Adult Female Rhesus Macaques Research Articles

Western-style diet in the presence of elevated circulating testosterone induces adipocyte hypertrophy without proinflammatory responses in rhesus macaques.

Anovulatory infertility is commonly associated with hyperandrogenemia (elevated testosterone, T), insulin resistance, obesity, and white adipose tissue (WAT) dysfunction associated with adipocyte hypertrophy. However, whether hyperandrogenemia and adipocyte hypertrophy per se induce a proinflammatory response is unknown. Young adult female rhesus macaques were exposed to an obesogenic Western-style diet (WSD) in the presence of elevated circulating testosterone (T+WSD) or a low-fat control diet with no exogenous T. Immune cells residing in visceral omental white adipose tissue (OM-WAT), corpus luteum and the contralateral ovary, endometrium, lymph nodes, bone marrow, and peripheral blood mononuclear cells were characterized by flow cytometry during the luteal phase of the reproductive cycle. Following one year of treatment, T+WSD animals became more insulin-resistant and exhibited increased body fat and adipocyte hypertrophy compared to controls. T+WSD treatment did not induce macrophage polarization toward a proinflammatory phenotype in the tissues examined. Additionally, T+WSD treatment did not affect TNFα production by bone marrow macrophages in response to toll-like receptor agonists. While the major lymphoid subsets were not significantly affected by T+WSD treatment, we observed a significant reduction in the frequency of effector memory CD8+ T-cells (Tem) in OM-WAT, but not in other tissues. Notably, OM-WAT Tem frequencies were negatively correlated with insulin resistance as assessed by the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR). This study shows that short-term T+WSD treatment induces weight gain, insulin resistance, and adipocyte hypertrophy, but does not have a significant effect on systemic and tissue-resident proinflammatory markers, suggesting that adipocyte hypertrophy and mild hyperandrogenemia alone are not sufficient to induce a proinflammatory response.

LBSUN86 Effects Of Hyperandrogenemia And Western-style Diet On Rhesus Macaque Tissue-resident Memory T-cells And Their Potential Role In Controlling Local Inflammation And Insulin Sensitivity

Abstract We have previously demonstrated that the combination of chronic hyperandrogenemia (elevated circulating testosterone; T) and an obesogenic Western-style diet (WSD) exerts synergistic effects on the metabolic and reproductive axis, leading to the development of insulin resistance, visceral obesity, and ovarian dysfunction in female rhesus macaques. The underlying cellular connection between T+WSD treatment and abnormal metabolic and reproductive function is not well understood. Tissue resident-memory T-cells (Trm) have emerged as potent regulators of local inflammation. Thus, we hypothesized that T+WSD treatment drives proinflammatory phenotypic changes in Trm cells. To address this hypothesis, we studied a cohort of adult female rhesus macaque exposed to a low-fat diet and no exogenous T (controls) versus those treated with T+WSD for one year. T+WSD animals became insulin resistant and had higher visceral adiposity than control animals. During the mid-luteal phase, all animals were necropsied and immune cells residing in visceral omental white adipose tissue (OM-WAT), the corpus luteum (CL), the contralateral ovary not containing the CL (OV), endometrium (ENDO), lymph nodes (LN), bone marrow (BM), and peripheral blood mononuclear cells (PBMC) were characterized by flow cytometry. Non-activated CD4+ and CD8+ naïve/central memory cells expressing CD28 were detected in all tissue examined. However, the tissue retention receptor CD69 was expressed only on CD4+ and CD8+ cells residing in OM-WAT, CL, OV, and ENDO. These Trm subsets expressed high levels of the C-C Motif Chemokine Receptor 5 (CCR5), whose role in inflammation has been previously reported. Combined T+WSD treatment resulted in a significant increase in CD8+ Trm cell populations in OM-WAT. Notably, the frequencies of Trm in OM-WAT were positively correlated with HOMA-IR, the indicator of insulin resistance. Collectively, our data indicate that combined hyperandrogenemia and WSD can lead to selective recruitment or local differentiation of Trm cells in visceral fat. Trm activation in fat may drive the development of a proinflammatory environment that negatively affects adipose metabolic function, leading to altered systemic metabolic parameters (i. e., hyperinsulinemia) that influence ovarian and uterine processes. Future studies are warranted to define how Trm cell-adipocyte interactions affect adipose metabolic processes. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Hormonal Suppression in Female Rhesus Macaques (Macaca mulatta) Implanted Subcutaneously with Deslorelin.

Providing effective contraception for nonhuman primates (NHP) is challenging. Deslorelin acetate is a commercially available gonadotropin-releasing hormone (GnRH) agonist that may provide a relatively noninvasive, long-lasting, and potentially reversible alternative to standard NHP contraception methods. This study evaluated the duration of suppression of progesterone and estradiol in 6 adult female rhesus macaques (Macaca mulatta) that received a single subcutaneous 4.7 mg deslorelin implant. We hypothesized that deslorelin would suppress production of these hormones for 6 mo with a correspond- ing cessation of menses. Prior to implantation, blood was collected over 1 mo for baseline hormone analyses. Macaques were sedated at the onset of the next menstrual cycle and a 4.7 mg deslorelin implant was placed in the interscapular region. Blood was collected over the subsequent month at the same intervals used for the baseline collection schedule, and then every 7 d thereafter. Results showed that estradiol and progesterone transiently increased 1 to 3 d after implantation, then fell to basal levels within 6 d of implantation. The duration of hormone suppression (progesterone <0.5 ng/mL) varied among animals. Two macaques returned to cyclicity by 96 d and 113 d after implantation, while hormones remained suppressed in the other 4 macaques at 6 mo after implantation. Cessation of menses correlated with hormone suppression except in 1 animal that continued to have sporadic vaginal bleeding despite progesterone remaining below 0.5 ng/mL. This study indicates that deslorelin is a noninvasive and long-lasting contraceptive method in female rhesus macaques. However, individual variation should be considered when determining reimplantation intervals.

Long-term effects of wildfire smoke exposure during early life on the nasal epigenome in rhesus macaques

BackgroundWildfire smoke is responsible for around 20% of all particulate emissions in the U.S. and affects millions of people worldwide. Children are especially vulnerable, as ambient air pollution exposure during early childhood is associated with reduced lung function. Most studies, however, have focused on the short-term impacts of wildfire smoke exposures. We aimed to identify long-term baseline epigenetic changes associated with early-life exposure to wildfire smoke. We collected nasal epithelium samples for whole genome bisulfite sequencing (WGBS) from two groups of adult female rhesus macaques: one group born just before the 2008 California wildfire season and exposed to wildfire smoke during early-life (n = 8), and the other group born in 2009 with no wildfire smoke exposure during early-life (n = 14). RNA-sequencing was also performed on a subset of these samples. ResultsWe identified 3370 differentially methylated regions (DMRs) (difference in methylation ≥ 5%, empirical p < 0.05) and 1 differentially expressed gene (FLOT2) (FDR < 0.05, fold of change ≥ 1.2). The DMRs were annotated to genes significantly enriched for synaptogenesis signaling, protein kinase A signaling, and a variety of immune processes, and some DMRs significantly correlated with gene expression differences. DMRs were also significantly enriched within regions of bivalent chromatin (top odds ratio = 1.46, q-value < 3 × 10-6) that often silence key developmental genes while keeping them poised for activation in pluripotent cells. ConclusionsThese data suggest that early-life exposure to wildfire smoke leads to long-term changes in the methylome over genes impacting the nervous and immune systems. Follow-up studies will be required to test whether these changes influence transcription following an immune/respiratory challenge.

The effects of delta-9-tetrahydrocannabinol exposure on female menstrual cyclicity and reproductive health in rhesus macaques

To determine the dose-dependent effect of contemporary marijuana exposure on female menstrual cyclicity and reproductive endocrine physiology in a nonhuman primate model. Research animal study. Research institute environment. Adult female rhesus macaques (6-12 years of age; n = 8). Daily delta-9-tetrahydrocannabinol (THC) edible at medically and recreationally relevant contemporary doses. Menstrual cycle length (MCL), anti-Müllerian hormone, prolactin, basal follicle-stimulating hormone (FSH), estradiol (E2) and progesterone, luteinizing hormone (LH), and thyroid-stimulating hormone. The average before THC weight was 6.9 kg (standard deviation, 0.8), and at the highest THC dosing, the average weight was 7.2 kg (standard deviation, 0.8). With increasing THC dosing, MCL and FSH concentrations increased, while basal E2 concentration was stable. The average MCL concentration increased 4.0 days for each mg/7 kg/day of THC (95% CI, 1.4-6.6 days). Follicle-stimulating hormone concentration increased significantly with increasing THC dose, 0.34 ng/mL for each mg/7 kg/day of THC (95% CI, 0.14-0.57 ng/mL). No significant trends were observed between THC dosing and average basal progesterone, anti-Müllerian hormone, prolactin, LH, or thyroid-stimulating hormone concentrations. In rhesus macaques, a dose response toward increased MCL and basal FSH concentrations but plateau of basal E2 and LH concentrations was observed with increasing THC dosing, suggesting ovulatory dysfunction. Further studies are needed to determine the effects of a longer duration of exposure and whether the significant increase in MCL and FSH concentrations results in reduced fecundity.

SARS‐CoV‐2‐associated neuropathology in non‐human primates

SARS‐CoV‐2 infection impacts multiple organ systems, including the central nervous system (CNS). Multiple reports have described a variety of neurological manifestations associated with infection that may contribute to worsening COVID‐19. The neuropathology of SARS‐CoV‐2 is not well understood, necessitating the development of relevant animal models for investigation. Here, we report marked neuropathology but with limited virus in the CNS of two non‐human primate models (NHPs) of SARS‐CoV‐2 infection.Adult male and female purpose‐bred Rhesus macaques (RMs; n = 4) and wild‐caught African green monkeys (AGMs; n = 4) were inoculated with the 2019‐nCoV/USA‐WA1/2020 strain of SARS‐CoV‐2 via multi‐route mucosal or aerosol challenge. SARS‐CoV‐2 nucleocapsid (SARS‐N) mRNA was detected in nasal swabs within the first week of inoculation, demonstrating infection of all study animals. All animals were euthanized at the study endpoint of 4 weeks post‐inoculation, with the exception of two AGMs that reached humane endpoints at 8‐ and 22‐days post‐challenge. Seven regions of the CNS were investigated for pathology and virus infection. Archival brain tissues from two non‐infected adult female RMs were used as aged‐matched controls.Mild, but chronic, hypoxemia with impaired gas exchange were suggested by SpO2 values that stayed at or below 95% and elevated blood CO2 in the majority of the study animals. Neuroinflammation was seen throughout the brain and brainstem but with limited virus detection by immunohistochemistry and RNAscope of fixed tissues and viral RNA detection using a highly sensitive CRISPR‐fluorescent detection system on RNA extracted from sectioned brain lysates. In addition, neuronal injury and death were suggested by pyknotic and karyolytic nuclei and cellular blebbing. Limited myelin vacuolation was revealed in two infected animals through Luxol Fast Blue staining. Neuronal cleaved caspase 3 positivity was seen at a greater frequency in infected animals compared to controls, suggesting increased apoptosis in infection. Microhemorrhages were larger and more frequent among infected NHPs, as compared to controls. Neuroinflammation, neuronal injury and death, and microhemorrhages were seen in animals that did not develop severe respiratory disease and may suggest neuropathology contributes to on‐going symptoms of convalesced patients.Our findings in NHPs are in agreement with human autopsy and neuroimaging studies and demonstrate this is a relevant animal model for investigating neuropathological changes associated with COVID‐19. Our results also suggest that hypoxic‐ischemic events leading to energy failure and neuronal injury, contribute to the neuropathological consequences of COVID‐19. Further studies are warranted to elucidate the mechanisms of SARS‐CoV‐2 neuropathogenesis.

Changes in glucocorticoid receptor sensitivity following long‐term ethanol self‐administration in male and female rhesus macaques

Stress can alter ethanol consumption and long-term use results in adaptation of the stress response, highlighting a bidirectional relationship that remains poorly understood. Non-human primates are an excellent model to investigate the interaction of stress and long-term ethanol use due to their similar neuroendocrine system to humans and propensity to chronically drink intoxicating amounts of alcohol. Here, circulating stress hormones (adrenocorticotropin hormone (ACTH) and cortisol) were measured in response to a low (17 mg/kg) and high (130 mg/kg) dose of dexamethasone, a potent glucocorticoid receptor (GR) agonist. The dexamethasone response was compared before (baseline) and after 7 months of daily 22 h/d ethanol self-administration in young adult male (n=6) and female (n=6) rhesus macaques. At both timepoints, the high dose dexamethasone challenge resulted in 90-100% suppression of both ACTH and cortisol with no difference between sexes. At baseline, low dose dexamethasone reduced both ACTH and cortisol, but to a lesser degree and with greater between-subject variability, 0 – 60%. Following seven-months of daily ethanol consumption (average intakes ranging from 0.6 – 4.0 g/kg/day) the response to low dose dexamethasone was greater. This effect was more robust in females compared to males. Additionally, the change in suppression between baseline and following long-term daily ethanol consumption correlated with daily intake, such that the magnitude of change over time was greater in heavier drinkers. Because stress increases reliance on putamen-dependent or habitual behavior and heavy ethanol drinking leads to excitatory-inhibitory neurotransmission imbalance within the putamen, we tested the hypothesis that long-term ethanol self-administration will alter striatal transmission via the GR. Therefore, we used whole cell patch clamp electrophysiology to investigate the effect of bath application of dexamethasone onto putamen medium spiny neurons (MSNs). Dexamethasone rapidly decreased the area and amplitude of spontaneous inhibitory postsynaptic currents among binge drinkers. These data show that acute dexamethasone, presumably via activation of the membrane bound GR, activated fewer GABAergic receptors among binge drinkers. These data highlight a unique role of the GR within the putamen that may mediate stress dependent changes contributing to addiction vulnerability, particularly among binge drinkers.

Effects of medroxyprogesterone acetate on social behavior in female rhesus macaques (Macaca mulatta) depend on male breeding season introductions.

Although medroxyprogesterone acetate (MPA) is used commonly as a contraceptive in women and female non-human primates, its effects on social behavior remain unclear. This study examined whether MPA treatment and introduction of new adult males during the breeding season influence the social behaviors of group-housed adult female rhesus macaques. Subjects were 12 MPA-treated and 12 matched case-control females. Aggressive, affiliative, and sexual behaviors were measured. MPA-treated females showed less affiliative and sexual behavior compared to matched controls during the breeding season. MPA treatment was associated with decreased aggression emitted toward and received from females during the breeding season. MPA treatment is associated with differences in social behavior of female rhesus macaques during the breeding season, when normal hormonal cycles are attenuated by the treatment, but there is no indication that MPA-treated females bring an additional risk for more aggression during the male introduction and breeding season.

Stage-dependent actions of antimüllerian hormone in regulating granulosa cell proliferation and follicular function in the primate ovary

To study the direct action and physiological role of antimüllerian hormone (AMH) in regulating ovarian follicular development and function in vivo in primates. Animals were assigned to six treatment sequences in a crossover design study. Intraovarian infusion was performed during the follicular phase of the menstrual cycle with agents including: control vehicle; recombinant human AMH (rhAMH); and neutralizing anti-human AMH antibody (AMHAb). Before ovariectomy after the final treatment, the animals received intravenous injections of bromodeoxyuridine (BrdU). National primate research center. Adult female rhesus macaques (Macaca mulatta). None. Cycle length, follicle cohorts, and serum steroid levels were assessed. Ovarian histology, as well as granulosa cell (GC) proliferation and oocyte viability, were evaluated. In vehicle-infused ovaries, a dominant follicle was observed at midcycle E2 peak. However, rhAMH-treated ovaries exhibited an increased number of small antral follicles, whereas AMHAb-treated ovaries developed multiple large antral follicles. Serum E2 levels in the follicular phase decreased after rhAMH infusion and increased after AMHAb infusion. The rhAMH infusion increased serum T levels. Whereas early-growing follicles of rhAMH-treated ovaries contained BrdU-positive GCs, antral follicles containing BrdU-positive GCs were identified in AMHAb-treated ovaries. Autophagy was observed in oocytes of early-growing and antral follicles exposed to AMHAb and rhAMH, respectively. AMH enhanced early-stage follicle growth, but prevented antral follicle development and function via its stage-dependent regulation of GC proliferation and oocyte viability. This study provides information relevant to the pathophysiology of ovarian dysfunction and the treatment of infertility.

Ethanol-Impaired Myogenic Differentiation is Associated With Decreased Myoblast Glycolytic Function.

Myopathy affects nearly half of individuals with alcohol use disorder (AUD), and impaired skeletal muscle regenerative potential is a probable contributing factor. Previous findings from our laboratory indicate that chronic in vivo and in vitro ethanol (EtOH) treatment decreases myogenic potential of skeletal muscle myoblasts. Myogenesis, a highly coordinated process, requires shifts in cellular metabolic state allowing for myoblasts to proliferate and differentiate into mature myotubes. The objective of this study was to determine whether alcohol interferes with myoblast mitochondrial and glycolytic metabolism and impairs myogenic differentiation. Myoblasts were isolated from vastus lateralis muscle excised from alcohol-naïve adult male (n=5) and female (n=5) rhesus macaques. Myoblasts were proliferated for 3days (day 0 differentiation; D0) and differentiated for 5days (D5) with or without 50mM EtOH. Metabolism was assessed using a mitochondrial stress test to measure oxygen consumption (OCR) and extracellular acidification (ECAR) rates at D0. Differentiation was examined at D5. Expression of mitochondrial and glycolytic genes and mitochondrial DNA (mtDNA) was measured at D0 and D5. Ethanol significantly (p<0.05) increased myoblast maximal OCR and decreased ECAR at D0, and decreased fusion index, myotubes per field, and total nuclei at D5. The EtOH-induced decrease in ECAR was associated with the EtOH-mediated decreases in fusion index and myotubes per field. EtOH did not alter the decrease in glycolytic gene expression and increase in mtDNA from D0 to D5. During myoblast proliferation, EtOH decreased glycolytic metabolism and increased maximal OCR, suggesting that myoblast metabolic phenotype was dysregulated with EtOH. The EtOH-induced decrease in ECAR was associated with decreased differentiation. These findings suggest that EtOH-mediated shifts in metabolic phenotype may underlie impaired differentiation, which has important clinical implications for myogenesis in those affected by alcoholic myopathy.

Ketamine-induced neuromuscular reactivity is associated with aging in female rhesus macaques.

Rhesus macaques represent an important species for translational and pre-clinical research studies across a multitude of disease and injury models, including aging. Ketamine anesthesia is used in humans and non-human primates but may be associated with adverse effects, including neuromuscular reactions. The effects of aging on ketamine adverse effects is not well characterized. Urodynamic recordings and electromyography (EMG) studies were performed in aged (>20 years old) and adult (3.9–14.9 years old) female rhesus macaques under an equal and light plane of sedation by constant rate infusion (CRI) of ketamine. A total of 4 of 41 adult subjects (9.7%) showed clinical signs of ketamine-induced abnormal neuromuscular reactivity, whereas a larger portion of 14 of 26 aged subjects showed similar ketamine-induced neuromuscular reactivity (53.8%; P< 0.001). The ketamine CRI rate was 19.8±0.9 mg/kg/h in adults and lower in aged subjects at 16.5±1.4 mg/kg/h (P<0.05). The ketamine CRI rate was negatively correlated with age (r = -0.30, P<0.05, n = 64). The incidence of ketamine reactivity or CRI rate was not different between aged pre-and post-menopausal females. EMG recordings during neuromuscular reactivity showed coordinated activation of multiple muscles, suggesting a central nervous system (CNS) mechanism for ketamine-associated neuromuscular reactivity. The incidence of ketamine-induced neuromuscular reactivity is age related but not affected by the estrous cycle in female rhesus macaques. A coordinated activation of multiple muscles, innervated by different peripheral nerves, suggests that ketamine-induced neuromuscular reactivity originates in the CNS.

Improved behavioral indices of welfare in continuous compared to intermittent pair-housing in adult female rhesus macaques (Macaca mulatta).

Limiting opportunities for captive nonhuman primates (NHPs) to express species-specific social behaviors may disrupt the adaptive drive for social companionship and may lead to increases in coping behaviors and inactivity. While captive NHPs show improved welfare when moving to pair-housing from single-housing, the impact of daily separation of pair-mates, as is implemented in intermittent pair-housing, is not fully understood. We compared behavioral indices of welfare exhibited by adult female rhesus macaques (Macaca mulatta) in two conditions: (1) intermittent pair-housing, involving daily overnight separation of pair-mates, and (2) continuous pair-housing, involving little separation of pair-mates. A within-subjects study design tested two groups of females experiencing both pairing conditions in an alternate order, switching either from continuous to intermittent pair-housing, or from intermittent to continuous pair-housing. Behavioral observations, recording activity state, self-directed, abnormal, and social behaviors, were conducted at midday when all females were paired, and in the afternoon when intermittent pairs were separated. Females exhibited higher levels of inactivity and self-directed behavior when separated due to intermittent pair-housing in comparison to continuous pair-housing. In addition, intermittently paired females showed higher levels of grooming and other types of affiliation when paired, than during the same time frame when they were continuously paired. These results suggest that females in the continuous presence of a social partner experience improved levels of activity and do not need to elevate levels of behavioral coping mechanisms (e.g., self-scratching, increased affiliation) as they receive the benefits associated with social companionship consistently throughout the day. Overall, this study provides the first evidence that continuous pair-housing affords better welfare than intermittent pair-housing in adult female rhesus macaques. Pair-housing options, such as continuous pairing, that reduce reliance on behavioral coping mechanisms and promote adaptive social behavior throughout the entirety of the day should be prioritized over husbandry care scheduled for convenience.

SAT-587 Molecular Markers of Beige Adipose Following ESR1 Knockdown in the Mediobasal Hypothalamus of Adult Female Rhesus Monkeys

Our studies in female marmoset monkeys show that the ablation of ovarian estradiol (E2) production fails to alter energy homeostasis or body fat accumulation. Peripheral E2 may therefore not play a crucial role in metabolic regulation in female primates. shRNA-mediated knockdown of ESR1 expression in the hypothalamic ventromedial nucleus (VMN) in adult female rodents, however, induces obesity and suggests ESR1 is a hypothalamic target for E2 regulation of energy homeostasis, and likely mediates thermogenesis in brown/beige adipose depots. In female primates, including humans, the hypothalamic estrogen receptor mediating metabolic regulation is unknown. To test the hypothesis that ESR1 mediates female primate regulation of energy homeostasis, 11 ovary intact, adult female rhesus macaques, pair housed with female peers, received five 12µl MRI-guided MBH infusions into the rostral-to-caudal extent of both right and left VMN. Each infusion comprised a gadolinium contrast agent and ~3–4 x 1010 adeno-associated virus 8 (AAV8) particles containing either an shRNA specific for ESR1 (n=6, ERaKD) or scrambled shRNA (n=5, control). Mid-surgery MRI scans identified targeting accuracy. ~ 1.5 yrs following AAV8 infusion, pronounced gain in BMI enabled conversion of 83% of ERaKD females to overweight/obese compared to 20% of controls (p=0.08). Percent increase in BMI remained intermittently greater (p<0.05) than controls thereafter. Adipose depots were harvested at necropsy ~2.5–3 yrs following treatment. Total RNA was isolated using the Qiagen AllPrep DNA/RNA/miRNA Universal kit. RNA was reverse transcribed with High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). All quantitative real-time PCR (qRT-PCR) were performed on a StepOnePlus System using Power SYBR Green master mix (Applied Biosystems). Primer sequences were designed using NCBI Primer-Blast. Expression of TATA-box binding protein (TBP) was used as the internal control housekeeping gene. The relative expression of target genes was measured using the comparative cycle threshold (Ct) method with results expressed as target mRNA expression relative to TBP using the formula 2^-delta Ct. Upper body beige adipose represents an organ system in primates, including humans, involved in thermogenesis. Axillary beige adipose depots in ERaKD females, however, did not exhibit significantly diminished gene expression for selected markers of beige adipocytes, including PAT2, CD137 and C/EBPβ, compared to control females. More crucially, thermogenically relevant UCP1 expression also did not differ between ERaKD females and controls. Taken together, these results suggest that knockdown of VMN ESR1 in adult female monkeys, while inducing modest weight gain after 1.5 years, may not markedly alter beige adipose gene expression of initially selected thermogenically relevant genes.

OR31-05 Emergence of Ovarian Hyperandrogenism and Luteal Insufficiency Following ESR1 Knockdown in the Mediobasal Hypothalamus of Adult Female Rhesus Monkeys

Diminished estradiol (E2) negative feedback action by neuronal ESR1 in the arcuate nucleus (ARC) of the mediobasal hypothalamus (MBH) is hypothesized to cause gonadotropin-releasing hormone (GnRH) hypersecretion, and thus LH excess, contributing to ovarian hyperandrogenism in polycystic ovary syndrome (PCOS). In primates, including humans, however, the mediating estrogen receptor is unknown. Thus, to test the hypothesis that diminished E2 action on ARC ESR1 contributes to female primate ovarian hyperandrogenism, eleven, ovary intact, adult female rhesus macaques, pair housed with female peers, received five 12µl MRI-guided MBH infusions into the rostral-to-caudal extent of both right and left ARC. Each infusion comprised gadolinium contrast agent and ~3-4 x 1010 adeno-associated virus 8 (AAV8) particles containing either a shRNA specific for ESR1 (n=6, ERaKD) or scrambled shRNA (n=5, control). Mid-surgery MRI scans identified targeting accuracy. 2-2.5 years following AAV8 infusion, EIA-determined P4 values were obtained from twice weekly serum samples; samples obtained during the follicular phase of menstrual cycles or anovulatory periods were submitted to liquid chromatography, tandem mass spectrometry (LCMS) for additional steroid hormones. LCMS-determined values were also obtained 0 hours (h) and 24 h following an IM injection of 200IU hCG. Both ERaKD (28.5 ± 1.3 days, mean ± SEM) and control (34.0 ± 3.3 days) female groups exhibited comparably regular menstrual cycles. ERaKD exhibited higher circulating levels of LH (2.8 ± 0.2 ng/ml, p=0.03), androstenedione (A4, 0.43 ± 0.03 ng/ml, p=0.03) and testosterone (T, 0.23 ± 0.03 ng/ml, p=0.09), and LH/FSH ratio (1.7 ± 0.2, p=0.05) compared to controls (LH, 2.1 ± 0.4; A4, 0.30 ± 0.05; T, 0.18 ± 0.01 ng/ml; LH/FSH 1.3 ± 0.2). Following an ovarian androgen-stimulating hCG injection, ERaKD 24-h peak levels for T (0.28 ± 0.01 ng/ml) were higher (p=0.03) compared to controls (0.21 ± 0.01 ng/ml). In addition, luteal insufficiency emerged in ERaKD females, with mean (2.4 ± 0.3 ng/ml), peak (3.6 ± 0.4 ng/ml) and area-under-the-curve (AUC, 23.2 ± 4.2 ng/ml/days) P4 values diminished compared to controls (mean, 3.6 ± 0.1, p=0.01; peak 5.7 ± 0.1 ng/ml, p=0.01; AUC, 43.7 ± 6.7 ng/ml/days, p=0.03). Taken together, these results suggest that knockdown of ARC ESR1 disrupts Gn stimulation of ovarian function, contributing to female monkey ovarian hyperandrogenism and menstrual cycle impairment emulating PCOS in women.

Selective attention for affiliative and agonistic interactions of dominants and close affiliates in macaques

Monitoring conspecifics is a crucial process in social learning and a building block of social cognition. Selective attention to social stimuli results from interactions of subject and stimulus characteristics with dominance rank often emerging as an important predictor. We extend previous research by providing as stimuli naturally occurring affiliative interactions between group members instead of pictorial or auditory representations of conflicts, and by extending to the affiliative relationship, i.e. social bond, between subject and stimulus instead of just their dominance relations. Our observational data on adult female rhesus macaques support the prediction that subjects pay more attention to affiliative interactions of others than to solitary controls. Exceedingly more attention was paid to conflicts unfolding in the group which can have more prompt and direct consequences than others’ friendly interactions. The valence of the stimulus (affiliative vs. agonistic) affected biases towards individuals dominant over the subject, but not the ubiquitous bias towards close affiliates of the subject. Keeping track of the whereabouts and interactions of key social partners has been proposed as a prerequisite for behavioral coordination among bonded partners. In groups of socially very active monkeys, social attention is gated by both social dominance and social bonding.

Conditionally reprogrammed macaque endocervical cells retain steroid receptor expression and produce mucus.

Cervical mucus produced by the endocervix plays an essential role as a hormonally induced regulator of female fertility. Cervical mucus fluctuates in both physical characteristics and in sperm penetrability in response to estrogens and progestogens. However, the mechanisms by which steroid hormones change mucus remains poorly understood. Current in vitro models have limited capability to study these questions as primary endocervical cells possess limited expansion potential, and immortalized cells lose in vivo characteristics such as steroid sensitivity. Here we overcome these limitations by establishing an in vitro primary endocervical cell culture model using conditionally reprogrammed cells (CRCs). CRC culture utilizes a Rho-kinase inhibitor and a fibroblast feeder layer to expand proliferative potential of epithelial cell types that have normally short in vitro life spans. In our studies, we produce CRC cultures using primary endocervical cells from adult female rhesus macaques (Macaca mulatta). We demonstrate that primary endocervical cells from the nonhuman primate can be robustly expanded using a CRC method, while retaining steroid receptor expression. Moreover, when removed from CRC conditions and switched to differentiation conditions, these cells are able to differentiate and produce mucus including MUC5B, the most prevalent mucin of the endocervix. We conclude that this method provides a promising in vitro platform for conducting mechanistic studies of cervical mucus regulation as well as for screening new therapeutic targets for fertility regulation and diseases of the endocervix.

Deconstructing sociality: the types of social connections that predict longevity in a group-living primate.

Many species use social interactions to cope with challenges in their environment and a growing number of studies show that individuals which are well-connected to their group have higher fitness than socially isolated individuals. However, there are many ways to be 'well-connected' and it is unclear which aspects of sociality drive fitness benefits. Being well-connected can be conceptualized in four main ways: individuals can be socially integrated by engaging in a high rate of social behaviour or having many partners; they can have strong and stable connections to favoured partners; they can indirectly connect to the broader group structure; or directly engage in a high rate of beneficial behaviours, such as grooming. In this study, we use survival models and long-term data in adult female rhesus macaques (Macaca mulatta) to compare the fitness outcomes of multiple measures of social connectedness. Females that maintained strong connections to favoured partners had the highest relative survival probability, as did females well-integrated owing to forming many weak connections. We found no survival benefits to being structurally well-connected or engaging in high rates of grooming. Being well-connected to favoured partners could provide fitness benefits by, for example, increasing the efficacy of coordinated or mutualistic behaviours.

Determining overweight and underweight with a new weight-for-height index in captive group-housed macaques.

Housing primates in naturalistic groups provides social benefits relative to solitary housing. However, food intake may vary across individuals, possibly resulting in overweight and underweight individuals. Information on relative adiposity (the amount of fat tissue relative to body weight) is needed to monitor overweight and underweight of group‐housed individuals. However, the upper and lower relative adiposity boundaries are currently only known for macaques living solitarily in small cages. We determined the best measure of relative adiposity and explored the boundaries of overweight and underweight to investigate their incidence in group‐housed adult male and female rhesus macaques and long‐tailed macaques living in spacious enclosures at the Biomedical Primate Research Centre (BPRC), the Netherlands. During yearly health checks different relative adiposity measures were obtained. For long‐tailed macaques, comparable data on founder and wild animals were also available. Weight‐for‐height indices (WHI) with height to the power of 3.0 (WHI3.0) for rhesus macaques and 2.7 (WHI2.7) for long‐tailed macaques were optimally independent of height and were highly correlated with other relative adiposity measures. The boundary for overweight was similar in group‐housed and solitary‐housed macaques. A lower boundary for underweight, based on 2% body fat similar to wild primates, gave a better estimate for underweight in group‐housed macaques. We propose that for captive group‐housed rhesus macaques relative adiposity should range between 42 and 67 (WHI3.0) and for long‐tailed macaques between 39 and 62 (WHI2.7). The majority of group‐housed macaques in this facility have a normal relative adiposity, a considerable proportion (17–23%) is overweight, and a few (0–3%) are underweight.

Alopecia in rhesus macaques (Macaca mulatta): Association with pregnancy and chronic stress.

Alopecia in captive non-human primates is often presented as a welfare issue. However, it is a complex condition with a number of possible causes. The purpose of this study was to assess the impact of pregnancy and hair cortisol concentrations on alopecia in rhesus macaques. Subjects were 113 socially housed adult female rhesus macaques (27 pregnant, 35 nursing infants, 51 controls). During routine physicals, photographs were taken for alopecia assessment and hair samples were collected for cortisol assay. Alopecia was more prevalent in pregnant than in control females, but there was no association between alopecia and hair cortisol. However, there was a significant effect of pregnancy on hair cortisol. Nursing females had higher hair cortisol levels than pregnant females, which had higher levels than control females. Although alopecia does not appear to be associated with hair cortisol, both alopecia and hair cortisol were associated with pregnancy.

13C-lutein is differentially distributed in tissues of an adult female rhesus macaque following a single oral administration: a pilot study

Despite the growing awareness regarding lutein's putative roles in eyes and brain, its pharmacokinetics and tissue distribution in primates have been poorly understood. We hypothesized that 13C-lutein will be differentially distributed into tissues of an adult rhesus macaque (Macaca mulatta) 3 days following a single oral dose. After a year of prefeeding a diet supplemented with unlabeled lutein (1 μmol/kg/d), a 19-year-old female was dosed with 1.92 mg of highly enriched 13C-lutein. Tissues of a nondosed, lutein-fed monkey were used as a reference for natural abundance of 13C-lutein. On the third day postdose, plasma and multiple tissues were collected. Lutein was quantified by high-performance liquid chromatography–photodiode array detector, and 13C-lutein tissue enrichment was determined by liquid chromatography quadrupole time-of-flight mass spectrometry. In the tissues of a reference monkey, 12C-lutein with natural abundance of 13C-lutein was detectable. In the dosed monkey, highly enriched 13C-lutein was observed in all analyzed tissues except for the macular and peripheral retina, with the highest concentrations in the liver followed by the adrenal gland and plasma. 13C-lutein accumulated differentially across 6 brain regions. In adipose depots, 13C-lutein was observed, with the highest concentrations in the axillary brown adipose tissues. In summary, we evaluated 13C-lutein tissue distribution in a nonhuman primate following a single dose of isotopically labeled lutein. These results show that tissue distribution 3 days following a dose of lutein varied substantially dependent on tissue type.