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Research Article |
Department of Physiology,3 Medical College of Georgia, Augusta, Georgia 30912
California National Primate Research Center (CNPRC),4 Davis, California 95616
Department of Obstetrics, Gynecology, and Reproductive Sciences,5 University of Maryland School of Medicine, Baltimore, Maryland 21201
ABSTRACT
Ovulationand luteal formation in primates are associated with the sustained synthesis of progesterone. The observed high intrafollicular concentrations of progesterone during the periovulatory interval raise the possibility that this steroid serves as a precursor for mineralocorticoids. The aim of this study was to determine if mineralocorticoids are synthesized by the luteinizing macaque follicle during controlled ovarian stimulation cycles in which follicular fluid and granulosa cell aspirates were obtained before or after an ovulatory hCG bolus. Follicular fluid concentrations of progesterone and 17alpha-hydroxyprogesterone increased within 3 h of an ovulatory hCG bolus. Their respective metabolites, 11-deoxycorticosterone (DOC) and 11-deoxycortisol, were not detectable before an ovulatory stimulus and increased starting at 6 h after hCG, while corticosterone and aldosterone were undetectable. Cortisol was present before and after hCG administration and had increased 2-fold at 24 h after an ovulatory stimulus. The expression of 21-hydroxylase (CYP21A2) mRNA increased within 3 h of hCG administration, while 11beta-hydroxylase-1 (CYP11B1) and 11beta-hydroxylase-2 (CYP11B2) mRNAs were not detectable. 11beta-Hydroxysteroid dehydrogenase-1 (HSD11B1) mRNA had increased at 12 h after hCG administration, and 11beta-hydroxysteroid dehydrogenase-2 (HSD11B2) had decreased by 3 h after hCG administration. Mineralocorticoid receptor mRNA levels did not change following hCG administration, while glucocorticoid receptor mRNA levels increased in response to an ovulatory stimulus.Treatment of granulosa cells with the mineralocorticoid receptor antagonist spironolactone blocked hCG-induced progesterone synthesis in vitro. These data indicate that macaque granulosa cells can synthesize mineralocorticoids in response to an ovulatory stimulus and that the mineralocorticoid receptor plays a key role in steroid synthesis associated with luteinization of macaque granulosa cells.
granulosa cells, luteinization, macaque, mineralocorticoid, ovary, ovulation, steroid hormones, steroidogenesis
INTRODUCTION
The complex processes of ovulation and corpus luteum formation are initiated by the midcycle surge of LH. The onset of the LH surge heralds the start of the periovulatory interval that extends until the extrusion of the fertilizable oocyte. A hallmark of the periovulatory interval is the rapid induction of progesterone that occurs within 30 min of an ovulatory stimulus to rhesus monkeys undergoing controlled ovarian stimulation [1]. The increase in progesterone during the periovulatory interval has peripheral and essential local actions that lead to ovulation and corpus luteum formation [2–4]. What remains underappreciated is the potential for high levels of progesterone to be locally metabolized into mineralocorticoids.
Progesterone and 17
-hydroxyprogesterone can be converted to 11-deoxycorticosterone (DOC) and 11-deoxycortisol, respectively, through the action of 21-hydroxylase (CYP21A2). DOC and 11-deoxycortisol can be further metabolized to corticosterone or cortisol by 11ß-hydroxylase-1 (CYP11B1). Unlike rats, in which corticosterone is the primary glucocorticoid, corticosterone in primates serves as a substrate for 11ß-hydroxylase-2 (CYP11B2) in the synthesis of aldosterone. Although (to our knowledge) no studies have yet examined the expression of CYP21A2 in the ovary, the presence of cortisol in human follicular fluid suggests that this enzyme is expressed and active in the human ovary [5].
The present study was designed to test the hypothesis that macaque granulosa cells synthesize mineralocorticoids in response to an ovulatory stimulus and that these steroids have a local role in luteinization. In addition, local metabolism of 17-hydroxyprogesterone to cortisol in the primate ovary has not been completely addressed, despite evidence that cortisol is present in follicular fluid [6–8]. In light of the findings that cortisol may be relevant to ovarian processes [9], we report herein the intrafollicular levels of glucocorticoids and the granulosa cell expression of glucocorticoid-synthesizing enzymes before and after an ovulatory stimulus. A controlled ovarian stimulation of rhesus macaques is used in conjunction with an in vitro model of granulosa cell luteinization to address these questions [10].
MATERIALS and METHODS
Animals
Adult female rhesus macaques (Macaca mulatta) were housed at the California National Primate Research Center (CNPRC) as previously described [11]. Animal protocols and experiments were approved by the CNPRC Animal Care and Use Committee, and investigations were conducted in accord with the Guide for the Care and Use of Laboratory Animals [12]. Following the onset of menstruation, adult female rhesus monkeys were treated with 37.5 IU i.m. of recombinant human FSH (r-hFSH) (Ares-Serono, Randolph, MA; or Organon, West Orange, NJ) twice a day for 7 days. Antide (5 mg/kg BW s.c.) (Ares-Serono) was administered once a day to prevent endogenous gonadotropin secretion. Follicles were aspirated the morning after the last dose of r-hFSH by an ultrasound-guided procedure as previously described for use in in vitro luteinization procedures [11]. A subset of animals received recombinant hCG (1000 IU s.c.; Ares-Serono) to initiate periovulatory events, and follicles were aspirated before hCG administration (at 0 h) and 3, 6, 12, and 24 h later (n = 3 at all time points, although follicular fluid was available from only two animals at 3 h after hCG administration and was not included in the statistical analyses). Additional animals received hCG for 33 h as part of an in vitro fertilization program. Aspirates from each animal were pooled, and granulosa cells were isolated as described previously [13].
In Vitro Luteinization of Macaque Granulosa Cells
Granulosa cells from individual animals (n = 3) were plated in duplicate overnight at 37°C with an initial seeding density of 2.5 x 105 viable cells/well in 24-well plates precoated with fibronectin (Biocoat; Becton Dickinson, Bedford MA) in Dulbecco modified Eagle medium/F12 supplemented with penicillin/streptomycin (50 U/ml), ITS (20 µl/ml), and hFSH (25 ng/ml) (F4021; Sigma-Aldrich). Following the initial overnight seeding period, media were changed to include 25 ng/ml of hFSH to maintain a nonluteinized phenotype (control samples) or 20 IU/ml of hCG (Sigma-Aldrich) to induce luteinization [13]. Cultures were terminated at 6 h or 24 h after the addition of hCG or hFSH. Total RNA was isolated using the RNAqueous Micro kit (Ambion Inc., Austin, TX), and media were assayed for steroids as described herein.
Mineralocorticoid Receptor Antagonism
To investigate the effects of mineralocorticoid receptor antagonism on steroid synthesis, granulosa cells were cultured as already described in the presence or absence of the mineralocorticoid receptor antagonist spironolactone (10 µM) for 24 h. Because spironolactone also acts as an androgen receptor antagonist [14], FSH or hCG with or without the androgen receptor antagonist flutamide (100 nM; Sigma-Aldrich) was used as a control. Culture media were removed for steroid analysis.
Granulosa Cell Viability
Cell viability of cultured granulosa cells was measured using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) per the manufacturer's instructions. Granulosa cells were cultured in a 96-well plate and treated with hCG or FSH with or without spironolactone or flutamide as already described for 48 h. This time point was chosen to ensure that spironolactone or flutamide did not induce loss of viability.
Real-Time RT-PCR
Granulosa cell RNA was reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Invitrogen), and semiquantitative real-time RT-PCR (Applied Biosystems, Inc., Foster City, CA) was performed for CYP21A2, mineralocorticoid receptor and glucocorticoid receptor, CYP11B1 and CYP11B2, and 11ß-hydroxysteroid dehydrogenase-1 (HSD11B1) and 11ß-hydroxysteroid dehydrogenase-2 (HSD11B2). Primers and 6FAM-labeled probes were synthesized by Applied Biosystems, Inc. Primers and ROX-labeled probe for internal control ribosomal protein L19 (RPL19) were synthesized by Biosearch Technologies (Novato, CA). Target genes and RPL19 were detected in the same reaction. Relative mRNA levels were quantified using a standard curve constructed with pooled macaque granulosa cell cDNA. PCR was carried out as previously described [10], and data were expressed as a ratio of the target gene to the internal control. H295R human adrenocortical cells treated with 10 µM forskolin for 5 h were used as a positive control. The primer and probe sequences were as follows:
CYP21A2 For CYP21A2, the sequences were: Forward: 5'-ATTGTGGACGTGATTCCCTTTCT (nt 633–655). Probe: 5'-6FAM-TCCGGAGGCTGAAGCAGGCCATAG (nt 679–702). Reverse: 5'-TCTCCACGATGTGATCCCTCTT (nt 726–705).
Mineralocorticoid receptor For mineralocorticoid receptor, the sequences were: Forward: 5'-CGGATTCTTCATTCTCAGTACCAATA (nt 1262–1287). Probe: 5'-6FAM-TCAACCAAGCATTCATGTTCAGGCACC (nt 1297–1323). Reverse: 5'-GGTTTACTGTTGGATTCCCTTTAAAA (nt 1351–1326).
CYP11B1 For CYP11B1, the sequences were: Forward: 5'-TGGGTGGCCTACAGACAACAT (nt 343–363). Probe: 5'-6FAM-TGAATCCAGAAGTGCTGTCGCCCAA (nt 428–452). Reverse: 5'-GGCCACTGCATCCACCAT (nt 492–475).
CYP11B2 For CYP11B2, the sequences were: Forward: 5'-ACGGTGACAACTGTATCCAGAAAAT (nt 800–824). Probe: 5'-6FAM-CGCCCTCAACACTACACAGGCATCGT (nt 847–872). Reverse: 5'-CTCCCTGCAGTGAGTTCCATAGA (nt 947–925).
Experiments for HSD11B1 (Hs00194153_m1), HSD11B2 (Hs00388669_m1), and glucocorticoid receptor (Hs00230818_m1) mRNAs were performed using Assays-on-Demand Gene Expression products (Applied Biosystems) per the manufacturer's instructions.
Steroid Assays
Gas chromatography and mass spectrophotometry Gas chromatography and mass spectrophotometry (GC/MS) was performed as previously described [15]. Steroids were from Steraloids (Newport, RI), and cortisol-d4 (used as an internal standard [IS]) was from CDN Isotopes (Pointe-Claire, QB, Canada). Samples were extracted using a SepPak C18 cartridge and eluted with methanol. After addition of 5 ng of cortisol-d4, samples were evaporated under N2 stream and derivatized by heating for 1 h at 55°C with 50 µl of 2% (w/v) methoxyamine hydrochloride in pyridine, followed by 16 h at 100°C with 50 µl of N-trimethylsilylimidazole. Three calibration samples were extracted in parallel. The final extracts were evaporated and redissolved in 40 µl of N,O-bis[trimethylsilyl]-trifluoroacetamide. The analyses were performed by injecting 3 µl of the final extracts in a gas chromatographer coupled to an ion trap mass spectrometer. A specific method was set up to optimize the sensitivity for 11-deoxycorticosterone, corticosterone, 11-deoxycortisol, and cortisol. GC/MS was carried out on a PolarisQ ion trap mass spectrometer (Thermofinnigan, San Jose, CA) interfaced with a TraceGC (Carlo Erba, Milan, Italy) gas chromatograph. Samples were injected with an AI3000 autosampler (Carlo Erba). The sterols were separated on a DB-1 crosslinked methylsilicone column (inner diameter, 15 m x 0.25 mm and film thickness, 0.25 µm; J&W Scientific, Folsom, CA). To evaluate the linearity and the precision, five calibration samples at three different concentrations (one replicate each at low and high concentrations and three replicates at medium concentration) were prepared and analyzed. Those calibration samples were extracted in parallel to the samples. A linear correlation was found in all cases, with mean determination coefficients (r2) better than 0.98. Intra-assay variability for all compounds was less than 15%.
ELISA Measurements of 17ß-estradiol, progesterone, and cortisol concentrations in culture media were made using commercially available ELISA kits (Alpco Diagnostics, Windham, NH).
Statistical Analysis
Data are presented as means ± SEM. Bartlett chi-square was used to test for heterogeneity of variance, and data were logarithmically transformed (log+2) if variances were significantly different. Data were analyzed by one-way ANOVA, followed by a Student-Newman-Keuls means comparison test (data from in vitro samples were analyzed with one repeated measure). Means were considered significantly different at P < 0.05. For analysis of in vitro experiments, raw data were used for statistical purposes, but for graphical presentation, the data from each animal were normalized to corresponding FSH controls and presented as the mean percentage of the FSH controls.
RESULTS
Follicular fluid aspirated from the ovaries of adult macaques undergoing controlled ovarian stimulation before or up to 24 h after hCG administration in vivo was assayed for steroid hormones by GC/MS (Fig. 1). Consistent with published data [1], intrafollicular progesterone increased 43-fold (P < 0.05) at 6 h after hCG administration and remained elevated through 24 h after hCG administration. DOC was undetectable in follicular fluid from any animal before hCG administration but was present in both animals at 3 h after hCG administration. DOC increased significantly at 6, 12, and 24 h after hCG administration (8.7 ± 4.1, 38.9 ± 18.1, and 198.5 ± 75.6 mg/ml, respectively; P < 0.05). Neither corticosterone nor aldosterone was detectable in follicular fluid at any time point in any animal before or after hCG injection. 17-Hydroxyprogresterone had increased 15-fold (18 ± 18 ng/ml) at 6 h after hCG administration (P < 0.05) and remained elevated at 6, 12, and 24 h after hCG administration (265.2 ± 98.4, 176.2 ± 95.2, and 421.6 ± 95.2 ng/ml, respectively). 11-Deoxycortisol was not detectable in follicular fluid before hCG administration (at 0 h) or at 3 h after hCG administration but had increased (P < 0.05) at 6 h and 12 h (5.6 ± 1.6 and 7.7 ± 3.2 mg/ml, respectively) after hCG administration, with a further increase (P < 0.05) at 24 h (31.8 ± 9.2 mg/ml) after hCG administration. Cortisol was present in all samples before and after hCG administration, and intrafollicular concentrations had increased 2-fold at 24 h after an ovulatory stimulus (64.3 ± 18.2 ng/ml at 0 h and 140.0 ± 21.5 ng/ml at 24 h).
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Treatment of nonluteinized macaque granulosa cells with hCG administration in vitro for 6 h resulted in a 3-fold increase (P < 0.05) in culture media levels of progesterone over FSH controls (Fig. 2A). GC/MS was used to determine the identity of the mineralocorticoids present in culture media at 6 h after FSH or hCG administration. Similar to follicular fluid, corticosterone was undetectable in FSH or hCG treatment, and given the absence of this precursor, aldosterone was not assayed. In contrast, hCG induced a 7-fold increase in DOC over FSH-treated cells (P < 0.05) (Fig. 2B). Two of three FSH-treated samples did not have detectable levels of DOC. Media concentrations of cortisol were low (<1 ng/ml) and did not change with treatment (Fig. 2C).
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CYP21A2 metabolizes progesterone and 17-hydroxyprogestserone to DOC and 11-deoxycortisol, respectively. In vivo administration of hCG induced 6-fold expression of CYP21A2 mRNA in granulosa cells within 3 h of hCG administration (P < 0.05) (Fig 3). Levels of CYP21A2 mRNA returned to control (0 h) values at 24 h after hCG administration. Treatment of cultured granulosa cells with hCG for 6 h increased levels of CYP21A2 mRNA 12-fold (P < 0.05) over FSH controls (Fig. 3). The expression of CYP21A2 mRNA at 24 h after addition of hCG to culture media was not different from FSH controls. CYP11B1 and CYP11B2 mRNAs were undetectable before and after hCG administration in vivo and in vitro. For both genes, H295R adrenocortical cells served as a positive control to verify the probe and primer sets.
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The enzyme HSD11B2 metabolizes cortisol to its inactive metabolite cortisone. In contrast, HSD11B1 metabolizes cortisone to cortisol. Levels of HSD11B1 mRNA were present in all samples collected before and after an ovulatory hCG bolus, albeit at low expression levels until 12 h after hCG administration, at which time mRNA levels increased 25-fold relative to control values (P < 0.05) (Fig. 3). An additional increase in HSD11B1 mRNA had occurred at 24 h after hCG administration (233-fold increase versus control values, P < 0.05). Similarly, HSD11B1 mRNA levels from in vitro cultured granulosa cells did not increase until 24 h after hCG administration (12-fold increase versus FSH control values, P < 0.05) (Fig. 3). Conversely, mRNA levels of HSD11B2 were highest before an ovulatory stimulus and declined within 3 h of hCG administration (2-fold decrease versus control values, P < 0.05), with an additional reduction in mRNA levels at 24 h after hCG administration (8-fold decrease versus control values, P < 0.05). Treatment of nonluteinized granulosa cells (NLGCs) with hCG reduced HSD11B2 mRNA levels 5-fold and 2.5-fold 6 h and 24 h later, respectively (P < 0.05).
Mineralocorticoid receptor mRNA was detectable in all samples in vitro and in vivo, and levels did not change in response to hCG administration (Fig. 4). Levels of glucocorticoid receptor mRNA had increased 5-fold (P < 0.05) at 33 h after an ovulatory hCG bolus, although this was not recapitulated by treatment of NLGCs with hCG for 24 h.
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The mineralocorticoid receptor antagonist spironolactone was used to elucidate the role of mineralocorticoids during luteinization of macaque granulosa cells. NLGCs treated with hCG for 24 h showed an expected increase (18-fold) in progesterone over FSH controls (P < 0.05) (Fig. 5A). Treatment of NLGCs with FSH with spironolactone did not alter progesterone synthesis significantly. In contrast, spironolactone completely blocked hCG-induced progesterone synthesis. The androgen receptor antagonist flutamide was used to demonstrate that the effects of spironolactone were through mineralocorticoid receptor rather than androgen receptor. Treatment of NLGCs with flutamide did not alter progesterone levels in the presence of FSH or hCG. Previous studies demonstrated that hCG treatment of NLGCs in vitro results in a transient increase in estrogen synthesis [1, 10]. Treatment of NLGCs with hCG for 24 h induced a 2-fold increase (P < 0.05) in media concentrations of estrogen (Fig. 5B). Similar to progesterone synthesis, spironolactone inhibited hCG-induced but not FSH-induced estrogen production. Flutamide did not alter estrogen levels during FSH or hCG treatment. To determine if the reduction in steroidogenesis following hCG administration was due to loss of cell viability, NLGCs were treated for 24 h with FSH or hCG in the presence or absence of spironolactone or flutamide. Figure 5C demonstrates that no significant changes were observed in cell viability as assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) cell viability assay.
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DISCUSSION
Although cortisol has been postulated to have actions on the ovary, notably in the regulation of steroidogenesis [16, 17], a similar role for mineralocorticoids has not been proposed, to our knowledge. In addition, observed high concentrations of intrafollicular progesterone following an ovulatory gonadotropin surge raise the possibility that progesterone metabolites such as mineralocorticoids may be present in the luteinizing follicle. The present study was undertaken to determine if follicles in macaques synthesize mineralocorticoids in response to an ovulatory bolus of hCG. Progesterone, DOC, 17-hydroxyprogesterone, and 11-deoxycortisol, but not corticosterone or aldosterone, are increased in follicular fluid following hCG administration. Cortisol also increases after hCG administration but in a modest fashion. Most important, cortisol is present in follicular fluid before hCG administration, while the direct precursor 11-deoxycortisol is not, suggesting that some or all of the cortisol is of nonovarian origin. The expression of steroidogenic enzymes is consistent with hCG-induced DOC production, as well as local conversion of cortisone to cortisol. Treatment of luteinizing granulosa cells with a mineralocorticoid receptor antagonist blocks hCG-induced progesterone synthesis. These data collectively support the hypothesis that hCG stimulates the synthesis of DOC and that mineralocorticoid receptors have an important role in periovulatory processes.
The administration of an ovulatory hCG stimulus to rhesus monkeys undergoing controlled ovarian stimulation results in a marked increase in the concentration of intrafollicular progesterone [1]. Treatment of NLGCs with hCG in vitro indicates that the progesterone increase in culture is essentially identical to that observed in vivo [10], underscoring the central importance of steroidogenesis as a part of the luteinization program. Progesterone is essential for follicle rupture, luteinization, and (in luteal phase species, e.g., primate and bovine) corpus luteum function [2, 4, 18, 19] and has the potential to serve as a substrate for glucocorticoids and mineralocorticoids. Levels of 17-hydroxyprogesterone also increase after hCG administration, although intrafollicular concentrations are approximately 10-fold lower than those of progesterone. Findings from previous studies suggest that expression of the P450c17 gene, the product of which metabolizes pregnenolone to dehydroepiandrosterone and progesterone to 17-hydroxyprogesterone, declines following hCG administration, although the presence of 17a-hydroxylase/17,20-lyase in human luteinized granulosa cells has been reported [20, 21]. The net result is the synthesis of progesterone and 17-hydroxyprogesterone following an ovulatory stimulus.
The synthesis of DOC following hCG administration supports the hypothesis that intrafollicular progesterone serves as a mineralocorticoid substrate. DOC is not detectable before hCG administration and increases as early as 3 h after an ovulatory stimulus, although concentrations even at 24 h after hCG administration are substantially lower than those of follicular progesterone. NLGCs treated with FSH in vitro have low levels of DOC, which were detectable in only one of three cultures. Treatment with hCG in vitro results in a marked increase in DOC, and similar to the intrafollicular milieu, levels are approximately 10-fold less than those of progesterone. Most important, granulosa cells express CYP21A2 mRNA, which increases following an ovulatory stimulus in a temporal profile similar to that of DOC, although mRNA levels at 24 h after hCG administration decline back to control levels before hCG administration. Whether this means that luteal levels of DOC decline after ovulation is unknown, although circulating levels of DOC change in tandem with those of progesterone in pregnant women, and the profile of serum DOC during the nonfertile luteal phase parallels that of progesterone [22]. Luteal phase levels of circulating DOC, but not cortisol, are refractory to dexamethasone suppression in normo-ovulatory women [23], suggesting an extraadrenal (possibly luteal) origin. The low levels of DOC in compared with those of progesterone during luteinization suggest that synthesis is limited by CYP21A2 rather than substrate. The expression of CYP21A2 in H295R adrenocortical cells is mediated by multiple transduction pathways, including protein kinase, protein kinase C, potassium, and angiotensin II [24], although it is unknown if the regulation of CYP21A2 mRNA in granulosa cells is similar to that in adrenal cells or perhaps is unique to the ovary. Most important, rat granulosa cells do not express CYP21A2 mRNA before or after hCG administration, nor are mineralocorticoids evident in follicular fluid in rats during eCG/hCG-induced follicular growth (unpublished results).
The ovary is also a site of 11-deoxycortisol synthesis. 11-Deoxycortisol requires 17-hydroxyprogesterone as a precursor, suggesting theca cells as a possible source. Because 11-deoxycortisol requires 21-hydroxylation, theca cells in primates could also express CYP21A2. Alternatively, theca-derived 17-hydroxyprogesterone could be metabolized to 11-deoxycortisol in granulosa cells. This latter scenario would be more consistent with the observed low levels of intrafollicular 11-deoxycortisol. The actions of 11-deoxycortisol are poorly understood, although some data suggest that this steroid transactivates mineralocorticoid receptor but not glucocorticoid receptor [25].
Cortisol is present in follicular fluid before and after an ovulatory bolus and exhibits a modest increase at 24 h after hCG administration, especially relative to (for example) 11-deoxycortisol. The fact that intrafollicular concentrations of cortisol are in the range of 60 ng/ml before hCG administration, while its immediate precursor 11-deoxycortisol is undetectable at that time, suggests that all or at least most of the preovulatory follicular cortisol derives from the adrenal gland. This is supported by the fact that granulosa cells do not express detectable levels of CYP11B1 mRNA. This hypothesis is in contrast with a recent report by Acosta et al. [6] in which bovine follicles were shown to synthesize cortisol. Similarly, there is an increase in bioactive cortisol in follicular fluid of women following the LH surge of the natural cycle [26]. Based on the lack of 11-deoxycortisol before hCG administration and the shift in the mRNA ratio of HSD11B1 to HSD11B2, it is hypothesized that the increase in follicular fluid cortisol stems from local metabolism of cortisone to cortisol.
Granulosa cells in vivo and in vitro express mineralocorticoid receptor mRNA at equivalent levels before and after hCG administration. This is in contrast to the hormonally stimulated immature rat, in which mineralocorticoid receptor mRNA is reduced following an ovulatory bolus [9]. Although DOC is a mineralocorticoid receptor agonist and has been shown to be almost as potent as aldosterone in transactivating mineralocorticoid receptor [27], it would be premature to draw a mechanistic link between hCG-induced DOC and mineralocorticoid receptor activity. More compelling in implicating mineralocorticoid receptor in normal periovulatory events is the fact that the mineralocorticoid receptor antagonist spironolactone inhibits hCG-induced progesterone synthesis. However, because spironolactone has androgen receptor antagonistic properties [14], the androgen receptor antagonist flutamide was used to verify specificity. No effects of flutamide were noted on steroid synthesis in response to hCG. Furthermore, spironolactone does not appear to reduce granulosa cell viability, although the reduction in progesterone would be expected ultimately to reduce granulosa cell survival [28]. An additional caveat to these data is the fact that progesterone may be a mineralocorticoid receptor antagonist, although there is considerable variation between species, among cell types, and in vitro versus in vivo, making it unclear if progesterone is a physiological antagonist of the mineralocorticoid receptor [29]. Finally, a recent study by Ge et al. [30] indicates that Leydig cells respond to aldosterone with increased testosterone synthesis. Therefore, although it is clear that mineralocorticoid receptor agonists can regulate gonadal steroids, the mechanisms remain obscure.
The presence of glucocorticoid receptors in human granulosa cells has been suggested [31], supporting a local role for cortisol. The expression of glucocorticoid receptor mRNA is increased at 33 h after ovulatory hCG bolus. Glucocorticoid receptor mRNA does not increase in vitro after hCG administration, suggesting two possibilities. First, glucocorticoid receptor mRNA may increase at 24 h after hCG administration; thus, the increase was not evident in vitro. Second, increased glucocorticoid receptor mRNA may be mediated by theca-derived factors that are absent in granulosa cell culture. In either event, the increase in glucocorticoid receptor mRNA supports the hypothesis that cortisol mediates the inflammatory events associated with ovulation [32].
Figure 6 summarizes the effects of an ovulatory hCG bolus on the synthesis of mineralocorticoids and glucocorticoids by macaque preovulatory follicles. The presence of mineralocorticoid receptor mRNA, along with the potent specific action of the mineralocorticoid receptor antagonist spironolactone in blocking hCG-induced steroidogenesis in vitro, suggests that mineralocorticoids play a key role in initiating or maintaining periovulatory progesterone synthesis in primates.
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ACKNOWLEDGMENTS
The authors are grateful to the laboratory of Dr. Cedric Shackleton, Children's Hospital Oakland Research Institute, Oakland, CA, for performing the GC/MS assays. Dr. Mary Cherian-Shaw, Jennifer Cannon, and Muraly Puttabyatappa provided technical assistance.
FOOTNOTES
1 Supported in part by grants HD047964 (K.N.F.), RR13439 (C.A.V.), HD043358 (C.L.C.), and RR00169 (CNPRC) from the National Institutes of Health and by a UNCF/Merck fellowship (K.N.F.). 
2 Correspondence: Charles L. Chaffin, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of Maryland School of Medicine, Bressler Research Laboratories 11–011, 655 W. Baltimore St., Baltimore, MD 21201. FAX: 410 706 5747; cchaffin{at}upi.umaryland.edu
Received: 26 April 2006.
First decision: 19 May 2006.
Accepted: 7 July 2006.
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