Reproductive Neuroendocrine Axis Research Articles

TAC3 regulates GnRH/gonadotropin synthesis in female chickens

Neurokinin B (NKB), a peptide encoded by the tachykinin 3 (TAC3), is critical for reproduction in all studied species. However, its potential roles in birds are less clear. Using the female chicken (c-) as a model, we showed that cTAC3 is composed of five exons with a full-length cDNA of 787 bp, which was predicted to generate the mature NKB peptide containing 10 amino acids. Using cell-based luciferase reporter assays, we demonstrated that cNKB could effectively and specifically activate tachykinin receptor 3 (TACR3) in HEK293 cells, suggesting its physiological function is likely achieved via activating cTACR3 signaling. Notably, cTAC3 and cTACR3 were predominantly and abundantly expressed in the hypothalamus of hens and meanwhile the mRNA expression of cTAC3 was continuously increased during development, suggesting that NKB-TACR3 may emerge as important components of the neuroendocrine reproductive axis. In support, intraperitoneal injection of cNKB could significantly promote hypothalamic cGnRH-Ι, and pituitary cFSHβ and cLHβ expression in female chickens. Surprisingly, cTAC3 and cTACR3 were also expressed in the pituitary gland, and cNKB treatment significantly increased cLHβ and cFSHβ expression in cultured primary pituitary cells, suggesting cNKB can also act directly at the pituitary level to stimulate gonadotropin synthesis. Collectively, our results reveal that cNKB functionally regulate GnRH/gonadotropin synthesis in female chickens.

24 Effects of Gestational and Postweaning Nutrition on Brain Development and Puberty in Beef Heifers

Abstract Approximately 5 million beef heifers enter the U.S. cow herd annually, and their lifetime productivity is largely dependent upon their ability to attain puberty and produce a calf by 24 mo of age. However, a considerable proportion of heifers within existing U.S. production systems fail to achieve these goals, particularly in southern regions where Bos indicus-influenced cattle predominate. Consequently, managerial approaches are needed that employ nutritional programming of the reproductive neuroendocrine axis, while minimizing feeding costs and optimizing the consistent attainment of puberty by approximately 14 mo of age. Our previous research has clearly demonstrated that increased BW gain between 4 and 8 mo of age facilitates pubertal development by programming hypothalamic centers that regulate gonadotropin releasing-hormone (GnRH) secretion. Among the different metabolic hormones, leptin has a critical role in conveying nutritional information to the hypothalamus and controlling puberty. Two hypothalamic neuronal populations that express the orexigenic peptide neuropeptide Y (NPY) and the anorexigenic peptide alpha melanocyte-stimulating hormone (αMSH; a product of the POMC gene) are key components of afferent pathways that convey inhibitory (NPY) and excitatory (αMSH) inputs to GnRH and kisspeptin neurons. Our studies have demonstrated that short-term increases in dietary energy intake after early weaning at 4 mo of age result in epigenetic, structural, and functional modifications in these hypothalamic pathways to promote high-frequency, episodic release of GnRH and luteinizing hormone. However, integrating the foundational knowledge of metabolic imprinting of the brain for early puberty with issues related to lifetime performance is complex. One approach has been to employ a novel stair-step nutritional regimen involving alternating periods of dietary energy-restriction and re-feeding during juvenile development. This approach is designed to support early onset of puberty by imprinting functional alterations in the hypothalamus during key periods of brain development while optimizing other aspects of growth and performance. Finally, while several key processes of fetal brain development occur during late gestation, neither maternal undernutrition (BCS 3.0-3.5) nor maternal obesity (BCS 7.5-8.0) during the last two trimesters of gestation impacted the organization of hypothalamic neurocircuitries controlling GnRH/LH secretion or age at puberty in the heifer offspring. Collectively, these results indicate that the adult reproductive phenotype in Bos indicus-influenced females is resilient to significant degrees of nutritional stress imposed during prenatal development, particularly if early gestation (first trimester) is avoided. These findings are in contrast with reports in Bos taurus females in which late gestation nutritional extremes result in delayed puberty in heifer offspring, suggesting that potential genotype-specific differences may exist. Research support: USDA-NIFA-AFRI (2018-67015-27595 and 2021-67015-33676).

The Role of RFRP Neurons in the Allostatic Control of Reproductive Function.

Reproductive function is critical for species survival; however, it is energetically costly and physically demanding. Reproductive suppression is therefore a physiologically appropriate adaptation to certain ecological, environmental, and/or temporal conditions. This 'allostatic' suppression of fertility enables individuals to accommodate unfavorable reproductive circumstances and safeguard survival. The mechanisms underpinning this reproductive suppression are complex, yet culminate with the reduced secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus, which in turn suppresses gonadotropin release from the pituitary, thereby impairing gonadal function. The focus of this review will be on the role of RFamide-related peptide (RFRP) neurons in different examples of allostatic reproductive suppression. RFRP neurons release the RFRP-3 peptide, which negatively regulates GnRH neurons and thus appears to act as a 'brake' on the neuroendocrine reproductive axis. In a multitude of predictable (e.g., pre-puberty, reproductive senescence, and seasonal or lactational reproductive quiescence) and unpredictable (e.g., metabolic, immune and/or psychosocial stress) situations in which GnRH secretion is suppressed, the RFRP neurons have been suggested to act as modulators. This review examines evidence for and against these roles.

SAT423 Regulation Of The Male Neuroendocrine Axis By Per- And Polyfluoroalkyl Substances (PFASs)

Abstract Disclosure: S. Bradley: None. J. Hayworth: None. B.T. Akingbemi: None. Introduction and Objectives. Per- and polyfluoroalkyl substances (PFASs) are man-made chemicals present in several consumer products. Unregulated disposal of PFASs into landfills contaminates groundwater sources for both drinking and agriculture and raise public health concerns. Short-chain or emerging PFASs, such as perfluorobutanoic acid (PFBA) and perfluorobutanesulfonic acid (PFBS), are thought to be safer than perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) due to decreased toxicity and rapid environmental degradation. The present study is designed to compare the endocrine-disrupting properties of legacy and emerging PFASs. Methods. Male Long-Evans rats (n=12) were fed test chemicals (PFOA, PFOS, PFBA, PFBS) in drinking water at 1, 10, 100, and 1000 ng/L from 21 to 35 days of age (acute or 14-day exposure) or at 10 or 100 ng/L from postnatal days 21 to 49 (chronic or 28-day exposure). At termination of chemical exposures, animals were sacrificed to obtain testicular tissue and Leydig cell fractions for incubation in DMEM/F-12 culture medium for 3 h for basal T or with 100 ng/mL ovine LH (NIDDK, NIH) to measure LH-stimulated testosterone (T) production by RIA. Testes, Leydig cells and pituitary glands were processed for western blot analyses of protein gene expression. Results. Acute exposures of male rats to PFBA at 10 ng/L increased basal (p<0.05) and LH-stimulated (p<0.00001) testicular T production just as basal testicular T production was increased by PFBS at 1 ng/L (p<0.05) and PFOS at 1000 ng/L (p<0.001). However, PFOS at 10 ng/L caused a decrease (p<0.001) in LH-stimulated testicular T production compared to control. Chronic exposures to PFOS and PFBS at 10 and 100 ng/L decreased (p< 0.001) basal testicular T production, but PFBA at 10 ng/L increased (p<0.05) LH-stimulated testicular T production. Testicular expression of the Mϋllerian Inhibiting Substance (MIS) protein was increased by PFOS and PFBA at 100 ng/L (p<0.05), whereas testicular inhibin-β protein was decreased (p<0.001) by all test chemicals at 10 and 100 ng/L doses except at the lower PFBA dose. Exposure to PFOA at 10 ng/L and PFBS at 100 ng/L increased (p<0.05) pituitary luteotropin subunit protein expression similar to PFBA at 100 ng/L and PFBS at 10 ng/L which increased (p<0.001) pituitary FSH-β protein. In Leydig cells, PFOA at 100 ng/L increased (p<0.0001) but PFBA at 100 ng/L decreased (p<0.05) the StAR protein. Cytochrome P450 17A1 enzyme protein was decreased by both PFBA and PFOS at 100 ng/L (p<0.05) in Leydig cells compared to control. Conclusions. These observations showed that both legacy and emerging PFASs can impact the male neuroendocrine-reproductive axis. PFASs increased as well as decreased gonadal T secretion with implications for germ cell development, sperm production and male fertility. Presentation: Saturday, June 17, 2023

Leptin, but not Estradiol, Signaling in PACAP Neurons Modulates Puberty Onset

Abstract The adipose-derived hormone leptin critically modulates reproductive function, such that its absence results in hypothalamic hypogonadism. Pituitary adenylate cyclase-activating polypeptide (PACAP)-expressing neurons are potential mediators of leptin's action on the neuroendocrine reproductive axis because they are leptin-sensitive and involved in both feeding behavior and reproductive function. In the complete absence of PACAP, male and female mice exhibit metabolic and reproductive abnormalities, yet there is some sexual dimorphism in the reproductive impairments. We tested whether PACAP neurons play a critical and/or sufficient role in mediating leptin's effects on reproductive function by generating PACAP-specific leptin receptor (LepR) knockout and rescue mice, respectively. We also generated PACAP-specific estrogen receptor alpha knockout mice to determine whether estradiol-dependent regulation of PACAP was critically involved in the control of reproductive function and whether it contributed to the sexually dimorphic effects of PACAP. We showed that LepR signaling in PACAP neurons is critically involved in the timing of female, but not male, puberty onset, but not fertility. Rescuing LepR-PACAP signaling in otherwise LepR-deficient mice was unable to rescue the reproductive deficits observed in LepR null mice but led to a marginal improvement in body weight and adiposity in females. Finally, PACAP-specific estrogen receptor alpha knockout did not lead to any changes in body weight or puberty onset compared with control mice. These data highlight that PACAP is a critical mediator of some of leptin's, but not estradiol's, influence on puberty onset in females, but is not critically involved in relaying leptin's effects in males or in adult females.

Review: Gestational and postnatal nutritional effects on the neuroendocrine control of puberty and subsequent reproductive performance in heifers.

Pubertal attainment is an intricate biological process that involves maturation of the reproductive neuroendocrine axis and increased pulsatile release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone. Nutrition is a critical environmental factor controlling the timing of puberty attainment. Nutrient restriction during early postnatal development delays puberty, whereas increased feed intake and adiposity during this period hasten pubertal maturation by imprinting the hypothalamus. Moreover, the dam's nutrition during gestation can program the neuroendocrine system in the developing fetus and has the potential to advance or delay puberty in the offspring. Leptin, a hormone produced primarily by adipose cells, plays an important role in communicating energy status to the brain and regulating sexual maturation. Leptin's regulation of GnRH release is mediated by an upstream neuronal network since GnRH neurons do not contain the leptin receptor. Two groups of neurons located in the arcuate nucleus of the hypothalamus that express neuropeptide Y (NPY), an orexigenic peptide, and alpha melanocyte-stimulating hormone (αMSH), an anorexigenic peptide, are central elements of the neural circuitry that relay inhibitory (NPY) and excitatory (αMSH) inputs to GnRH neurons. Moreover, KNDy neurons, neurons in the arcuate nucleus that co-express kisspeptin, neurokinin B (NKB), and dynorphin, also play a role in the metabolic regulation of puberty. Our studies in beef heifers demonstrate that increased rates of BW gain during early postweaning (4-9 mo of age) result in reduced expression of NPY mRNA, increased expression of proopiomelanocortin and kisspeptin receptor mRNA, reduced NPY inhibitory inputs to GnRH neurons, and increased excitatory αMSH inputs to KNDy neurons. Finally, our most recent data demonstrate that nutrition of the cow during the last two trimesters of gestation can also induce transcriptional and structural changes in hypothalamic neurocircuitries in the heifer progeny that likely persist long-term after birth. Managerial approaches, such as supplementation of the dam during gestation (fetal programming), creep feeding, early weaning, and stair-step nutritional regimens have been developed to exploit brain plasticity and advance pubertal maturation in heifers.

What do we know about kisspeptin?

Kisspeptins are a group of peptide fragments that are expressed by the neurons of the hypothalamus mainly in the arcuate, dorsal-medial and antero ventricular, periventricular nuclei of the hypothalamus besides the amygdala.1 Initially, Kisspeptin via its G protein-coupled receptor 54 (GPR54) was introduced to have an important role in cancer biology with its metastasis suppression effect. In light of recent research, Kisspeptin is found to have a major role in the neuroendocrine reproductive axis through the Kisspeptin 1 (KISS1) gene. The KISS1 gene that encodes Kisspeptin was first discovered in 1996 by researchers from the United States of America. Kisspeptinreceptors are also expressed by the same neurons. Kisspeptin has a pivotal role in reproduction through the hypothalamic-hypophysial axis while exerting an extra hypothalamic function on sexual and emotional behavior.

Systemic treatment with GnRH agonist produces antidepressant-like effects in LPS induced depression male mouse model

Gonadotropin-releasing hormone (GnRH) is at the head of the neuroendocrine reproductive axis. However, the non-reproductive functions of GnRH expressed in various tissues, including hippocampus, are still not known. Here, we unveil a previously unknown effect of GnRH, which mediates depression-like behaviors through the modulation of microglia function during immune challenge. Specifically, we found that either systemic treatment with GnRH agonist or over-expression of endogenous hippocampal GnRH via viral tool abolished the depression-like behavior after LPS challenges in mice. And the anti-depressant of GnRH was dependent on the hippocampal GnRHR signaling, since antagonizing GnRHR by drug treatment or by hippocampal GnRHR knockdown could block the antidepressant-effect of GnRH agonist. Interestingly, we found that the peripheral GnRH treatment prevented the microglia activation mediated inflammation in the hippocampus of mice. In light of the research findings presented here, we propose that, at least in the hippocampus, GnRH appears to act on GnRHR to regulate higher order non-reproductive functions associated with the microglia mediated neuroinflammation. These findings also provide insights into the function and cross-talk of GnRH, a known neuropeptide hormone, in neuro-immune response.

Deletion of androgen receptors from kisspeptin neurons prevents PCOS features in a letrozole mouse model.

Polycystic ovarian syndrome (PCOS) is the leading cause of anovulatory infertility and is a heterogenous condition associated with a range of reproductive and metabolic impairments. While its etiology remains unclear, hyperandrogenism and impaired steroid negative feedback have been identified as key factors underpinning the development of PCOS-like features both clinically and in animal models. We tested the hypothesis that androgen signaling in kisspeptin-expressing neurons, which are key drivers of the neuroendocrine reproductive axis, is critically involved in PCOS pathogenesis. To this end, we used a previously validated letrozole (LET)-induced hyperandrogenic mouse model of PCOS in conjunction with Cre-lox technology to generate female mice exhibiting kisspeptin-specific deletion of androgen receptor (KARKO mice) to test whether LET-treated KARKO females are protected from the development of reproductive and metabolic PCOS-like features. LET-treated mice exhibited hyperandrogenism, and KARKO mice exhibited a significant reduction in the coexpression of kisspeptin and androgen receptor mRNA compared to controls. In support of our hypothesis, LET-treated KARKO mice exhibited improved estrous cyclicity, ovarian morphology, and insulin sensitivity in comparison to LET-treated control females. However, KARKO mice were not fully protected from the effects of LET-induced hyperandrogenism and still exhibited reduced corpora lutea numbers and increased body weight gain. These data indicate that increased androgen signaling in kisspeptin-expressing neurons plays a critical role in PCOS pathogenesis, but highlight that other mechanisms are also involved.

Adult Male Syrian Hamsters (Mesocricetus auratus) Exhibit Daily Oscillations in Their Serum Levels of Melatonin and Leptin As Well As in the Expression of the GnRH, GnIH, and Kisspeptin Genes

The complex neuronal and hormonal interaction between the brain and gonads controls the neuroendocrine reproductive axis. GnRH, GnIH, and kisspeptin are important neuropeptides in this relationship. Although seasonal variations of these neuropeptides have been demonstrated in photoperiodic animals, there is no clear evidence in their daily rhythms. Melatonin and leptin hormones are also two important hormones in reproductive regulation. In our study, the relationship of melatonin and leptin hormones with daily rhythm with GnIH, GnRH, and kisspeptin gene expressions and protein oscillations was examined. Adult male Syrian hamsters were exposed to the long photoperiod and at the end of the 30-day experimental period, blood and tissue samples from each group were collected at 04:00 h, 12:00 h, 20:00 h, and 00:00 h. Daily rhythms of melatonin and leptin hormones were determined by ELISA. Quantitative analysis of GnRH, GnIH, Kisspeptin, and β-actin genes was performed with the corresponding primers in Real-Time PCR. Protein expressions were determined by the Western Blot technique. Serum melatonin and leptin levels showed an inverse rhythmic relationship. Leptin level was found to be low while melatonin was high in the dark. Daily rhythms were observed in GnIH, GnRH, and kisspeptin mRNA expressions and protein oscillations. As a result, our findings could imply that all of the relationships between melatonin, leptin, GnIH, GnRH, and kisspeptin are not reproductive, but rather metabolic in nature.

A mammalian gonadotropin-inhibitory hormone homolog RFamide-related peptide 3 regulates pain and anxiety in mice.

RFamide-related peptide (RFRP) is a homologous neuropeptide to gonadotropin-inhibitory hormone (GnIH), which is a hypothalamic neuropeptide that negatively regulates the hypothalamic-pituitary-gonadal axis. RFRP/GnIH is thought to be the mediator of stress because various stressors increase RFRP/GnIH mRNA expression and/or RFRP/GnIH neuronal activities. RFRP/GnIH may also directly regulate behavior, because RFRP/GnIH neuronal fibers and RFRP/GnIH receptor are widely expressed in the brain. Here, we create a RFRP/GnIH knockout (GnIH-KO) mice and conduct various behavioral tests. Dense RFRP/GnIH neuronal fibers are located in the limbic system and broad areas in the thalamus, hypothalamus, and midbrain in wild-type mice but not in RFRP/GnIH-KO mice. Spatial working memory is not improved in GnIH-KO mice as shown by Y-maze test. GnIH-KO mice perform intensive wheel running exercise for several hours after light-off. Hot plate test shows that GnIH-KO mice have decreased sensitivity to pain and central administration of RFRP3 to GnIH-KO mice recovers pain sensitivity. Elevated plus maze test shows that GnIH-KO mice have decreased level of anxiety and central administration of RFRP3 to GnIH-KO mice recovers anxiety level. These results indicate that RFRP3 regulates pain and anxiety in mice. RFRP3 may be involved in the negative regulation of spontaneous activity in addition to negatively regulating the reproductive neuroendocrine axis in stressful conditions.

Neuroendocrine mechanisms underlying estrogen positive feedback and the LH surge.

A fundamental principle in reproductive neuroendocrinology is sex steroid feedback: steroid hormones secreted by the gonads circulate back to the brain to regulate the neural circuits governing the reproductive neuroendocrine axis. These regulatory feedback loops ultimately act to modulate gonadotropin-releasing hormone (GnRH) secretion, thereby affecting gonadotropin secretion from the anterior pituitary. In females, rising estradiol (E2) during the middle of the menstrual (or estrous) cycle paradoxically “switch” from being inhibitory on GnRH secretion (“negative feedback”) to stimulating GnRH release (“positive feedback”), resulting in a surge in GnRH secretion and a downstream LH surge that triggers ovulation. While upstream neural afferents of GnRH neurons, including kisspeptin neurons in the rostral hypothalamus, are proposed as critical loci of E2 feedback action, the underlying mechanisms governing the shift between E2 negative and positive feedback are still poorly understood. Indeed, the precise cell targets, neural signaling factors and receptors, hormonal pathways, and molecular mechanisms by which ovarian-derived E2 indirectly stimulates GnRH surge secretion remain incompletely known. In many species, there is also a circadian component to the LH surge, restricting its occurrence to specific times of day, but how the circadian clock interacts with endocrine signals to ultimately time LH surge generation also remains a major gap in knowledge. Here, we focus on classic and recent data from rodent models and discuss the consensus knowledge of the neural players, including kisspeptin, the suprachiasmatic nucleus, and glia, as well as endocrine players, including estradiol and progesterone, in the complex regulation and generation of E2-induced LH surges in females.

Role of Melatonin in Temperature-Induced Activation of the Neuroendocrine Reproductive Axis in Garter Snakes

An animal’s ability to respond optimally to changing environmental conditions is paramount to successfully reproducing and thus maximizing fitness. Studies on photoperiod-induced changes in neural thyroid hormone metabolism have conclusively linked environmental cues to the neuroendocrine reproductive axis of birds and mammals. Whether this conserved mechanism also transduces changes in environmental temperature, however, has not been fully addressed. We investigated whether the hormone melatonin mediates the effects of low-temperature dormancy on thyroid hormone metabolism within the hypothalamus of red-sided garter snakes (Thamnophis sirtalis parietalis). To address this question, we used immunohistochemistry to assess changes in thyroid-stimulating hormone (TSH) in the infundibulum of the pituitary and deiodinase 3 (Dio3) and gonadotropin-releasing hormone (GnRH) in the hypothalamus. We also asked if changes in TSH, Dio3, and/or GnRH immunoreactivity are associated with changes in male courtship behavior. In contrast to our predictions, 6 weeks of dormancy at 4°C significantly decreased the number of TSH-labeled cells in the infundibulum. It is possible that the observed decrease in TSH is related to the release of snakes from temperature refractoriness, but this idea needs further testing. Treatment of snakes with the melatonin precursor 5-hydroxytryptophan during dormancy at 4°C both reversed the temperature-induced change in TSH immunoreactivity and disrupted the temporal pattern of male courtship behavior. These results suggest that TSH cells within the infundibulum are both modulated by temperature and sensitive to changes in melatonin. As predicted, male snakes hibernated at an elevated temperature of 12°C for 6 weeks and treated with vehicle showed no change in TSH-, Dio3-, or GnRH-immunoreactive cell number. Treatment of snakes with the melatonin receptor antagonist luzindole was not sufficient in rescuing the effects of dormancy at 12°C on TSH immunoreactivity or courtship behavior. However, luzindole-treated snakes showed a significant increase in GnRH-immunoreactive cell number, suggesting that melatonin exerts an inhibitory effect on GnRH in garter snakes. In summary, our results provide critical insights into the mechanisms that mediate the effects of temperature on reproductive physiology and behavior.

Mouse Cre drivers: tools for studying disorders of the human female neuroendocrine-reproductive axis†.

Benign disorders of the human female reproductive system, such primary ovarian insufficiency and polycystic ovary syndrome are associated with infertility and recurrent miscarriage, as well as increased risk of adverse health outcomes, including cardiovascular disease and type 2 diabetes. For many of these conditions, the contributing molecular and cellular processes are poorly understood. The overarching similarities between mice and humans have rendered mouse models irreplaceable in understanding normal physiology and elucidating pathological processes that underlie disorders of the female reproductive system. The utilization of Cre-LoxP recombination technology, which allows for spatial and temporal control of gene expression, has identified the role of numerous genes in development of the female reproductive system and in processes, such as ovulation and endometrial decidualization, that are required for the establishment and maintenance of pregnancy in mammals. In this comprehensive review, we provide a detailed overview of Cre drivers with activity in the neuroendocrine-reproductive axis that have been used to study disruptions in key intracellular signaling pathways. We first summarize normal development of the hypothalamus, pituitary, ovary, and uterus, highlighting similarities and differences between mice and humans. We then describe human conditions resulting from abnormal development and/or function of the organ. Finally, we describe loss-of-function models for each Cre driver that elegantly recapitulate some key features of the human condition and are associated with impaired fertility. The examples we provide illustrate use of each Cre driver as a tool for elucidating genetic and molecular underpinnings of reproductive dysfunction.

Hypothalamic and Cell-Specific Transcriptomes Unravel a Dynamic Neuropil Remodeling in Leptin-Induced and Typical Pubertal Transition in Female Mice.

SummaryEpidemiological and genome-wide association studies (GWAS) have shown high correlation between childhood obesity and advance in puberty. Early age at menarche is associated with a series of morbidities, including breast cancer, cardiovascular diseases, type 2 diabetes, and obesity. The adipocyte hormone leptin signals the amount of fat stores to the neuroendocrine reproductive axis via direct actions in the brain. Using mouse genetics, we and others have identified the hypothalamic ventral premammillary nucleus (PMv) and the agouti-related protein (AgRP) neurons in the arcuate nucleus (Arc) as primary targets of leptin action in pubertal maturation. However, the molecular mechanisms underlying leptin's effects remain unknown. Here we assessed changes in the PMv and Arc transcriptional program during leptin-stimulated and typical pubertal development using overlapping analysis of bulk RNA sequecing, TRAP sequencing, and the published database. Our findings demonstrate that dynamic somatodendritic remodeling and extracellular space organization underlie leptin-induced and typical pubertal maturation in female mice.

Hypothalamic and Cell-Specific Transcriptomes Unravel a Dynamic Neuropil Remodeling in Leptin-Induced and Typical Pubertal Transition

Epidemiological and genome-wide association studies (GWAS) have shown high correlation between childhood obesity and advance in puberty. Early age at menarche is associated with a series of morbidities, including breast cancer, cardiovascular diseases, type 2 diabetes and obesity. The adipocyte hormone leptin signals the amount of fat stores to the neuroendocrine reproductive axis via direct actions in the brain. Using mouse genetics, we and others have identified the hypothalamic ventral premammillary nucleus (PMv) and the agouti-related protein (AgRP) neurons in the arcuate nucleus (Arc) as the primary targets of leptin action in pubertal maturation. However, the molecular mechanisms underlying leptin’s effects remain unknown. Here we assessed changes in the PMv and Arc transcriptional program during leptin-stimulated and typical pubertal development using overlapping analysis of bulk RNAseq, TRAPseq and the published database. Our findings demonstrate that dynamic somatodendritic remodeling and extracellular space organization underlie leptin-induced and typical pubertal maturation in mice.

SUN-263 In Vitro Modeling of Brain-Bone Communication: Factors Secreted from Immortalized Female Kisspeptin Neurons Modulate Gene Expression in Osteoblasts

While the sex steroid hormone estrogen (E2) is essential for maximum reproductive fitness, circulating E2 is also involved in maintaining skeletal homeostasis in females. E2 in male and female animals has been shown to stimulate cancellous bone formation via activation of estrogen receptor alpha (ERα), and it was widely thought that this effect was mediated directly at the level of bone. A recent study, however, demonstrated a large increase in bone density in female mice in which ERα was deleted from specific neuroendocrine neurons in the arcuate nucleus of the hypothalamus, specifically those expressing kiss1, a population required for fertility and pubertal progression. Bone from transgenic Kiss1-cre X ERα floxed females showed higher osteoblast functioning, accompanied by increases in the expression of sp7 and runx2, positing the existence of a direct neural-bone regulatory axis that is altered by circulating E2 at the level of the brain (Herber et al., Nat Commun 2019).Our laboratory recently published a study demonstrating that GnRH and Kisspeptin, typically thought to act primarily within the neuroendocrine reproductive axis, are synthesized and secreted in an autocrine fashion by canine osteosarcoma cells in vitro, and that these neuropeptides can stimulate tumor cell proliferation (BMC Cancer 2019). Separately, our lab has also recently generated two immortalized Kiss1-expressing and –secreting cell lines, KTaR-1 (representative of female arcuate Kiss-1 neurons) and KTaV-3 cells (representative of female AVPV Kiss-1 neurons) (Endocrinology 2017). In the current study, we have combined these two in vitro models to explore if factors secreted by female ARC-derived KTaR-1 cells may affect multiple parameters of osteoblast function, including sp7 and runx2 expression (evaluated by qPCR), and ability to form bone matrix (evaluated by Alizarin Red assay). Preliminary results suggest that exposure canine osteosarcoma (COS) cells to conditioned media from KTaR-1 cells leads to increases in sp7 expression in an E2-dependent manner, such that 24h E2-deprivation of these neurons stimulates secretion of osteogenic factors. Additionally, media from both KTaR-1 and KTaV-3 cells stimulated Ca2+ production from cultured osteoblasts, as evaluated by Alizarin Red. We are continuing to explore these in vitro interactions using COS cells, as well as normal osteoblasts (cNOB) and immortalized cNOBs, and are evaluating media and exosomal proteomics to further characterize putative factors. While further study is required, these initial results suggest that our immortalized neuronal KP cell models may provide useful molecular tools to explore the regulation of this newly-proposed neural-bone axis.

Chemogenetic Suppression of GnRH Neurons during Pubertal Development Can Alter Adult GnRH Neuron Firing Rate and Reproductive Parameters in Female Mice.

Gonadotropin-releasing hormone (GnRH) neurons control anterior pituitary, and thereby gonadal, function. GnRH neurons are active before outward indicators of puberty appear. Prenatal androgen (PNA) exposure mimics reproductive dysfunction of the common fertility disorder polycystic ovary syndrome (PCOS) and reduces prepubertal GnRH neuron activity. Early neuron activity can play a critical role in establishing circuitry and adult function. We tested the hypothesis that changing prepubertal GnRH neuron activity programs adult GnRH neuron activity and reproduction independent of androgen exposure in female mice. Activating (3Dq) or inhibitory (4Di) designer receptors exclusively activated by designer drugs (DREADDs) were targeted to GnRH neurons using Cre-lox technology. In control studies, the DREADD ligand clozapine n-oxide (CNO) produced the expected changes in GnRH neuron activity in vitro and luteinizing hormone (LH) release in vivo. CNO was administered to control or PNA mice between two and three weeks of age, when GnRH neuron firing rate is reduced in PNA mice. In controls, reducing prepubertal GnRH neuron activity with 4Di increased adult GnRH neuron firing rate and days in diestrus but did not change puberty onset or GABA transmission to these cells. In contrast, activating GnRH neurons had no effect on reproductive parameters or firing rate and did not rescue reproductive phenotypes in PNA mice. These studies support the hypothesis that prepubertal neuronal activity sculpts elements of the adult reproductive neuroendocrine axis and cyclicity but indicate that other PNA-induced programming actions are required for full reproductive phenotypes and/or that compensatory mechanisms overcome activity-mediated changes to mitigate reproductive changes in adults.

33 Nutritional programming of puberty in Bos indicus-influenced heifers

Abstract Approximately 5 million beef heifers enter the U.S. cow herd annually and their lifetime productivity is heavily-dependent upon their ability to attain puberty and produce a calf by 24 mo of age. However, a significant proportion of heifers within existing U.S. production systems fail to achieve these goals, particularly in southern regions where Bos indicus-influenced cattle predominate. Therefore, approaches are needed that facilitate nutritional programming of the reproductive neuroendocrine axis, while minimizing feeding costs and optimizing the consistent establishment of puberty by 14–15 mo of age. Increased BW gain between 4 and 9 mo of age facilitates pubertal development by programming hypothalamic centers that regulate GnRH secretion. Among the different metabolic hormones, leptin plays a critical role in conveying nutritional information to the brain and controlling puberty. Two hypothalamic neuronal populations that express the orexigenic peptide neuropeptide Y (NPY) and the anorexigenic peptide alpha melanocyte-stimulating hormone (αMSH) are key components of afferent pathways that convey inhibitory (NPY) and excitatory (αMSH) inputs to GnRH neurons. Our studies have demonstrated that short-term increases in dietary energy intake during juvenile development result in epigenetic, structural, and functional modifications in these hypothalamic pathways to promote high-frequency, episodic release of GnRH/LH. However, integrating the foundational knowledge of metabolic imprinting of the brain for early puberty with issues related to lifetime performance is complex. One approach has been to employ a novel stair-step nutritional regimen involving alternating periods of dietary energy-restriction and re-feeding during juvenile development. This approach is designed to support early onset of puberty by imprinting functional alterations in the hypothalamus during key periods of brain development while optimizing other aspects of growth and performance. Finally, our recent findings suggest that maternal nutrition during gestation can also induce neuroendocrine changes that are likely to persist and influence reproductive performance throughout adulthood in cattle.

205 Neuroendocrine pathways and the nutritional control of puberty in heifers

Abstract Approximately 5 million beef heifers enter the U.S. cow herd annually, and their lifetime productivity is heavily-dependent upon their ability to attain puberty and produce a calf by 24 mo of age. However, a significant proportion of heifers within existing U.S. production systems fail to achieve these objectives. Therefore, approaches are needed that facilitate nutritional programming of the reproductive neuroendocrine axis, while minimizing feeding costs and optimizing the consistent establishment of puberty by 14–15 mo of age. Accelerated rates of BW gain during early calfhood, particularly between 4 and 9 mo of age, facilitate pubertal development by programming hypothalamic centers that regulate GnRH secretion. Among the different metabolic hormones, leptin plays a critical role in conveying nutritional information to the brain and controlling pubertal progression. Two hypothalamic neuronal populations that express the orexigenic peptide neuropeptide Y (NPY) and the anorexigenic peptide alpha melanocyte-stimulating hormone (αMSH) are key components of afferent pathways that convey inhibitory (NPY) and excitatory (αMSH) inputs to GnRH neurons. Our studies have demonstrated that short-term increases in dietary energy intake during juvenile development result in epigenetic, structural, and functional modifications in these hypothalamic pathways to promote high-frequency, episodic release of GnRH/LH. However, integrating the foundational knowledge of metabolic imprinting of the brain for early puberty with issues related to lifetime performance is complex. One approach has been to employ a novel stair-step nutritional regimen involving alternating periods of dietary energy-restriction and re-feeding during juvenile development. This approach is designed to support early onset of puberty by imprinting genomic, biochemical and morphologic alterations in the hypothalamus during key periods of brain development while optimizing other aspects of growth and performance. Finally, our recent findings suggest that maternal nutrition during gestation can also induce neuroendocrine changes that are likely to persist long after puberty and influence reproductive performance throughout adulthood in cattle.