Molecular Human Reproduction, Vol. 6, No. 1, 11-18,
January 2000
© 2000 European Society of Human Reproduction and Embryology
Endocrinology |
Hormonal regulation of steroidogenic enzyme expression in granulosa cells during the peri-ovulatory interval in monkeys
1 Division of Reproductive Sciences, 2 Division of Neuroscience, Oregon Regional Primate Research Center, 505 NW 185th Ave, Beaverton, OR 97006, and 3 Department of Physiology and Pharmacology, Oregon Health Sciences University, 3181 Sam Jackson Park Road, Portland, OR 97201, USA
Abstract
Although progesterone plays an essential role in ovulation and the luteiniziation of the primate follicle, the expression of cellular components required for progesterone synthesis and their control is not well defined. This study was designed to determine the time course and gonadotrophin versus steroid regulation of the transcription of genes involved in progesterone synthesis in peri-ovulatory follicles. Granulosa cells or whole ovaries were obtained from macaques undergoing controlled ovarian stimulation either before (0 h) or up to 36 h following the administration of an ovulatory human chorionic gonadotrophin (HCG) bolus with or without a 3ß-hydroxysteroid dehydrogenase (3ß-HSD) inhibitor, with or without a non-metabolizable progestin. Granulosa cell concentrations of low density lipoprotein receptor (LDL-R) and steroidogenic acute regulatory protein (StAR) mRNA increased transiently 12 h following HCG administration (P < 0.05) at which time steroid depletion tended to reduce StAR mRNA (P = 0.06). At 36 h post-HCG progesterone suppressed the LDL-R mRNA levels (P < 0.05). P450 side-chain cleavage (P450scc) mRNA decreased in a time-dependent fashion up to 24 h, whereas 3ß-HSD mRNA increased within 12 h of HCG administration (P < 0.05) in a steroid-independent manner. Whole ovarian 17
-hydroxylase (P450c17) and granulosa cell P450 aromatase (P450arom) mRNA declined in a time-dependent fashion; by 36 h after HCG administration, steroid depletion increased P450arom mRNA, although progestin replacement did not return aromatase to control values (P < 0.05). These data demonstrate diverse patterns of steroidogenic enzyme expression that generally reflect the conversion of the macaque peri-ovulatory follicle from an oestrogen to progesterone producing gland. Although mRNAs associated with progesterone synthesis and metabolism are primarily regulated by gonadotrophins, cholesterol uptake and utilization may be modulated locally by steroids in luteinizing granulosa cells.
granulosa cells/luteinization/macaque/peri-ovulatory interval/steroidogenic enzymes
Introduction
During the 36–38 h peri-ovulatory interval in primates, follicular steroidogenesis shifts from predominantly oestrogen and androgen to progesterone production. In both natural (Weick et al., 1973
; Hoff et al., 1983) and hormonally controlled (Chaffin et al., 1999a) menstrual cycles, an ovulatory gonadotrophin stimulus increases circulating concentrations of progesterone, androstenedione, and oestradiol within 12 h. However, progesterone concentrations remain elevated throughout the peri-ovulatory interval, leading to follicular rupture and luteal formation (Hibbert et al., 1996), whereas androstenedione and oestradiol decline toward the expected time of ovulation (Weick et al., 1973; Hoff et al., 1983; Chaffin et al., 1999a). While primate corpora lutea express low density lipoprotein receptor (LDL-R), steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage (P450scc), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17-hydroxylase (P450c17), and P450 aromatase (P450arom) (Benyo et al., 1993; Pollack et al., 1997; Sanders and Stouffer, 1997), few data exist regarding the regulation of these key components in the steroidogenic process in primate peri-ovulatory follicles. Recent studies in rhesus monkeys suggest that the principal determinant for the peri-ovulatory rise in progesterone is increased P450scc activity in granulosa cells rather than changes in 3ß-HSD or cholesterol utilization (Chaffin et al., 1999a). However, the relationship between granulosa cell enzyme activity and mRNA expression is unknown in primates.
While peri-ovulatory events are initiated by the mid-cycle gonadotrophin surge, it is unclear whether changes in steroid synthesis are due to luteinizing hormone (LH)/human chorionic gonadotrophin (HCG) acting directly or indirectly via gonadotrophin-stimulated steroid production in the luteinizing follicle. For example, gonadotrophins promote the expression of P450scc in granulosa and luteal cells (Goldring et al., 1987; Ravindranath et al., 1992; Yong et al., 1994), and several reports suggest that progesterone increases 3ß-HSD activity (Dimattina et al., 1986; Tanaka et al., 1993; Donath et al., 1997). Thus, it is postulated that progesterone stimulates its own synthesis (Rothchild, 1981) during the peri-ovulatory interval by regulating the expression of key steroidogenic enzymes in the mature follicle.
The current study was designed to examine the regulation of mRNAs relating to cholesterol utilization (LDL-R, StAR) and steroid synthesis (P450scc, 3ß-HSD and P450arom) in granulosa cells aspirated from rhesus monkeys (Macaca mulatta) undergoing controlled ovarian stimulation before (0 h), 12, 24, or 36 h following an ovulatory stimulus of HCG. In addition, whole ovaries obtained from hormonally-stimulated pigtail macaques (Macaca nemistrina) before (0), 3, 6, 24, or 36 h after HCG were used for analysis of P450c17 mRNA. These models provide multiple large antral follicles and permit analyses at precise time-points after administering the ovulatory gonadotrophin bolus in order to elucidate the time course for the shift from oestrogen/androgen to progesterone production (Chaffin et al., 1999a). In addition, the intrafollicular steroid milieu can be controlled using a 3ß-hydroxysteroid dehydrogenase (HSD) inhibitor during the peri-ovulatory interval (Hibbert et al., 1996), permitting the analysis of local steroid regulation of steroidogenic enzyme genes.
Materials and methods
Animals
The general care and housing of monkeys at the Oregon Regional Primate Research Center (ORPRC) was described previously (Wolf et al., 1996). Animal protocols and experiments were approved by the ORPRC Animal Care and Use Committee, and studies were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Adult female rhesus monkeys exhibiting normal menstrual cycles of ~28 days were stimulated with recombinant human gonadotrophins [rh follicle stimulating hormone (FSH), 30 IU i.m. twice daily for 8 days; rhLH, 30 IU twice daily on days 7 and 8; Laboratoires Serono SA, Aubonne, Switzerland] beginning 1–3 days after the onset of menses in order to promote the development of multiple pre-ovulatory follicles. Monkeys also received a daily s.c. injection of the gonadotrophin-releasing hormone (GnRH) antagonist Antide (08:00, 0.5 mg/kg body weight in propylene glycol:water, 1:1; Laboratoires Serono SA, Aubonne, Switzerland) throughout the stimulation protocol to prevent an endogenous LH surge. This or comparable protocols have been utilized by our research group to study peri-ovulatory events in the primate, including the effects of steroid depletion (Chaffin et al., 1999a; Chaffin and Stouffer, 1999). Animals were assigned randomly to receive no ovulatory stimulus, or 1000 IU rHCG (single injection i.m.; Laboratoires Serono SA, Aubonne, Switzerland) to initiate peri-ovulatory events. Pre-ovulatory follicles (4–7 mm) were aspirated using a 22 gauge needle during laparotomy of anaesthetized animals either the morning after the last LH/FSH treatment (0 h) or 12, 24 or 36 h following administration of 1000 IU rHCG (n = 3–5 monkeys/time-point). An additional group of monkeys (n = 3/time-point) was stimulated in an identical fashion, but also received the 3ß-HSD inhibitor Trilostane (TRL; Sanofi Research Division, Malvern, PA, USA) orally [1 g in 8 ml orange Kool-Aid; Kraft General Foods Inc, White Plains, NY, USA containing 1% (w/v) gum tragacanth; Sigma] beginning 4 h prior to HCG administration and for every 12 h thereafter until the time of follicular aspiration. A third group of animals (n = 3/time-point) received TRL plus the non-metabolizable progestin R5020 (Promegestrone; DuPont/NEN; Boston, MA, USA, 2.5 mg in sesame oil, s.c., once daily starting at the time of HCG). Follicles from the TRL and TRL+R5020 groups were aspirated only at 12 and 36 h post-HCG, representing respectively, the time-point when follicular fluid progesterone is substantially increased and just prior to follicular rupture (Weick et al., 1973, Hoff et al., 1983, Chaffin et al., 1999a).
Pigtail macaques being utilized for unrelated experiments underwent controlled ovarian stimulation as detailed above. Whole ovaries (n = 2/time-point) were removed during laparotomy as described above before (0 h), 3, 6, 24, or 36 h following the ovulatory HCG (1500 IU) bolus, and snap-frozen on dry ice for subsequent RNA isolation.
Daily blood samples were obtained from unanaesthetized animals by saphenous venipuncture from the beginning of gonadotrophin treatment. Serum oestradiol and progesterone concentrations were determined using specific radioimmunoassays, and follicular growth was monitored using serum steroid concentrations and ultrasonography performed on day 6–7 of stimulation (Wolf et al., 1996). The steroid milieu in follicular fluid collected from rhesus monkeys during the peri-ovulatory interval with or without TRL ± R5020 was reported by this laboratory (Chaffin et al., 1999a; Chaffin and Stouffer, 1999). Published data indicate marked depletion of progesterone and oestradiol at both 12 and 36 h after TRL administration. In addition, we reported that administration of R5020 at concentrations equivalent to the current study were sufficient to restore ovulation and luteinization in TRL-treated monkeys (Hibbert et al., 1996). Serum concentrations of bioactive LH were determined for the 3 days prior to and including the day of follicle aspiration using an in-vitro mouse Leydig cell bioassay (Chaffin et al., 1999a); the results confirm the absence of an endogenous LH surge.
Follicle aspiration and granulosa cell preparation
Granulosa cells were obtained by follicle aspiration during laparotomy of anaesthetized animals. Cells were removed from follicular fluids by centrifugation at 277 g for 15 min (4°C), the pellet resuspended in Tyrode's albumin lactate pyruvate (TALP)–HEPES. Oocytes were removed for use in other studies, and the remaining aspirate was enriched for granulosa cells as follows. Cells were centrifuged at 190 g (10 min, 4°C) and resuspended in Ham's F-10 medium (Life Technologies, Grand Island, NY, USA). The resuspension was layered onto a gradient of 40% Percoll (Sigma Chemical, St Louis, MO, USA) and 60% Hank's balanced salt solution (HBSS) with 0.1% bovine serum albumin and centrifuged at 470 g for 30 min at 4°C. The resulting layer of granulosa cells was resuspended in Ham's F-10 media, cell numbers were determined using a haemacytometer, and cell viability was assessed by Trypan Blue exclusion.
Total RNA isolation and reverse transcription–polymerase chain reaction (RT–PCR)
In order to maximize the amount of information obtainable from limited numbers of granulosa cells, an RT–PCR assay was employed. Total RNA was isolated from 104–105 granulosa cells using the Trizol reagent (BRL, Gaithersburg, MD, USA) as per manufacturer's instructions. Total RNA from whole ovaries was isolated using the guanidinium–acid phenol method as detailed previously (Dissen et al., 1996). Quality and quantity of RNA was determined by electrophoresis of samples against known concentrations of total ovarian RNA in a 2% agarose gel stained with ethidium bromide (granulosa cells) or spectrophotometry (whole ovarian RNA). RNA (0.5–1.0 µg in 10 µl) was treated with RNase-free DNase I (BRL, Gaithersburg, MD, USA) for 15 min at room temperature to remove contaminating genomic DNA. Reverse transcription and PCR were performed as previously described (Chaffin and Stouffer, 1999), and the concentration of MgCl2, primers, cDNA, and the number of cycles were determined empirically as part of the validation process. Temperature profile of the PCR was denaturation at 94°C for 30 s, annealing at 60°C for 1 min, and extension at 72°C for 1 min. Oligonucleotides used for PCR were synthesized by the Molecular Biology Core Laboratory at the Oregon Regional Primate Research Center. Table I lists the primer sequences with the corresponding optimal MgCl2 concentration and number of cycles. Aliquots of each PCR reaction (20 µl) were electrophoresed through a 2% agarose gel stained with 0.1 µg/ml ethidium bromide. Gels were visualized on a UV transilluminator and photographed using 667 Polaroid film. The photographs were scanned using an Afga flatbed scanner, and densitometry was performed using NIH image analysis. All values were normalized to the internal standard ß2-microglobulin (ß2MG; Chaffin and Stouffer, 1999); no apparent changes were observed in granulosa cell expression for ß2MG between time-points following administration of HCG. In order to conserve limited samples, RNA from TRL+R5020-treated animals was not assayed unless significant differences were observed between control and TRL groups within a time-point.
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Validation of the RT–PCR assay was performed using RNA from granulosa cells aspirated 27 h following HCG during routine in-vitro fertilization (IVF) laparoscopy (Wolf et al., 1996
; data not shown). In brief, the amount of co-amplified product for experimental and internal standard primer sets was linear and parallel with increasing amount of cDNA, and both sets of primers were in the exponentially increasing phase relative to the number of cycles. In order to control for inter-assay variability, total RNA from granulosa cells of three monkeys was combined and reverse transcribed as described to form a pool that was amplified in triplicate during each PCR with the appropriate set of primers. Intra-assay variability calculated using the triplicate pool samples was typically <15%. Sequence analysis by the Molecular Biology Core Laboratory (ORPRC) confirmed the identity of the PCR products. Because data for each set of primers was collected in 2–3 rounds of PCR reactions, the pool triplicates were also used to normalize data between reactions.
Statistical analysis
In order to test for heterogeneity of variance, data were subjected to a Bartlett's 
2 test, and subsequently transformed (to log+2) prior to analyses. Control values at various time-points were analysed by one-way analysis of variance (ANOVA), followed by Newman–Keuls test for comparison between means. Because TRL and TRL+R5020 data were collected at only 12 and 36 h post-HCG, separate comparisons were made between treatments within a time-point. Data from control and TRL groups were compared using unpaired t-tests. When significant differences were observed between control and TRL groups, data from the TRL+R5020 group was included and a one-way ANOVA followed by Newman–Keuls test was performed. P < 0.05 was considered to be significant and values are presented as mean ± SEM.
Results
Cholesterol utilization
Figure 1 depicts changes in LDL-R mRNA values in granulosa cells aspirated from peri-ovulatory follicles. LDL-R mRNA was detected by RT–PCR in all samples prior to the HCG bolus, and increased 4-fold by 12 h post-HCG. LDL-R mRNA subsequently declined at 24 h to values equivalent to those at 0 h and remained at this value 36 h post-HCG. TRL treatment increased LDL-R mRNA 3-fold at 36 h (but not 12 h) post-HCG; co-administration of TRL and R5020 returned LDL-R mRNA to 36 h baseline values. In contrast, StAR mRNA was not detectable until a marked increase was observed 12 h after HCG (P < 0.05; Figure 2). StAR mRNA was again undetectable 24 h post-HCG, and at 36 h was present at low concentrations compared with 12 h (P < 0.05). StAR mRNA values tended (P = 0.06) to be reduced by TRL 12 h after HCG, but was not affected at 36 h (Figure 2).
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Steroidogenic enzymes
P450scc mRNA values were highest prior to HCG, and decreased 2.4-fold by 12 h (P < 0.05) and 20-fold by 24 h post-HCG (P < 0.05 versus 0 h; Figure 3
). By 36 h post-HCG, P450scc mRNA was equivalent to 12 h values. Steroid depletion did not alter P450scc mRNA values at 12 or 36 h after HCG administration.
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3ß-HSD mRNA was present in one out of three granulosa cell samples prior to HCG administration, but increased 17-fold within 12 h (P < 0.05; Figure 4
). At 24 h post-HCG, 3ß-HSD mRNA was intermediate between 0 and 12 h (P < 0.05 versus 0 and 12 h), and at 36 h, was not different from 12 or 24 h samples. 3ß-HSD mRNA was not affected by steroid depletion at either time-point examined.
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Based on the two-cell theory for oestrogen production by the follicle, we postulated that macaque granulosa cells from pre-ovulatory follicles would contain little P450c17 mRNA, but appreciable P450arom mRNA. Figure 5a
(inset) demonstrates that only very low values of P450c17 mRNA were detectable in granulosa cell preparations. In order to evaluate further any changes to P450c17 mRNA during the peri-ovulatory interval, RNA was extracted from whole ovaries obtained from macaques undergoing controlled ovarian stimulation. Values of ovarian P450c17 mRNA were highest prior to HCG, and progressively decreased during the 36 h following the ovulatory stimulus (Figure 5b). In contrast, P450arom mRNA was easily detectable in granulosa cell preparations; values were highest prior to HCG administration (0 h), and then declined in a time-dependent fashion until 24–36 h after HCG (P < 0.05; Figure 6). Trilostane treatment increased granulosa cell P450arom mRNA values at 36 h, but not 12 h post-HCG, and R5020 did not reverse this effect.
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Discussion
This study details changes in mRNAs for enzymes associated with cholesterol utilization (LDL-R, StAR) and steroidogenesis (P450scc, 3ß-HSD, P450arom, P450c17) in granulosa cells of peri-ovulatory follicles from monkeys undergoing controlled ovarian stimulation. Three distinct patterns of mRNA expression were observed following administration of an ovulatory stimulus: (i) a transient increase (LDL-R, StAR); (ii) a progressive decrease with time (P450scc, P450arom); and (iii) a sustained increase (3ß-HSD). Although little P450c17 mRNA expression was detected in granulosa cells, amounts declined progressively in whole ovaries after HCG injection. Whereas all of these mRNAs change in a gonadotrophin-dependent manner, only LDL-R, P450arom, and possibly StAR mRNAs appear regulated by steroids during the peri-ovulatory interval (steroid regulation of P450c17 was not investigated in the current study).
LDL-R mRNA in granulosa cells increased transiently 12 h after administering HCG, declining to pre-exposure concentrations by 24–36 h. The function of this transient increase during the peri-ovulatory interval is not known, although it may reflect a lack of receptor-mediated LDL usage at later points during the peri-ovulatory interval. In recent studies, acute (2 h) incubations of macaque granulosa cells obtained before or after HCG adminstration determined that these cells do not utilize LDL as a source of cholesterol for progesterone production (Chaffin et al., 1999a). However, luteinizing granulosa cells from monkeys and women that are cultured for 
24 h reportedly utilize LDL as a substrate for progesterone production, mediated via increased LDL-R expression (Golos and Strauss, 1987; Brannian et al., 1992; Reaven et al., 1995; Murata et al., 1998). These data support the hypothesis that LDL is an important substrate for steroidogenesis in the developing corpus luteum rather than in peri-ovulatory granulosa cells (Bramley et al., 1987; Reaven et al., 1994; Sanders and Stouffer, 1995). Interestingly, the progesterone synthesis inhibitor trilostane prevented, and exogenous progestin (R5020) restored, the decline in LDL-R gene expression near the time of ovulation. The expression of progesterone receptor (PR) mRNA 12 h after HCG administration along with sustained high intrafollicular concentrations of progesterone (Chaffin et al., 1999b) suggest that this steroid plays a role in the transient expression of LDL-R mRNA during the peri-ovulatory interval. The relationship between progesterone and LDL-R expression may represent a a local negative feedback loop by which progesterone regulates its own synthesis between 12–36 h post-HCG.
The expression of StAR mRNA in granulosa cells was also transient following the ovulatory stimulus. In human, equine, and rat follicles, StAR increases in response to the LH surge/cAMP (Sandhoff and McLean, 1996; Pollack et al., 1997; Ronen-Fuhrmann et al., 1998; Kerban et al., 1999; reviewed in Reinhart et al., 1999), supporting a role for this gene in the enhanced steroidogenesis that occurs during the peri-ovulatory interval. Through the use of acute incubations of granulosa cells, we have demonstrated that the conversion of exogenous cholesterol to progesterone (e.g. StAR activity) is not clearly increased during the peri-ovulatory interval (Chaffin et al., 1999a), although increased StAR activity during this time period may be masked by the overall rise in steroidogenic activity. Although there is no evidence to date that progesterone regulates StAR, steroid depletion tended to reduce StAR mRNA by macaque granulosa cells during the early stages of the peri-ovulatory interval. Other steroids, e.g. oestrogen, reportedly stimulate StAR expression in the rabbit corpus luteum (Townson et al., 1996); thus further studies are needed to determine if an important role exists for steroids in regulating StAR expression and activity during the peri-ovulatory interval.
The expression of mRNA encoding P450scc decreased in granulosa cells between 0–24 h and then increased at 36 h post-HCG to a value equivalent with 12 h values. In contrast, we reported recently that progesterone concentrations in follicular fluid are dramatically elevated within 12 h of HCG, and granulosa cell P450scc activity, as measured by in-vitro conversion of a soluble cholesterol analogue to progesterone, increases in this same time frame (Chaffin et al., 1999a). Thus, changes in P450scc mRNA and activities are divergent in monkey granulosa cells following the ovulatory stimulus. Notably, thecal and granulosa cell P450scc mRNA declines following an ovulatory stimulus in bovine and porcine ovaries (Voss and Fortune, 1993a; Guthrie et al., 1994), but increases following HCG in rats (Oonk et al., 1989; Ronen-Fuhrmann et al., 1998). These species differences may relate either to the length of the peri-ovulatory interval or the presence of a functional corpus luteum. In primates, expression of P450scc in the corpus luteum is gonadotrophin-dependent, while in rats, P450scc expression is independent of gonadotrophins by 5–6 h after HCG (Oonk et al., 1989; Ravindranath et al., 1992; Yong et al., 1994). Thus, it is possible that changing values of P450scc mRNA parallel granulosa cell responsiveness to gonadotrophins (e.g. Segaloff et al., 1990), although we cannot exclude the possibility that HCG down-regulates P450scc in the primate follicle. Furthermore, P450scc is not a steroid-regulated gene during the peri-ovulatory interval in macaques.
In contrast to P450scc mRNA, values of 3ß-HSD mRNA increase dramatically following an ovulatory HCG bolus. However, we have recently demonstrated that 3ß-HSD activity in granulosa cells, measured by conversion of exogenous pregnenolone to progesterone during acute incubations of granulosa cells obtained before or after an ovulatory stimulus, does not change following HCG (Sasano et al., 1990; Chaffin et al., 1999a). Other reports on the ovine and human (Juengel et al., 1998; Duncan et al., 1999) corpus luteum indicate an uncoupling of 3ß-HSD message and protein/activity. Further, despite the fact that progesterone increases following an ovulatory stimulus in all species yet examined, 3ß-HSD mRNA values drop in bovine follicles (Voss and Fortune, 1993a). The fact that only one of three samples expressed 3ß-HSD mRNA prior to HCG may indicate differences between mural and antral granulosa cells, as cells along the antrum are more likely to be collected during aspiration (Tabarowski and Szoltys, 1987). Thus, the regulation of 3ß-HSD is complex and occurs at multiple levels, and is most likely not the sole determinant for the peri-ovulatory rise in progesterone (Chaffin et al., 1999a).
A variety of reports have either supported or refuted the hypothesis that progesterone regulates 3ß-HSD activity. These results can be placed into two groups depending on the method of progesterone signal modulation. The first group, using PR antagonists, reported reduced values of 3ß-HSD (Dimattina et al., 1986; Tanaka et al., 1993; Donath et al., 1997). The next group depleted progesterone using synthesis inhibitors in vivo, and did not find effects on 3ß-HSD, either activity or mRNA (Espey et al., 1990; current study). Thus, either progesterone synthesis inhibitors are less efficacious in reducing the progesterone signal than receptor antagonists (which is unlikely, as in all cases, synthesis inhibitors blocked ovulation, which was restored by progestin administration), or PR antagonists, e.g. RU486, may be capable of receptor-mediated action to down-regulate 3ß-HSD (Meyer et al., 1990; Rothchild, 1996).
P450c17 mRNA is present at very low to undetectable values in granulosa cells before or after an ovulatory stimulus. However, when whole ovaries were tested, mRNA concentrations were highly expressed, presumably by theca–interstitial cells (Sanders and Stouffer, 1997). The administration of HCG results in a time-dependent decrease in P450c17 mRNA values that begins within 3 h. Intrafollicular concentrations of androstenedione (Chaffin et al., 1999a) and dihydroepiandrostenedione (DHEA; unpublished data) increase 12 h after HCG, and fall to 0 h values thereafter. Thus, decreasing concentrations of P450c17 may lead to the reduced concentration of follicular androgens and consequently reduced substrate for oestrogen synthesis during the peri-ovulatory interval. A number of other reports have described decreased P450c17 in response to an ovulatory stimulus in the bovine (Voss and Fortune, 1993b; Conley et al., 1995), porcine (Guthrie et al., 1994), hamster (Johnson, 1987), and rat (e.g. Tsafriri and Eckstein, 1983; Ronen-Fuhrmann et al., 1998) ovary. While steroid depletion and replacement was not undertaken in the study that provided whole ovaries, there are reports that progestins inhibit C17,20-lyase activity in vitro (Mahajan and Samuels, 1975), and regulates 17-hydroxylase activity (Johnson, 1987). Oestrogens are also reported to inhibit P450c17 activity in rats (Tsafriri and Eckstein, 1983; Johnson and Crane, 1995). Both of these regulatory pathways are plausible and whether they participate in the peri-ovulatory decrease in P450c17 mRNA in the monkey ovary remains to be determined.
Similar to P450c17 mRNA in theca–interstitial tissue, P450arom mRNA values in granulosa cells were high prior to the ovulatory stimulus. Consistent with other species, aromatase mRNA concentrations in monkey granulosa cells declined in a time-dependent manner after exposure to an ovulatory stimulus (Voss and Fortune, 1993b; Fitzpatrick et al., 1997; Ronen-Fuhrmann et al., 1998). Within the follicle, concentrations of 17ß-oestradiol transiently increase following HCG administration before dropping to near pre-ovulatory concentrations as the time of ovulation approaches (Chaffin et al., 1999a). However, in both primate and hamster granulosa cells, aromatase activity following an ovulatory stimulus does not change, indicating that the concentration of peri-ovulatory oestrogen is dependent upon the amount of aromatizable androgen (Johnson, 1987; Chaffin et al., 1999a).
P450arom mRNA values were increased following steroid depletion 36 h post-HCG. Since replacement with the non-metabolizable progestin R5020 did not return aromatase to control (i.e. HCG only) concentrations, we postulate that either androgen, oestrogen, or perhaps glucocorticoids (Tetsuka et al., 1999), exert a suppressive effect on aromatase near the time of follicle rupture. While the presence of oestrogen receptor (ER) protein in primate granulosa cells remains equivocal, we have recently reported that both ER and ß, as well as androgen receptor, mRNA are present in late peri-ovulatory granulosa cells (Hild-Petito et al., 1991; Chaffin et al., 1999b), suggesting that either of these steroids may repress aromatase expression in late peri-ovulatory granulosa cells.
In summary, this study characterizes the expression patterns of enzymes associated with cholesterol metabolism (LDL-R, StAR) and steroidogenesis (P450scc, 3ß-HSD, P450arom) in granulosa cells or whole ovaries (P450c17) before, and up to 36 h after the administration of an ovulatory bolus of HCG. The patterns of expression support the conversion of the follicle from oestrogen/androgen to progesterone production, and suggest that the ratio of 3ß-HSD:P450c17 changes in favour of progesterone synthesis (Conley and Bird, 1997). Steroid depletion and progestin replacement do not support a major role of steroids in pathways regulating steroidogenic enzyme expression, but may regulate cholesterol uptake and utilization, especially during the late phase of the peri-ovulatory interval. Additional studies of thecal cell steroidogenesis are warranted to further clarify the dynamics of peri-ovulatory steroidogenesis in the primate follicle.
Acknowledgments
The authors appreciate the expert service provided by the Division of Animal Resources and the surgical team of Dr John Fanton, the Endocrine Services Core Laboratory, the Assisted Reproductive Technology Core, and the Molecular Biology Core Laboratory. Recombinant human LH, FSH, CG, and Antide were generously provided by Ares Advanced Technology, Inc., a member of the Ares-Serono Group. Trilostane was graciously supplied by Sanofi Pharmaceutical Inc, Great Valley, Malvern, PA, USA. This work was supported by NIH HD20869 (RLS), RR-00163, HD-18185, HD-8302 (CLC), HD-24870 (GAD).
Notes
4 To whom correspondence should be addressed at: Oregon Regional Primate Research Center, 505 NW 185th Ave., Beaverton, OR 97006, USA 
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Submitted on June 30, 1999; accepted on October 4, 1999.
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