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Department of Physiology and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
Address all correspondence and requests for reprints to: Anthony J. Zeleznik, Ph.D., 830 Scaife Hall, University of Pittsburgh School of Medicine, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261. E-mail: zeleznik{at}pitt.edu.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dihydrotestosterone (DHT) leads to follicular atresia and alterations in the cell cycle of granulosa cells (4, 5). Moreover, administration of an androgen antagonist amplifies the stimulatory effects of FSH-human chorionic gonadotropin (hCG) treatment in hypophysectomized rats, indicating that endogenous androgens are antagonistic to gonadotropin-stimulated ovarian function as well (6). To date, most of our information regarding the actions of androgens on the ovary has been obtained using rodents, and the extent to which these results can be extrapolated to the primate is uncertain. Recently, a series of studies in macaques has suggested that androgens appear to be positive regulators of follicular development, as treatment of rhesus monkeys with T or DHT increases the abundance of mRNA for the FSH receptor in granulosa cells and increases the growth of small follicles (7, 8). However, whether androgens influence the responsiveness of ovaries to gonadotropins in vivo in primates remains to be determined. A limitation in identifying the physiological effects of steroids on ovarian function in vivo is the fact that treatment with steroid hormones results in feedback inhibition of FSH and LH secretion with a concomitant diminution in ovarian function. To circumvent this problem, we developed a gonadotropin clamp model in rhesus monkeys for investigation of the actions of potential autocrine and paracrine agents on ovarian function. In this model, potential effects on gonadotropin secretion are eliminated by the pulsatile infusion of exogenous gonadotropins (9). Herein we report on the use of this model system to explore the effects of androgens (DHT and T) on gonadotropin-dependent steroid production in rhesus monkeys.
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Materials and Methods |
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Adult female rhesus monkeys (Macaca mulatta), 5–8 kg in body weight, with normal menstrual cycle histories were used in this study and were housed under standard husbandry conditions at the University of Pittsburgh Primate Research Laboratory. All experimental procedures were approved by the University of Pittsburgh institutional animal care and use committee. Each animal was equipped with two catheters inserted into the jugular or femoral veins that were exteriorized through a small incision in the scapular region. The catheters were protected by a vest and a flexible stainless steel cable and were connected to a three-channel swivel device (Spalding Medical Products, Arroyo Grand, CA) mounted to the roof of the cage.
Experimental design
On the day of catheterization, each animal began receiving im injections of a GnRH receptor antagonist (acyline; 60 µg/kg in 5% mannitol) every 2 d until the completion of the study to suppress endogenous gonadotropin secretion. Acyline was synthesized by Bioqual (Rockville, MD) and was provided by the Contraception and Reproductive Health Branch, Center for Population Research, NICHHD, NIH. Seven days later, by which time ovarian suppression was achieved (estradiol, <50 pg/ml; progesterone, <200 pg/ml), animals (three per group) were administered DHT, T, or saline. For DHT administration, each animal received an sc implant of a DHT-containing pellet (Innovative Research of America, Sarasota, FL), which was designed to deliver 25 mg DHT over a period of 21 d, yielding an average DHT dose of 180 µg/kg·d. T was administered by the sc placement of 10 5-cm-long capsules made from SILASTIC brand medical grade tubing (Dow Corning, Midland, MI; inner diameter, 0.132 in.; outer diameter, 0.183 in.) containing crystalline T (Sigma-Aldrich Corp., St. Louis, MO). Resultant serum concentrations of DHT and T were verified by RIA. Control animals (n = 3) received an iv infusion of saline as described previously (9).
An intermittent infusion of human (h) FSH and hLH at a frequency of one 3-min pulse/h was initiated 5 d after the initiation of DHT, T, or saline treatment. The hFSH (AFP-8792B; 1685 IU/mg) and hLH (AFP 7572B; 4500 IU/mg) were provided by Dr. A. Parlow and the National Hormone and Pituitary Program, NIDDK, NIH. Stock solutions were diluted into saline containing 0.1% rhesus monkey serum, 100 U/ml penicillin, and 100 µg/ml streptomycin and were infused in a total volume of 0.5 ml for each 3-min pulse. The hLH infusion was set at a dosage of 75 ng/kg·pulse, which was estimated to produce a serum LH concentration of 6–8 mIU/ml (9). The hFSH infusion was set at 54 ng/kg·pulse, which was estimated to produce a serum FSH concentration of 7.5 mIU/ml (9). Approximately 48 h after initiating the gonadotropin infusion, serum concentrations of hFSH and hLH were determined by RIA, and if necessary, the amounts of FSH and LH administered were adjusted by direct proportion to achieve the desired serum concentrations. Thereafter, the amount of LH delivered per pulse remained constant throughout the entire 15-d treatment interval. The amount of FSH delivered per pulse was adjusted every 3 d in an attempt to produce stepwise elevations in serum FSH concentrations of 7.5, 10, 12.5, 15, and 17.5 mIU/ml over the 15-d treatment interval. On the fifth day of gonadotropin infusion, the animals were anesthetized with 2% isoflurane, and the left ovaries were removed through a midventral incision, weighed, and frozen. On the final day of gonadotropin infusion, the right ovaries were removed, weighed, and frozen.
RIAs
Serum levels of hFSH, hLH, estradiol, and androstenedione were measured by kits purchased from Diagnostic Products Corp. (Los Angeles, CA). DHT was measured with a kit purchased from Diagnostic Systems Laboratories (Webster, TX). Progesterone and T were measured as described previously (10, 11). The interassay coefficients of variation were as follows: DHT, 11.71% (two assays); androstenedione, 3.7% (two assays); estradiol, 1.4% (two assays); and FSH, 8.1% (four assays). All T samples were analyzed in a single assay with a 7.2% intraassay coefficient of variation.
Statistics
Serum levels of estradiol and androstenedione during the interval of gonadotropin infusions were analyzed for statistical differences using ANOVA with repeated measures (StatView version 4.5, Abacus Concepts, Inc., Berkeley, CA). Data from control animals used in the statistical analysis were reported previously (9). Each of the control animals received the same preparations of hFSH and hLH as the T- and DHT-treated animals.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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illustrates serum DHT and T concentrations in monkeys that received either sc DHT-containing pellets or T-filled SILASTIC brand capsules. DHT levels were below the limits of detectability before the insertion of DHT pellets in the experiment group. Upon insertion of DHT pellets, serum DHT concentrations rose to approximately 30 ng/ml (70 nM) and gradually declined over the course of the study. In animals that received T capsules, T levels were below the limit of detectability before the insertion of T capsules. Upon insertion of T capsules, serum concentrations rose to approximately 30–40 ng/ml (150 nM) and remained elevated throughout the remainder of the study.
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Figure 2 (top panel) illustrates serum FSH and LH concentrations in control animals and in animals that received DHT. As reported previously (9), serum FSH levels in control animals exhibited a stepwise elevation in response to the stepwise increments in FSH infusion dosage. Our attempt to reproduce this exact pattern of FSH concentrations in DHT-treated animals failed. Instead of producing stepwise increments in FSH concentrations during d 3–9 of infusion, FSH concentrations were greater than those in control animals. Thereafter (d 10–15), FSH levels in DHT-treated animals were similar to those in controls. LH concentrations in DHT-treated animals were slightly greater than those in control animals (Fig. 2, center panel). Despite being exposed to slightly greater levels of FSH and LH, estrogen production by animals treated with DHT were significantly reduced (P < 0.01) compared with the control group throughout the entire gonadotropin infusion period (Fig. 2, lower panel). Serum progesterone concentrations were less than 0.2 ng/ml before and continuing throughout the FSH and LH infusion.
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Three animals were treated with T before and during gonadotropin infusion. Data from individual animals are presented because of differences in their responses. Figure 3 shows the results for animal 2992. Serum FSH concentrations (top panel) and LH concentrations (center panel) were slightly greater than those in control animals during the first 6 d of gonadotropin treatment and thereafter were similar to those in control animals. As shown in the lower panel, this animal failed to produce estrogen in response to the exogenously administered FSH and LH. Serum progesterone concentrations were less than 0.2 ng/ml before and continuing throughout the FSH and LH infusion.
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illustrates results from animal 2993. As with the other animals of this study, serum FSH and LH concentrations were slightly greater than those in the control animals during the first 6 d of gonadotropin treatment and thereafter were similar to those in the control animals. As shown in the lower panel of Fig. 4, estrogen production in response to the gonadotropin infusion was similar to that in the control animals. Serum progesterone concentrations were less than 0.2 ng/ml before and continuing throughout the FSH and LH infusion.
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illustrates results from animal 2995. As with the other animals of this study, serum FSH and LH concentrations were slightly greater than those in the control animals during the first 6 d of gonadotropin treatment and, as shown in the lower panel, these concentrations of FSH and LH stimulated estrogen production comparable to levels seen in the control animals. On d 6 of gonadotropin infusion, the infusion rate of FSH and LH was reduced in an attempt to reduce gonadotropin levels similar to those in control animals. However, this led to an acute decline in estrogen production. When FSH and LH were subsequently increased on d 10, estrogen production recommenced. Serum progesterone concentrations were less than 0.2 ng/ml before and continuing throughout the FSH and LH infusion.
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Serum androstenedione levels before and during gonadotropin infusion were similar in control and DHT-treated animals throughout the duration of the studies (Fig. 6). In T-treated animals, androstenedione levels rose upon insertion of T capsules and remained elevated throughout the remainder of the study. Infusion of FSH and LH did not further increase the apparent conversion of T to androstenedione in these animals.
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One ovary was collected and weighed from each animal on the fifth day of FSH and LH infusion, and the remaining ovary was collected and weighed on the final day (d 15) of gonadotropin infusion to provide indexes of the early responses (d 5) and later responses (d 15) of gonadotropic stimulation. Ovarian weights on d 5 did not differ among the three treatment groups (control, 588 ± 185 mg; DHT, 490 ± 154 mg; T, 395 ± 118 mg). Ovarian weights on the final day of gonadotropin treatment from DHT-treated animals were significantly less (P < 0.05) than those of control animals (763 ± 268 vs. 1915 ± 159 mg), whereas the weights of ovaries of T-treated animals did not differ (P > 0.05) from those of controls (1611 ± 1067 vs. 1915 ± 159 mg).
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Discussion |
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Given the uncertainty regarding the actions of androgens on the ovary, our goal was to identify the effects of androgens on the responsiveness of the primate ovary to FSH and LH in vivo. To the best of our knowledge, this is the first report of the effects of chronic administration of androgens on gonadotropin-stimulated ovarian function in primates. The most significant finding is that we found no evidence for enhanced estrogen production in response to a pulsatile infusion of FSH and LH by either DHT or T. In these studies we achieved levels of DHT (5 x 10-8 M) and T (1.5 x 10-7 M) in the peripheral circulation and presumably within the follicle. These concentrations of T and DHT have been previously shown by Harlow et al. (13) to be effective with cultured marmoset granulosa cells in amplifying the ability of FSH to stimulate estrogen and progesterone production. The level of T achieved in our infusions was comparable to that in human follicular fluid (1–3 x 10-7M) (14, 15), whereas the level of DHT in our studies was slightly higher than that in normal human follicular fluid (1–2 x 10-8 M) (16) and was comparable to that previously shown to stimulate the growth of preantral follicles and the expression of mRNA for IGF-I and the IGF-I receptor in monkeys (8, 17).
In contrast to observing a stimulatory effect on ovarian function, our findings clearly show that DHT exerted a pronounced suppressive effect on gonadotropin-stimulated estradiol production and the augmentation of ovarian weight in the rhesus monkey in response to exogenously administered FSH and LH. This finding is consistent with previous in vivo studies in rodents that androgens are inhibitory to gonadotropin-stimulated follicular development (4, 5, 6). Our results also indicate that the effects of DHT and T were not identical, because treatment of monkeys with T interfered with gonadotropin-stimulated estradiol production in only one of three animals, whereas the other T-treated animals responded to exogenous FSH and LH in a manner similar to that of saline-treated control animals. It is possible that there is a threshold effect of androgens on follicular function such that antagonistic actions of androgens may be manifested at elevated concentrations, and the differences in responses between DHT and T may reflect the fact that DHT has a greater affinity than T for the androgen receptor, which, in turn, results in enhanced sensitivity to DHT (18). The concept of an androgen threshold is also supported by in vitro studies of rat granulosa cells. In this regard, culture of granulosa cells with 90 ng/ml DHT suppresses FSH-stimulated cyclin D2 mRNA levels (5), whereas culture of granulosa cells with 30 ng/ml T does not (3). It is also possible that there may be subsets of genes that are differentially regulated by DHT and T (19), or that metabolism of DHT and T by target tissues may differ, which could differentially alter intracellular concentrations of DHT and T or hypothetically result in the differential production of biologically active androgen metabolites (20).
The mechanisms by which DHT inhibits FSH-stimulated estrogen secretion in vivo is not known. 5-Reduced androgens are known to competitively inhibit aromatase activity of human and rat granulosa cells in vitro (12, 21). In those studies the 5-androstenedione was shown to be a more potent inhibitor than DHT when either androstenedione or T was used as a substrate for aromatase. When both androstenedione and T were combined as substrates, 10-5 M DHT did not reduce the aromatase activity of human granulosa cells (12). In our current studies the plasma DHT level was approximately 5 x 10-8 M, well below the concentration of DHT necessary to inhibit aromatase activity in in vitro studies. Therefore, although we are unable to completely rule this out, it is highly unlikely that the suppression of FSH-stimulated estrogen production was due to a direct inhibition of aromatase by DHT. In further support of this, serum concentrations of androstenedione, the primary substrate for aromatase, were not increased in DHT-treated animals, and others have shown that the administration of an aromatase inhibitor rapidly elevates peripheral androstenedione concentrations in monkeys (22).
There are a number of physiological implications of our current study. First, these studies provide pertinent information regarding the relationship between hyperandrogenism and anovulation. Agarwal et al. (12) demonstrated that 5-reduced androgens function as competitive antagonists of aromatase in human granulosa cells, and that 5-androstenedione concentrations are elevated in follicular fluids from women with PCOS. Based upon this observation, these researchers proposed that intrafollicular 5-reduced androgens may interfere with FSH-stimulated follicular development by inhibiting aromatase. Our current findings demonstrate that DHT significantly reduced estrogen production in response to infusion of exogenous FSH and LH, consistent with the idea that a high androgenic microenvironment in PCOS may be causal to anovulation. However, as noted above, the lack of estrogen production in DHT-treated animals in our current study did not appear to be due to a direct inhibition of aromatase. Rather, it appears from our current data that DHT treatment caused ovarian resistance to FSH and LH, as concentrations of these gonadotropins that were effective in stimulating estrogen production and increasing ovarian weight in control animals were ineffective in DHT-treated animals. However, unlike what we observed in DHT-treated animals, it does not appear that follicles in women with PCOS are resistant to FSH, as ovarian responsiveness to exogenous FSH stimulation, as reflected by increments in follicle size and systemic estrogen levels, appears to be either normal or exaggerated in PCOS (23, 24, 25).
In regard to the normal menstrual cycle, recent studies in humans have indicated that LH may play an important role in the selection of the dominant follicle during the mid to late follicular phase of the menstrual cycle by protecting the maturing follicle from its self-imposed reduction in FSH secretion (26). Subsequent studies demonstrated that administration of LH or low doses of hCG also reduced the number of smaller follicles, which has led to an additional suggestion that LH may promote monoovulation by both reducing the number of smaller follicles and maintaining the growth of the lead follicle (27). These observations are reminiscent of earlier studies in rodents in which it was demonstrated that low doses of hCG or LH reduced ovarian weights and that this inhibition was overridden by treatment with antiandrogens (28). Indeed, McNatty et al. (29) demonstrated that T levels in antral fluids of small (<8-mm diameter) follicles increased during the mid and late follicular phases of the menstrual cycle in humans. This interval of the menstrual cycle is also associated with rising concentrations of LH owing to increased LH pulse frequency. It is therefore possible that the mechanism by which LH may reduce the growth of small follicles during the mid to late follicular phase of the menstrual cycle is by stimulation of androgen production. In this regard it has been proposed that there is an LH ceiling with respect to the deleterious effects of LH on follicular growth, such that elevated LH concentrations may interfere with FSH-stimulated follicular development (30). This is also supported by the findings of Williams and Hodgen (31) that administration of hCG during the late follicular phase before the spontaneous LH surge resulted in atresia of the dominant follicle and a delay in the onset of cyclic ovarian function in five of 11 monkeys. If the deleterious effects of LH/hCG on follicular function are mediated by androgens, the concept of an LH ceiling could be extended to an androgen ceiling, which would account for our current observation that T was less effective than DHT in reducing FSH responsiveness in vivo. Confirmation of this idea, however, would require a more extensive study with multiple doses of T and LH in the presence and absence of antiandrogens. Finally, our previous studies have highlighted the concept that small changes in the blood levels of FSH are of major importance for both the initiation of preovulatory folliculogenesis at the beginning of the follicular phase of the menstrual cycle as well as selection of the preovulatory follicle during the late follicular phase (32, 33). In this regard, the responses of T-treated monkey 2995 shown in Fig. 5
are of interest, because as a result of our downward adjustment of FSH concentrations, estrogen production ceased despite the fact that FSH and LH levels remained within the range that was effective in stimulating estrogen production in control animals. This raises the possibility that there could be concentration-dependent interactions between FSH and T, such that the ability of T to interfere with FSH-stimulated ovarian function may be overridden by elevated levels of FSH.
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Acknowledgments |
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Footnotes |
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Abbreviations: DHT, Dihydrotestosterone; h, human; hCG, human chorionic gonadotropin; PCOS, polycystic ovarian syndrome; T, testosterone.
Received July 24, 2003.
Accepted November 10, 2003.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-dihydrotestosterone interact differently with the androgen receptor to enhance transcription of the MMTV-CAT reporter gene. Mol Cell Endocrinol 88:15–22[CrossRef][Medline]
-reduced C19 steroids inhibit the aromatase reaction in vitro. J Endocrinol 84:409–419[Abstract]
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