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.

Influence of female hormonal state on rhesus sexual behavior varies with space for social interaction.

The sexual behavior of rhesus monkeys in 15 male-female pairings was observed in both a large and a small area during the follicular and luteal phases of the female's cycle. Males ejaculated in all tests at the follicular phase of the female's cycle and in 53 percent of tests at the luteal phase. However, a significant decline in ejaculation during tests at the luteal phase occurred in the large, but not in the small area. Thus the degree to which the pair's sexual behavior was influenced by the female's hormonal state depended on the spatial conditions of the test.

Mating behavior: facilitation in the female rat after cortical application of potassium chloride.

Mating behavior in ovariectomized female rats treated with estrogen can be greatly enhanced by subcutaneous injections of progesterone. Application of potassium chloride to the cortex of females previously treated with estrogen can also induce greatly increased sexual receptivity as indicated by the lordosis response. This facilitation of mating behavior by a treatment known to cause functional decortication suggests that mechanisms mediating female mating behavior are under tonic inhibition by an inhibitory system which involves the cerebral cortex.

Prenatal androgen blockade accelerates pubertal development in male rhesus monkeys.

Aspects of the prenatal environment, including steroid hormones, modulate the timing of puberty onset in many mammalian species. This study tested whether prenatal androgen manipulations altered pubertal development in male rhesus macaques (Macaca mulatta). Pregnant females received testosterone enanthate (TE), the androgen receptor blocker flutamide, or vehicle during one of two periods of gestation, and their male offspring were observed for morphological, endocrine, and behavioral development from 3 to 4.5 years of age. Males exposed to flutamide early in gestation had a greater response to exogenous GnRH prepubertally, and greater testes volume, elevated testosterone, and elevated LH at age 3.5 than did control subjects. Males exposed to flutamide late in gestation also had greater testes volumes at age 3.5 than did control males. However, these differences between flutamide treated males and control males did not persist postpubertally. By 4.5 years of age, development in control males had reached comparable levels to that of flutamide-treated males. Late gestation treatment with TE had no effect on morphological pubertal development but early TE treatment altered some aspects of endocrine function during puberty. None of the prenatal androgen manipulations affected sexual behavior. These findings suggest that prenatal androgens, in conjunction with social factors, masculinize pubertal timing in rhesus monkey males.

Desire and ability: hormones and the regulation of female sexual behavior.

The distinction between the ability to copulate and the desire to copulate is used to understand species differences in hormonal regulation of female sexual behavior. Evidence is presented demonstrating that ovarian hormones modulate female sexual motivation in both rodent and primate females. The thesis is developed that rodent females differ from primate females primarily in their dependence upon hormones for the ability to mate. Thus, apparent differences between the two groups of females in the extent to which hormones control copulatory behavior does not stem from differences in hormonal regulation of female sexual motivation but from the physical ability of primate, but not rodent, females to mate without hormonal stimulation. This emancipation of the ability to copulate from hormonal influence makes female sexual motivation the primary regulator of mating in primates. Dependence upon female sexual motivation means that the copulatory behavior of primate females is easily influenced by their physical and social environment. Because primate females can mate without hormonal input, female sexual initiation, not copulation, is argued to be the only valid indicator of female sexual motivation.

Blockade of neonatal activation of the pituitary-testicular axis: effect on peripubertal luteinizing hormone and testosterone secretion and on testicular development in male monkeys.

The objective of this study was to examine the effect of blockade of neonatal activation of the pituitary-testicular axis, using a GnRH agonist, on sexual development in male rhesus monkeys. Monkeys were treated with either a GnRH agonist (10 micrograms/day; n = 8) or vehicle (n = 9) for 112 days using osmotic minipumps beginning at 10-13 days of age. In control monkeys serum LH and testosterone concentrations during the first 3 postnatal months were similar to those in adults; they then declined to very low levels. GnRH agonist administration caused an immediate and precipitous decline in serum LH and testosterone concentrations to very low levels, and both remained low throughout the rest of the agonist administration period. Neither group had any significant elevation in serum LH or testosterone concentrations during the next 2 yr. In the control monkeys serum LH and testosterone began to rise during the third year, with a rapid increase occurring during the fall coincident with the breeding season. This peripubertal rise of LH and testosterone secretion was associated with rapid enlargement of the testes and the appearance of sperm in ejaculates. The monkeys who had received GnRH agonist had subnormal serum LH and testosterone increases, and testicular enlargement was also attenuated compared to that in the control animals during the third year of life. Semen samples were recovered from only 50% of the GnRH agonist-treated monkeys during this period, and the sperm count per ejaculate was suppressed. The serum LH responses of the GnRH agonist-treated monkeys to an iv bolus dose of GnRH (5 micrograms/kg BW) during the third year were normal. These results suggest that the induction of reversible hypogonadotropin-hypogonadism in neonatal male monkeys alters subsequent testicular development and peripubertal endocrine changes. Thus, neonatal activation of the pituitary-testicular axis may be a critical developmental event in the process of sexual development in male primates.

Neonatal treatment of male monkeys with a gonadotropin-releasing hormone agonist alters differentiation of central nervous system centers that regulate sexual and skeletal development.

Male rhesus monkeys treated continuously with a GnRH agonist for the first 4 months of postnatal life exhibited a delay in the onset of puberty and an attenuated peripubertal rise in testosterone (T) secretion. The objectives of the current study were to determine whether these effects on sexual development were permanent and whether the hypothalamic-pituitary-testicular axis was functioning normally in these animals as adults. Neonatal GnRH agonist treatment delays but does not permanently block sexual maturation in male monkeys. Treated animals that did not show a pubertal rise in serum T during the breeding season of their 4th year exhibited a seasonal but subnormal elevation of serum T during the subsequent breeding season. Growth of the skeleton was diminished as evidenced by shorter adult crown-rump, tibia, and femur length and reduced bone mineral density of the humerus and lumbar spine. The magnitude of the serum LH and T response to iv pulses of GnRH [50 ng/kg body weight (BW)] and naloxone (1 mg/kg BW) did not differ between control and treated animals during the nonbreeding or breeding season at 6 yr of age. Conversely, treated animals showed a subnormal serum LH and T response to N-methyl-D,L-aspartic acid (5 mg/kg BW iv) during the nonbreeding season. These data suggest that adult monkeys treated neonatally with a GnRH agonist exhibit subnormal sensitivity of the central nervous system to one or more excitatory amino acids (e.g. aspartate or glutamate). Thus, abolishing neonatal activation of the pituitary-testicular axis with a GnRH agonist may permanently alter differentiation of central nervous system centers that are either involved in GnRH secretion or govern this process.

Blockade of neonatal activation of the pituitary-testicular axis with continuous administration of a gonadotropin-releasing hormone agonist in male rhesus monkeys.

The objective of this study was to determine the effects of continuous GnRH agonist (Ag) treatment on neonatal activation of the pituitary-testicular axis in male rhesus monkeys. Five infants were treated continuously with Ag(10 micrograms/day; Wy-40972) for 112 days using osmotic minipumps beginning at 10-13 days of age. Two of five age-matched control animals were implanted sc with Silastic implants of comparable size to the minipumps; three did not receive sham implants. Ag treatment caused a fall in serum LH (bioassay) values to undetectable levels (much less than 0.1 micrograms/ml) within 3 weeks, where they remained throughout Ag treatment. Mean serum testosterone (T) levels fell from pretreatment values of 1.52 +/- 0.45 to 0.38 +/- 0.09 (+/- SE) ng/ml after 3 weeks of Ag treatment. The level of T never exceeded 0.60 ng/ml throughout the subsequent course of Ag treatment. In contrast, serum LH and T were elevated to levels that approached adult values during the first 2 postnatal months in control infants with or without sham implants. Both LH and T then gradually declined, and by 4 months of age, T levels were indistinguishable from those in Ag-treated animals. Control infants had an increase in serum LH from 0.56 +/- 0.10 to 2.67 +/- 0.49 micrograms/ml within 60 min of administration of 5 micrograms GnRH/kg BW at 60 days of age. Serum T values rose from 2.35 +/- 1.00 to 9.48 +/- 3.15 ng/ml during the same period. Seven weeks of Ag treatment abolished the LH and T responses to GnRH. Thirty days after the termination of Ag treatment (approximately 150 days of age), Ag-treated and control infants had comparable serum LH and T responses to GnRH, although the responses were reduced relative to the responses in controls at 60 days of age. These results suggest that continuous administration of Ag desensitizes the pituitary of the male infant rhesus monkey to GnRH and blocks neonatal activation of the pituitary-testicular axis.

Neonatal treatment with luteinizing hormone-releasing hormone analogs alters peripheral lymphocyte subsets and cellular and humorally mediated immune responses in juvenile and adult male monkeys.

We examined the effect of treatment with a LH-releasing hormone (LHRH) agonist (Ag), antagonist (Ant), or Ant and androgen (Ant/And) for the first 4 months of postnatal life on lymphocyte subsets and cellular and humorally mediated immune responses in juvenile and adult male monkeys. We also determined the effect of 9 weeks of Ant treatment on lymphocyte subsets in adult male monkeys. Adult male monkeys that had been treated neonatally with an Ag had increased levels of CD8-positive (CD8+) T-cells and reduced levels of B-cells compared to vehicle-treated controls. Lymphocytes from these animals also showed an elevated proliferative response to a variety of mitogens compared to cells from control animals. Antibody production in response to tetanus toxoid was normal in treated animals. Other neonates treated with Ant/And exhibited subnormal levels of lymphocytes, CD8+ T-cells, and B-cells at 4 months of age. Similar changes, but of lesser magnitude, were observed in animals treated with Ant alone. At 6 months of age, lymphocytes from both groups of Ant-treated monkeys exhibited an above normal proliferative response to streptolysin-O, but not to other mitogens. At 18 months of age, animals treated with Ant alone produced more antitetanus antibody in response to a tetanus toxoid booster than the controls or Ant/And-treated animals. Ant treatment was without major effect on lymphocyte subsets in adult monkeys. Serum LH and testosterone levels declined, and there was a small but significant increase in B-cells, lymphocytes expressing the interleukin-2 receptor, and the CD4+/CD8+ T-cell ratio during treatment, but these parameters normalized during the posttreatment period. The data suggest that chronic neonatal treatment with an Ag or Ant alters the development of immune system responses in male primates. The significance of these changes and their impact on the ability of these animals to respond to pathogenic agents is under investigation.

Inhibin-B in the male rhesus monkey: impact of neonatal gonadotropin-releasing hormone antagonist treatment and sexual development.

We examined the effect of reversibly suppressing pituitary-testicular function during the neonatal period on developmental changes in inhibin-B and FSH secretion in male rhesus monkeys. Infants were treated with either vehicle, a GnRH antagonist (Ant) or the Ant and androgen (Ant/And) for the first 4 postnatal months, and the effects on serum inhibin-B and FSH were monitored during the neonatal and peripubertal periods. In neonates, Ant or Ant/And treatment lowered both serum FSH and inhibin-B levels. By 12 months of age, inhibin-B concentrations no longer differed across treatment groups. A major increase in inhibin-B occurred between 27-36 months of age (late prepubertal period) in all groups, but levels were lower at 33 and 36 months of age in Ant/And-treated animals than in controls. These differences most likely were related to fewer Ant/And-treated animals achieving sexual maturity during their fourth year of life. Regardless of treatment, inhibin-B levels were higher in those that were destined to become mature (in year 4) than in those that were not. During the late prepubertal period, serum inhibin-B was positively correlated with age and testicular volume, but not with serum LH or testosterone. After this period (39-52 months of age), inhibin-B no longer correlated with these parameters. FSH levels were near or below detection limits in most peripubertal animals, but FSH was detectable in fewer samples from control than treated animals. The data suggest that inhibin-B secretion in the neonate is driven by gonadotropin secretion, but during the juvenile hiatus in gonadotropin secretion, the monkey testis continues to produce substantial amounts of this hormone.

Advancement of ovulation in the guinea-pig with exogenous progesterone and related effects on length of the oestrous cycle and life span of the corpus luteum.

A single subcutaneous injection of progesterone (0-4 mg) was given to intact female guinea-pigs on either day 1, 7, 12, 13, 14, 15, or 16 of the behavioral oestrous cycle (day of oestrus is day 0). Injections given on either day 1 or day 7 had little effect, although there was a suggestion that injection on day 7 produced a lengthening of the cycle. Animals injected on either day 12 or day 13 underwent cycles of 16-9 and 18-1 days average length, respectively, which were significantly longer than the mean of 15-8 days for uninjected control females. Injection of progesterone on days 14, 15, or 16 was associated with one of three distinct sequelae: (1) simple prolongation of the cycle associated with a return to spontaneous oestrus 4--7 days later; (2) advancement of ovulation, formation of abnormal corpora lutea, and return to spontaneous oestrus 9--13 days later, and (3) return to spontaneous oestrus 14--16 days after the progesterone injection. These findings suggest that progesterone can cause the release of ovulatory amounts of gonadotropin following a period of endogenous oestrogen conditioning of the gonadotrophic system. If progesterone is administered before oestrogen conditioning is complete, then it inhibits or delays the conditioning process, and spontaneous oestrus and ovulation are postponed.

Sexual maturation in male rhesus monkeys: importance of neonatal testosterone exposure and social rank.

In a 5-year longitudinal study, we examined the effect of disrupting the neonatal activity of the pituitary-testicular axis on the sexual development of male rhesus monkeys. Animals in a social group under natural lighting conditions were treated with a GnRH antagonist (antide), antide and androgen, or both vehicles, from birth until 4 months of age. In antide-treated neonates, serum LH and testosterone were near or below the limits of detection throughout the neonatal period. Antide + androgen-treated neonates had subnormal serum LH, but above normal testosterone concentrations during the treatment period. From 6 to 36 months of age, serum LH and testosterone were near or below the limits of detection. Ten of 12 control animals reached puberty during the breeding season of their 4th year, compared with five of 10 antide- and three of eight antide + androgen-treated animals. Although matriline rank was balanced across treatment groups at birth, a disruption within the social group during year 2 resulted in a marginally lower social ranking of the two treated groups compared with the controls. More high (78%) than low (22%) ranking animals reached puberty during year 4. During the breeding season of that year, serum LH, testosterone and testicular volume were positively correlated with social rank. Thus the lower social rank of treated animals may have contributed to the subnormal numbers of these animals reaching puberty during year 4. However, of those animals achieving puberty during year 4, the pattern of peripubertal changes in serum testosterone and testicular volume differed between control and antide-treated animals. The results appear to suggest that the disruption of normal activity of the neonatal pituitary--testicular axis retarded sexual development, but that social rank is a key regulatory factor in setting the timing of sexual maturation in male rhesus monkeys. The effect of neonatal treatment with antide and low social rank on sexual development could not be reversed by neonatal exposure to greater than normal concentrations of androgen.

Preoptic lesions increase the display of lordosis by male rats.

Male rats do not normally show feminine patterns of sexual behavior even when injected with the ovarian hormones estrogen and progesterone. We find that brain lesions which damage the preoptic-anterior hypothalamic continuum augment the display of lordosis in hormone-treated male rats. The most effectively feminizing brain lesions are ones which bilaterally destroy a substantial portion of the medial preoptic area encompassing the sexually dimorphic nucleus of the preoptic area (SDN-POA). Males with particularly large preoptic lesions are receptive following estrogen treatment and show a progesterone facilitation of receptivity. In this respect, they cannot be behaviorally distinguished from females. Thus, axons originating in and/or passing through the preoptic area apparently inhibit the display of feminine sexual behaviors in males. Preoptic development and lordosis are each predictably affected by perinatal stimulation by testicular hormones, and hormone-stimulated preoptic development may form the neurological basis for some of the defeminizing effects of perinatal hormonal exposure. Our results raise the possibility that the site of this behavioral defeminization is the SDN-POA.

Interest in infants varies with reproductive condition in group-living female pigtail macaques (Macaca nemestrina).

Previous studies of macaques have failed to show a relationship between female reproductive condition and infant-directed behavior. This has led to the view that maternal responsiveness in nonhuman primates is independent from hormonal variables and is mainly regulated by social and experiential factors. The present study reports longitudinal data on interactions between group-living adult pigtail macaque females and other females' infants less than 12 weeks of age during the 6 weeks prior to conception, the 24 weeks of pregnancy, and the first 12 weeks of lactation. These periods represent different hormonal conditions in the reproductive cycle. Infant-directed behavior increased in frequency during early and middle pregnancy, decreased around the time of parturition, and increased again during lactation. The frequency of infant-directed behavior also increased significantly in the 2 weeks after infant loss during lactation. These results are in accord with recent evidence in New World monkeys indicating that maternal responsiveness in nonhuman primates develops during pregnancy and it is probably hormonally regulated.

Social complexity and hormonal influences on sexual behavior in rhesus monkeys (Macaca mulatta).

Rhesus females in multi-animal groups mate only during the mid-follicular and periovulatory portions of their ovarian cycle, whereas females in pair-tests often mate at all cycle phases. We investigated the influence of social context on hormonal mediation of rhesus sexual behavior by observing the same males and females in both pair-tests and in single male/multi-female group-tests. Five intact adult female rhesus were tested with each of four males during their follicular, periovulatory, and luteal cycle phases as verified by steroid radioimmunoassay. Male initiated behaviors of approach, hiptouch, mount, and ejaculation were significantly above luteal levels during periovulatory group-tests, with periovulatory approach and hiptouch frequencies also significantly above follicular levels. Approach did not vary with the female's cycle phase in pair-tests and both follicular and periovulatory frequencies of hiptouch, mount, and ejaculation were higher than luteal levels. Pair-test frequencies were greater than group-test frequencies at all cycle phases, except for male approach, where the difference depended upon female cycle phase. When group-tested, females approached the male, presented, and handslapped most frequently during periovulatory tests. In pair-tests, females approached and handslapped equally during follicular and periovulatory tests and lower luteally, but presents did not vary cyclically. Females approached males significantly more frequently during pair- than group-tests, but there were no consistent differences between the two social conditions for present and handslap. In group-tests, periovulatory females were approached and threatened by other group females significantly more often that they were at other cycle phases.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of male rhesus testicular function and sexual behavior by a gonadotropin-releasing-hormone agonist.

Castrated rhesus monkeys tested in pair tests in small areas mate up to 6 yr after castration, though there is high individual variability (22,23). The generalizability of these findings to social groups in larger areas is unknown. The sexual behavior of 4 adult rhesus males tested singly with a group of 9 intact adult females was examined during short-term, counterbalanced, gonadotropin-releasing-hormone(GnRH)-agonist-induced testicular suppression and control treatment. GnRH-agonist treatment suppressed testosterone to less than 0.6 ng/ml within 16 days. Ten days later (e.g., after 26 days of GnRH-agonist treatment) males were observed for 7 days. The frequency of hiptouches, mounts, intromissons and ejaculations were significantly reduced by testicular suppression. This behavioral reduction was more marked than previously reported in pair-tested castrates during a similar time-period, suggesting testicular suppression more profoundly affects behavior in multifemale groups in larger areas. Males differed in the extent that testicular suppression reduced their sexual behavior. Male ejaculations were completely unaffected in one male and completely eliminated in another. Sexual behavior was reduced less in males with high control levels of testosterone and behavior. Behavioral suppression was unrelated to differences in female behavior and appeared to result from reduced male sexual responsiveness or motivation.

Lordosis behavior in males of two inbred strains of guinea pig.

The lordosis behavior of male guinea pigs from inbred strains 2 and 13 was examined. Significantly more isolated gonadally intact males of strain 2 than strain 13 displayed lordosis. Castration did not decrease the display of lordosis. In castrated strain 2 males, those which showed lordosis did not have higher plasma androgen, estrone or estradiol levels than those which did not show lordosis. They also did not differ hormonally from ovariectomized strain 2 females even though strain 2 females never showed lordosis without hormone replacement. Although the lordosis shown by strain 2 males was not related to endogenous gonadal hormone levels, estradiol benzoate (EB) administration facilitated lordosis. EB had no clear effect on lordosis in strain 13 males. Progesterone after EB priming had no further facilitative effect in males of either strain. These results indicate that lordosis is more readily elicited from strain 2 than strain 13 males. Furthermore, lordosis in strain 2 males is not dependent upon gonadal hormones for its display although it is facilitated by EB (but not progesterone).