is 0.10 nmol.
Statistical Analysis
Homogeneity of variances was assessed by Levene's test. When variances were not homogeneous, a natural logarithm transformation was applied. When variances remained inhomogeneous after transformation, a nonparametric test was used. Analysis of variance followed by Fisher's least significant difference test for determination of differences among groups was used to analyze differences among more than two groups. Alternatively, the Kruskal-Wallis test was used for nonparametric data. For comparisons of the means of two groups, the independent samples t-test for equal or nonequal variances, as appropriate, was used. Results were considered significantly different if P < 0.05. Statistical analyses were carried out using SPSS 11.0 for Macintosh OSX.
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RESULTS |
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Ovarian GSH concentrations and GCL subunit levels. Ovarian GSH concentrations were significantly higher at 24 and 48 h after PMSG treatment than with saline (P < 0.001, P = 0.003, respectively; Fig. 1A). Ovarian GCLm protein levels increased significantly by 8 h after PMSG and remained elevated relative to saline controls at 24 and 48 h (P = 0.034, P = 0.002, P < 0.001, respectively; Fig. 1B). There was no concomitant increase in gclm mRNA levels after PMSG. In fact, both gclc and gclm mRNA levels were significantly lower at 24 h after PMSG than at 24 h after saline (P = 0.008, P = 0.003, respectively). Ovarian GCLc protein levels were significantly increased at 24 h and 48 h after PMSG relative to saline (P = 0.023, P = 0.018; Fig. 1B). Ovarian gclc mRNA levels were 1.3-fold higher at 4 h after PMSG than at 4 h after saline (P = 0.029) and 2.0-fold higher at 48 h after PMSG than at 48 h after saline (P = 0.027; Fig. 1C).
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Ovarian GSH and GSSG and serum estradiol and progesterone concentrations. There were no significant changes in ovarian total GSH or in GSSG concentrations after hCG treatment (Fig. 4A). Serum estradiol levels declined and serum progesterone levels increased (Table 2), consistent with the previously described time course of changes in ovarian progesterone and estradiol levels after hCG (8). Ovarian weights also increased significantly (Table 2).
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Quantitation of gclm mRNA using a fluorogenic 5'-nuclease assay (real-time PCR) showed a 10-fold increase in gclm mRNA levels normalized to gapdh at 4 h after hCG, followed by a decline to threefold over baseline levels at 6 h, and a further decline to below baseline levels at 12 h (P = 0.002, effect of time after hCG). In contrast, gclc mRNA levels normalized to gapdh did not change significantly after hCG (P = 0.072, effect of time after hCG). The change in the gclm to gclc mRNA ratio, from 1.1 ± 0.2 at 0 h to 6.5 ± 2.1 at 4 h, and to 1.0 ± 0.4 at 12 h (Fig. 4D), highlights the divergent effects of hCG on expression of the two subunits (P = 0.009, effect of time after hCG). We estimated the relative copy numbers of gclm and gclc mRNA present in the samples at the start of PCR using the equation derived by Wilkening and Bader (52): N0 = 1.12 x 1010/ECN, where N0 is the initial copy number, 1.12 x 1010 is the number of copies of a PCR product at the crossing point; E is the PCR efficiency, and CN is the cycle number at the crossing point. The estimated number of copies of gclm relative to copies of gclc in the control (0 h) samples from two different experiments were 1.9 and 12.4, indicating that gclm mRNA was more abundant than gclc in the ovaries at 0 h.
Statistical analyses carried out on the nonnormalized data yielded essentially the same results as analyses on the normalized data for both mRNA and protein levels (data not shown).
Intraovarian localization of gclm and gclc mRNA. Expression of gclm mRNA expression at 0 h was prominent in oocytes and theca cells of secondary and antral follicles and in theca cells of preovulatory follicles (Fig. 5B). The lowest levels of gclm expression at 0 h were observed in the granulosa cells of preovulatory follicles (Fig. 5B). gclc mRNA expression at 0 h was highest in the theca cells of growing follicles (Fig. 6B). At 4 h after hCG (Fig. 5, D, F, and H), there was a pronounced increase in gclm mRNA expression in the mural granulosa cells of preovulatory follicles, with minimal or no increase in the cumulus cells (Fig. 5H). gclm mRNA expression also increased greatly in theca cells of growing and preovulatory follicles and in areas of interstitium (Fig. 5F). In contrast, granulosa cell gclm expression did not increase in primary, secondary, and smaller antral follicles. At 6 h after hCG, gclm expression was qualitatively similar to 4 h, but less intense (data not shown). Ovarian gclc expression did not change among the various time points (Fig. 6, B, D, and F).
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Ovarian GSH. Total ovarian GSH concentrations increased between 0900 on proestrus and 0900 on estrus (Fig. 7A), as we have previously reported (28). Although the effect of estrous cycle time was not statistically significant by ANOVA (P = 0.110), a test for linear trend showed that ovarian GSH concentrations increased significantly between 0900 proestrus and 0900 estrus (P = 0.027).
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Effect of PMSG and hCG on Ovarian GCL Enzymatic Activity
Ovarian GSH concentrations increased concomitantly with ovarian GCL enzymatic activity at 48 h after PMSG treatment (Table 3). Consistent with previous observations (3, 28), GSH concentration per milligram ovarian weight (P < 0.001, effect of treatment) and GSH concentration per milligram protein (P = 0.011, effect of treatment) increased at 48 h after PMSG compared with saline. There was no further increase at 4 h after hCG. Similarly, ovarian GCL enzymatic activity per milligram ovarian weight increased significantly at 48 h after PMSG administration compared with saline, with no further increase in GCL activity 4 h after hCG (P = 0.046, effect of treatment by ANOVA). In contrast, there was no increase in GCL activity per milligram protein at 48 h after PMSG or at 4 h after hCG (P = 0.96, effect of treatment by ANOVA).
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DISCUSSION |
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Our observation of increases in GCLm protein and mRNA levels after an ovulatory gonadotropin stimulus without a concomitant increase in GCLc is an intriguing finding. Others have reported an increase in GCL mRNA levels peaking at 4 h after hCG by Northern blotting; however, it was not specified which GCL subunit was measured (7). GCLm is not known to possess functionality alone; it is known to heterodimerize with GCLc to increase the Ki for GSH and decrease the Km for glutamate, leading to increased enzymatic activity of GCLc (13, 45). Our real-time PCR results show that, 48 h after PMSG, ovarian gclm mRNA is somewhat more abundant than gclc mRNA, suggesting that increasing gclm alone will not increase levels of GCL holoenzyme unless the two subunits are translated at different rates. Our Western blotting data show that, like the mRNA, GCLm protein increased after hCG, whereas GCLc protein did not. Ovarian GCL enzymatic activity and GSH concentrations did not increase after hCG treatment, suggesting that the increase in GCLm subunit expression did not result in higher levels of GCL holoenzyme. Future experiments will manipulate expression of GCLm alone in cell lines to investigate roles of this protein in the absence of GCLc.
Our results extend and add specificity to previous observations that GCL mRNA (subunit not specified) expression increased in granulosa and theca cells of preovulatory follicles 4 after hCG (7). Our observations from in situ hybridization using an antisense gclm riboprobe show a similar time course and localization pattern to those reported for "GCL" in that study. In contrast, our observations using an antisense gclc riboprobe show very different patterns. The upregulation of gclm expression in granulosa and theca cells of preovulatory follicles by hCG may constitute part of an antioxidative stress response within preovulatory follicles. Although we did not observe increased GCL enzymatic activity or GSH concentrations in whole ovary homogenates after hCG, it is possible that an increase in GSH synthesis was localized to preovulatory follicles. Future experiments will test the effect of hCG treatment on cultured PMSG-primed preovulatory follicles to evaluate this possibility. Similar to the present observations with gclm mRNA, manganese superoxide dismutase mRNA expression increases markedly in theca cells of preovulatory follicles after hCG administration in PMSG-primed rats (40). This antioxidant response may serve to limit potentially destructive effects of ROS generated by the periovulatory influx of leukocytes into the preovulatory follicle (14, 22).
The mechanism by which an hCG/LH stimulus upregulates gclm mRNA expression remains to be determined. LH receptors are present in theca cells of growing and preovulatory follicles, in granulosa cells of preovulatory follicles, and in interstitial cells (21), all the cell types in which we observed increased gclm mRNA expression after hCG administration. LH binding to its receptors on granulosa cells and theca cells is well known to activate the cyclic AMP-protein kinase A intracellular signaling pathway (10). Neither the rat gclc promoter nor the rat gclm promoter contains a cyclic AMP response element (53, 54), suggesting that cyclic AMP response element-binding protein (CREB)-mediated regulation is not the mechanism that increases gclm transcription. It was recently reported that estrogen regulates antioxidant response element (ARE)-mediated gene transcription (2). AREs are important in transcriptional regulation of the human and mouse gclc and gclm genes (30, 32, 44); however, they have not been found in the sequenced portions of the rat promoters (53, 54). Ansell et al. (2) found that estradiol downregulated ARE reporter gene expression in COS-1 cells and decreased glutathione-S-transferase and quinone reductase enzymatic activity in mouse uterus in vivo. They found no significant effects of estradiol on these enzymes in other organs, but they did not measure activity in ovary. Estradiol has been reported to upregulate gclc mRNA expression in human MCF7 cells via an ARE-mediated mechanism (31). Estradiol has also been reported to increase uterine GSH concentrations in rats (46), but the effect on GCL was not examined in that study. In the in vivo model system used in the current studies, ovarian estradiol synthesis increases with follicular growth induced by PMSG and declines after hCG administration (Table 2 and Ref. 8). However, the time course of change in estradiol was different from that of gclm mRNA. We observed no increase in gclm mRNA levels after PMSG treatment but a dramatic increase in gclm mRNA levels at 4 h after hCG followed by declines at 6 and 12 h. In contrast, serum estradiol levels were maximal at 48 h after PMSG, remained elevated at 4 and 6 h after hCG, and declined at 12 h after hCG. Another possibility is that the stimulatory effects of hCG on gclm expression are mediated by IGF-I. Insulin upregulates gclc expression, but not gclm or glutathione synthetase expression, in cultured rat hepatocytes (4, 17). Gonadotropins and estradiol stimulate granulosa cell production of IGF-I (16).
In contrast to the increase in both GCLm mRNA and protein levels observed after an ovulatory gonadotropin stimulus, stimulation of follicular development by PMSG increased ovarian GCLc and GCLm protein levels and GCL enzymatic activity without increasing gclm mRNA levels and only minimally increasing gclc mRNA levels (present studies and Ref. 28). These results are consistent with the conclusion that the increasing ovarian GCLc and GCLm protein levels observed at 8, 24, and 48 h after PMSG are likely caused by posttranscriptional effects of PMSG on GCL subunits. An increase in protein levels in the absence of an increase in mRNA levels could be due to increased translation or decreased degradation of GCLc and GCLm. Experiments are currently ongoing in our laboratory to determine whether gonadotropin suppresses GCL protein degradation or increases translation. The rate of protein degradation could be affected by alterations in ubiquitylation. Effects of gonadotropins on ubiquitin-related proteins have been reported. For example, granulosa cell levels of small ubiquitin-related modifier-1 protein are decreased at 48 h after PMSG administration (43). Recent evidence also shows that both LH and FSH can alter translation of some proteins without affecting their transcription. LH was reported to downregulate the rate of translation of connexin-43 in cultured ovarian follicles by a mechanism involving both protein kinase A and mitogen-activated protein kinase signaling (19). FSH stimulation increased translation of hypoxia-inducible factor-1 in granulosa cells by a mechanism involving phosphatidylinositol 3-kinase/Akt signaling (1).
We have demonstrated herein that, during follicular growth stimulated by PMSG, ovarian GSH concentrations, GCL subunit protein levels, and GCL enzymatic activity increase without a concomitant increase in GCL subunit mRNA levels. Within the ovary, PMSG treatment increases follicular GCLc protein in growing follicles. In contrast, an ovulatory gonadotropin stimulus in PMSG-primed rats dramatically increases ovarian gclm mRNA and protein levels without a concomitant increase in ovarian GCLc expression, GCL enzymatic activity, or GSH concentrations. We also showed that an ovulatory LH stimulus upregulates gclm mRNA expression, but not gclc mRNA, in mural granulosa cells and theca cells of preovulatory follicles and in theca cells of growing follicles. Thus gclc and gclm expression are differentially regulated by an ovulatory gonadotropin stimulus, and gclm expression is differentially regulated in follicles of different stages by an ovulatory gonadotropin stimulus. Our results also show that ovarian GSH synthesis is tightly regulated via gonadotropin stimulation of protein and/or mRNA expression of its rate-limiting enzyme in a follicle stage-dependent manner.
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ACKNOWLEDGMENTS |
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Portions of this work were previously presented in abstract form at the Annual Meeting of the Society of Toxicology in 2003 and at the Annual Meeting of the Society for the Study of Reproduction in 2004.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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