Microglia: sculptors of the polycystic ovary syndrome (PCOS)-like brain?

In brief: Neuroendocrine dysfunction and transgenerational susceptibility associated with polycystic ovary syndrome (PCOS) suggest that programmed changes within the brain contribute to adult development of the syndrome. This review discusses a potentially important role for microglia in mediating prenatal androgen-programmed changes in the female brain that contribute to PCOS-like features. Abstract: Several lines of evidence support a role for the brain in both the development and maintenance of polycystic ovary syndrome (PCOS), the most common cause of anovulatory infertility worldwide. Persistently elevated luteinizing hormone secretion and impaired gonadal steroid hormone feedback in PCOS patients suggest impairments within the neuronal networks that regulate the reproductive axis. Evidence from preclinical models has linked androgen excess during prenatal life with altered structure and function of the developing female brain that might underpin syndrome development in adulthood. Studies investigating the mechanisms by which excess androgens program changes in the female brain have highlighted an important role for microglia. This review discusses how these non-neuronal cells shape the developing female brain in response to excess androgens and focuses on how microglia may be involved in the development of the neuroendocrine dysfunctions associated with PCOS.

Maternal androgen excess significantly impairs sexual behavior in male and female mouse offspring: Perspective for a biological origin of sexual dysfunction in PCOS.

Introduction: Polycystic ovary syndrome (PCOS) is the most common infertility disorder worldwide, typically characterised by high circulating androgen levels, oligo- or anovulation, and polycystic ovarian morphology. Sexual dysfunction, including decreased sexual desire and increased sexual dissatisfaction, is also reported by women with PCOS. The origins of these sexual difficulties remain largely unidentified. To investigate potential biological origins of sexual dysfunction in PCOS patients, we asked whether the well-characterized, prenatally androgenized (PNA) mouse model of PCOS exhibits modified sex behaviours and whether central brain circuits associated with female sex behaviour are differentially regulated. As a male equivalent of PCOS is reported in the brothers of women with PCOS, we also investigated the impact of maternal androgen excess on the sex behaviour of male siblings. Methods: Adult male and female offspring of dams exposed to dihydrotestosterone (PNAM/PNAF) or an oil vehicle (VEH) from gestational days 16 to 18 were tested for a suite of sex-specific behaviours. Results: PNAM showed a reduction in their mounting capabilities, however, most of PNAM where able to reach ejaculation by the end of the test similar to the VEH control males. In contrast, PNAF exhibited a significant impairment in the female-typical sexual behaviour, lordosis. Interestingly, while neuronal activation was largely similar between PNAF and VEH females, impaired lordosis behaviour in PNAF was unexpectedly associated with decreased neuronal activation in the dorsomedial hypothalamic nucleus (DMH). Conclusion: Taken together, these data link prenatal androgen exposure that drives a PCOS-like phenotype with altered sexual behaviours in both sexes.

Enhanced pup retrieval behaviour in a mouse model of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common endocrinopathy to affect women of reproductive-age world-wide. Hyperandrogenism is both a hallmark feature of PCOS, and is hypothesised to be an underlying mechanism driving the development of the condition in utero. With circulating hormones known to profoundly influence maternal responses in females, we aimed to determine whether maternal behaviour is altered in a well-described prenatally androgenised (PNA) mouse model of PCOS. Mouse dams were administered with dihydrotestosterone or vehicle on days 16, 17 and 18 of pregnancy. Maternal responses were assessed in both the dihydrotestosterone-injected dams following parturition and in their adult female PNA offspring. Exposure of dams to excess androgens during late pregnancy had no detrimental effects on pregnancy outcomes, including gestation length, pup survival and gestational weight gain, or on subsequent maternal behaviour following parturition. By contrast, PNA virgin females, modelling PCOS, exhibited enhanced maternal behaviour when tested in an anxiogenic novel cage environment, with females rapidly retrieving pups and nesting with them. In comparison, most control virgin females failed to complete this retrieval task in the anxiogenic environment. Assessment of progesterone receptor and oestrogen receptor α immunoreactivity in the brains of virgin PNA and control females revealed increased numbers of oestrogen receptor α positive cells in the brains of PNA females in regions well known to be important for maternal behaviour. This suggests that increased oestrogenic signalling in the neural circuit that underlies maternal behaviour may be a possible mechanism by which maternal behaviour is enhanced in PNA female mice.

Unusual suspects: Glial cells in fertility regulation and their suspected role in polycystic ovary syndrome.

Gonadotropin-releasing-hormone (GnRH) neurons sitting within the hypothalamus control the production of gametes and sex steroids by the gonads, therefore ensuring survival of species. As orchestrators of reproductive function, GnRH neurons integrate information from external and internal cues. This occurs through an extensively studied neuronal network known as the "GnRH neuronal network." However, the brain is not simply composed of neurons. Evidence suggests a role for glial cells in controlling GnRH neuron activity, secretion and fertility outcomes, although numerous questions remain. Glial cells have historically been seen as support cells for neurons. This idea has been challenged by the discovery that some neurological diseases originate from glial dysfunction. The prevalence of infertility disorders is increasing worldwide, with one in four couples being affected; therefore, it remains essential to understand the mechanisms by which the brain controls fertility. The "GnRH glial network" could be a major player in infertility disorders and represent a potential therapeutic target. In polycystic ovary syndrome (PCOS), the most common infertility disorder of reproductive aged women worldwide, the brain is considered a prime suspect. Recent studies have demonstrated pathological neuronal wiring of the "GnRH neuronal network" in PCOS-like animal models. However, the role of the "GnRH glial network" remains to be elucidated. In this review, I aim to propose glial cells as unusual suspects in infertility disorders such as PCOS. In the first part, I state our current knowledge about the role of glia in the regulation of GnRH neurons and fertility. In the second part, based on our recent findings, I discuss how glial cells could be implicated in PCOS pathology.

Morphological evidence indicates a role for microglia in shaping the PCOS-like brain.

Although polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility worldwide, the aetiology of the disorder remains poorly defined. Animal-based evidence highlights the brain as a prime suspect in both the development and maintenance of PCOS. Prenatally androgenised (PNA) models of PCOS exhibit excessive GABAergic wiring associated with PCOS-like reproductive deficits in adulthood, with aberrant brain wiring detected as early as postnatal day (P) 25, prior to disease onset, in the PNA mouse. The mechanisms underlying this aberrant brain wiring remain unknown. Microglia, the immune cells of the brain, are regulators of neuronal wiring across development, mediating both the formation and removal of neuronal inputs. Here, we tested the hypothesis that microglia play a role in the excessive GABAergic wiring that leads to PCOS-like features in the PNA brain. Using specific immunolabelling, microglia number and morphology associated with activation states were analysed in PNA and vehicle-treated controls across developmental timepoints, including embryonic day 17.5, P0, P25 and P60 (n = 7-14 per group), and in two regions of the hypothalamus implicated in fertility regulation. At P0, fewer amoeboid microglia were observed in the rostral preoptic area (rPOA) of PNA mice. However, the greatest changes were observed at P25, with PNA mice exhibiting fewer total microglia, and specifically fewer "sculpting" microglia, in the rPOA. Based on these findings, we assessed microglia-mediated refinement of GABAergic synaptic terminals at two developmental stages of peak synaptic refinement: P7 and P15 (n = 7 per group). PNA mice showed a reduction in the uptake of GABAergic synaptic material at P15. These findings reveal time-specific changes in the microglia population and refinement of GABAergic inputs in a mouse model of PCOS driven by prenatal androgen excess and suggest a role for microglia in shaping the atypical brain wiring associated with the development of PCOS features.

Reproductive health research in Australia and New Zealand: highlights from the Annual Meeting of the Society for Reproductive Biology, 2019.

The 2019 meeting of the Society for Reproductive Biology (SRB) provided a platform for the dissemination of new knowledge and innovations to improve reproductive health in humans, enhance animal breeding efficiency and understand the effect of the environment on reproductive processes. The effects of environment and lifestyle on fertility and animal behaviour are emerging as the most important modern issues facing reproductive health. Here, we summarise key highlights from recent work on endocrine-disrupting chemicals and diet- and lifestyle-induced metabolic changes and how these factors affect reproduction. This is particularly important to discuss in the context of potential effects on the reproductive potential that may be imparted to future generations of humans and animals. In addition to key summaries of new work in the male and female reproductive tract and on the health of the placenta, for the first time the SRB meeting included a workshop on endometriosis. This was an important opportunity for researchers, healthcare professionals and patient advocates to unite and provide critical updates on efforts to reduce the effect of this chronic disease and to improve the welfare of the women it affects. These new findings and directions are captured in this review.

Activation of arcuate nucleus GABA neurons promotes luteinizing hormone secretion and reproductive dysfunction: Implications for polycystic ovary syndrome.

BACKGROUND: Enhanced GABA activity in the brain and a hyperactive hypothalamic-pituitary-gonadal axis are associated with polycystic ovary syndrome (PCOS), the most common form of anovulatory infertility. Women with PCOS exhibit elevated cerebrospinal fluid GABA levels and preclinical models of PCOS exhibit increased GABAergic input to GnRH neurons, the central regulators of reproduction. The arcuate nucleus (ARN) is postulated as the anatomical origin of elevated GABAergic innervation; however, the functional role of this circuit is undefined. METHODS: We employed a combination of targeted optogenetic and chemogenetic approaches to assess the impact of acute and chronic ARN GABA neuron activation. Selective acute activation of ARN GABA neurons and their fiber projections was coupled with serial blood sampling for luteinizing hormone secretion in anesthetized male, female and prenatally androgenised (PNA) mice modelling PCOS. In addition, GnRH neuron responses to ARN GABA fiber stimulation were recorded in ex vivo brain slices. Chronic activation of ARN GABA neurons in healthy female mice was coupled with reproductive phenotyping for PCOS-like features. FINDINGS: Acute stimulation of ARN GABA fibers adjacent to GnRH neurons resulted in a significant and long-lasting increase in LH secretion in male and female mice. The amplitude of this response was blunted in PNA mice, which also exhibited a blunted LH response to GnRH administration. Infrequent and variable GABAA-dependent changes in GnRH neuron firing were observed in brain slices. Chronic activation of ARN GABA neurons in healthy females impaired estrous cyclicity, decreased corpora lutea number and increased circulating testosterone levels. INTERPRETATION: ARN GABA neurons can stimulate the hypothalamic-pituitary axis and chronic activation of ARN GABA neurons can mimic the reproductive deficits of PCOS in healthy females. Unexpectedly blunted HPG axis responses in PNA mice may reflect a history of high frequency GnRH/LH secretion and reduced LH stores, but also raise questions about impaired function within the ARN GABA population and the involvement of other circuits.

Synaptic Innervation of the GnRH Neuron Distal Dendron in Female Mice.

GnRH neuron cell bodies are scattered throughout the basal forebrain but funnel their projections to the median eminence to release GnRH into the pituitary portal system to control fertility. Prior studies have shown that GnRH neurons located in the anterior hypothalamus send projections to the median eminence that have characteristics of both dendrites and axons. These unusual structures have been termed "dendrons." To address whether the dendron is unique to anterior hypothalamic GnRH neurons or is also a characteristic of more rostral GnRH neurons, we used viral vector‒mediated GnRH neuron‒specific tract-tracing coupled with CLARITY optical clearing. Individual rostral preoptic area GnRH neurons in female mice were identified to elaborate processes up to 4 mm in length that exhibited spines and projected all the way to the median eminence before branching into multiple short axons. The synaptic innervation patterns of distal GnRH neuron dendrons and their short axons in the vicinity of the median eminence were examined using electron microscopy. This revealed the presence of a high density of synaptic inputs to distal dendrons at the border of the median eminence. In contrast, no synapses were detected on any GnRH neuron axons. These studies demonstrate that GnRH neurons in the rostral preoptic area project dendrons to the edge of the median eminence, whereupon they branch into multiple short axons responsible for GnRH secretion. The dense synaptic innervation of these distal dendrons likely represents an efficient mechanism for controlling GnRH secretion required for fertility.

Female sexual behavior in mice is controlled by kisspeptin neurons.

Sexual behavior is essential for the survival of many species. In female rodents, mate preference and copulatory behavior depend on pheromones and are synchronized with ovulation to ensure reproductive success. The neural circuits driving this orchestration in the brain have, however, remained elusive. Here, we demonstrate that neurons controlling ovulation in the mammalian brain are at the core of a branching neural circuit governing both mate preference and copulatory behavior. We show that male odors detected in the vomeronasal organ activate kisspeptin neurons in female mice. Classical kisspeptin/Kiss1R signaling subsequently triggers olfactory-driven mate preference. In contrast, copulatory behavior is elicited by kisspeptin neurons in a parallel circuit independent of Kiss1R involving nitric oxide signaling. Consistent with this, we find that kisspeptin neurons impinge onto nitric oxide-synthesizing neurons in the ventromedial hypothalamus. Our data establish kisspeptin neurons as a central regulatory hub orchestrating sexual behavior in the female mouse brain.

Defining Subpopulations of Arcuate Nucleus GABA Neurons in Male, Female, and Prenatally Androgenized Female Mice.

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, ß-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 ß-endorphin (<1%), and only a small proportion of kisspeptin (∼10%) or ß-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.

Potential contribution of progesterone receptors to the development of sexual behavior in male and female mice.

We previously showed that estradiol can have both defeminizing and feminizing effects on the developing mouse brain. Pre- and early postnatal estradiol defeminized the ability to show lordosis in adulthood, whereas prepubertal estradiol feminized this ability. Furthermore, we found that estradiol upregulates progesterone receptors (PR) during development, inducing both a male-and female-typical pattern of PR expression in the mouse hypothalamus. In the present study, we took advantage of a newly developed PR antagonist (ZK 137316) to determine whether PR contributes to either male- or female-typical sexual differentiation. Thus groups of male and female C57Bl/6j mice were treated with ZK 137316 or OIL as control: males were treated neonatally (P0-P10), during the critical period for male sexual differentiation, and females were treated prepubertally (P15-P25), during the critical period for female sexual differentiation. In adulthood, mice were tested for sexual behavior. In males, some minor effects of neonatal ZK treatment on sexual behavior were observed: latencies to the first mount, intromission and ejaculation were decreased in neonatally ZK treated males; however, this effect disappeared by the second mating test. By contrast, female mice treated with ZK during the prepubertal period showed significantly less lordosis than OIL-treated females. Mate preferences were not affected in either males or females treated with ZK during development. Taken together, these results suggest a role for PR and thus perhaps progesterone in the development of lordosis behavior in female mice. By contrast, no obvious role for PR can be discerned in the development of male sexual behavior.

GnRH Episodic Secretion Is Altered by Pharmacological Blockade of Gap Junctions: Possible Involvement of Glial Cells.

Episodic release of GnRH is essential for reproductive function. In vitro studies have established that this episodic release is an endogenous property of GnRH neurons and that GnRH secretory pulses are associated with synchronization of GnRH neuron activity. The cellular mechanisms by which GnRH neurons synchronize remain largely unknown. There is no clear evidence of physical coupling of GnRH neurons through gap junctions to explain episodic synchronization. However, coupling of glial cells through gap junctions has been shown to regulate neuron activity in their microenvironment. The present study investigated whether glial cell communication through gap junctions plays a role in GnRH neuron activity and secretion in the mouse. Our findings show that Glial Fibrillary Acidic Protein-expressing glial cells located in the median eminence in close vicinity to GnRH fibers expressed Gja1 encoding connexin-43. To study the impact of glial-gap junction coupling on GnRH neuron activity, an in vitro model of primary cultures from mouse embryo nasal placodes was used. In this model, GnRH neurons possess a glial microenvironment and were able to release GnRH in an episodic manner. Our findings show that in vitro glial cells forming the microenvironment of GnRH neurons expressed connexin-43 and displayed functional gap junctions. Pharmacological blockade of the gap junctions with 50 µM 18-α-glycyrrhetinic acid decreased GnRH secretion by reducing pulse frequency and amplitude, suppressed neuronal synchronization and drastically reduced spontaneous electrical activity, all these effects were reversed upon 18-α-glycyrrhetinic acid washout.

Development and Aging of the Kisspeptin-GPR54 System in the Mammalian Brain: What are the Impacts on Female Reproductive Function?

The prominent role of the G protein coupled receptor GPR54 and its peptide ligand kisspeptin in the progression of puberty has been extensively documented in many mammalian species including humans. Kisspeptins are very potent gonadotropin-releasing hormone secretagogues produced by two main populations of neurons located in two ventral forebrain regions, the preoptic area and the arcuate nucleus. Within the last 2 years a substantial amount of data has accumulated concerning the development of these neuronal populations and their timely regulation by central and peripheral factors during fetal, neonatal, and peripubertal stages of development. This review focuses on the development of the kisspeptin-GPR54 system in the brain of female mice, rats, sheep, monkeys, and humans. We will also discuss the notion that this system represents a major target through which signals from the environment early in life can reprogram reproductive function.

The intimate relationship of gonadotropin-releasing hormone neurons with the polysialylated neural cell adhesion molecule revisited across development and adult plasticity.

The neurohormone gonadotropin-releasing hormone (GnRH) is critical for all the aspects of reproductive life in vertebrates. GnRH is secreted by a small number of neurons dispersed within the preoptic-hypothalamic region. These neurons are derived from the embryonic olfactory pit. They then migrate along olfactory, vomeronasal and terminal nerves to their final destination. Classical approaches to study the regulation of GnRH secretion during the reproductive cycle have focused on the various neuronal inputs on GnRH neurons and their regulation by ovarian steroids. However, it is well known that steroids will change the microenvironment of neuronal networks and can induce plasticity and functional changes. In this review, we will focus on the intimate relationship of developing and adult GnRH neurons with the polysialylated form of neural cell adhesion molecule (PSA-NCAM), a major molecular actor in the morphogenesis and adult plasticity of the nervous system. We will first recapitulate the spatiotemporal relationship between PSA-NCAM and migrating GnRH neurons during embryogenesis of various vertebrate species and discuss its importance for GnRH neuron development as shown by various loss of function studies. In the adult, we will review the relationships between PSA-NCAM and GnRH neurons across various physiological states, and open the discussion to the use of new model systems that can help to unravel the function and mechanism of action of PSA-NCAM on GnRH neuronal network activity and GnRH release.