Clustering of PCOS-like traits in naturally hyperandrogenic female rhesus monkeys.

Study question: Do naturally occurring, hyperandrogenic (≥1 SD of population mean testosterone, T) female rhesus monkeys exhibit traits typical of women with polycystic ovary syndrome (PCOS)? Summary answer: Hyperandrogenic female monkeys exhibited significantly increased serum levels of androstenedione (A4), 17-hydroxyprogesterone (17-OHP), estradiol (E2), LH, antimullerian hormone (AMH), cortisol, 11-deoxycortisol and corticosterone, as well as increased uterine endometrial thickness and evidence of reduced fertility, all traits associated with PCOS. What is known already: Progress in treating women with PCOS is limited by incomplete knowledge of its pathogenesis and the absence of naturally occurring PCOS in animal models. A female macaque monkey, however, with naturally occurring hyperandrogenism, anovulation and polyfollicular ovaries, accompanied by insulin resistance, increased adiposity and endometrial hyperplasia, suggests naturally occurring origins for PCOS in nonhuman primates. Study design, size, duration: As part of a larger study, circulating serum concentrations of selected pituitary, ovarian and adrenal hormones, together with fasted insulin and glucose levels, were determined in a single, morning blood sample obtained from 120 apparently healthy, ovary-intact, adult female rhesus monkeys (Macaca mulatta) while not pregnant or nursing. The monkeys were then sedated for somatometric and ultrasonographic measurements. Participants/materials, setting, methods: Female monkeys were of prime reproductive age (7.2 ± 0.1 years, mean ± SEM) and represented a typical spectrum of adult body weight (7.4 ± 0.2 kg; maximum 12.5, minimum 4.6 kg). Females were defined as having normal (n = 99) or high T levels (n = 21; ≥1 SD above the overall mean, 0.31 ng/ml). Electronic health records provided menstrual and fecundity histories. Steroid hormones were determined by tandem LC-MS-MS; AMH was measured by enzymeimmunoassay; LH, FSH and insulin were determined by radioimmunoassay; and glucose was read by glucose meter. Most analyses were limited to 80 females (60 normal T, 20 high T) in the follicular phase of a menstrual cycle or anovulatory period (serum progesterone <1 ng/ml). Main results and the role of chance: Of 80 monkeys, 15% (n = 12) exhibited classifiable PCOS-like phenotypes. High T females demonstrated elevations in serum levels of LH (P < 0.036), AMH (P < 0.021), A4 (P < 0.0001), 17-OHP (P < 0.008), E2 (P < 0.023), glucocorticoids (P < 0.02-0.0001), the serum T/E2 ratio (P < 0.03) and uterine endometrial thickness (P < 0.014) compared to normal T females. Within the high T group alone, anogenital distance, a biomarker for fetal T exposure, positively correlated (P < 0.015) with serum A4 levels, while clitoral volume, a biomarker for prior T exposure, positively correlated (P < 0.002) with postnatal age. Only high T females demonstrated positive correlations between serum LH, and both T and A4. Five of six (83%) high T females with serum T ≥2 SD above T mean (0.41 ng/ml) did not produce live offspring. Large scale data: N/A. Limitations, reasons for caution: This is an initial study of a single laboratory population in a single nonhuman primate species. While two biomarkers suggest lifelong hyperandrogenism, phenotypic expression during gestation, prepuberty, adolescence, mid-to-late reproductive years and postmenopause has yet to be determined. Wider implications of the findings: Characterizing adult female monkeys with naturally occurring hyperandrogenism has identified individuals with high LH and AMH combined with infertility, suggesting developmental linkage among traits with endemic origins beyond humans. PCOS may thus be an ancient phenotype, as previously proposed, with a definable pathogenic mechanism(s). Study funding/competing interest(s): Funded by competitive supplement to P51 OD011106 (PI: Mallick), by P50 HD028934 (PI: Marshall) and by P50 HD044405 (PI: Dunaif). The authors have no potential conflicts of interest.

Breath carbon stable isotope ratios identify changes in energy balance and substrate utilization in humans.

Rapid detection of shifts in substrate utilization and energy balance would provide a compelling biofeedback tool for individuals attempting weight loss. As a proof of concept, we tested whether the natural abundance of exhaled carbon stable isotope ratios (breath δ(13)C) reflects shifts between negative and positive energy balance. Volunteers (n=5) consumed a 40% energy-restricted diet for 6 days followed by 50% excess on day 7. Breath was sampled immediately before and 1 h and 2 h after breakfast, lunch and dinner. Exhaled breath δ(13)C values were measured by cavity ring-down spectroscopy. Using repeated measures analysis of variance (ANOVA) followed by Dunnett's contrasts, pre-breakfast breath values on days 2-6 were compared with day 1, and postprandial day 7 time points were compared with pre-breakfast day 7. Energy restriction diminished pre-breakfast breath δ(13)C by day 3 (P<0.05). On day 7, increased energy intake was first detected immediately before dinner (-23.8±0.6 vs -21.9±0.7‰, P=0.002 (means±s.d.)), and breath δ(13)C remained elevated at least 2 h post dinner. In conclusion, when shifting between negative and positive energy balance, breath δ(13)C showed anticipated isotopic changes. Although additional research is needed to determine specificity and repeatability, this method may provide a biomarker for marked increases in caloric intake.

Adrenal androgen concentrations increase during infancy in male rhesus macaques (Macaca mulatta).

This study investigated adrenal androgens (AA), gonadotropins, and cortisol in castrated and gonad-intact male rhesus macaques from birth through infancy. Blood samples were collected longitudinally from castrated (n = 6; weekly, 1-40 wk) and intact (n = 4; every other week, 1-17 wk) males. Plasma concentrations of AA were determined by liquid chromatography-tandem mass spectrometry, and plasma concentrations of cortisol and gonadotropins were determined by RIA. Dehydroepiandrosterone sulfate (DHEAS) concentrations increased almost threefold (to 8 wk), dehydroepiandrosterone (DHEA) increased more than eightfold (to 11 wk), and androstenedione doubled (to 15 wk) in five castrated infant males and declined continuously thereafter. A sixth castrated male had markedly different temporal patterns and concentrations (many times more than 2 SDs from the cohort mean) of AA and gonadotropins from first sampling (3 wk) and was excluded from analysis. Cortisol increased over 16 wk but correlated poorly with DHEAS. Luteinizing and follicle-stimulating hormones increased to peaks at 3 and 7 wk, respectively. Testis-intact males exhibited similar profiles, but with earlier peaks of DHEAS (5 wk) and DHEA and androstenedione (7 wk). Peak concentrations of DHEAS were lower and those of DHEA and androstenedione were higher in intact than castrated infants. Testosterone was undetectable in castrated males and >0.5 ng/ml in intact males but was not correlated with DHEA or DHEAS. These are the first data documenting a transient increase in AA secretion during infancy in an Old World primate and are consistent with the previously documented time course of zona reticularis development that accompanies increases in androgen synthetic capacity of the adrenal. The rhesus is a promising model for androgen secretion from the human adrenal cortex.

Early prenatal androgenization results in diminished ovarian reserve in adult female rhesus monkeys.

BACKGROUND: Early prenatal androgenization (PA) accelerates follicle differentiation and impairs embryogenesis in adult female rhesus monkeys (Macaca mulatta) undergoing FSH therapy for IVF. To determine whether androgen excess in utero affects follicle development over time, this study examines whether PA exposure, beginning at gestational days 40-44 (early treated) or 100-115 (late treated), alters the decline in serum anti-Mullerian hormone (AMH) levels with age in adult female rhesus monkeys and perturbs their ovarian response to recombinant human FSH (rhFSH) therapy for IVF. METHODS: Thirteen normal (control), 11 early-treated and 6 late-treated PA adult female monkeys had serum AMH levels measured at random times of the menstrual cycle or anovulatory period. Using some of the same animals, basal serum AMH, gonadotrophins and steroids were also measured in six normal, five early-treated and three late-treated PA female monkeys undergoing FSH therapy for IVF during late-reproductive life (>17 years); serum AMH also was measured on day of HCG administration and at oocyte retrieval. RESULTS: Serum AMH levels in early-treated PA females declined with age to levels that were significantly lower than those of normal (P < or = 0.05) and late-treated PA females (P < or = 0.025) by late-reproductive life. Serum AMH levels positively predicted numbers of total/mature oocytes retrieved, with early-treated PA females having the lowest serum AMH levels, fewest oocytes retrieved and lowest percentage of females with fertilized oocytes that cleaved. CONCLUSIONS: Based on these animals, early PA appears to program an exaggerated decline in ovarian reserve with age, suggesting that epigenetically induced hormonal factors during fetal development may influence the cohort size of ovarian follicles after birth.

Anogenital distance in newborn daughters of women with polycystic ovary syndrome indicates fetal testosterone exposure.

Polycystic ovary syndrome (PCOS) affects ~7% of reproductive age women. Although its etiology is unknown, in animals, excess prenatal testosterone (T) exposure induces PCOS-like phenotypes. While measuring fetal T in humans is infeasible, demonstrating in utero androgen exposure using a reliable newborn biomarker, anogenital distance (AGD), would provide evidence for a fetal origin of PCOS and potentially identify girls at risk. Using data from a pregnancy cohort (The Infant Development and Environment Study), we tested the novel hypothesis that infant girls born to women with PCOS have longer AGD, suggesting higher fetal T exposure, than girls born to women without PCOS. During pregnancy, women reported whether they ever had a PCOS diagnosis. After birth, infant girls underwent two AGD measurements: anofourchette distance (AGD-AF) and anoclitoral distance (AGD-AC). We fit adjusted linear regression models to examine the association between maternal PCOS and girls' AGD. In total, 300 mother-daughter dyads had complete data and 23 mothers reported PCOS. AGD was longer in the daughters of women with a PCOS diagnosis compared with daughters of women with no diagnosis (AGD-AF: ß=1.21, P=0.05; AGD-AC: ß=1.05, P=0.18). Results were stronger in analyses limited to term births (AGD-AF: ß=1.65, P=0.02; AGD-AC: ß=1.43, P=0.09). Our study is the first to examine AGD in offspring of women with PCOS. Our results are consistent with findings that women with PCOS have longer AGD and suggest that during PCOS pregnancies, daughters may experience elevated T exposure. Identifying the underlying causes of PCOS may facilitate early identification and intervention for those at risk.

Gonadotrophin-releasing hormone (GnRH) release in marmosets II: pulsatile release of GnRH and pituitary gonadotrophin in adult females.

Unlike other mammals, including rodents, Old World primates and humans, common marmosets and probably all other New World primates synthesise and release chorionic gonadotrophin (CG), and not luteinising hormone (LH) from pituitary gonadotrophs. However, little is known about the physiological dynamics of gonadotrophin-releasing hormone (GnRH)-regulated CG release from gonadotrophs and whether such CG release has pulsatile release characteristics similar to those of LH in other mammalian species. Consequently, we performed a series of in vivo and in vitro studies in ovariectomised laboratory rats and female marmosets to compare GnRH-induced pituitary LH and CG release characteristics, respectively. Exogenous GnRH stimulated a slower onset of release of marmoset pituitary CG, both in vivo and in vitro, and induced an approximately 400% greater increase in the duration of marmoset pituitary CG release compared to that for rat LH. Not surprisingly, hypothalamic pulsatile release of GnRH in vivo was not obviously concordant with endogenous episodic changes in circulating levels of CG in marmosets, in contrast to the clear concordance observed between in vivo GnRH and LH release previously demonstrated in rats and other mammals. Pituitary CG release in marmosets thus demonstrates considerable divergence from the timely hypothalamic GnRH-regulated LH release in other female mammals, implying potentially different physiological dynamics in gonadotrophin regulation of marmoset ovarian function.

Gonadotrophin-releasing hormone (GnRH) release in marmosets I: in vivo measurement in ovary-intact and ovariectomised females.

In vivo hypothalamic gonadotrophin-releasing hormone (GnRH) release was characterised for the first time in a New World primate. A nonterminal and repeatable push-pull perfusion (PPP) technique reliably measured GnRH in conscious common marmoset monkeys. Nineteen adult females (n = 8 ovary-intact in the mid-follicular phase; n = 11 ovariectomised) were fitted with long-term cranial pedestals, and a push-pull cannula was temporarily placed in unique locations within the pituitary stalk-median eminence (S-ME) 2 days prior to each PPP session. Marmosets underwent 1-3 PPPs (32 PPPs in total) lasting up to 12 h. Plasma cortisol levels were not elevated in these habituated marmosets during PPP, and PPP did not disrupt ovulatory cyclicity or subsequent fertility in ovary-intact females. GnRH displayed an organised pattern of release, with pulses occurring every 50.0 +/- 2.6 min and lasting 25.4 +/- 1.3 min. GnRH pulse frequency was consistent within individual marmosets across multiple PPPs. GnRH mean concentration, baseline concentration and pulse amplitude varied predictably with anatomical location of the cannula tip within the S-ME. GnRH release increased characteristically in response to a norepinephrine infusion and decreased abruptly during the evening transition to lights off. Ovary-intact (mid-follicular phase) and ovariectomised marmosets did not differ significantly on any parameter of GnRH release. Overall, these results indicate that PPP can be used to reliably assess in vivo GnRH release in marmosets and will be a useful tool for future studies of reproductive neuroendocrinology in this small primate.

Contested dominance modifies the anovulatory consequences of social subordination in female marmosets.

Dominance status among female marmosets is reflected in agonistic behavior and ovarian function. Socially dominant females receive submissive behavior from subordinates, while exhibiting normal ovulatory function. Subordinate females, however, receive agonistic behavior from dominants, while exhibiting reduced or absent ovulatory function. Such disparity in female fertility is not absolute, and groups with two breeding females have been described. The data reported here were obtained from 8 female-female pairs of captive female marmosets, each housed with a single unrelated male. Pairs were classified into two groups: "uncontested" dominance (UD) and "contested" dominance (CD), with 4 pairs each. Dominant females in UD pairs showed significantly higher frequencies (4.1) of agonism (piloerection, attack and chasing) than their subordinates (0.36), and agonistic behaviors were overall more frequently displayed by CD than by UD pairs. Subordinates in CD pairs exhibited more agonistic behavior (2.9) than subordinates in UD pairs (0.36), which displayed significantly more submissive (6.97) behaviors than their dominants (0.35). The data suggest that there is more than one kind of dominance relationship between female common marmosets. Assessment of progesterone levels showed that while subordinates in UD pairs appeared to be anovulatory, the degree of ovulatory disruption in subordinates of CD pairs was more varied and less complete. We suggest that such variation in female-female social dominance relationships and the associated variation in the degree and reliability of fertility suppression may explain variations of the reproductive condition of free-living groups of common marmosets.

Developmental programming: rescuing disruptions in preovulatory follicle growth and steroidogenesis from prenatal testosterone disruption.

BACKGROUND: Prenatal testosterone (T) excess from days 30-90 of gestation disrupts gonadotropin surge and ovarian follicular dynamics and induces insulin resistance and functional hyperandrogenism in sheep. T treatment from days 60-90 of gestation produces a milder phenotype, albeit with reduced fecundity. Using this milder phenotype, the aim of this study was to understand the relative postnatal contributions of androgen and insulin in mediating the prenatal T induced disruptions in ovarian follicular dynamics. METHODS: Four experimental groups were generated: 1) control (vehicle treatment), 2) prenatal T-treated (100 mg i.m. administration of T propionate twice weekly from days 60-90 of gestation), 3) prenatal T plus postnatal anti-androgen treated (daily oral dose of 15 mg/kg/day of flutamide beginning at 8 weeks of age) and 4) prenatal T and postnatal insulin sensitizer-treated (daily oral dose of 8 mg/day rosiglitazone beginning at 8 weeks of age). Follicular response to a controlled ovarian stimulation protocol was tested during their third breeding season. Main outcome measures included the determination of number and size of ovarian follicles and intrafollicular concentrations of steroids. RESULTS: At the end of the controlled ovarian stimulation, the number of follicles approaching ovulatory size (≥6 mm) were ~35 % lower in prenatal T-treated (6.5 ± 1.8) compared to controls (9.8 ± 2.0). Postnatal anti-androgen (10.3 ± 1.9), but not insulin sensitizer (5.0 ± 0.9), treatment prevented this decrease. Preovulatory sized follicles in the T group had lower intrafollicular T, androstenedione, and progesterone compared to that of the control group. Intrafollicular steroid disruption was partially reversed solely by postnatal insulin sensitizer treatment. CONCLUSIONS: These results demonstrate that the final preovulatory follicular growth and intrafollicular steroid milieu is impaired in prenatal T-treated females. The findings are consistent with the lower fertility rate reported earlier in these females. The finding that final follicle growth was fully rescued by postnatal anti-androgen treatment and intrafollicular steroid milieu partially by insulin sensitizer treatment suggest that both androgenic and insulin pathway disruptions contribute to the compromised follicular phenotype of prenatal T-treated females.

Insights into the development of polycystic ovary syndrome (PCOS) from studies of prenatally androgenized female rhesus monkeys.

The developmental pathophysiology of polycystic ovary syndrome (PCOS) is unknown. However, prenatally androgenized female rhesus monkeys exhibit ovarian and endocrinological features that mimic those found in women with PCOS. Thus, prenatal androgen excess may provide an etiology for hyperandrogenism and anovulation in adulthood.

Developmental programming: exposure to testosterone excess disrupts steroidal and metabolic environment in pregnant sheep.

Gestational exposure to excess T leads to intrauterine growth restriction, low birth weight, and adult metabolic/reproductive disorders in female sheep. We hypothesized that as early mediators of such disruptions, gestational T disrupts steroidal and metabolic homeostasis in both the mother and fetus by both androgenic and metabolic pathways. Maternal blood samples were measured weekly for levels of insulin, glucose, and progesterone from four groups of animals: control; gestational T (twice weekly im injections of 100 mg of T propionate from d 30 to d 90 of gestation); T plus an androgen antagonist, flutamide (15 mg/kg·d oral; T-Flutamide); and T plus the insulin sensitizer, rosiglitazone (0.11 mg/kg·d oral; T-Rosi) (n = 10-12/group). On day 90 of gestation, maternal and umbilical cord samples were collected after a 48-hour fast from a subset (n = 6/group) for the measurement of steroids, free fatty acids, amino acids, and acylcarnitines. Gestational T decreased maternal progesterone levels by 36.5% (P < .05), which was prevented by flutamide showing direct androgenic mediation. Gestational T also augmented maternal insulin levels and decreased medium chained acylcarnitines, suggesting increased mitochondrial fatty acid oxidation. These changes were prevented by rosiglitazone, suggesting alterations in maternal fuel use. Gestational T-induced increases in fetal estradiol were not prevented by either cotreatment. Gestational T disrupted associations of steroids with metabolites and progesterone with acylcarnitines, which was prevented either by androgen antagonist or insulin sensitizer cotreatment. These findings suggest a future combination of these treatments might be required to prevent alteration in maternal/fetal steroidal and metabolic milieu(s).

Timing of prenatal androgen excess determines differential impairment in insulin secretion and action in adult female rhesus monkeys.

This study determined whether timing of prenatal androgen excess resulted in differential impairment of insulin-glucose homeostasis in adult female rhesus monkeys. Ten female rhesus monkeys exposed to testosterone propionate starting on gestational day 40 (early treated), 9 females exposed to testosterone propionate starting between gestational days 100-115 (late treated), and 15 control females were studied. The modified minimal model was used to examine various measures derived from an i.v. glucose tolerance test, with regression analysis performed between these variables and body mass index. In addition, the disposition index (DI) and the hyperbolic relationship between insulin sensitivity (S(I)) and acute insulin response to glucose were examined. Early treated females demonstrated impaired pancreatic beta-cell function, as shown by diminished DI and decreased percentile ranking for the hyperbolic relationship between S(I) and acute insulin response to glucose. In contrast, late treated females exhibited both an increase in DI and a negative relationship between body mass index and S(I). These results suggest that prenatal androgen excess in female rhesus monkeys, regardless of gestational timing, perturbs insulin-glucose homeodynamics, with androgen excess in early and late gestation impairing pancreatic beta-cell function and altering insulin sensitivity, respectively.

Skeletal effects of aging and menopausal status in female rhesus macaques.

To further define the nonhuman primate as a model of the adult human skeleton, we explored the impact of growth, natural menopause, and osteoarthritis on bone mass, serum markers of bone turnover (osteocalcin and C-terminal telopeptide of type I collagen) and measures of skeletal relevance (PTH, 25-hydroxyvitamin D, total alkaline phosphatase, calcium, phosphorus, creatinine, and albumin). Fifty-eight female (aged 4-30 yr) rhesus macaques were defined as growing (G; n = 12; < or = 10 yr old), adult premenopausal (APre; n = 30; > 10 yr old; eumenorrheic, high serum estradiol and low FSH), or postmenopausal (Post; n = 16; amenorrheic for at least 1 yr, with low serum estradiol and high FSH). Total body and posterior-anterior spinal bone masses were lower in G than APre animals (P < 0.05). Post females had lower total body, distal radius, and spinal bone mass than premenopausal animals (P < 0.05). Osteocalcin was higher in Post than APre animals (P < 0.01). Other measures showed no relationship with menopausal status. In older monkeys, spinal osteoarthritis became common, causing increased dual-energy x-ray absorptiometry-measured bone mass in the lumbar spinal posterior-anterior projection. In conclusion, after natural menopause, rhesus monkeys have lower bone mass and higher skeletal turnover without alteration of the calcium-vitamin D axis. As such, they are an excellent model of human estrogen-depletion bone loss.

Relationship between dominance hierarchy, cerebrospinal fluid levels of amine transmitter metabolites (5-hydroxyindole acetic acid and homovanillic acid) and plasma cortisol in monkeys.

The dominance hierarchy has repercussions for a monkey's sexual behaviour and endocrine state. Here we report on neural mechanisms that are sensitive to a monkey's status in the social hierarchy, and which may regulate not only its endocrine function but its sexual responsiveness to its own hormones. During the initial phase of group formation, 5-hydroxyindole acetic acid, the metabolite of serotonin, increases in the cerebrospinal fluid of monkeys which become subordinate (all groups), but decreases in monkeys which become dominant (two out of three groups) and shows no changes in intermediate-ranking animals (five out of seven). Homovanillic acid, a metabolite of dopamine, may also increase in the cerebrospinal fluid of monkeys that become subordinate (two out three groups). In the initial period of group formation these changes in transmitter metabolites do not parallel changes in cortisol. However, in the established social groups, both 5-hydroxyindole acetic acid and plasma cortisol are related to the social hierarchy, being greater in those monkeys that are subordinate, but homovanillic acid shows no consistent change. Although subordinate monkeys receive more aggression than others in their group, fluctuations in 5-hydroxyindole acetic acid do not correlate with aggressive behaviour, and are equally high on days when no aggression occurs. Dominant males, however, had higher 5-hydroxyindole acetic acid levels on days when they were involved in agonistic encounters. In the established social hierarchy therefore, elevated levels of the serotonin metabolite in cerebrospinal fluid seem reflect a "state"-dependent consequence of occupying a position of low social status.

Developmental origin of polycystic ovary syndrome - a hypothesis.

Polycystic ovary syndrome (PCOS) is a common but complex endocrine disorder and is a major cause of anovulation and consequent subfertility. It is also associated with a metabolic disturbance, characterized by hyperinsulinaemia and insulin resistance that carries an increased risk of type 2 diabetes in later life. Despite its prevalence little is known about its aetiology, but there is increasing evidence for an important genetic involvement. On the basis of experimental observations in the prenatally androgenized sheep and rhesus monkey, and supported by data from human studies, we propose that the clinical and biochemical features of PCOS can arise as a consequence of genetically determined hypersecretion of androgens by the ovary during, or very likely long before, puberty. The resulting hyperandrogenism results in 'programming' of the hypothalamic-pituitary unit to favour excess LH secretion, and encourages preferential abdominal adiposity that predisposes to insulin resistance. The severity of hyperinsulinaemia and insulin resistance (which has a profound influence on the phenotype of PCOS) is further influenced by both genetic factors (such as polymorphism in the insulin gene regulatory region) and environmental factors, notably obesity. This hypothesis therefore suggests a unifying, 'linear' model to explain the aetiology of the heterogeneous phenotype.

Social status controls LH secretion and ovulation in female marmoset monkeys (Callithrix jacchus).

The suppression of ovulation in subordinate female marmosets was associated with suppressed pituitary LH secretion and reduced pituitary LH response to gonadotrophin-releasing hormone (GnRH). In subordinate females, basal plasma LH concentrations were commonly below 2 IU/l (n = 5) (maximum 10.7 IU/l). Plasma oestrogen concentrations were similarly low (maximum 0.62 nmol/l) and plasma progesterone concentrations of below 30 nmol/l confirmed the anovulatory condition. This infertility condition was rapidly reversed when subordinate females (n = 5) were removed from their social groups and housed singly, when plasma LH (maximum 140.0 IU/l) and oestrogen (maximum 7.84 nmol/l) concentrations increased preceding ovulation. Infertility was rapidly reimposed when these singly housed females were re-introduced to subordinate status in new social groups, when plasma LH concentrations fell to their previous low values within 4 days; no ovulation occurred thereafter. Plasma oestrogen levels also fell, but less dramatically. The luteal phases of three of the subordinate females were shortened following the re-instatement of subordinate status. The maximum LH response of subordinate females to the highest dose of GnRH (200 ng) was only 19.1 +/- 6.7 IU/l (mean +/- S.E.M.; n = 8): this contrasted with that in dominant females in either the follicular phase (40.0 +/- 13.3 IU/l; n = 6) or the luteal phase (126.7 +/- 24.9 IU/l; n = 10) of the ovarian cycle. These results suggest that the social suppression of fertility in subordinate female marmosets is mediated by impaired hypothalamic GnRH secretion. Such an immediate and precise behavioural control of LH secretion and ovulation is without equal in anthropoid primates.

The passage of 5 alpha-dihydrotestosterone from serum into cerebrospinal fluid and LH negative feedback in castrated rhesus monkeys.

Seven castrated monkeys were given either 50 or 100 micrograms 5 alpha-dihydrotestosterone (DHT) propionate/kg per day. There was no correlation between serum and cerebrospinal fluid (CSF) levels of DHT, which remained very low in the CSF (0.3-0.6% of blood levels) despite the presence of high, supraphysiological amounts in the circulation. There was also no relation between unbound DHT in the blood and the CSF, in which all DHT is unbound. These results differ from previous work on testosterone, the metabolic precursor of DHT. 5 alpha-Dihydrotestosterone propionate at the higher dose maintained suppressed levels of serum LH; LH in two out of four monkeys treated at the lower dose increased to levels observed in castrated, untreated rhesus monkeys. There was no predictable relationship between the amount of DHT in the CSF and levels of LH in the blood: by contrast, DHT in the blood was correlated with serum levels of LH. Levels of LH rose in monkeys in which total blood DHT fell below about 68 nmol/l and, even more obviously, if unbound DHT decreased to less than about 2 nmol/l. Differences between the distribution of testosterone and DHT between blood and CSF cannot be explained by serum binding, lipid solubility or clearance from the brain, and suggest that there may be some mechanism for excluding DHT from the CSF. Though DHT reaches the CSF from the blood in small amounts, levels there do not relate predictably to those in the vascular compartment. It seems unlikely, therefore, that levels of intracerebral DHT are controlled by changes in those of the blood.

Social suppression of cortisol in female marmoset monkeys: role of circulating ACTH levels and glucocorticoid negative feedback.

Behaviorally subordinate female common marmoset monkeys (Callithrix jacchus) exhibit pronounced, chronic reductions of circulating cortisol levels. Cortisol suppression in these animals is mediated in part by adrenocortical hyporesponsiveness to adrenocorticotropic hormone (ACTH). In addition, we hypothesized that social subordination may activate a central, neurally mediated mechanism to further inhibit hypothalamo-pituitary-adrenal function. In this study, therefore, we evaluated basal plasma cortisol and ACTH concentrations, as well as cortisol and ACTH responses to dexamethasone (DEX), in dominant and subordinate females to initially characterize such a mechanism. Morning plasma cortisol and ACTH levels were determined before, and 1, 2, and 3 days following administration of DEX (0.5, 1.0, or 5.0 mg/kg, IM) or saline. Baseline cortisol concentrations prior to DEX treatment were significantly lower in subordinate females than in dominants, as previously reported. However, ACTH concentrations in the same blood samples did not differ between the two groups. Furthermore, dominant and subordinate females showed similar cortisol and ACTH responses to DEX. These results indicate that reduced circulating cortisol levels in subordinate females are not associated with either altered circulating ACTH concentrations or enhanced responsiveness to glucocorticoid negative feedback. However, the finding that basal ACTH levels are not elevated in subordinate females as compared to dominants, in spite of low circulating cortisol concentrations, suggests that ACTH secretion in subordinate females is restrained by a steroid-independent inhibitory mechanism operating at the level of the brain or pituitary.

Reduced adrenocortical responsiveness to adrenocorticotropic hormone (ACTH) in socially subordinate female marmoset monkeys.

Socially subordinate female common marmoset monkeys undergo pronounced, chronic reductions in basal plasma cortisol levels, which appear to result both from socially induced suppression of reproductive hormones and from direct effects of social subordination. In this study, we tested the hypothesis that this cortisol suppression is mediated by reduced adrenocortical responsiveness to adrenocorticotropic hormone (ACTH). Dominant, subordinate, and ovariectomized females were given dexamethasone (5 mg/kg, IM), followed the next morning by human ACTH(1-39) (10 microg/kg, IV) or sterile saline (0.5 ml/kg, IV); blood samples were collected at -20 through 150 min from ACTH or saline treatment and assayed for cortisol. ACTH, but not saline, caused a marked elevation of plasma cortisol levels. Prior to ACTH treatment, dominant females tended to have higher dexamethasone-suppressed cortisol levels than subordinate and ovariectomized females. After ACTH treatment, dominant females had significantly higher cortisol concentrations, as well as higher peak and net integrated cortisol responses to ACTH, than did subordinate and ovariectomized animals; the latter two groups showed comparable cortisol responses to ACTH. These results suggest that dampened adrenocortical responsiveness to ACTH contributes to chronic reductions in cortisol levels in subordinate female marmosets and may be mediated by suppression of reproductive hormones.

Prenatal exposure of female rhesus monkeys to testosterone propionate increases serum luteinizing hormone levels in adulthood.

OBJECTIVE: To determine whether prenatal androgenization of the developing primate hypothalamohypophyseal unit induces irreversible changes in LH secretion. DESIGN: Prospective nonrandomized study. SETTING: An academic research environment. ANIMALS: Forty-one adult ovulatory female rhesus monkeys. INTERVENTION(S): Seventeen female rhesus monkeys exposed prenatally to testosterone propionate (female pseudohermaphrodites) and 24 normal females underwent blood sampling over two ovulatory menstrual cycles. MAIN OUTCOME MEASURE(S): Serum FSH, LH, E2, and T were determined by RIA; P was determined by enzyme immunoassay. Serum bioactive LH was measured by mouse Leydig cell bioassay. RESULT(S): Tonic immunoactive LH hypersecretion and normal FSH release occurred in female pseudohermaphrodites compared with normal females. Periovulatory immunoactive LH and FSH secretion was similar in both female types, whereas a relative increase in the amount of circulating bioactive LH to immunoactive LH was found at midcycle in female pseudohermaphrodites versus normal females. The length of the follicular phase was unaffected by prenatal androgen exposure, but the slopes of serum T and E2 concentrations versus follicular phase cycle day were significantly lower in female pseudohermaphrodites than normal females. Luteal phase length and P secretion were comparable in both types of females. CONCLUSION(S): Androgen exposure during primate neural differentiation may alter permanently the pattern of LH secretion in the presence of cyclic gonadotropin release.