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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-0691
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 12 6630-6637
Copyright © 2005 by The Endocrine Society

Adrenal Hyperandrogenism Is Induced by Fetal Androgen Excess in a Rhesus Monkey Model of Polycystic Ovary Syndrome

Rao Zhou, Ian M. Bird, Daniel A. Dumesic and David H. Abbott

National Primate Research Center (R.Z., D.A.D., D.H.A.), Department of Obstetrics and Gynecology (I.M.B., D.H.A.), and Endocrinology-Reproductive Physiology Program (R.Z., I.M.B., D.H.A.), University of Wisconsin, Madison, Wisconsin 53715; and Reproductive Medicine and Infertility Associates (D.A.D.), Woodbury, Minnesota 55125

Address all correspondence and requests for reprints to: Rao Zhou, 3009 Da Vinci Drive, Westfield, Indiana 46074. E-mail: rzhou{at}wisc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: Adrenal androgen excess is found in approximately 25–60% of women with polycystic ovary syndrome (PCOS), but the mechanisms underlying PCOS-related adrenal androgen excess are unclear.

Objective: The objective of this study was to determine whether adrenal androgen excess is manifest in a nonhuman primate model for PCOS.

Participants: Six prenatally androgenized (PA) and six control female rhesus monkeys of similar age, body weight, and body mass index were studied during d 2–6 of two menstrual cycles or anovulatory 30-d periods.

Interventions: Predexamethasone adrenal steroid levels were assessed in the first cycle (cycle 1). In a subsequent cycle (cycle 2), occurring one to three cycles after cycle 1, adrenal steroids were determined 14.5–16.0 h after an im injection of 0.5 mg/kg dexamethasone (postdexamethasone levels) and after an iv injection of 50 µg ACTH-(1–39).

Results: Both before and after dexamethasone, serum levels of dehydroepiandrosterone (DHEA) in PA females exceeded those in controls. After ACTH injection, PA females exhibited higher circulating levels of DHEA, androstenedione, and corticosterone but comparable levels of 17{alpha}-hydroxyprogesterone, cortisol, the sulfoconjugate of DHEA, and testosterone compared with controls.

Conclusion: Enhanced basal and ACTH-stimulated adrenal androgen levels in PA female monkeys may reflect up-regulation of 17,20 lyase activity in the adrenal zona reticularis, causing adrenal androgen excess comparable with that found in PCOS women with adrenal androgen excess. These findings open the possibility that PCOS adrenal hyperandrogenism may have its origins in fetal androgen excess reprogramming of adrenocortical function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
POLYCYSTIC OVARY SYNDROME (PCOS) is a heterogeneous reproductive and metabolic disorder found in 6–7% of reproductive-aged women (1, 2, 3) and represents the most common human female endocrinopathy (4, 5, 6). The PCOS accounts for 82% of women presenting with hyperandrogenism (7) and 75% of women requiring treatment for anovulatory infertility (6). Characteristics of PCOS include amenorrhea/oligomenorrhea, infertility, hyperinsulinemia from insulin resistance, LH hypersecretion, and androgen excess (8), with many of these features of PCOS worsened by coexistent obesity (4, 5, 6). The diagnosis of PCOS requires the presence of oligomenorrhea or amenorrhea together with hyperandrogenism of ovarian origin (9) or, more recently, two of three criteria that additionally include visualization of polycystic ovaries by ultrasound (10), excluding phenotypically similar but mechanistically different disorders, such as classical and nonclassical 21-hydroxylase deficiency.

Androgen excess is the most consistent defect underlying PCOS (11, 12, 13). Although the ovary is the principal source of androgen excess in PCOS women, about 25–60% of women with PCOS also demonstrate elevated levels of adrenal androgens, particularly dehydroepiandrosterone (DHEA), its sulfoconjugate (DHEAS), and androstenedione (14, 15, 16). Adrenocortico-steroidogenesis may thus provide an additional, but separate, contribution to hyperandrogenism in some PCOS women and may be an inherited, stable trait (17). Although many studies have been conducted to examine possible causes of adrenal hyperandrogenism in women with PCOS (18, 19, 20), the underlying mechanisms remain unclear.

As an established nonhuman primate model for PCOS (21, 22, 23), the prenatally androgenized (PA) female rhesus monkey provides an opportunity to examine whether exposure to androgen excess during fetal life provides a developmental origin for adrenal androgen excess in adulthood. Such female rhesus monkeys, exposed to androgen excess in utero, not only exhibit anovulation (21), enlarged multifollicular ovaries (24), and ovarian hyperandrogenism (25) but also demonstrate elevated circulating levels of DHEAS, a conjugated androgen of adrenal origin (25). Similar to 25–60% of PCOS women (14, 15, 16), androgen biosynthesis may thus be enhanced in the adrenal cortex of PA female monkeys, in addition to the hyperandrogenism demonstrated for the PA monkey ovary. Currently, acute adrenal stimulation by parenteral ACTH administration is considered as the preferred method to study adrenocortical enzymatic activities in vivo (26, 27). In this study, we use a combined dexamethasone-ACTH (Dex-ACTH) test, similar to those widely used to assess adrenal steroidogenic function in humans and animal models (27, 28), to identify profound adrenal androgen excess in PA female rhesus monkeys.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

The 12 adult female rhesus monkeys (Macaca mulatta) used in this study (February 2000 to June 2002) were captive born and were housed at the National Primate Research Center (University of Wisconsin, Madison, WI) in accordance with routine care, management, and assessment protocol (29, 30). None of the females had experienced any previous long-term, postnatal treatment (i.e. prolonged steroid therapy). The health and general behavior of all monkeys were assessed daily, and each monkey was fed once daily with a meal of 16–30 biscuits (~96–180 g) of Purina Monkey Chow (product 5038; Ralston Purina, St. Louis, MO). The meal was supplemented with either one or two pieces of fresh fruit or bread. The number of biscuits given was varied so that at least one to three biscuits were found when all remaining food in an animal’s cage was removed between 1700 and 1800 h. To provide social enrichment, eight of the 12 animals were reunited with an adult female cage mate overnight. The remaining four female monkeys (all controls) were not socially housed because of incompatibility with available cage partners. The Institutional Animal Care and Use Committee of the University of Wisconsin-Madison approved all of the procedures used in this study. Animal maintenance was in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals and Animal Welfare Act with its subsequent amendments.

As described previously, six PA female monkeys were exposed in utero to fetal male serum levels of testosterone, averaging 1–3 ng/ml (31). This fetal androgen excess was achieved when their dams received daily sc injections of 10 mg of testosterone propionate for 15–35 d, starting at d 40–44 of gestation. Body weights of the sires and dams of PA females were typical for adult rhesus monkeys (data not shown). The six control female monkeys that were unexposed to prenatal androgen excess were of similar reproductive age (control, 19.29 ± 0.98 yr; PA, 21.52 ± 0.79 yr; mean ± SEM), body weight (control, 9.16 ± 0.65 kg; PA, 9.11 ± 0.46 kg), and body mass index (control, 38.67 ± 3.08 kg/m2; PA, 39.47 ± 1.35 kg/m2) to PA female monkeys to prevent any confounds of body mass index influencing adrenal sensitivity to ACTH (32) or of age contributing to circulating levels of cortisol (33, 34), DHEA, DHEAS, or androstenedione (35, 36, 37). Because rhesus females reach menarche and menopause at about 2.5 and 26–28 yr of age, respectively (38), all female monkeys in the present study were in their mid to late reproductive years, approximately 5–7 yr before the predicted onset of menopause.

Experimental design

Predexamethasone hormone level assessment during cycle 1. Initial assessment was performed on d 2–6 (all control and three PA female monkeys) or d 8 (one PA female monkey with a follicular phase >20 d) of the menstrual cycle or on d 30 of an anovulatory cycle (two PA female monkeys). A single saphenous vein blood sample was drawn to determine basal circulating adrenal steroid levels. The sample was taken at 0700–0900 h after a 14.5–16.0 h overnight fast. Blood was subsequently centrifuged at approximately 2500 rpm for 10 min, and serum was stored at –20 C before assay.

Postdexamethasone hormone level assessment and combined Dex-ACTH testing during cycle 2. After the initial assessment, a combined Dex-ACTH test was performed during the follicular phase (d 2–6) after one to three menstrual cycles or during a period of amenorrhea greater than 30 d in duration (two PA female monkeys). Dexamethasone (0.5 mg/kg body weight; American Regent Laboratories, Shirley, NY) was given as an im injection at 1600–1700 h on the day before the ACTH infusion. At 0730–0800 h the next day, all monkeys were anesthetized with an im injection of ketamine (ketamine HCl; 15 mg/kg body weight). A venous catheter (polyethylene tubing, Intramedic TM, PE60; Becton Dickinson, Sparks, MD) was inserted through the saphenous vein, and its tip was positioned in the inferior vena cava for the entire procedure. ACTH [50 µg (~5.5 µg/kg), human ACTH-(1–39); Organon Pharmaceuticals, West Orange, NJ] was infused as a bolus through the catheter at 0 min, with blood samples (4 ml) withdrawn immediately before (i.e. postdexamethasone levels) and at 15, 30, and 60 min after ACTH infusion. The ACTH dose administered to our female monkeys was greater than that reported in other nonhuman primate studies (i.e. 10 ng/kg) (39) but was within a range of ACTH doses previously administered to adult humans (i.e. 0.1–16.2 µg/kg) (18, 40). Such supraphysiological amounts of infused ACTH are required to provide evidence of adrenal P450c17 enzyme dysregulation (18, 41).

Assay procedures

All hormones were assayed in National Primate Research Center Assay Services laboratories, as described previously (42, 43, 44). Assays for DHEA, androstenedione, testosterone, and corticosterone were performed after diethyl ether extraction of serum and solvent fraction separation by celite chromatography. 17{alpha}-Hydroxyprogesterone (45), cortisol (45), DHEA (46), DHEAS (45), androstenedione (43), and corticosterone (47) were determined using RIAs. Testosterone (42) was assayed by enzyme immunoassay. Intraassay and interassay coefficients of variation for quality control preparation (QC) values were as follows: DHEA: QC1, 13.4 ± 0.8 ng/ml, 7.7 and 9.7%, respectively; QC2, 3.4 ± 0.2 ng/ml, 6.5 and 9.9%, respectively; androstenedione: QC1, 98.7 ± 4.0 pg/ml, 4.6 and 7.0%, respectively; QC2, 14.4 ± 1.7 pg/ml, 13.75 and 20.9%, respectively; testosterone: QC1, 106.0 ± 5.8 ng/ml, 1.3 and 13.5%, respectively; QC2, 26.6 ± 1.5 ng/ml, 2.6 and 13.6%, respectively; corticosterone: QC, 458.7 ± 50.5 ng/ml, 3.4 and 19.1%, respectively; 17{alpha}-hydroxyprogesterone: QC1, 3.5 ± 0.2 ng/ml, 3.7 and 14%, respectively; QC2, 0.6 ± 0.1 ng/ml, 5.3 and 17.4%, respectively; cortisol: QC, 23.0 ± 1.9 µg/dl, 7.0 and 7.3%, respectively; and DHEAS: QC, 27.3 ± 0.8 ng/ml, 1.1 and 7.5%, respectively.

Statistical analysis

Circulating concentrations, area-under-the-curve (AUC) values, and hormone ratios were log transformed to achieve normality and homogeneity of variance and to increase linearity (48), except for those related to Dex-ACTH test corticosterone values. Differences in circulating steroid values between 0 and 60 min after an iv injection of ACTH were not transformed because of negative values. Pre- and postdexamethasone hormonal variables were compared by paired t test, whereas all Dex-ACTH test hormonal variables were analyzed using two-way ANOVA, with fetal androgen exposure and time from ACTH as independent variables. When significant (P < 0.05) statistical interactions were identified by ANOVA, post hoc univariate analyses were performed on the variables (Systat, version 5.2 for Macintosh; Systat, Evanston, IL). Dex-ACTH test serum corticosterone levels were analyzed using nonparametric statistical comparisons, because log transformation failed to normalize the data distribution. Log-transformed data, non-log-transformed data, and Dex-ACTH test serum corticosterone levels are expressed as back transformed means ± 95% confidence limits, mean ± SEM, and median ± interquartile interval, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Pre- and postdexamethasone circulating steroid levels

Serum DHEA levels were higher in PA than control female monkeys before (P < 0.03) and after (P < 0.001) dexamethasone treatment, whereas serum 17{alpha}-hydroxyprogesterone, androstenedione, testosterone, cortisol, corticosterone, and DHEAS levels were comparable between female monkey groups under similar conditions (P > 0.05) (Table 1Go). Serum levels of 17{alpha}-hydroxyprogesterone, cortisol, DHEA, and testosterone were suppressed by dexamethasone therapy in both control and PA female monkeys (17{alpha}-hydroxyprogesterone: control, P < 0.001; PA, P < 0.002; cortisol: control, P < 0.001; PA, P < 0.01; DHEA: control, P < 0.001; PA, P < 0.006; and testosterone: control, P < 0.006; PA, P < 0.004). In contrast, serum corticosterone levels were significantly suppressed by dexamethasone therapy in control (P < 0.007) but not in PA female monkeys. The ratio of corticosterone/cortisol after dexamethasone therapy (~0.01; data not shown), however, was similar in both control and PA female monkeys. Serum androstenedione and DHEAS levels did not significantly decline after dexamethasone therapy in either female monkey group, possibly due to sufficient ovarian contribution to the former and longer half-life of the latter.


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  		TABLE 1. Mean (95% confidence interval) circulating levels of steroid hormones and DHEAS in PA and control female rhesus monkeys before and after im injection of 0.5 mg/kg dexamethasone

 
Circulating steroid responses to ACTH infusion after dexamethasone therapy

Adrenocortical steroidogenic responses to ACTH stimulation reflect ACTH-induced adrenal steroid biosynthesis in nonhuman primates and are illustrated in Figs. 1Go and 2Go. Serum DHEA levels were greater (P < 0.001) in PA compared with control female monkeys throughout the Dex-ACTH test. Serum DHEA levels reached their post-ACTH maximum by 15 min (P < 0.001) in control female monkeys but continued to increase at least up to 60 min (P < 0.05) in PA female monkeys.



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  		FIG. 1. Steroid biosynthesis in the mid to inner adrenal cortical zones, zona fasciculata (Z.f.) and zona reticularis, of Old World primates and humans [modified from Conley et al. (51 ) and Pattison et al. (58 )]. Steroids in bold represent the predominant pathway for androgen biosynthesis, steroids within boxes represent the predominant pathway for cortisol biosynthesis, and underlined steroids represent the predominant pathway for corticosterone biosynthesis. The darker arrows reflect the proposed enhanced 3ß-HSD II and 17,20 lyase enzymatic function in PA female monkeys, and the lighter arrows reflect relatively low enzymatic activity in both control and PA females. 17OHP5, 17{alpha}-Hydroxypregnenelone; 17OHP4, 17{alpha}-hydroxyprogesterone; CYP11A, P450scc; CYP 17, P450c17; ST, sulfotransferase; CYP21, P450c21; CY11B1, P450c11.

 


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  		FIG. 2. Serum steroid levels in PA (filled bars) and control (open bars) adult female rhesus monkeys after 0.5 mg/kg dexamethasone im at –16 to –14.5 h and 50 mg ACTH iv at 0 min [means and upper 95% confidence limits (median and upper quartile range for corticosterone values)]. A, 17{alpha}-Hydroxyprogesterone: a, P < 0.001 vs. 0 min; b, P < 0.02 vs. 15 min. B, DHEA: A, P < 0.01 vs. control females at respective time points; B, P < 0.05 vs. control females at 30 min; c, p < 0.001 vs. 0 min; d, P < 0.005 vs. 0 min; e, P < 0.005 vs. 15 min; f, P < 0.05 vs. 30 min. C, Androstenedione: A, P < 0.03 vs. control females at respective time points; B, P < 0.001 vs. control females at 30 min; c, P < 0.05 vs. 0 min; d, P < 0.01 vs. 0 min; e, P < 0.05 vs. 15 min. D, Corticosterone: A, P < 0.05 vs. control females at respective time points; b, P < 0.05 vs. 0 min; c, P < 0.05 vs. 15 min. E, Cortisol: a, P < 0.001 vs. 0 min; b, P < 0.05 vs. 15 or 30 min. F, DHEAS: a, P < 0.01 vs. 0 min; b, P < 0.05 vs. 15 or 30 min, all females combined. G, Testosterone: a, P < 0.001 vs. 0 min; b, P < 0.03 vs. 15 min; c, P < 0.04 vs. 30 min, all females combined. Conversion to SI units: 17{alpha}-hydroxyprogesterone x 3.0257 nmol/liter; cortisol x 27.59 nmol/liter; DHEA x 3.47 nmol/liter; DHEAS x 0.02714 µmol/liter; androstenedione x 3.49 pmol/liter; corticosterone x 2.886 nmol/liter; testosterone x 3.47 nmol/liter.

 
Although both control and PA female monkeys demonstrated increased serum androstenedione levels after ACTH stimulation (P < 0.001), androstenedione levels in PA female monkeys were higher than those in control females at 15 min, 60 min (P < 0.03), and 30 min (P < 0.001) after ACTH injection. Serum androstenedione levels reached their maximum after 15 min (P < 0.05) in controls but increased until 30 min (P < 0.05) in PA female monkeys. Serum DHEAS and testosterone levels were all similarly increased after ACTH injection in both control and PA female monkeys, with all values progressively increasing until 60 min (P < 0.05–0.001) after ACTH injection (Fig. 2Go).

Serum 17{alpha}-hydroxyprogesterone levels increased after ACTH injection in both female monkey groups (control and PA, P < 0.001), with maximum elevations achieved by controls after 15 min (P < 0.001) vs. 30 min (P < 0.02) for PA female monkeys. Serum cortisol levels increased after ACTH injection in both control and PA female monkeys, demonstrating an abrupt rise at 15 min that increased again at 60 min (P < 0.05–0.001) after ACTH (Fig. 2Go). Serum corticosterone levels, however, were higher at both 30 and 60 min (P < 0.05) after ACTH injection in PA compared with control female monkeys. Furthermore, serum corticosterone levels reached their maximum levels after 15 min (P < 0.05) in control female monkeys compared with 30 min (P < 0.05) in PA female monkeys. Thus, in PA female monkeys, ACTH-induced increases in corticosterone, although delayed, were ultimately greater and more prolonged than those of control female monkeys.

By 60 min after ACTH injection, PA female monkeys demonstrated a greater increase from baseline in circulating levels of DHEA, androstenedione, and corticosterone compared with controls (DHEA, P < 0.005; androstenedione, P < 0.05; corticosterone, P < 0.01) but exhibited similar increases to controls for cortisol, 17{alpha}-hydroxyprogesterone, DHEAS, and testosterone (Table 2Go).


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  		TABLE 2. Differences in circulating adrenal steroid values between 0 and 60 min after an iv injection of 50 µg ACTH in control and PA female rhesus monkeys

 
Ratios of adrenal steroid hormone responses during the Dex-ACTH test

After dexamethasone suppression of adrenal steroidogenesis and after ACTH infusion, the ratio for serum androstenedione/DHEA was lower in PA compared with control female monkeys (P < 0.01) (Fig. 3Go). After ACTH infusion, the serum androstenedione/DHEA ratio decreased similarly in both female monkey groups (control, P < 0.005; PA, P < 0.05). The serum DHEAS/DHEA ratio, in contrast, was lower in PA vs. control female monkeys after dexamethasone suppression (P < 0.01) and then decreased after ACTH injection in both control (P < 0.005) and PA (P < 0.05) female monkeys, although still remaining lower (P < 0.01) in PA female monkeys throughout.



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  		FIG. 3. Mean (+ 95% confidence interval) ratios of serum adrenal steroid hormones after an iv injection of 50 µg ACTH in control and PA female rhesus monkeys. A, Androstenedione/DHEA (x 10–2): A, P < 0.012 vs. control female, all time points combined; B, P < 0.001 vs. control female at 0 min; a, P < 0.005 vs. 0 min; b, P < 0.05 vs. 0 min. B, DHEAS/DHEA: C, P < 0.001 vs. control females, all time combined; D, P < 0.01 vs. control females at respective time points; c, P < 0.005 vs. 0 min; d, P < 0.05 vs. 0 min. C, DHEA/17{alpha}-hydroxyprogesterone: E, P < 0.02 vs. control females, all time points combined; F, P < 0.05 vs. control females at respective time points; e, P < 0.03 vs. 0 min; f, P < 0.009 vs. 0 min. D, Corticosterone/cortisol (x 10–2): G, P < 0.04 vs. control females, all time points combined; H, P < 0.01 vs. control females at respective time points; g, P < 0.005 vs. 0 min. E, 17{alpha}-Hydroxyprogesterone/testosterone: h, P < 0.001 vs. 0 min control and PA females combined.

 
Although serum DHEA and 17{alpha}-hydroxyprogesterone levels were elevated in both female monkey groups after ACTH stimulation, the serum DHEA/17{alpha}-hydroxyprogesterone ratio decreased in PA, but failed to change in control, female monkeys and remained higher throughout (P < 0.02) in PA compared with control female monkeys (Fig. 3Go). Serum 17{alpha}-hydroxyprogesterone/testosterone ratios, conversely, increased after ACTH stimulation (P < 0.001) in both control and PA female monkeys and did not differ between the two female monkey groups. Together, the ratios of DHEA to other steroid hormones after ACTH injection indicate relatively greater DHEA responses to ACTH in PA female monkeys than exhibited by control females.

Although changes in circulating levels of corticosterone paralleled those of cortisol in controls, showing approximately an 8-fold increase in response to ACTH stimulation at 60 min, the serum corticosterone response to ACTH in PA female monkeys (~14-fold increase) was clearly greater. Serum corticosterone/cortisol ratios were similar in PA compared with control female monkeys after dexamethasone suppression, but, after ACTH stimulation, they showed a greater increase in PA female monkeys by 30 and 60 min (PA vs. control female monkeys, 128 vs. 71% and 94 vs. 35%, respectively; P < 0.01) (Fig. 3Go).

Net increase AUC of adrenal steroid changes after ACTH stimulation

The net increase of AUC steroid responses to ACTH stimulation was illustrated in Table 3Go. The increase of AUC responses of serum corticosterone (P < 0.002), DHEA (P < 0.007), and androstenedione to ACTH infusion (P < 0.005) were higher in PA compared with control female monkeys. The increase of AUC responses of serum 17{alpha}-hydroxyprogesterone, cortisol, DHEAS, and testosterone to ACTH infusion, however, were similar in the two female monkey groups.


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  		TABLE 3. Net increase of mean ± SEM AUCs for serum steroid levels following an iv injection of 50 µg ACTH in control and PA female rhesus monkeys

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The primate adrenal cortex is unique in possessing a morphologically and functionally distinct inner zone, the zona reticularis (49). The adult zona reticularis either originates or is remodeled from another unique primate attribute, the adrenal fetal zone (50). Both the primate fetal zone and zona reticularis strongly express the androgenic biosynthetic enzyme P450c17 (17{alpha}-hydroxylase/17,20 lyase), its catalytic accessory protein, cytochrome b5, and the relevant sulfo-conjugating enzyme, while having greatly reduced expression of 3ß-hydroxysteroid dehydrogenase II (3ß-HSD II) compared with the outer cortical zones, the zona fasciculata and zona glomerulosa (49, 50, 51) (Fig. 1Go). It is, therefore, not surprising that both the fetal zone and the zona reticularis are normally responsible for the relatively high circulating levels of DHEA and DHEAS typical of primates (49, 51).

As typical primates, rhesus monkeys and baboons undergo adrenarche (51), the phenomenon of increased adrenal DHEA and DHEAS secretion, similar to that manifest in humans and Great Apes (52). The developmental timing of this adrenal androgenization differs, however, with rhesus monkeys and baboons having a neonatal (51) rather than a prepubertal to adolescent progressive (51, 53) differentiation of the zona reticularis found in humans. Also, senescence of the zona reticularis is seen after the third month of life in rhesus monkeys and baboons compared with the third decade of life in humans, and, thereafter, circulating DHEAS levels progressively decline in both sexes (51). Additionally, similar to humans, 17{alpha}-hydroxyprogesterone is an inefficient substrate for 17,20 lyase in nonhuman primates (51), with negligible conversion of 17{alpha}-hydroxyprogesterone to androstenedione (54) (Fig. 1Go). Thus, rhesus monkeys provide a close approximation to human adrenal androgen physiology and, in the present study, provide a viable opportunity to investigate the fetal origins of pathological adrenal androgen excess in PCOS women.

Our findings demonstrate that female rhesus monkeys, exposed to experimentally induced androgen excess during early gestation, manifest endogenous adrenal androgen excess in adulthood. In this regard, they closely resemble approximately 25–60% of PCOS women with adrenal androgen excess (7, 20). Although many studies have examined adrenal hyperandrogenism in PCOS women (11, 18, 19, 20, 26), the mechanisms underlying PCOS-related adrenal androgen excess are still unclear. Azziz et al. (18) demonstrated in PCOS patients with adrenal androgen excess specific androstenedione and DHEA hyperandrogenic responses of the zona reticularis to ACTH, which appeared to be unaccompanied by other abnormalities of the adrenal (i.e. zona glomerulosa or zona fasciculata) or the hypothalamic-pituitary axis. PCOS women with adrenal androgen excess vary in their presentation of adrenocortical hyperandrogenism (55) but are most commonly identified by increased secretion of DHEA in response to ACTH (11, 20, 41) and increased basal levels of DHEAS (16, 18). In addition, PCOS women with adrenal androgen excess demonstrate enhanced ACTH-stimulated androstenedione levels (18, 20, 41). Female PA monkeys closely emulate these clinical findings because basal serum DHEA levels were increased, whereas ACTH stimulation caused exaggerated DHEA and androstenedione elevations.

Basal serum DHEAS levels, however, were not elevated in PA female monkeys compared with controls, although they were increased in PA monkeys when studied 3–4 yr earlier (25). These findings suggest that the absence of elevated serum DHEAS levels in the present PA female monkeys may represent an age-related decline in DHEAS levels. In this regard, if PA female rhesus monkeys are a nonhuman equivalent of PCOS women with adrenal androgen excess, then loss of elevated basal DHEAS levels with age may closely parallel that found in PCOS women over a similar time interval of 3–5 yr (17). Predexamethasone serum DHEAS levels in the present PA and control female rhesus monkeys are typical for their age (51), and those in PA females are approximately 13% less than those reported previously (25), whereas control female monkeys demonstrated no such decline. Predexamethasone serum 17{alpha}-hydroxyprogesterone, cortisol, and testosterone levels, however, are similar to those reported previously (25). No other age comparisons are possible because additional steroid hormone measurements were not performed in the previous PA female monkey study (25).

The most likely cause of the excessive adrenal androgen secretion appears to be abnormal regulation of the 17{alpha}-hydroxylase and 17,20 lyase activities of P450c17, the rate-limiting step in androgen biosynthesis (Fig. 1Go). Conventionally considered localized to the primate zona reticularis, promotion of 17,20 lyase activity, without enhancement of 17{alpha}-hydroxylase, requires phosphorylation of the serine residues of P450c17 (56) and the presence of cytochrome b5, which allosterically enables the interaction of P450c17 with P450 oxidoreductase, an obligate electron donor (51, 57). Adrenal androgen excess in the PA monkeys of this study, and in PCOS women, appears consistent with enhanced 17,20 lyase activity in the zona fasciculata (increased DHEA and androstenedione), in addition to 17,20 lyase activity in the zona reticularis, without enhanced 17{alpha}-hydroxylase activity (suggested by normal 17{alpha}-hydroxyprogesterone and cortisol). Increased serine phosphorylation of P450c17 (56), increased cytochrome b5 activity, or the combination of the two might well provide the cellular and molecular basis for the DHEA excess observed in both PA female monkeys and PCOS women with adrenal androgen excess.

To account for all three excessive steroidogenic responses (corticosterone, DHEA, and androstenedione) after ACTH stimulation in PA monkeys, however, we need to propose additional enzymatic changes in adrenal steroid biosynthesis and metabolism beyond enhanced activity of 17,20 lyase in the zonae fasciculata and reticularis. Corticosterone, produced primarily in the primate zona fasciculata (51, 57) (Fig. 1Go), is elevated after ACTH stimulation in PA compared with control female monkeys, and its ratio to cortisol is also elevated in PA female monkeys. Increased 3ß-HSD II activity may be present in these female monkeys, because 1) pregnenolone is the preferred substrate for cortisol biosynthesis in primates (Fig. 1Go), 2) progesterone is the key precursor to corticosterone biosynthesis (Fig. 1Go), and 3) there is a 3–4% decrease in the efficiency of cortisol biosynthesis in the zona fasciculata (derived from the increased ratio of corticosterone/cortisol at 60 min after ACTH injection) (58) of PA female monkeys. Such specific enhancement of 3ß-HSD II enzymatic activity in PA female monkeys would direct a small, but significant, increase in substrate metabolism toward progesterone instead of 17{alpha}-hydroxypregnenelone (Fig. 1Go), thus providing increased substrate for corticosterone biosynthesis and other zona fasciculata products that would not normally be present. Our Dex-ACTH test most likely demonstrated such a shift in steroidogenesis via increased 3ß-HSD II activity because the preferred P450c17 pathway was probably saturated after the high dose of ACTH used. The approximate 14-fold increase in corticosterone in PA female monkeys after ACTH injection compared with only an approximate 8-fold increase in cortisol further supports the notion of increased 3ß-HSD II activity in the zona fasciculata of these animals.

In conclusion, prenatal exposure of female rhesus monkeys to androgen excess induces irreversible physiological changes in adrenal cortex function, namely hypersecretion of adrenal DHEA, androstenedione, and corticosterone. The hyperandrogenic findings closely resemble those observed in 25–60% of PCOS women who have adrenal hyperandrogenism (14, 15, 16). Fetal androgen excess may result in the elevation of circulating concentrations of adrenal androgens through a variety of defects in steroidogenic enzyme function or regulation. Because experimentally induced fetal programming of female monkeys can so closely mimic adrenal androgen excess found in 25–60% of women with PCOS, our findings suggest that differentiation of the fetal adrenal cortex in a hyperandrogenic environment may permanently up-regulate its androgenic function, probably in the zona reticularis and zona fasciculata. Such reprogramming is then retained in the development of mature, postnatal cortex zonation. Whether this adult outcome of fetal programming additionally involves altered hypothalamic-pituitary regulation of adrenal function remains to be determined.


   Acknowledgments
 
We thank the following: E. J. Peterson, J. M. Turk, K. Hable, S. DeBruin, and R. D. Medley for technical assistance; F. Wegner, D. Wittwer, S. Jacoris, and Assay Services of the National Primate Research Center, University of Wisconsin-Madison, for hormone assay expertise; D. Florence, D.V.M., I. Bolton, D.V.M., K. Brunner, D.V.M., and D. Welner-Kern for veterinary care; D. Wade and S. Maves for animal care; and J. C. Pattison for comments on this manuscript.


   Footnotes
 
This work was supported by National Institutes of Health Grants R01 RR013635 (to D.H.A.) and P51 RR000167 (to Wisconsin National Primate Research Center). This research was conducted at a facility constructed with support from Research Facilities Improvement Program Grants RR15459-01 and RR020141-01.

First Published Online September 20, 2005

Abbreviations: AUC, Area under the curve; Dex-ACTH, combined dexamethasone-ACTH test; DHEA, dehydroepiandrosterone; DHEAS, DHEA sulfate; 3ß-HSD II, 3ß-hydroxysteroid dehydrogenase II; NS, not significant; PA, prenatally androgenized; PCOS, polycystic ovary syndrome; QC, quality control preparation.

Received March 29, 2005.

Accepted September 9, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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