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Hyperandrogenemia is associated with several clinical disorders in which both reproductive dysfunction and metabolic changes may coexist [i.e. polycystic ovary syndrome (PCOS), obesity and congenital adrenal hyperplasia]. Moreover, there is growing evidence that the elevated levels of circulating androgens in obese girls may lead to an increased neuroendocrine drive to the reproductive axis, similar to that associated with PCOS.To test whether androgen exposure in the childhood and adolescent period could lead to pubertal alterations in LH secretory patterns, female rhesus monkeys received subcutaneous testosterone implants prepubertally beginning at 1 year of age, maintaining a 3.7-fold increase (P = 0.001) in circulating testosterone levels over cholesterol-implant controls (n = 6/group) into the post-pubertal period. In early adulthood, pulsatile secretion of LH was measured over 12 h during the early follicular phase of a menstrual cycle, and responsiveness of the pituitary to gonadotrophin-releasing hormone was determined. In addition, ultrasounds were performed to assess ovarian morphology and glucose tolerance testing was performed to assess insulin sensitivity.The timing of menarche was similar between groups. Testosterone-treated animals had a significantly greater LH pulse frequency during the early follicular phase compared with controls (P = 0.039) when measured at 5 years of age. There was a larger LH response to GnRH when testosterone-treated animals were 4 years of age (P = 0.042), but not when the animals were 5 years old (P = 0.57). No differences were seen in insulin sensitivity or ovarian morphology, and the groups showed similar rates of ovulation in early adulthood.Exposure to increased levels of androgens over the course of pubertal development appears to trigger physiological changes in the neural drive to the reproductive axis that resemble those of obese hyperandrogenemic girls in early adulthood and are characteristic of PCOS.

Most biomedical facilities that use rhesus macaques (Macaca mulatta) limit the amount of blood that may be collected for experimental purposes. These limits typically are expressed as a percentage of blood volume (BV), estimated by using a fixed ratio of blood (mL) per body weight (kg). BV estimation ratios vary widely among facilities and typically do not factor in variables known to influence BV in humans: sex, age, and body condition. We used indicator dilution methodology to determine the BV of 20 adult rhesus macaques (10 male, 10 female) that varied widely in body condition. We measured body composition by using dual-energy X-ray absorptiometry, weight, crown-to-rump length, and body condition score. Two indicators, FITC-labeled hydroxyethyl starch (FITC-HES) and radioiodinated rhesus serum albumin ((125)I-RhSA), were injected simultaneously, followed by serial blood collection. Plasma volume at time 0 was determined by linear regression. BV was calculated from the plasma volume and Hct. We found that BV calculated by using FITC-HES was consistently lower than BV calculated by using (125)I-RhSA. Sex and age did not significantly affect BV. Percentage body fat was significantly associated with BV. Subjects categorized as having 'optimal' body condition score had 18% body fat and 62.1 mL/kg BV (by FITC-HES; 74.5 mL/kg by (125)I-RhSA). Each 1% increase in body fat corresponded to approximately 1 mL/kg decrease in BV. Body condition score correlated with the body fat percentage (R(2) = 0.7469). We provide an equation for calculating BV from weight and body condition score.

Anxiety is a normal aspect of human personality, which can manifest in a variety of disorders and other negative traits. The primary treatment for anxiety is the class of drugs known as the selective serotonin reuptake inhibitors (SSRIs), which bind to the serotonin reuptake transporter. The upstream region of the gene that codes for this transporter contains a polymorphism that is an insertion/deletion event that in turn, produces long (l) and short (s) alleles in the population. This particular polymorphism in the serotonin transporter, the 5HTTLPR (serotonin transporter linked polymorphic region), is thought to be involved in the genesis of anxious traits and disorders. Most studies with human subjects have examined adult behavior, which may derive from diverse experiential and environmental backgrounds, as well as genetic differences. To better isolate the effect of genetics, we genotyped 128 infant and juvenile monkeys for the 5HTTLPR and tested for behavioral response in four testing paradigms designed to elicit fearful-anxious behaviors: a free play, remote-controlled car, human intruder, and novel fruit test. The s/s monkeys were found to be behaviorally inhibited in the free play test, engaged in more fear behaviors in the remote-controlled car test, and threatened more in the stare portion of the human intruder test, even though a small number of monkeys were assessed. There was no difference between genotypes of either sex in the prolactin response to fenfluramine. These data indicate greater anxiety in the s/s monkeys for distinct facets of anxious behavior, which are independent of a global neurohormonal challenge test. These neurobehavioral data support recent neuroimaging findings in humans indicating the importance of the 5HTTLPR for amygdala-dependent anxious behavior.

Many patients with hyperandrogenemia are overweight or obese, which exacerbates morbidities associated with polycystic ovary syndrome (PCOS). To examine the ability of testosterone (T) to generate PCOS-like symptoms, monkeys received T or cholesterol (control) implants ( n = 6/group) beginning prepubertally. As previously reported, T-treated animals had increased neuroendocrine drive to the reproductive axis [increased luteinizing hormone (LH) pulse frequency] at 5 yr, without remarkable changes in ovarian or metabolic features. To examine the combined effects of T and obesity, at 5.5 yr (human equivalent age: 17 yr), monkeys were placed on a high-calorie, high-fat diet typical of Western cultures [Western style diet (WSD)], which increased body fat from

Noradrenaline plays a key role in the initiation of ovulation in nonprimate species. A similar noradrenaline role in the primate has not been established experimentally. We utilized the ovariectomized-oestrogen-supplemented (OVX + E) rhesus macaque to examine the effects of intravenous (i.v.) infusion of oestradiol-17beta (E2) on the activity of the brain noradrenaline system. Experiment 1 established the induction of a preovulatory surge-like release of luteinizing hormone in OVX + E monkeys by i.v. infusion of E2 (OVX + E + E2). In experiment 2, a marked increase in hypothalamic microdialysate noradrenaline concentrations occurred after identical E2 infusion into the OVX + E monkeys that were used in experiment 1. In experiment 3, tyrosine hydroxylase (TH) mRNA expression in the locus coeruleus of the brainstem increased at various times after E2 infusion as determined by semiquantitative in situ hybridization. The amount of TH mRNA in OVX + E + E2 animals was higher (P0.05) than that in either the OVX + E or OVX monkeys; no difference was found in the latter two groups. Moreover, selected locus coeruleus sections from E2-infused monkeys were examined for the localization of oestrogen receptors (ER) by in situ hybridization. Both ER-alpha and ER-beta mRNAs were expressed in the locus coeruleus, although the expression was greater for ER-alpha than for ER-beta. We conclude that i.v. infusion of E2, which induces a preovulatory surge-like release of LH, stimulates brain noradrenaline activity; this enhanced activity likely involves an ER-mediated process and is reflected by hypothalamic noradrenaline release and locus coeruleus TH mRNA expression. The results support the concept that noradrenaline can influence the E2-stimulated ovulation in nonhuman primates and that the brainstem is one of the components in this neuroendocrine process.

Leptin acts as a metabolic activator of the neuroendocrine reproductive axis in several rodent species, but whether leptin plays a similar role in primates is unknown. To explore this question, we examined the effects of leptin on gonadotropin and testosterone secretion in male rhesus monkeys that were fasted for 2 days. Mean plasma levels of LH and FSH, LH pulse frequency, and LH pulse amplitude were significantly higher in leptin-treated animals compared with saline-treated controls during the second day of the fast. To identify targets for leptin's action, we used in situ hybridization and computerized imaging to map leptin receptor (Ob-R) messenger RNA (mRNA) distribution. Ob-R mRNA was observed in the anterior pituitary and several areas of the brain, including the arcuate and ventromedial nuclei of the hypothalamus. Ob-R mRNA was coexpressed in both POMC and neuropeptide Y neurons in the arcuate nucleus, whereas little or no coexpression of Ob-R mRNA was evident in GnRH neurons. These results suggest that leptin is a metabolic signal to the reproductive axis in primates and imply that both POMC and neuropeptide Y neurons are involved in mediating leptin's effects in the brain.

Certain brain peptides and catecholamines function in activating the hypothalamohypophysial-ovarian axis in both rabbits and rhesus monkeys. The natural stimulus for a surge release of GnRH is coitus in rabbits, whereas the initial excitatory signal is ovarian steroids in monkeys. Despite this contrast in initial signals, specific neurochemicals may serve as common stimuli for GnRH secretion in both species. Evidence is presented that one such substance is norepinephrine (NE), which is released from the mediobasal hypothalamus before, or simultaneously with, GnRH, although the latency in time between stimulus (coitus/estrogen) and response (GnRH/LH release) is very different. Moreover, both stimuli activate NE gene expression in cells located in the brainstem. We suggest that the brainstem is an extra-hypothalamic site where preovulatory signals for GnRH surges are developed in both rabbits and primates.

The mammalian testis is innervated by extrinsic catecholaminergic nerves and responds to catecholamines with steroid secretion. Although the primate testis has also been shown to be innervated, potential differences in the density of this innervation between immature and sexually developed individuals have not been described. A recent study demonstrated that the primate ovary contains a network of neuron-like cells and that some of these cells are catecholaminergic. It is thus possible that the male gonad is also endowed with a similar intragonadal source of catecholamines. The present study addresses these two issues. Catecholaminergic nerves were identified as such by their content of immunoreactive tyrosine hydroxylase (TH; the rate-limiting step in catecholamine biosynthesis), and in some cases by glyoxylic acid histochemistry. Fibers containing TH were abundant in testes from juvenile animals (1-2 yr of postnatal life), but the density of this innervation was not maintained in adult animals, whose testis showed only a few TH-positive fibers scattered in the interstitial tissue. Testicular norepinephrine (NE) concentration was much lower in adult than in juvenile animals, suggesting that the marked increase in testicular weight that occurs with the attainment of sexual maturity is not accompanied by corresponding changes in NE content. At the ultrastructural level, testicular nerve fibers contained pleiomorphic, dense-core and clear vesicles, suggesting the presence of catecholamines and other neurotransmitters. In addition to this extrinsic catecholaminergic innervation, prepubertal testes, but not adult gonads, contained an intrinsic population of TH-immunopositive neuron-like elements, identified as cells by confocal scanning laser microscopy. To determine whether the prepubertal monkey testis indeed expresses the TH gene, testicular RNA was subjected to reverse transcriptase polymerase chain reaction to amplify the 5' end of TH mRNA, which encodes the regulatory domain of the enzyme. The cDNA that was obtained predicts an amino acid sequence similar, but not identical, to that encoded by the alternatively spliced type 1 TH mRNA form present in the adrenal gland. These results indicate 1) that the primate testis receives a dual catecholaminergic input, one provided by the extrinsic innervation and the other by neuron-like cells located within the gonad itself, and 2) that the influence exerted by both sources on testicular function may be more prominent during the prepubertal period than in adulthood. The presence in the testis of a TH mRNA variant encoding amino acid substitutions in its 5' end suggests that regulation of testicular TH enzyme activity may include a gonad-specific component.

Concomitant fluctuations in median eminence perfusate GnRH and plasma LH occur in rhesus macaques during the periovulatory period and after ovariectomy. The association between GnRH and LH pulses during the follicular and luteal phases of the monkey menstrual cycle is less clearly defined. However, observed LH patterns suggest higher amplitude and slower pulses of GnRH in the luteal than in the follicular phase of the menstrual cycle. The present studies were planned to compare the GnRH/LH patterns in individual monkeys by simultaneous push-pull perfusion (PPP) and blood sampling during different ovarian steroid milieus. In the initial trial, placement of two push-pull cannulae (PPCs) in the median eminence and a jugular vein catheter caused immediate loss of regular menstrual cycles in 3 monkeys, although cycles resumed over 3-6 mo postoperatively. After the return of normal reproductive cycles, PPP was performed for 12 h on either Day 7, 8, or 9 of the luteal phase. The results showed an unexpected and profound decline in LH and progesterone (P4) concentrations during the initial 4 h. No pulses of LH or P4 were observed in the remaining 8 h. All 3 monkeys exhibited menstrual bleeding 2-3 days after PPP. In subsequent trials, we continuously infused the opioid receptor antagonist nalmefene (Nmf, 1 mg/h, i.v.), starting the fourth day after PPC implantation into 11 monkeys. Menstrual cycles with accompanying fluctuations of circulating estradiol-17 beta (E2) and P4 returned in less than 40 days in these macaques and continued without further Nmf treatment. Trials of 12-h PPP/blood sampling were performed during the follicular phase with (n = 4) or without (n = 4) Nmf, and during the luteal phase with (n = 6) or without (n = 3) Nmf. Endocrine data from the 3 animals without Nmf during the luteal phase were combined with the hormonal values that were obtained in the initial trial because all 6 animals exhibited similar GnRH, LH, and P4 profiles, i.e., low levels and infrequent or absent pulses. Treatment with Nmf during luteal sampling enhanced hypothalamic GnRH secretion (10-fold increase in mean GnRH levels over those without Nmf) and reinitiated distinctive serum LH and P4 pulses. In contrast, patterns of hypothalamic GnRH and serum LH during the follicular phase were similar with or without Nmf treatment. These GnRH/LH profiles consisted of low-amplitude hourly pulses. Collectively, the observations suggest that stress-induced activation of opiatergic neurons can inhibit the GnRH pulse generator and that these neuronal systems are more sensitive to such inhibition in the presence of elevated levels of circulating P4. However, our observation that Nmf accelerated the reinstatement of ovarian cycles after surgery, when circulating E2 and P4 were very low, suggests that GnRH secretions are influenced by activation of different opioid receptor subtypes in response to different stresses. Some of these GnRH/opioid interactions are independent of P4.

Twenty-four-hour pulsatile patterns of bioactive luteinizing hormone (LH) and immunoactive estradiol (E2) and progesterone (P4) were measured during the mid-follicular (FP, Days 5, 6, or 7) and mid-luteal (LP, Days 7, 8, 9, or 10) phases of the menstrual cycle in six intact and in four ovariectomized (OVX) rhesus monkeys. Blood samples were collected remotely at 7.5-min (FP) and 15-min (LP, OVX) intervals via catheters in freely moving, vested monkeys. Hormonal patterns were analyzed by the MUNRO computer program. A significant coupling of LH and E2 pulses was observed during the FP and between the LH and P4 pulses during the LP. No diurnal rhythm of LH pulse frequency was observed during the FP or in OVX monkeys. In contrast, day/night differences in frequency were seen during the luteal phases (0.35 +/- 0.03 vs. 0.24 +/- 0.05 pulses/h, day vs. night, p less than 0.05). Naloxone (NAL), an opiate antagonist, was continuously infused (2 mg/h.i.v.) for 12 h at night during the LP. NAL treatment increased nighttime LH pulse frequency to 0.33 +/- 0.07 pulses/h, which was not different from the daytime LH pulse frequency, thereby abolishing the normal diurnal pattern. Interestingly, NAL treatment did not achieve the approximately 1 pulse/h frequency seen in the FP or OVX condition. These results support the hypothesis that P4 modulates the activity of an opioid system that is responsible both for a slowing of LH pulse frequency during the LP and for an additional nightly reduction in hypothalamic LH pulse-generating activity.