Intact Male Rhesus Research Articles

Short-term testosterone manipulations do not affect cognition or motor function but differentially modulate emotions in young and older male rhesus monkeys

Human aging is characterized by declines in cognition and fine motor function as well as improved emotional regulation. In men, declining levels of testosterone (T) with age have been implicated in the development of these age-related changes. However, studies examining the effects of T replacement on cognition, emotion and fine motor function in older men have not provided consistent results. Rhesus monkeys (Macaca mulatta) are excellent models for human cognitive aging and may provide novel insights on this issue. We tested 10 aged intact male rhesus monkeys (mean age=19, range 15–25) on a battery of cognitive, motor and emotional tasks at baseline and under low or high T experimental conditions. Their performance was compared to that of 6 young males previously tested in the same paradigm (Lacreuse et al., 2009; Lacreuse et al., 2010). Following a 4-week baseline testing period, monkeys were treated with a gonadotropin releasing hormone agonist (Depot Lupron, 200μg/kg) to suppress endogenous T and were tested on the task battery under a 4-week high T condition (injection of Lupron+T enanthate, 20mg/kg, n=8) or 4-week low T condition (injection of Lupron+oil vehicle, n=8) before crossing over to the opposite treatment. The cognitive tasks consisted of the Delayed Non-Matching-to-Sample (DNMS), the Delayed Response (DR), and the Delayed Recognition Span Test (spatial-DRST). The emotional tasks included an object Approach-Avoidance task and a task in which monkeys were played videos of unfamiliar conspecifics in different emotional context (Social Playbacks). The fine motor task was the Lifesaver task that required monkeys to remove a Lifesaver candy from rods of different complexity.T manipulations did not significantly affect visual recognition memory, working memory, reference memory or fine motor function at any age. In the Approach-Avoidance task, older monkeys, but not younger monkeys, spent more time in proximity of novel objects in the high T condition relative to the low T condition. In both age groups, high T increased watching time of threatening social stimuli in the Social Playbacks. These results suggest that T affects some aspects of emotional processing but has no effect on fine motor function or cognition in young or older male macaques. It is possible that the duration of T treatment was not long enough to affect cognition or fine motor function or that T levels were too high to improve these outcomes. An alternative explanation for the discrepancies of our findings with some of the cognitive and emotional effects of T reported in rodents and humans may be the use of a chemical castration, which reduced circulating gonadotropins in addition to T. Further studies will investigate whether the luteinizing hormone LH mediates the effects of T on brain function in male primates.

Short-term testosterone manipulations modulate visual recognition memory and some aspects of emotional reactivity in male rhesus monkeys

The role of testosterone (T) in modulating cognitive function and emotion in men remains unclear. The paucity of animal studies has likely contributed to the slow progress in this area. In particular, studies in nonhuman primates have been lacking. Our laboratory has begun to address this issue by pharmacologically manipulating T levels in intact male rhesus monkeys, using blind, placebo-controlled, crossover designs. We previously found that T-suppressed monkeys receiving supraphysiological T for 4weeks had lower visual recognition memory for long delays and enhanced attention to videos of negative social stimuli (Lacreuse et al., 2009, 2010) compared to when treated with oil. To further delineate the conditions under which T affects cognition and emotion, the present study focused on the short-term effects of physiological T. Six intact males were treated with the gonadotropin-releasing hormone antagonist degarelix (3mg/kg) for 7days and received one injection of T enanthate (5mg/kg) followed by one injection of oil vehicle 7days later (n=3), or the reverse treatment (n=3). Performance on two computerized tasks, the Delayed-non-matching-to-sample (DNMS) with random delays and the object-Delayed Recognition Span test (object-DRST) and one task of emotional reactivity, an approach/avoidance task of negative, familiar and novel objects, was examined at baseline and 3–5days after treatment. DNMS performance was significantly better when monkeys were treated with T compared to oil, independently of the delay duration or the nature (emotional or neutral) of the stimuli. Performance on the object-DRST was unaffected. Interestingly, subtle changes in emotional reactivity were also observed: T administration was associated with fewer object contacts, especially on negative objects, without overt changes in anxious behaviors. These results may reflect increased vigilance and alertness with high T. Altogether, the data suggest that changes in general arousal may underlie the beneficial effects of T on DNMS performance. This hypothesis will require further study with objective measures of physiological arousal.

Limited effects of β-endorphin compared to loperamide or fentanyl in a neuroendocrine biomarker assay in non-human primates

The in vivo pharmacodynamics of the opioid neuropeptide beta-endorphin (a major endogenous agonist at the mu-opioid receptor) is difficult to determine in non-human primate models with translational value, or in humans. The present studies therefore employed a neuroendocrine biomarker assay, prolactin release, to systematically compare the in vivo profile of i.v. beta-endorphin (0.01-0.32 mg/kg; i.v.) in gonadally intact male rhesus monkeys (n=4) to that of the peripherally selective mu-agonist loperamide (0.01-0.32 mg/kg; i.v.) and the centrally penetrating mu-agonist fentanyl (0.0056-0.018 mg/kg; i.v.). Studies utilized a standardized time course design (measuring prolactin levels 5-120 min after agonist administration). Beta-endorphin displayed only limited effectiveness in causing prolactin release when tested over this 30-fold dose range, compared to loperamide or fentanyl. Furthermore, two of the four subjects were only minimally responsive to beta-endorphin. This differential responsiveness was not due to the presence of a previously described single nucleotide polymorphism at the OPRM1 gene (C77G), known to affect beta-endorphin pharmacodynamics in vitro. In vivo biotransformation studies with MALDI-mass spectrometry determined that full-length beta-endorphin was detectable in all subjects up to at least 5 min after i.v. administration. Thus, the relative ineffectiveness of i.v. beta-endorphin in this assay does not appear to be principally due to rapid generation of non-opioid fragments of this neuropeptide.

Medroxyprogesterone Acetate and Dihydrotestosterone Induce Coronary Hyperreactivity in Intact Male Rhesus Monkeys

Coronary hyperreactivity (CH), characterized by persistent severe vasoconstrictions in response to vasoconstrictor challenge, is oppositely influenced by progesterone (P) and medroxyprogesterone acetate (MPA) treatment in surgically menopausal primates. In this study we tested whether multiweek MPA or dihydrotestosterone (DHT) exposure induced CH in intact male rhesus monkeys. Coronary angiographic experiments with intracoronary serotonin and the thromboxane A(2) analog U46619 stimulated brief vasoconstriction (for 1-3 min) in large epicardial coronaries in untreated male monkeys. In contrast, MPA- and DHT-treated monkeys displayed long-duration constrictions (>5 min), with significantly greater reductions in the minimal diameters of epicardial coronaries. Immunocytochemistry demonstrated androgen receptors (AR) and P receptors in aorta and coronary arteries, and immunocytochemistry and Western blotting showed AR and P receptors in rhesus coronary vascular muscle cells. In vivo, MPA or DHT increased thromboxane prostanoid (TP) receptor expression in the aorta. In vitro, MPA or DHT increased, whereas P did not change, TP receptor expression in primary coronary vascular muscle cell. This MPA- or DHT-mediated increase in TP receptor expression was attenuated by the AR antagonist flutamide. MPA or DHT induction of CH in intact adult male primates, hypothesized to occur via androgenic up-regulation of vascular muscle TP receptor expression, could predispose to CH-mediated myocardial ischemia.

Differential effects of N-methyl-D-aspartate receptor stimulation on growth hormone secretion at specific stages of postnatal development of the male rhesus monkey

The present study attempts to examine the role of N-methyl-D-aspartate (NMDA) receptor in the central regulation of growth hormone (GH) secretion during specific stages of pubertal development of the male rhesus monkey ( Macaca mulatta). Infantile (n=4), prepubertal (n=5), peripubertal (n=5) and adult (n=5) intact male rhesus monkeys were given an agonist of NMDA receptor, N-methyl-D,L-aspartate (NMA) (15 mg/kg BW) through a teflon cannula implanted in the saphenous vein. Blood samples were collected 20-60 min before and 40-80 min after the injection of the drug at 10-20 min intervals. NMA was dissolved in normal saline immediately before use and passed through a 0.22 μm filter at the time of injection. All bleedings were carried out under ketamine hydrochloride anesthesia (initial dose 5 mg/kg BW, im followed by 2.5 mg/kg at 30 min intervals). The plasma levels of GH and testosterone (T) were determined by using specific assay systems. The hypothalamic-somatotrope activity under basal conditions was studied by averaging all the GH concentrations obtained before NMA injection, whereas the sensitivity of NMDA receptor to NMA stimulation was determined by comparing basal GH levels immediately before NMA injection at 0 min and GH concentrations obtained 10 min after the injection. The mean basal plasma concentrations of GH in the four groups of animals showed marked age-related differences. The levels of GH were found to be higher in infantile and peripubertal monkeys as compared to those of prepubertal and adult animals. A single iv injection of NMA produced differential effects on GH secretion during specific stages of postnatal development depending upon the level of GH secretion under basal conditions. Whereas NMA had no demonstrable effect on GH secretion in infantile and peripubertal animals in which the basal GH levels were high, it produced pronounced effects on GH secretion in prepubertal and adult monkeys wherein baseline GH concentrations were low. In conclusion, the present study suggests that the glutamatergic component of the control system that governs GH secretion by utilizing NMDA receptor may participate in regulation of age-related changes in the secretion of GH in the male rhesus monkey.

Circadian Rhythm of Oxytocin in the Cerebrospinal Fluid of Rhesus and Cynomolgus Monkeys: Effects of Castration and Adrenalectomy and Presence of a Caudal-Rostral Gradient

Oxytocin and vasopressin (AVP) were previously reported to have a diurnal rhythm in cerebrospinal fluid (CSF) collected from the cervical cistern of chaired, intact male rhesus monkeys. In the present study, we continuously sampled CSF from temporary indwelling catheters placed in the spinal subarachnoid space of unanesthetized monkeys maintained on tether and swivel systems. CSF was collected from intact and castrate female rhesus monkeys and intact female and castrate, adrenalectomized male cynomolgus monkeys to determine if oxytocin and AVP rhythms are expressed in spinal subarachnoid CSF, if the magnitude of the CSF rhythm displays a rostral-caudal gradient, and if the rhythm is present in adrenalectomized and castrate monkeys, or is specific to the sex or species of macaque. Monkeys, maintained on a 12-hour light/dark cycle with lights on from 06.00 to 18.00 h, had 19-gauge epidural catheters introduced at the L4-L5 intervertebral space and advanced cephalad in the subarachnoid space. The proximal end of the catheter was connected to a peristaltic pump for continuous removal of CSF (0.5 ml/h) and hourly CSF samples were radioimmunoassayed for oxytocin and AVP. For rostral-caudal studies, the distal tip of the catheter was repositioned every few days to collect CSF from 3 levels of the spinal subarachnoid space: C5-6, T5-6, T12-L1. Each animal had a diurnal CSF oxytocin rhythm with peak and trough oxytocin levels during early light and dark periods, respectively. The magnitude of the oxytocin rhythm differed among animals, but was consistent in an animal from day to day.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of gonadectomy and hormone replacement on steroid hormone receptors and 5 alpha-reductase activity in pituitaries of male rhesus macaques.

We measured androgen, estrogen, and progestin receptors and 5 alpha-reductase activity in the anterior and posterior pituitary gland of intact and 6-week castrate adult male rhesus monkeys and castrate males which were treated with testosterone (T) or estradiol (E) from time of surgery. Saturation analysis of anterior pituitary tissues from monkeys receiving various treatments revealed an apparent mean dissociation constant (Kd) of 0.53 +/- 0.17 (+/- SE) X 10(-10) mol/L (n = 3) for [3H]R1881 (androgen) binding to cytosol, 2.6 +/- 0.50 X 10(-10) mol/L (n = 3) for [3H]R2858 (estrogen) binding to cytosol, 1.7 X 10(-10) mol/L (n = 2) for [3H]R5020 (progestin) binding to cytosol, and 6.2 X 10(-10) mol/L (n = 2) for [3H )R1881 binding to cell nuclear extract. The highest levels of nuclear androgen receptor (AR) were found in intact males [37.1 +/- 3.5 (+/- SE) fmol/mg DNA; n = 7] and castrated males treated with T for 6 weeks (89.7 +/- 30.2 fmol/mg DNA; n = 5). High levels of nuclear AR corresponded to serum T levels and low serum LH levels. Nuclear AR was undetectable in castrated males and castrated males treated with E. Significantly greater levels of cytosolic AR were detected in intact males (27.5 +/- 1.6 fmol/mg protein) compared to all other groups (P less than 0.05). T or E treatment had no effect on cytosolic AR. Increased levels of cytosolic progestin receptor were found in intact monkeys and after E or T treatment compared to levels in untreated castrates. No differences in 5 alpha-reductase activity were found between any treatment groups. These data indicate that anterior pituitary nuclear androgen receptor is correlated with serum LH levels and support the hypothesis of a direct action of T on anterior pituitary LH secretion. In addition, it appears that cytosolic progestin receptor, but not AR, is regulated by estrogen in intact male rhesus monkeys. In the posterior pituitary, AR dynamics followed a profile in which cytosolic AR increased after castration and decreased after T treatment. Nuclear AR decreased after castration and increased after T treatment. The presence of a dynamic AR system in the posterior pituitary suggests hormonal regulation of its function by androgens.

Sexual behavior correlates with the diurnal plasma testosterone range in intact male rhesus monkeys.

There is evidence that androgens are necessary for the full expression of sexual behavior in male primates, but it has proved difficult to relate sexual activity to circulating androgens levels in comparisons between intact males. In the present study, 4423 behavior tests of 32 pairs of rhesus monkeys were conducted in a constant photoperiod over a 2-year period, and there was no significant relationship between the frequency of ejaculation and plasma testosterone levels in samples collected at 0800, 1600 or 2200 h. However, the magnitude of the diurnal range between the lowest and highest levels correlated negatively with sexual behavior. As the seasonal increase in sexual activity occurred, there was a corresponding decrease in the diurnal range of plasma testosterone. Furthermore, those males with the highest numbers of ejaculations showed the smallest diurnal plasma testosterone ranges. An additional experiment with 32 males revealed that neither behavior testing nor the occurrence of ejaculation influenced the diurnal testosterone range. Consequently, we have concluded that if any causality operated it would be in the direction of a hormonal influence on behavior. These findings suggest that increased nocturnal levels of testosterone do not enhance behavior and that a threshold level maintained throughout the 24 h may be a critical endocrine factor.

Low potency of intact male rhesus monkeys after long-term visual contact with their female partners.

We have examined the possibility that prolonged visual contact between future sexual partners, in the absence of any direct sexual contact and mating activity, is sufficient to produce the familiar-female phenomenon and is associated loss of male potency. For 1 year, 32 intact, feral-reared male rhesus monkeys were housed so that 14 males were in constant visual contact with their four future testing females, and 18 of the males were without any visual contact with females. After 1 year, each male was then tested once with each ovariectomized, estrogen-treated female (128 tests). Males in the group having prolonged prior visual contact (group II) were significantly less potent than males without prior visual contact (group I), and this difference was not related to plasma testosterone levels, prior history, time since capture, or presumed age. Group II males had fewer ejaculations during tests and ejaculated with fewer partners, and this appeared to be because they required significantly more stimulation (mounting and thrusting) to reach ejaculation. The data suggested that the familiar-partner phenomenon was not restricted to the male and was associated with increased social affinity and decreased agonistic tension between partners. Under natural conditions, the phenomenon may encourage troop transfers and outbreeding, and in laboratory studies, prior visual contact between testing partners should be regarded as a potentially uncontrolled source of variation.

Increases in testosterone levels and in copulatory behavior of male rhesus monkeys following treatment with human chorionic gonadotrophin

The sexual behavior of five male rhesus monkeys was observed before, during, and after administration of human chorionic gonadotropin (HCG) sufficient to induce increases in serum testosterone. Each male was given 12 half-hour pair tests with an estrogen-implanted ovariectomized female during each of the three phases of the study (Pretreatment, Treatment, Post-treatment). Summaries of selected categories of sexual and other social behaviors were tabulated for each phase of the study. Grooming, mount latency, and ejaculation frequency, as well as other behavioral measures, were unaffected by hormone treatment. However, latency to ejaculation decreased by 40% ( P < 0.01) and mount rate increased by 71% ( P < 0.05) during hormone administration. Sexual presentations by the female decreased by 38% ( P < 0.05). Limb shaking decreased 55% during HCG treatment ( P < 0.05). These findings demonstrate that increases in testosterone secretion subsequent to gonadotrophin injection in intact male rhesus monkeys can produce significant alterations in male and female sexual behavior.

Diurnal changes in plasma testosterone and studies on plasma corticosteroids in non-anaesthetized male rhesus monkeys (Macaca mulatta).

SUMMARY The assays of testosterone and corticosteroids in plasma from adult male rhesus monkeys using competitive protein-binding and radioimmunoassay techniques are described. The radioimmunoassay for testosterone was conducted without chromatography and, therefore, additionally estimated 17β-hydroxy-5α-androstan-3-one (dihydrotestosterone). Levels of testosterone in the peripheral plasma of 14 intact male rhesus monkeys showed marked fluctuations over a period of 24 h. Concentrations of testosterone at 22.00 h (1776 ± 814 ( ± s.d.) ng/100 ml) were approximately double those at 08.00 h (858 ± 407 ng/100 ml), 12.00 h (898 ± 316 ng/100 ml) and 16.00 h (784 ± 530 ng/100 ml). Castration resulted in low plasma testosterone levels (85 ± 29 ng/100 ml), and the increases at 22.00 h were no longer observed. In intact males, the 'basal' plasma corticosteroidlevel(08.00 h) was 22·4 ± 6·0 μg/100 ml. Administration of synthetic corticotrophin raised plasma corticosteroid levels without changing plasma testosterone concentration. Because plasma testosterone levels were not related to changes in adrenocortical activity, the noctural rises appear to be due to changes in testicular secretion.