Most temperate animals exhibit seasonal rhythms in reproductive physiology and behaviour. Gonadotropin-releasing hormone (GnRH) cells in the preoptic area (POA) and kisspeptin (Kiss) cells in the anteroventral periventricular (AvPv) nucleus are critical for timing photoperiod-induced changes in seasonal reproduction. In response to prolonged exposure to photoperiod cues, many rodents exhibit endogenously generated programmed changes in physiology that reflect a circannual timing mechanism. Here we used transcriptome sequencing coupled with measurements of surface body temperature and testes mass across a simulated circannual interval timer to characterize the molecular changes involved in the seasonal control of reproduction and surface body temperature. Adult male Djungarian hamsters were collected after exposure to short photoperiod on 4-week intervals from 4 to 32 weeks. The POA and AvPv transcriptomes from short-photoperiod hamsters were compared to a long photoperiod reference group. Our analyses confirmed robust photoperiodic regulation of Kiss1 mRNA expression in the AvPv. Weighted gene co-expression network analyses (WGCNAs) identified hundreds (POA: 211, AvPv: 415) of transcripts that were associated with testes mass. Gene set enrichment analysis (GSEA) identified 'gonadotropin secretion' and 'glial cell proliferation' as the primary pathways associated with circannual interval timing. Vimentin had a strong negative association with testes mass, indicating AvPv glial morphology may increase during gonadal involution. In the POA, pathways associated with 'regulation at synapse' and 'regulation of synaptic plasticity' were highly enriched and transcripts positively associated with gonadal involution indicated that ribosomal plasticity and intracellular calcium signalling are key mechanisms involved in seasonal reproduction. WGCNAs identified 567 transcripts in the POA that were significantly associated with surface body temperature. GSEA discovered thyrotrophin-releasing hormone was significantly negatively associated with surface body temperature. Overall, the POA and AvPv transcriptome datasets provide the foundation to expand our understanding of the molecular representation of seasonal time in the mammalian hypothalamus.

Animals respond to environmental cues to time phenological events, but the intrinsic mechanism of circannual timing remains elusive. We used transcriptomic sequencing and frequent sampling of multiple hypothalamic nuclei in Djungarian hamsters to examine the neural and molecular architecture of circannual interval timing. Our study identified three distinct phases of transcript changes, with deiodinase type-3 (Dio3) expression activated during the early induction phase. Subsequent work demonstrated that targeted mutation of Dio3 using CRISPR–Cas resulted in a shorter period for circannual interval timing. Hamsters that are non-responsive to short photoperiods and fail to show any winter adaptations do not display changes in Dio3 expression and do not show any change in body mass or pelage. Our work demonstrates that changes in Dio3 induction are essential for setting the period of circannual interval timing.

A series of well-described anabolic and catabolic neuropeptides are known to provide short-term, homeostatic control of energy balance. The mechanisms that govern long-term, rheostatic control of regulated changes in energy balance are less well characterized. Using the robust and repeatable seasonal changes in body mass observed in Siberian hamsters, this report examined the role of prolactin in providing long-term rheostatic control of body mass and photoinduced changes in organ mass (ie, kidney, brown adipose tissue, uterine, and spleen). Endogenous circannual interval timing was observed after 4 months in a short photoperiod, indicated by a significant increase in body mass and prolactin mRNA expression in the pituitary gland. There was an inverse relationship between body mass and the expression of somatostatin (Sst) and cocaine- and amphetamine-regulated transcript (Cart). Pharmacological inhibition of prolactin release (via bromocriptine injection), reduced body mass of animals maintained in long photoperiods to winter-short photoperiod levels and was associated with a significant increase in hypothalamic Cart expression. Administration of ovine prolactin significantly increased body mass 24 hours after a single injection and the effect persisted after 3 consecutive daily injections. The data indicate that prolactin has pleiotropic effects on homeostatic sensors of energy balance (ie, Cart) and physiological effectors (ie, kidney, BAT). We propose that prolactin release from the pituitary gland acts as an output signal of the hypothalamic rheostat controller to regulate adaptive changes in body mass.

Social environments modulate endocrine function, yet it is unclear whether individuals can become like their social partners in how they physiologically respond to stressors. This social transmission of hypothalamicpituitary-adrenal (HPA) axis reactivity could have long-term consequences for health and lifespan of individuals if their social partners react to stressors with an exaggerated HPA axis response. We tested whether glucocorticoid levels in response to stress of breeding partners changes after breeding depending on whether partners had similar or dissimilar postnatal conditions. We manipulated postnatal conditions by mimicking early life stress in zebra finch chicks (Taeniopygia guttata) via postnatal corticosterone exposure. When they reached adulthood, we created breeding pairs where the female and male had experienced either the same or different early life hormonal treatment (corticosterone or control). Before and after breeding, we obtained blood samples within 3 min and after 10 min or 30 min of restraint stress (baseline, cort10, cort30). We found that corticosterone levels of individuals in response to restraint were affected by their own and their partner's early life conditions, but did not change after breeding. However, across all pairs, partners became more similar in cort30 levels after breeding, although differences between partners in cort10 remained greater in pairs with a corticosterone-treated female. Thus, we show that HPA axis response to stressors in adulthood can be modulated by reproductive partners and that similarity between partners is reduced when females are postnatally exposed to elevated glucocorticoids.

Annual cycles in daylength provide an initial predictive environmental cue that plants and animals use to time seasonal biology. Seasonal changes in photoperiodic information acts to entrain endogenous programs in physiology to optimize an animal’s fitness. Attempts to identify the neural and molecular substrates of photoperiodic time measurement in birds have, to date, focused on blunt changes in light exposure during a restricted period of photoinducibility. The objectives of these studies were first to characterize a molecular seasonal clock in Japanese quail and second, to identify the key transcripts involved in endogenously generated interval timing that underlies photosensitivity in birds. We hypothesized that the mediobasal hypothalamus (MBH) provides the neuroendocrine control of photoperiod-induced changes in reproductive physiology, and that the pars distalis of the pituitary gland contains an endogenous internal timer for the short photoperiod-dependent development of reproductive photosensitivity. Here, we report distinct seasonal waveforms of transcript expression in the MBH, and pituitary gland and discovered the patterns were not synchronized across tissues. Follicle-stimulating hormone-β (FSHβ) expression increased during the simulated spring equinox, prior to photoinduced increases in prolactin, thyrotropin-stimulating hormone-β, and testicular growth. Diurnal analyses of transcript expression showed sustained elevated levels of FSHβ under conditions of the spring equinox, compared to autumnal equinox, short (<12L) and long (>12L) photoperiods. FSHβ expression increased in quail held in non-stimulatory short photoperiod, indicative of the initiation of an endogenously programmed interval timer. These data identify that FSHβ establishes a state of photosensitivity for the external coincidence timing of seasonal physiology. The independent regulation of FSHβ expression provides an alternative pathway through which other supplementary environmental cues, such as temperature, can fine tune seasonal reproductive maturation and involution.

Most animals in the temperate zone exhibit robust seasonal rhythms in neuroendocrine, physiological and behavioral processes. The integration of predictive and supplementary environmental cues (e.g., nutrients) involves a series of discrete, and interconnected brain regions that span hypothalamic, thalamic, mesencephalic, and limbic regions. Species-specific adaptive changes in these neuroendocrine structures and cellular plasticity have likely evolved to support seasonal life-history transitions. Despite significant advances in our understanding of ecological responses to predictive and supplementary environmental cues, there remains a paucity of literature on how these diverse cues impact the underlying neural and cellular substrates. To date, most scientific approach has focused on neuroendocrine responses to annual changes in daylength, referred to as photoperiod, due to the robust physiological changes to light manipulations in laboratory settings. In this review, we highlight the relatively few animal models that have been effectively used to investigate how predictive day lengths, and supplementary cues are integrated across hypothalamic nuclei, and discuss key findings of how seasonal rhythms in physiology are governed by adaptive neuroendocrine changes. We discuss how specific brain regions integrate environmental cues to form a complex multiunit or 'modular' system that has evolved to optimize the timing of seasonal physiology. Overall, the review aims to highlight the existence of a modular network of neural regions that independently contribute to timing seasonal physiology. This paper proposes that a multi-modular neuroendocrine system has evolved in which independent neural 'units' operate to support species-specific seasonal rhythms.

Temperate zone animals exhibit seasonal variation in multiple endocrine systems. In most cases, peripheral organs display robust switches in tissue involution and recrudescence in mass. Our understanding of the molecular control of tissue-specific changes in seasonal function remains limited. Central to this problem is the lack of information on the nucleic acid structure, and distribution of transcripts across tissues in seasonal model organisms. Here we report the transcriptome profile of nine endocrine tissues from Siberian hamsters. Luteinizing hormone receptor expression was localized to gonadal tissues and confirmed previous distribution analyses. Assessment of the prolactin receptor reveal relatively high abundance across tissues involved in reproduction, energy, and water homeostasis. Neither melatonin receptor-1a, nor -1b, were found to be expressed in most tissues. Instead, the closely related G-protein coupled receptor Gpr50 was widely expressed in peripheral tissues. Epigenetic enzymes such as DNA methyltransferase 3a, was widely expressed and the predominant DNA methylation enzyme. Quantitative PCR analyses revealed some sex- and tissue-specific differences for prolactin receptor and DNA methyltransferase 3a expression. These data provide significant information on the distribution of transcripts, relative expression levels and nucleic acid sequences that will facilitate molecular studies into the seasonal programs in mammalian physiology.

In most animals, annual rhythms in environmental cues and internal programs regulate seasonal physiology and behavior. Prolactin, an evolutionarily ancient hormone, serves as a molecular correlate of seasonal timing in most species. Prolactin is highly pleiotropic with a wide variety of well-documented physiological effects; in a seasonal context prolactin is known to regulate annual changes in pelage and molt. While short-term homeostatic variation of prolactin secretion is under the control of the hypothalamus, long-term seasonal rhythms of prolactin are programmed by endogenous timers that reside in the pituitary gland. The molecular basis of these rhythms is generally understood to be melatonin dependent in mammals. Prolactin rhythmicity persists for several years in many species, in the absence of hypothalamic signaling. Such evidence in mammals has supported the hypothesis that seasonal rhythms in prolactin derive from an endogenous timer within the pituitary gland that is entrained by external photoperiod. In this review, we describe the conserved nature of prolactin signaling in birds and mammals and highlight its role in regulating multiple diverse physiological systems. The review will cover the current understanding of the molecular control of prolactin seasonality and propose a mechanism by which long-term rhythms may be generated in amniotes.

Polycystic ovary syndrome (PCOS), the most common form of anovulatory infertility, is associated with altered signaling within the hormone-sensitive neuronal network that regulates gonadotropin-releasing hormone (GnRH) neurons, leading to a pathological increase in GnRH secretion. Circuit remodeling is evident between GABAergic neurons in the arcuate nucleus (ARN) and GnRH neurons in a murine model of PCOS. One-third of ARN GABA neurons co-express neuropeptide Y (NPY), which has a known yet complex role in regulating GnRH neurons and reproductive function. Here, we investigated whether the NPY-expressing subpopulation (NPYARN) of ARN GABA neurons (GABAARN) is also affected in prenatally androgenized (PNA) PCOS-like NPYARN reporter mice [Agouti-related protein (AgRP)-Cre;τGFP]. PCOS-like mice and controls were generated by exposure to di-hydrotestosterone or vehicle (VEH) in late gestation. τGFP-expressing NPYARN neuron fiber appositions with GnRH neurons and gonadal steroid hormone receptor expression in τGFP-expressing NPYARN neurons were assessed using confocal microscopy. Although GnRH neurons received abundant close contacts from τGFP-expressing NPYARN neuron fibers, the number and density of putative inputs was not affected by prenatal androgen excess. NPYARN neurons did not co-express progesterone receptor or estrogen receptor α in either PNA or VEH mice. However, the proportion of NPYARN neurons co-expressing the androgen receptor was significantly elevated in PNA mice. Therefore, NPYARN neurons are not remodeled by prenatal androgen excess like the wider GABAARN population, indicating GABA-to-GnRH neuron circuit remodeling occurs in a presently unidentified non-NPY/AgRP population of GABAARN neurons. NPYARN neurons do, however, show independent changes in the form of elevated androgen sensitivity.

INTRODUCTION:The central regulation of fertility is carefully coordinated with energy homeostasis, and infertility is frequently the outcome of energy imbalance. Neurons in the hypothalamus expressing neuropeptide Y and agouti-related peptide (NPY/AgRP neurons) are strongly implicated in linking metabolic cues with fertility regulation.OBJECTIVE:We aimed here to determine the impact of selectively activating NPY/AgRP neurons, critical regulators of metabolism, on the activity of luteinizing hormone (LH) pulse generation.METHODS:We employed a suite of in vivo optogenetic and chemogenetic approaches with serial measurements of LH to determine the impact of selectively activating NPY/AgRP neurons on dynamic LH secretion. In addition, electrophysiological studies in ex vivo brain slices were employed to ascertain the functional impact of activating NPY/AgRP neurons on gonadotropin-releasing hormone (GnRH) neurons.RESULTS:Selective activation of NPY/AgRP neurons significantly decreased post-castration LH secretion. This was observed in males and females, as well as in prenatally androgenized females that recapitulate the persistently elevated LH pulse frequency characteristic of polycystic ovary syndrome (PCOS). Reduced LH pulse frequency was also observed when optogenetic stimulation was restricted to NPY/AgRP fiber projections surrounding GnRH neuron cell bodies in the rostral preoptic area. However, electrophysiological studies in ex vivo brain slices indicated these effects were likely to be indirect.CONCLUSIONS:These data demonstrate the ability of NPY/AgRP neuronal signaling to modulate and, specifically, reduce GnRH/LH pulse generation. The findings suggest a mechanism by which increased activity of this hunger circuit, in response to negative energy balance, mediates impaired fertility in otherwise reproductively fit states, and highlight a potential mechanism to slow LH pulsatility in female infertility disorders, such as PCOS, that are associated with hyperactive LH secretion.

Homeostatic processes like metabolism and reproduction are tightly regulated by neural circuits, and any disruption of these neural circuits causes energy imbalance and infertility, respectively. Furthermore, in conditions of energy imbalance, such as anorexia or obesity, there is also reproductive dysfunction. This causal relationship between metabolism and reproduction, strongly suggests an interdependence of these neuronal networks. In order to better understand the mechanisms underlying infertility caused by metabolic imbalance, it is important to identify the specific circuitry governing the coordination of energy balance and reproductive function. Neuropeptide Y (NPY) / Agouti related peptide (AgRP) expressing neurons in the arcuate nucleus (ARN) are likely to play an important role as they are crucial in regulating food intake and also affect gonadotropin-releasing hormone (GnRH) neuronal activity and luteinizing hormone (LH) secretion. However, the precise impact of the ARN NPY/AgRP circuit on GnRH neuron activity and fertility remains unclear. To assess this, we used the stimulatory DREADD in AgRP-Cre mice to specifically activate ARN NPY/AgRP neurons and measured pulsatile LH secretion, as a readout of GnRH neuronal activity, in conscious male and female mice. We found that activation of NPY/AgRP neurons by injection of the DREADD ligand, clozapine-N-oxide (CNO), decreased post-castration LH pulse secretion in both sexes, suggesting a slowing of GnRH pulse generation. To map the specific circuit associated with slowing of LH secretion following ARN NPY/AgRP neuron activation, region specific activation of NPY/AgRP fibres was achieved with optogenetics. Selective optogenetic stimulation of NPY/AgRP terminals surrounding GnRH neuron cell bodies in the rostral preoptic area (rPOA) was capable of decreasing GnRH neuron activity in ex vivo slices and significantly decreased LH pulsatility in conscious animals, indicating a functional ARN NPY/AgRP to GnRH neuron circuit capable of slowing pulse generation. Chemogenetic and optogenetic activation of ARN NPY/AgRP neurons was also able to significantly reduce LH pulse frequency in a prenatally androgenised (PNA) model of polycystic ovary syndrome that exhibits high LH pulse frequency, thereby demonstrating therapeutic potential. However, chronic activation of ARN NPY/AgRP neurons with CNO delivery in drinking water over 2 weeks did not ameliorate the PCOS-like reproductive pathology in PNA mice. Together, these data show that activation of ARN NPY/AgRP neurons can slow GnRH/LH pulse generation, potentially through a direct circuit to GnRH neurons in the rPOA. These findings identify a specific mechanism by which ARN NPY/AgRP neurons mediate nutritional infertility and highlight the therapeutic potential of modulating ARN NPY/AgRP activity in infertile conditions associated with high GnRH/ LH pulsatility. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Polycystic ovary syndrome (PCOS), the most common form of anovulatory infertility, is associated with a breakdown in signaling within the hormone sensitive neural network that regulates gonadotropin-releasing hormone (GnRH) neurons, ultimately increasing GnRH neural output. Circuitry between GABAergic neurons in the arcuate nucleus (ARN) and GnRH neurons is remodeled in mice that recapitulate PCOS, implicating their role in this disorder1. One-third of ARN GABA neurons co-express neuropeptide Y (NPY)2, which itself has a known, yet complex role in regulating GnRH neurons and reproductive function. This project aimed to examine whether this subpopulation of NPY expressing ARN GABA neurons are also altered in a PCOS-like state. To determine whether innervation to GnRH neurons by ARN NPY neurons is altered, prenatally androgenized, PCOS-like mice were generated in mice expressing green fluorescent protein (GFP) in agouti-related peptide neurons (AgRP-Cre;τGFP mice) by administration of dihydrotestosterone (PNA) or a vehicle (VEH) control, on days 16-18 of gestataion3. Immunohistochemistry (IHC) against GnRH and GFP was carried out, and confocal microscopy was used to assess the density of contacts to GnRH neurons made by ARN NPY fibres. This revealed that innervation from ARN NPY neurons to GnRH neurons was not different between VEH (n=5) and PNA (n=8) mice. Sensitivity to steroid hormones was also assessed by IHC detection of progesterone receptor (PR), estrogen receptor alpha (ERα) and androgen receptor (AR) within GFP-expressing ARN NPY neurons. PR and ERα expression was almost completely absent from ARN NPY neurons in both VEH and PNA mice (n=5/group). However, the proportion of ARN NPY neurons expressing AR was significantly greater in PNA mice (33.2 ± 5.34%) compared with VEH controls (18.9 ± 1.83%, n=4/group, p<0.05), suggesting heightened androgen sensitivity in an already hyper-androgenic environment. In addition, gene transcription changes in the hypothalamus were investigated using a sensitive NanostringTM assay in tissue microdissected from the rostral preoptic area (rPOA), rostral periventricular area of the third ventricle (RP3V), and ARN in VEH and PNA animals (n=12/group). While transcription of Npy was unchanged in the ARN, Npy1r expression was significantly decreased in the RP3V (0.82 ± 0.07 expression of mRNA relative to VEH, p < 0.05). This may indicate reduced NPY signalling to kisspeptin neurons via the Y1 receptor in the RP3V, and therefore an indirect reduction in inhibitory NPYergic signalling to GnRH neurons in a PCOS-like state. 1Moore et al., PNAS, 2015; 112(2): 596-601. 2Marshall et al., Neuroendocrinology, 2017; 105(2): 157-169. 3Sullivan & Moenter, PNAS, 2004; 101: 7129-7134.

Background/Aims: Arcuate nucleus (ARN) γ-aminobutyric acid (GABA) neurons are implicated in many critical homeostatic mechanisms, from food intake to fertility. To determine the functional relevance of ARN GABA neurons, it is essential to define the neurotransmitters co-expressed with and potentially co-released from ARN GABA neurons. Methods: The present study investigated the expression of markers of specific signaling molecules by ARN GABA neurons in brain sections from male, female, and, in some cases, prenatally androgen-treated (PNA) female, vesicular GABA transporter (VGaT)-ires-Cre/tdTomato reporter mice. Immunofluorescence for kisspeptin, β-endorphin, neuropeptide Y (NPY), tyrosine hydroxylase (TH) and neuronal nitric oxide synthase (nNOS) was detected by confocal microscopy, and co-localization with tdTomato VGaT reporter expression throughout the ARN was quantified. Results: GABA neurons rarely co-localized with kisspeptin (<2%) or β-endorphin (<1%), and only a small proportion of kisspeptin (∼10%) or β-endorphin (∼3%) neurons co-localized with VGaT in male and female mice. In contrast, one-third of ARN GABA neurons co-localized with NPY, and nearly all NPY neurons (>95%) co-localized with VGaT across groups. Both TH and nNOS labeling was co-localized with ∼10% of ARN GABA neurons. The proportion of TH neurons co-localized with VGaT was significantly greater in males than either control or PNA females, and the proportion of nNOS neurons co-localizing VGaT was higher in control and PNA females compared with males. Conclusion: These data highlight NPY as a significant subpopulation of ARN GABA neurons, demonstrate no significant impact of PNA on signal co-expression, and, for the first time, show sexually dimorphic co-expression patterns of TH and nNOS with ARN GABA neurons.

Polycystic ovarian syndrome (PCOS), the leading cause of female infertility, is associated with an increase in luteinizing hormone (LH) pulse frequency, implicating abnormal steroid hormone feedback to gonadotropin-releasing hormone (GnRH) neurons. This study investigated whether modifications in the synaptically connected neuronal network of GnRH neurons could account for this pathology. The PCOS phenotype was induced in mice following prenatal androgen (PNA) exposure. Serial blood sampling confirmed that PNA elicits increased LH pulse frequency and impaired progesterone negative feedback in adult females, mimicking the neuroendocrine abnormalities of the clinical syndrome. Imaging of GnRH neurons revealed greater dendritic spine density that correlated with increased putative GABAergic but not glutamatergic inputs in PNA mice. Mapping of steroid hormone receptor expression revealed that PNA mice had 59% fewer progesterone receptor-expressing cells in the arcuate nucleus of the hypothalamus (ARN). To address whether increased GABA innervation to GnRH neurons originates in the ARN, a viral-mediated Cre-lox approach was taken to trace the projections of ARN GABA neurons in vivo. Remarkably, projections from ARN GABAergic neurons heavily contacted and even bundled with GnRH neuron dendrites, and the density of fibers apposing GnRH neurons was even greater in PNA mice (56%). Additionally, this ARN GABA population showed significantly less colocalization with progesterone receptor in PNA animals compared with controls. Together, these data describe a robust GABAergic circuit originating in the ARN that is enhanced in a model of PCOS and may underpin the neuroendocrine pathophysiology of the syndrome.