Female Rhesus Research Articles

SAT-411 Prepubertal Tonic Gamma-Amino Butyric Acid (GABA) Inhibition Is Upstream of Neurokinin B (NKB), Kisspeptin, and Gonadotropin Releasing Hormone (GnRH) Neuronal Network in Male Rhesus Monkeys

The concept that GnRH release during the prepubertal period is under tonic central inhibition is well established in primates. Although it has been reported that the makorin RING finger protein 3 (MKRN3) gene plays a role in this process (1), neuronal substrates responsible for prepubertal tonic inhibition remain unclear. A series of studies in this lab have shown that GABA neurons are consistently inhibitory to GnRH and kisspeptin neurons in female prepubertal rhesus monkeys (2, 3, 4). Because we recently found that release of GnRH, kisspeptin and NKB in prepubertal male monkeys is significantly lower than those in pubertal monkeys and the pubertal increase in release of these neuropeptides is gonadal steroid independent, in the present study, we ask the question of whether GABA neurons play a role in central inhibition of GnRH release before puberty onset in male monkeys. As described previously, a microdialysis probe was inserted into the median eminence of the hypothalamus in 4 gonadally intact prepubertal male monkeys (15.1 ± 1.9 months of age) and the effects of GABAA receptor antagonist, bicuculline, on release of GnRH, kisspeptin and NKB were examined. The results indicate that bicuculline at 1 µM, but not vehicle, dramatically stimulated release of GnRH, kisspeptin, and NKB in prepubertal male monkeys. Subsequently, we examined whether the bicuculline-induced release of GnRH and kisspeptin was affected by the presence of NKB antagonist, SB222200, as previously we found that NKB signaling is upstream of kisspeptin and GnRH neurons in male monkeys (5). We found that inhibition of NKB signaling attenuated the bicuculline-induced release of GnRH and kisspeptin, suggesting that NKB neurons, in part, relay inhibitory GABA signals to GnRH and kisspeptin neurons. These results are interpreted to mean that in male monkeys, inhibitory GABA signaling plays a role in central inhibition during the prepubertal period and that inhibitory GABA signaling is upstream of the NKB, kisspeptin, and GnRH neuronal pathway. How “Central GABA Inhibition” is regulated by genes involved in this process, such as MKRN3, remains to be investigated.

The effects of fluoxetine on socioemotional behavior in female rhesus monkeys is influenced by social status

BackgroundChronic adverse social experience is a major environmental variable linked to multiple psychiatric disorders. The use of social subordination in nonhuman primates represents a translational model of chronic psychosocial stress. While social dominance is associated with a resistance to a range of stressors and social subordination produces stress‐susceptible phenotypes, the neural mechanisms that underlie status‐induced differences in susceptibility remain unclear. The current study assessed the effects of increasing serotonin (5HT) activity by administering fluoxetine on rates of socioemotional behavior (including agonistic, affiliative, and anxiety‐like behaviors) in female rhesus monkeys. We hypothesized that fluoxetine would increase rates of aggression in dominant but not subordinate animals.MethodsSubjects were gonadally intact adult female rhesus monkeys (n=14) housed in small social groups of four to six females each (6 dominant and 8 subordinate females). Using a counterbalanced design, subjects received both a 14‐day treatment with fluoxetine (SC dose of 2.8 mg/kg/day) and vehicle (50% polyethylene glycol in sterile saline) separated by a three‐week washout period. Behavioral observations were collected every two days using a standard monkey ethogram during each treatment, and CSF samples were collected at the end of each treatment for assessment of central 5‐hydroxyindoleacetic (5HIAA).ResultsThe amount of aggression directed at group mates was influenced by a status by treatment interaction (p=0.05, η2=0.27), with fluoxetine enhancing aggression in dominant females and decreasing aggression in subordinate females. As expected, subordinate females emitted significantly higher rates of submission behavior than did dominant females and this was unaffected by treatment (p=0.001). Affiliation emitted towards others was influenced by a status by treatment interaction (p=0.05, η2=0.27), as fluoxetine treatment decreased affiliation emitted in dominant females and increased it in subordinates. Anxiety‐like behavior was also influenced by a status by treatment interaction (p=0.011), as anxiety‐like behaviors were reduced only in subordinate females by fluoxetine treatment (p=0.012). CSF 5HIAA concentrations were reduced by fluoxetine (p<0.001) regardless of social status.ConclusionThe data show that increased 5HT activity following fluoxetine treatment in female monkeys alters socioemotional behavior in a status‐dependent manner. Notably, subordinate females are less aggressive and more affiliative during fluoxetine treatment. Future studies include determining whether these effects of fluoxetine are sex dependent and whether subordinate males respond similarly to subordinate females.Support or Funding InformationThis research was supported by an NIMH grant MH110212.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of ketamine treatment on cocaine-induced reinstatement and disruption of functional connectivity in unanesthetized rhesus monkeys.

Substance use disorders are characterized by a loss of executive control over reward-based decision-making, and disruption of fronto-striatal connectivity has been implicated in this process. Sub-anesthetic ketamine has recently been shown to bolster fronto-striatal connectivity in drug-naïve subjects. The influence of ketamine treatment was examined on the disruptive effects of cocaine on functional connectivity (FC) and on cocaine-seeking behavior in female rhesus monkeys. Three female rhesus were trained for unanesthetized MRI scanning. Each received three drug-naïve/abstinent pharmacological MRI scans with acute injections of saline, cocaine (0.3mg/kg i.v.), and cocaine (0.3mg/kg i.v.) 48-h after a ketamine treatment (low dose =0.345mg/kg bolus + 0.256mg/kg/h for 1h; i.v.), and a fourth scan with saline injection following 2months of daily cocaine self-administration. A separate cohort of five rhesus (4 female), all with extensive histories of cocaine exposure, underwent reinstatement testing 48h after ketamine (or vehicle) treatment. Two sub-anesthetic doses were tested: low dose and high dose = 0.69mg/kg + 0.512mg/kg/h for 1h. Ketamine treatment attenuated the effects of cocaine on both global and fronto-striatal FC in drug-naïve/abstinent subjects. Two months of daily cocaine self-administration led to prolonged disruption of both global and fronto-striatal FC. Cocaine-seeking behavior during reinstatement was reduced following ketamine treatment at the low dose, but not high dose. These findings illustrate the disruptive effects of cocaine on functional connectivity and provide evidence for the potential efficacy of ketamine as a treatment for stimulant use disorder.

Glucocorticoid Negative Feedback in Regulation of the Hypothalamic-Pituitary-Adrenal Axis in Rhesus Monkeys With Various Types of Adaptive Behavior: Individual and Age-Related Differences.

The study of the mechanisms underlying the increased vulnerability of the individual to stressful environmental factors in different age periods is of great relevance for prevention and effective treatment of stress-dependent diseases that are widespread in the population of aging individuals. The purpose of our study was to investigate the individual and age-related features of the glucocorticoid negative feedback in regulation of the hypothalamic-pituitary-adrenal (HPA) axis, the key adaptive neuroendocrine system, in experiments with physically healthy young and old female rhesus monkeys with administration of mineracorticoid receptor (fludrocortisone) and glucocorticoid receptor (dexamethasone) agonists. We studied the monkeys with increased trait anxiety and depression-like behavior (DAB) characterized, as previously was shown, by the increased vulnerability to acute stress and the animals with normal standard behavior (SB) as the control. The pronounced individual differences in the reaction of HPA axis to fludrocortisone and dexamethasone in young animals were found. Young animals with DAB showed a lower sensitivity of HPA axis to the inhibitory effect of both fludrocortisone and dexamethasone compared with young animals with SB. At the same time, there were no significant intergroup differences in the concentration of ACTH and cortisol in response to placebo injection, i.e., in basal conditions. The old individuals with DAB demonstrated the essential relative resistance of HPA axis to fludrocortisone test and higher basal plasma levels of cortisol and ACTH in the evening (the period of HPA axis low circadian activity) compared to old SB animals. In the same time, the intergroup differences in the response of HPA axis to dexamethasone administration were leveled due to age-related increase in sensitivity of HPA axis to dexamethasone in animals with DAB. These data testify the pronounced intergroup and age differences in the feedback regulation of HPA axis, presumably resulting from unequal individual, and age-related changes in the activity of mineralcorticoid and glucocorticoid receptors in the brain structures supporting the functions of HPA axis. The maximum age disorders in functioning of the negative feedback mechanism in the regulation of HPA axis are characteristic of animals with DAB, which, apparently, underlie the increased vulnerability of these animals to stress exposure.

Rise of the Visible Monkey: Sectioned Images of Rhesus Monkey.

BackgroundGross anatomy and sectional anatomy of a monkey should be known by students and researchers of veterinary medicine and medical research. However, materials to learn the anatomy of a monkey are scarce. Thus, the objective of this study was to produce a Visible Monkey data set containing cross sectional images, computed tomographs (CTs), and magnetic resonance images (MRIs) of a monkey whole body.MethodsBefore and after sacrifice, a female rhesus monkey was used for 3 Tesla MRI and CT scanning. The monkey was frozen and sectioned at 0.05 mm intervals for the head region and at 0.5 mm intervals for the rest of the body using a cryomacrotome. Each sectioned surface was photographed using a digital camera to obtain horizontal sectioned images. Segmentation of sectioned images was performed to elaborate three-dimensional (3D) models of the skin and brain.ResultsA total of 1,612 horizontal sectioned images of the head and 1,355 images of the remaining region were obtained. The small pixel size (0.024 mm × 0.024 mm) and real color (48 bits color) of these images enabled observations of minute structures.ConclusionDue to small intervals of these images, continuous structures could be traced completely. Moreover, 3D models of the skin and brain could be used for virtual dissections. Sectioned images of this study will enhance the understanding of monkey anatomy and foster further studies. These images will be provided to any requesting researcher free of charge.

Neuromuscular control pattern in rhesus monkeys during bipedal walking.

Walking is characterized by repetitive limb movements associated with highly structured patterns of muscle activity. The causal relationships between the muscle activities and hindlimb segments of walking are difficult to decipher. This study investigated these particular relationships and clarified whether they are correlated with speed to further understand the neuromuscular control pattern. Four adult female rhesus monkeys (Macaca mulatta) were selected to record gait parameters while walking on a bipedal treadmill at speeds of 0.2, 0.8, 1.4, and 2.0 km/h. We recorded 3 ipsilateral hindlimb muscles by surface recording. In this study, we calculated the correlations between electromyography (EMG) and kinematic parameters (24 EMG*17 kinematic parameters). Of the 408 calculated coefficients, 71.6% showed significant linear correlations. Significant linear correlations were found between muscle activity, such as burst amplitudes and the integral of muscle activity, and the corresponding kinematic parameters of each joint. Most of these relationships were speed independent (91.7% of all variables). Through correlation analysis, this study demonstrated a causal association between kinematic and EMG patterns of rhesus monkey locomotion. Individuals have particular musculoskeletal control patterns, and most of the relationships between hindlimb segments and muscles are speed independent. The current findings may enhance our understanding of neuromusculoskeletal control strategies.

In utero Androgen Excess: A Developmental Commonality Preceding Polycystic Ovary Syndrome?

In utero androgen excess reliably induces polycystic ovary syndrome (PCOS)-like reproductive and metabolic traits in female monkeys, sheep, rats, and mice. In humans, however, substantial technical and ethical constraints on fetal sampling have curtailed safe, pathogenic exploration during gestation. Evidence consistent with in utero origins for PCOS in humans has thus been slow to amass, but the balance now leans toward developmental fetal origins. Given that PCOS is familial and highly heritable, difficulties encountered in discerning genetic contributions to PCOS pathogenesis are puzzling and, to date, accounts for <10% of PCOS presentations. Unaccounted heritability notwithstanding, molecular commonality in pathogenic mechanisms is emerging, suggested by co-occurrence at the same gene loci of (1) PCOS genetic variants (PCOS women), (2) epigenetic alterations in DNA methylation (PCOS women), and (3) bioinformatics, gene networks-identified, epigenetic alterations in DNA methylation (female rhesus monkeys exposed to testosterone (T) in utero). In addition, naturally occurring hyperandrogenism in female monkeys singles out individuals with PCOS-like reproductive and metabolic traits accompanied by somatic biomarkers of in utero T exposure. Such phenotypic and molecular convergence between highly related species suggests not only dual genetic and epigenetic contributions to a developmental origin of PCOS but also common molecular pathogenesis extending beyond humans.

Synaptic distributions of pS214-tau in rhesus monkey prefrontal cortex are associated with spine density, but not with cognitive decline.

Female rhesus monkeys and women are subject to age- and menopause-related deficits in working memory, an executive function mediated by the dorsolateral prefrontal cortex (dlPFC). Long-term cyclic administration of 17β-estradiol improves working memory, and restores highly plastic axospinous synapses within layer III dlPFC of aged ovariectomized monkeys. In this study, we tested the hypothesis that synaptic distributions of tau protein phosphorylated at serine 214 (pS214-tau) are altered with age or estradiol treatment, and couple to working memory performance. First, ovariectormized young and aged monkeys received vehicle or estradiol treatment, and were tested on the delayed response (DR) test of working memory. Serial section electron microscopic immunocytochemistry was then performed to quantitatively assess the subcellular synaptic distributions of pS214-tau. Overall, the majority of synapses contained pS214-tau immunogold particles, which were predominantly localized to the cytoplasm of axon terminals. pS214-tau was also abundant within synaptic and cytoplasmic domains of dendritic spines. The density of pS214-tau immunogold within the active zone, cytoplasmic, and plasmalemmal domains of axon terminals, and subjacent to the postsynaptic density within the subsynaptic domains of dendritic spines, were each reduced with age. None of the variables examined were directly linked to cognitive status, but a high density of pS214-tau immunogold particles within presynaptic cytoplasmic and plasmalemmal domains, and within postsynaptic subsynaptic and plasmalemmal domains, accompanied high synapse density. Together, these data support a possible physiological, rather than pathological, role for pS214-tau in the modulation of synaptic morphology in monkey dlPFC.

Genetic and environmental factors in the intergenerational transmission of maternal care in rhesus macaques: Preliminary findings.

Early life experiences reorganize the brain and behavior of the developing infant, often with lifelong consequences. There is perhaps no more potent developmental influence than the quality of parental care: it is an experience common to all mammals, and its effects have been observed across species. The effects of parental care can be particularly difficult to abolish, as levels of care are often perpetuated across generations. However, genetic relatedness between parents can obscure the true mechanism of transgenerational cycles of parental care, because in intact families, genes, and environment are confounded. We examined the transmission of maternal care quality in biologically reared (n = 21) and cross fostered (n = 6) female rhesus monkeys. Interactions between female infant subjects and their mothers were observed from subjects' birth to 12 weeks of age. Females were then observed 4-5 years later for the quality of care they displayed toward their own newborn offspring. Maternal protectiveness in the first and second generations were correlated in both biologically reared and cross-fostered females. However, other aspects of maternal care, such as aggressiveness and sensitivity, were transmitted differently depending on foster status. These data provide preliminary findings in a small sample that the intergenerational transmission of maternal care may arise from complex genetic and environmental mechanisms in rhesus monkeys.

Methylome profiling of healthy and central precocious puberty girls

BackgroundRecent studies demonstrated that changes in DNA methylation (DNAm) and inactivation of two imprinted genes (MKRN3 and DLK1) alter the onset of female puberty. We aimed to investigate the association of DNAm profiling with the timing of human puberty analyzing the genome-wide DNAm patterns of peripheral blood leukocytes from ten female patients with central precocious puberty (CPP) and 33 healthy girls (15 pre- and 18 post-pubertal). For this purpose, we performed comparisons between the groups: pre- versus post-pubertal, CPP versus pre-pubertal, and CPP versus post-pubertal.ResultsAnalyzing the methylome changes associated with normal puberty, we identified 120 differentially methylated regions (DMRs) when comparing pre- and post-pubertal healthy girls. Most of these DMRs were hypermethylated in the pubertal group (99%) and located on the X chromosome (74%). Only one genomic region, containing the promoter of ZFP57, was hypomethylated in the pubertal group. ZFP57 is a transcriptional repressor required for both methylation and imprinting of multiple genomic loci. ZFP57 expression in the hypothalamus of female rhesus monkeys increased during peripubertal development, suggesting enhanced repression of downstream ZFP57 target genes. Fourteen other zinc finger (ZNF) genes were related to the hypermethylated DMRs at normal puberty. Analyzing the methylome changes associated with CPP, we demonstrated that the patients with CPP exhibited more hypermethylated CpG sites compared to both pre-pubertal (81%) and pubertal (89%) controls. Forty-eight ZNF genes were identified as having hypermethylated CpG sites in CPP.ConclusionMethylome profiling of girls at normal and precocious puberty revealed a widespread pattern of DNA hypermethylation, indicating that the pubertal process in humans is associated with specific changes in epigenetically driven regulatory control. Moreover, changes in methylation of several ZNF genes appear to be a distinct epigenetic modification underlying the initiation of human puberty.

Effect of menstrual cycle on ethanol drinking in rhesus monkeys

BackgroundSex differences in the abuse-related effects of alcohol have been demonstrated in the clinic and in preclinical animal models. Less is known about the influence of menstrual cycle phase on drinking. MethodsIn this study, we examined the relationship between menstrual cycle phase and intake of ethanol (EtOH) in five adult female rhesus monkeys. Subjects consumed a 4% EtOH solution in their home cage 6 h per day, 5 days per week and pressed a lever to receive food pellets during the drinking session. Menstrual cycle was determined with vaginal swabs 5–7 days per week. To facilitate comparison with previous studies, the cycle was divided three different ways for analysis. ResultsFirst, no significant difference was observed when EtOH intake was compared between phases defined as “follicular” (days 5–10) and “luteal” (19–24). Second, when the cycle was further divided into four phases [early follicular (days 1–7), late follicular (8–14), early luteal (15–21) and late luteal (22-next cycle)], significant differences were detected, with intake highest in phases that bracket menses and lowest in the late follicular phase. Finally, EtOH intake during “mid-cycle” (days 12–16) was significantly lower than during “menses” (days 1–5) and “late luteal” (last 5 days). Effect sizes were small to moderate, although absolute differences in EtOH intake (g/kg) were <15%. Food-maintained responding was not different across phases. ConclusionsMenstrual cycle has modest but statistically significant and selective effects on EtOH drinking, with higher EtOH intake observed in the peri-menstrual period compared to the middle of the cycle.

Effects of lorcaserin and buspirone, administered alone and as a mixture, on cocaine self-administration in male and female rhesus monkeys.

Cocaine use disorder is a serious public health issue for which there is no effective pharmacotherapy. One strategy to speed development of medications for cocaine use disorder is to repurpose drugs already approved for use in humans based on their ability to interact with targets known to be important for addiction. Two such drugs, lorcaserin (Belviq; a drug with serotonin [5-HT]2C receptor agonist properties) and buspirone (Buspar; a drug with 5-HT1A receptor partial agonist and dopamine D3/D4 receptor antagonist properties) can produce modest decreases in cocaine self-administration in rhesus monkeys. The current study evaluated the effectiveness of mixtures of lorcaserin and buspirone (at fixed dose ratios of 3:1, 1:1, and 1:3 relative to each drug's ID50) to reduce responding for 0.032 mg/kg/inf cocaine under a progressive ratio schedule of reinforcement in 2 male and 2 female rhesus monkeys. Dose addition analyses were used to determine if the effects of the drug mixtures differed from those predicted for an additive interaction between lorcaserin and buspirone. Dose-dependent reductions of cocaine self-administration were observed when lorcaserin and buspirone were administered alone, as well as when they were administered as 3:1, 1:1, and 1:3 fixed ratio mixtures of lorcaserin + buspirone. The effects of the 1:1 mixture of lorcaserin + buspirone on cocaine self-administration were supraadditive, whereas the effects of 3:1 and 1:3 mixtures were additive. Together, these results indicate that a combination therapy containing a mixture of lorcaserin and buspirone might be more effective than either drug alone at treating cocaine use disorder. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

Effects of long-term high-fat food or methamphetamine intake and serotonin 2C receptors on reversal learning in female rhesus macaques.

Perseverative behavior has been highly implicated in addiction. Activation of serotonin 2C receptors (5-HT2CRs) attenuates cocaine and high caloric food intake, but whether a 5-HT2CR agonist can reduce high caloric diet (HCD) or methamphetamine (METH) intake and response perseveration remains unknown. Clarifying the role of 5-HT2CRs in these behaviors will improve knowledge of neurochemical processes that regulate flexible decision-making and whether improvements in decision-making are accompanied by decreases in HCD or METH intake. This study evaluated the effects of long-term HCD and METH intake on reversal learning in female rhesus monkeys. The effects of the 5-HT2CR agonist WAY163909 on reversal learning before and after extended HCD or METH intake, and on food intake, was also tested. Moreover, we examined whether the 5-HT2CR is necessary for the effects of WAY163909. WAY163909 was given prior to reversal learning at baseline and after extended HCD or METH intake, and prior to measures of food intake. Extended intake of METH or the HCD increased perseverative errors during reversal. WAY163909 increased correct responses and decreased perseverative errors, both before and after extended HCD or METH intake. Similarly, WAY163909 decreased consumption of a HCD, but not a low caloric diet. The effects of WAY163909 on all these measures were blocked by co-administration with a 5-HT2CR antagonist. These data indicate that long-term HCD or METH intake disrupts flexible decision-making. Further, the results suggest that reductions in food intake produced by WAY163909 are associated with parallel improvements in decision-making strategies, underscoring the role of the 5-HT2CR for these behavioral effects.

Development and In Vivo Preclinical Imaging of Fluorine-18-Labeled Synaptic Vesicle Protein 2A (SV2A) PET Tracers.

Synaptic vesicle protein 2A (SV2A) serves as a biomarker of synaptic density and positron emission tomography (PET) imaging of SV2A could provide a tool to assess progression of neurodegenerative diseases. Two tracers have primarily been reported and characterized in vivo: [11C]UCB-J and [18F]UCB-H. In early human studies, [11C]UCB-J showed promising results, while its F-18-labeled analogue [18F]UCB-H showed suboptimal specific signal in comparison to [11C]UCB-J. Considering the limited use of [11C]UCB-J to facilities with a cyclotron, having a F-18 variant would facilitate large, multicenter imaging trials. We have screened several F-18 derivatives of UCB-J in non-human primates and identified a promising F-18 PET candidate, [18F]MNI-1126, with additional investigations of the racemate [18F]MNI-1038, affording a signal comparable to [11C]UCB-J. F-18 derivatives of UCB-J and UCB-H were synthesized and administered to non-human primates for microPET imaging. Following screenings, [18F]MNI-1038 (racemate) and [18F]MNI-1126 (R-enantiomer) were identified with the highest signal and favorable kinetics and were selected for further imaging. Kinetic modeling with one- and two-tissue compartmental models, and linear methods were applied to PET data using metabolite-corrected arterial input function. Pre-block scans with levetiracetam (LEV, 10, 30mg/kg, iv) were performed to determine the tracers' in vivo specificity for SV2A. Two whole-body PET studies were performed with [18F]MNI-1038 in one male and one female rhesus, and radiation absorbed dose estimates and effective dose (ED, ICRP-103) were estimated with OLINDA/EXM 2.0. All compounds screened displayed very good brain penetration, with a plasma-free fraction of ~ 40%. [18F]MNI-1126 and [18F]MNI-1038 showed uptake and distribution the most consistent with UCB-J, while the other derivatives showed suboptimal results, with similar or lower uptake than [18F]UCB-H. VT of [18F]MNI-1126 and [18F]MNI-1038 was high in all gray matter regions (within animal averages ~ 30ml/cm3) and highly correlated with [11C]UCB-J (r > 0.99). Pre-blocking of [18F]MNI-1126 or [18F]MNI-1038 with LEV showed robust occupancy across all gray matter regions, similar to that reported with [11C]UCB-J (~ 85% at 30mg/kg, ~ 65% at 10mg/kg). Using the centrum semiovale as a reference region, BPND of [18F]MNI-1126 reached values of up to ~ 30 to 40% higher than those reported for [11C]UCB-J. From whole-body imaging average ED of [18F]MNI-1038 was estimated to be 22.3μSv/MBq, with tracer being eliminated via both urinary and hepatobiliary pathways. We have identified a F-18-labeled tracer ([18F]MNI-1126) that exhibits comparable in vivo characteristics and specificity for SV2A to [11C]UCB-J in non-human primates, which makes [18F]MNI-1126 a promising PET radiotracer for imaging SV2A in human trials.

Transcriptome analysis of rhesus monkey failed-to-mature oocytes: deficiencies in transcriptional regulation and cytoplasmic maturation of the oocyte mRNA population.

Which different pathways and functions are altered in rhesus monkey oocytes that fail to mature after an ovulatory stimulus? Failed to mature (FTM) oocytes complete a large portion of the transition in transcriptome composition associated with normal maturation, but also manifest numerous differences that indicate incomplete transcriptional repression and cytoplasmic maturation affecting multiple processes. Oocyte maturation defects contribute to unexplained female infertility. Failure of some oocytes to undergo germinal vesicle breakdown or progress to second meiotic metaphase in response to an ovulatory stimulus can limit the number of high quality oocytes available for ART. The transcriptome of rhesus monkey oocytes that failed to mature (FTM; n = 11, 5 donors) in response to an ovulatory stimulus in vivo was compared to those of normal germinal vesicle stage (GV, n = 7, 2 donors) and metaphase II stage (MII, n = 7, 5 donors) oocytes by RNA-sequencing (RNAseq). Female rhesus monkeys of normal breeding age (6-12 years old) and with regular menstrual cycles were used. Animals underwent a controlled ovarian stimulation protocol for the collection of oocytes by ultrasound-guided needle aspiration of follicles. We obtained a high quality RNAseq dataset consisting of n = 7, n = 7, and n = 11 libraries for normal GV, normal MII and FTM oocytes, respectively. Total reads acquired were an average of 34 million for each GV sample, 41 million for each FTM sample and 59 million for each MII oocyte sample. Approximately 44% of the total reads were exonic reads that successfully aligned to the rhesus monkey genome as unique non-rRNA gene transcript sequences, providing high depth of coverage. Approximately 44% of the mRNAs that undergo changes in abundance during normal maturation display partial modulations to intermediate abundances, and 9.2% fail to diverge significantly from GV stage oocytes. Additionally, a small group of mRNAs are grossly mis-regulated in the FTM oocyte. Differential expression was seen for mRNAs associated with mitochondrial functions, fatty acid beta oxidation, lipid accumulation, meiosis, zona pellucida formation, Hippo pathway signaling, and maternal mRNA regulation. A deficiency DNA methyltransferase one mRNA expression indicates a potential defect in transcriptional silencing. All RNAseq data are published in the Gene Expression Omnibus Database (GSE112536). These results do not establish cause of maturation failure but reveal novel correlates of incompetence to mature. Transcriptome studies likely do not capture all post-transcriptional or post-translational events that inhibit maturation, but do reveal mRNA expression changes that lie downstream of such events or that are related to effects on upstream regulators. The use of an animal model allows the study of oocyte maturation failure independent of covariates and confounders, such as pre-existing conditions of the female, which is a significant concern in human studies. Depending on the legislation, it may not be possible to collect and study oocytes from healthy women; and using surplus oocytes from patients undergoing ART may introduce confounders that vary from case to case. FTM oocytes were at various stages of meiotic progression, so correlates of specific times of arrest are not revealed. All the FTM oocytes failed to respond appropriately to an ovulatory stimulus in vivo. Therefore, this analysis informs us about common transcriptome features associated with meiotic incompetence. These results reveal that some diagnostic markers of oocyte quality may not reflect developmental competence because even meiotically incompetent oocytes display many normal gene expression features. The results also reveal potential mechanisms by which maternal and environmental factors may impact transcriptional repression and cytoplasmic maturation, and prevent oocyte maturation. This work was supported by grants from the National Institutes of Health Office of Research Infrastructure Programs Division of Comparative Medicine Grants R24 [OD012221 to K.E.L., OD011107/RR00169 (California National Primate Research Center), and OD010967/RR025880 to C.A.V.]; the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under the award number T32HD087166; MSU AgBioResearch, Michigan State University. Authors have nothing to disclose.

Estradiol treatment in a nonhuman primate model of menopause preserves affective reactivity.

Human affective life changes with age, becoming more positive and less negative in later life. This change occurs even as aging leads to declines in health and cognitive outcomes. Despite these well-documented effects in humans, the extent to which affective processes change as a result of aging in nonhuman animals, particularly nonhuman primates, is unclear. As a first step toward developing an animal model for human affective aging, we tested aged, surgically menopausal aged and middle-aged gonadally intact female rhesus monkeys (Macaca mulatta) on a classic index of affective reactivity in monkeys, the Human Intruder Task. The Human Intruder Task evaluates behavioral responses to varying levels of threat. Aged, surgically menopausal monkeys received hormone replacement therapy consisting of a cyclic estradiol regimen or vehicle injections as a control. Average responsivity to threat did not vary by condition, but middle-aged monkeys and aged monkeys on estradiol were more reactive to the most potent level of threat than to a moderate level of threat, replicating previously published results in other age groups and male monkeys. In contrast, aged monkeys who were not on estradiol did not show such calibration to threat level. These findings suggest that estrogen may be important for maintaining more youthful affective responding. They also illustrate the utility of behavioral assays of affective reactivity in nonhuman primate models of cognitive and reproductive aging in humans. (PsycINFO Database Record

Timing of cyclic estradiol treatment differentially affects cognition in aged female rhesus monkeys.

Some evidence suggests that there may be a limited "window of opportunity" for beneficial effects of hormone therapy after menopause in women. We tested whether the timing of cyclic estradiol (E2) treatment impacted its effect on cognitive function in aged, surgically menopausal (ovariectomized) rhesus monkeys. Monkeys were assigned to one of four treatment conditions after ovariectomy: either vehicle or E2 treatment for the duration of the protocol, vehicle for the first 2 years of the protocol followed by E2 for the remainder (delayed treatment), or E2 for the first 11 months of the protocol followed by vehicle for the remainder (withdrawn treatment). Delayed treatment addressed the hypothesis that E2 treatment initiated more than 2 years postovariectomy would have a reduced effect on cognitive function. Withdrawn treatment mirrored current clinical advice to women to use hormone therapy in the initial postmenopausal period then discontinue it. Two periods of cognitive testing assessed treatment effects on cognition over time. E2 treatment predominantly affected a prefrontal cortex-dependent test of spatiotemporal working memory (delayed response). Monkeys with delayed E2 treatment modestly improved in delayed response performance over time, whereas vehicle-treated monkeys declined. Monkeys with withdrawn E2 treatment maintained their performance across assessments, as did monkeys treated with E2 across the entire protocol. These findings suggest that a "window of opportunity" for hormone treatment after cessation of ovarian function, if present in nonhuman primates, lasts longer than 2 years. They also support the notion that beneficial effects of hormone therapy may persist after discontinuation of treatment. (PsycINFO Database Record

Tissue engineering using 3D printed nano-bioactive glass loaded with NELL1 gene for repairing alveolar bone defects.

The purposes of this study were to construct a novel tissue engineered bone composed of 3D-printed bioactive glass block/chitosan nanoparticles (BD/CSn) composites loaded with Nel-like Type I molecular-1 DNA (pDNA-NELL1) and/or bone marrow mesenchymal stem cells (BMSCs), and study their osteogenic activities by repairing bone defects in rhesus monkeys. CSn with NELL1 gene plasmid and rhesus monkey BMSCs were composited with a BD scaffold to prepare the tissue-engineered bone. Four adult female rhesus monkeys with 10- to 12-years old and 5–7 kg in weight were used in animal experiments. The first and second premolar teeth from four regions of each monkey were removed to form bone defects with size of 10 × 10 × 5 mm, which were then implanted with above-mentioned tissue engineered bone. At 12 weeks after the implantation, gross observations, X-ray and micro-CT observations revealed that the new bone was extremely close to normal bone in mass, density, hardness, and structure. The bony cortex was smooth and closely connected to the surrounding normal bone. Histological observations revealed moderate inflammation in the repair area, and the new bone tissues were similar to normal ones. In conclusion, tissue engineered bone of this study exhibited good osteoconductivity for promoting the formation of new alveolar bone tissue, and NELL1 gene played a promotional role in bone regeneration.

Social status predicts response to dietary cycling in female rhesus monkeys

With the prevalence of obesity among women the United States surpassing 40%, it is critical to understand how environmental factors influence appetite, body fat accumulation, and the ability to lose weight and maintain weight loss. Psychosocial stress exposure is a risk factor for increased consumption of calorically dense diets (CDD), which are high in fat and sugars and promote both increased food intake and weight gain. However, it remains unclear how appetite is affected by psychosocial factors when people striving to lose weight restrict intake of unhealthy, calorically dense foods. Using a translational non-human primate model of chronic psychosocial stressor exposure in females (n = 16), mediated by social subordination, we examined ad libitum food intake, weight change, and social behavior during three consecutive, 15-week dietary conditions: 1) obesogenic, dietary choice; 2) chow-only; and 3) a switch back to dietary choice. Data showed that a choice dietary environment that includes both chow and CDD promotes increased calorie consumption of CDD in subordinate female rhesus monkeys during the baseline choice and back-to-choice phases (p = 0.016). Removal of the CDD during the chow-only phase resulted in mild inappetence (p = 0.005) and a loss in body weight (p < 0.001) in subordinate females. Reintroduction of the CDD to subordinate, but not dominant, females was associated with increased calorie intake that surpassed baseline intake (p < 0.001), and greater body weight gain (p = 0.026). There were no effects of diet cycling on total food intake and body weight change in dominant females (p's > 0.05). Overall, our results suggest that adverse psychosocial experience is associated with increased preference for highly palatable, calorically dense food in a choice dietary environment.

Neuroanatomical Relationship of Neuronal Nitric Oxide Synthase to Gonadotropin-Releasing Hormone and Kisspeptin Neurons in Adult Female Sheep and Primates

Background: Neuronal intermediates that communicate estrogen and progesterone feedback to gonadotropin-releasing hormone (GnRH) neurons are essential for modulating reproductive cyclicity. Individually, kisspeptin and nitric oxide (NO) influence GnRH secretion. However, it is possible these 2 neuronal intermediates interact with one another to affect reproductive cyclicity. Methods: We investigated the neuroanatomical relationship of one isoform of the enzyme that synthesizes NO, neuronal NO synthase (nNOS), to kisspeptin and GnRH in adult female rhesus monkeys and sheep using dual-label immunofluorescence. Additionally, we evaluated if the phase of the reproductive cycle would affect these relationships. Results: Overall, no effect of the stage of cycle was observed for any variable in this study. In the arcuate nucleus (ARC) of sheep, 98.8 ± 3.5% of kisspeptin neurons colocalized with nNOS, and kisspeptin close-contacts were observed onto nNOS neurons. In contrast to ewes, no colocalization was observed between kisspeptin and nNOS in the infundibular ARC of primates, but kisspeptin fibers were apposed to nNOS neurons. In the preoptic area of ewes, 15.0 ± 4.2% of GnRH neurons colocalized with nNOS. In primates, 38.8 ± 10.1% of GnRH neurons in the mediobasal hypothalamus colocalized with nNOS, and GnRH close-contacts were observed onto nNOS neurons in both sheep and primates. Conclusion: Although species differences were observed, this work establishes a neuroanatomical framework between nNOS and kisspeptin and nNOS and GnRH in adult female nonhuman primates and sheep.