Accepted Manuscript Title: The neuroendocrine genesis of polycystic ovary syndrome: A role for arcuate nucleus GABA neurons Author: Aleisha M. Moore Rebecca E. Campbell PII: DOI: Reference:

S0960-0760(15)30104-7 http://dx.doi.org/doi:10.1016/j.jsbmb.2015.10.002 SBMB 4537

To appear in:

Journal of Steroid Biochemistry & Molecular Biology

Received date: Revised date: Accepted date:

16-6-2015 25-8-2015 2-10-2015

Please cite this article as: Aleisha M.Moore, Rebecca E.Campbell, The neuroendocrine genesis of polycystic ovary syndrome: A role for arcuate nucleus GABA neurons, Journal of Steroid Biochemistry and Molecular Biology http://dx.doi.org/10.1016/j.jsbmb.2015.10.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The neuroendocrine genesis of polycystic ovary syndrome: A role for arcuate nucleus GABA neurons Aleisha M. Moore & Rebecca E. Campbell Centre for Neuroendocrinology and Department of Physiology, School of Medical Sciences, University of Otago, Dunedin, New Zealand 9054

Corresponding author: Dr Rebecca Campbell Centre for Neuroendocrinology Department of Physiology University of Otago School of Medical Sciences PO Box 913 Dunedin, New Zealand 9054 1

Telephone: +64-3-479-7343 Fax: +64-3-479-7323 E-mail: [email protected] Highlights    

The common endocrine disorder polycystic ovary syndrome (PCOS) may be, in part, a disease of the brain Androgen actions leading to disrupted progesterone signalling is implicated in impaired negative feedback in PCOS Prenatal androgen exposure recapitulates the neuroendocrine features of PCOS in animal models Arcuate nucleus GABA neurons are implicated in mediating the neuroendocrine impairments of PCOS

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Abstract Polycystic ovary syndrome (PCOS) is a prevalent and distressing endocrine disorder lacking a clearly identified aetiology. Despite its name, PCOS may result from impaired neuronal circuits in the brain that regulate steroid hormone feedback to the hypothalamo-pituitary-gonadal axis. Ovarian function in all mammals is controlled by the gonadotropin-releasing hormone (GnRH) neurons, a small group of neurons that reside in the pre-optic area of the hypothalamus. GnRH neurons drive the secretion of the gonadotropins from the pituitary gland that subsequently control ovarian function, including the production of gonadal steroid hormones. These hormones, in turn, provide important feedback signals to GnRH neurons via a hormone sensitive neuronal network in the brain. In many women with PCOS this feedback pathway is impaired, resulting in the downstream consequences of the syndrome. This review will explore what is currently known from clinical and animal studies about the identity, relative contribution and significance of the individual neuronal components within the GnRH neuronal network that contribute to the pathophysiology of PCOS. We review evidence for the specific neuronal pathways hypothesised to mediate progesterone negative feedback to GnRH neurons, and discuss the potential mechanisms by which androgens may evoke disruptions in these circuits at different developmental time points. Finally, this review discusses data providing compelling support for disordered progesterone-sensitive GABAergic input to GnRH neurons, originating specifically within the arcuate nucleus in prenatal androgen induced forms of PCOS.

Keywords gonadotropin-releasing hormone; fertility; tract-tracing; progesterone receptor; negative feedback; mouse; prenatal androgen model

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1. Introduction Polycystic ovary syndrome (PCOS), also known as polycystic ovarian syndrome, is the most common form of anovulatory infertility (2) and estimated to affect more than 100 million women worldwide (3). The cardinal features of PCOS, first described by Stein and Leventhal (4), include menstrual dysfunction, androgenic features and polycystic ovaries. Currently, the diagnostic criteria of PCOS are a matter of some debate due to the heterogeneous presentation of the syndrome, with the most commonly used criteria stemming from the National Institutes of Health (NIH) conference and the Rotterdam workshop (5). According to the NIH consensus, PCOS

diagnosis

requires

the

presence

of

oligo-

or

amenorrhea

and

hyperandrogenaemia (HA) in the absence of other explanatory disorders that cause HA (6). Under these criteria, 6-10% of women of reproductive age are diagnosed with PCOS (7,8). The broader Rotterdam criteria define PCOS by the presence of two of the three following features: oligo-or amenorrhea, HA and polycystic ovaries by ultrasound imaging (9). Under these criteria, up to an estimated 19% of women of reproductive age are diagnosed with PCOS (10,11). Patients with PCOS often present with reproductive dysfunction accompanied by other distressing overt symptoms including excess body hair, acne, male-pattern baldness and obesity (12). Although not required for diagnosis, PCOS is strongly associated

with

a

metabolic

syndrome,

including

insulin

resistance

and

hyperinsulinemia (13-15). The presence of metabolic symptoms increase risk factors for developing cardiovascular disease and PCOS patients are seven times more likely to develop myocardial infarction than age-matched referents (16). Additionally, women with PCOS have an increased occurrence of ovarian, endometrial and breast cancers (17,18), and frequently suffer from depression and anxiety (19). Despite the prevalence and broad impact that PCOS has on female health, the genesis of the syndrome remains largely unknown. There are a number of competing hypotheses concerning the origin of the syndrome, including heritable genetic polymorphisms (20-27), hyperinsulinemia and obesity (13,28-32), and elevated androgen exposure during critical developmental windows (14,33-37). It also remains unclear whether PCOS is caused by a single factor, or a mixture of influences that contribute to the equally diverse spectrum of disease expression (38). While much remains unknown about the aetiology and manifestation of the syndrome, a clear shift from a focus on peripheral organs to the brain is becoming apparent in recent 4

literature. Classically, PCOS has been characterised as an ovarian disease, however, since the elegant review of Marshall and Eagleson in 1999 emphasizing the neuroendocrine defects in PCOS (39), a growing body of clinical and basic scientific research has supported the idea that the problem may lie upstream in the central nervous system regulation of gonadal function. There are many, excellent reviews on the The following review will highlight research elucidating the role that central brain circuits play in the development of PCOS and the specific role of GABA neurons within the arcuate nucleus of the hypothalamus. 2. PCOS: a neuroendocrine disorder 2.1 The healthy hypothalamic-pituitary gonadal axis Steroidogenesis and folliculogenesis, the primary events within the ovary, are entirely dependent upon the hypothalamic-pituitary-gonadal (HPG) axis; a neuroendocrine axis driven by the gonadotropin-releasing hormone (GnRH) neuronal network. This network is comprised of the GnRH neurons, a small, scattered population of neurons in the rostral forebrain, and the large neuronal network that communicates essential hormonal and neurochemical information from multiple areas of the brain (40). The GnRH peptide is released in a pulsatile manner from GnRH nerve terminals into the median eminence and hypophyseal portal system to drive the secretion of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), from the pituitary gland (41-43). Changes in the pulse frequency and amplitude of GnRH secretion drive the specific amplitude and frequency of LH and FSH release (44), which subsequently provides specific trophic signals to mediate follicular development and steroid hormone synthesis in the ovary over the ovarian cycle (45-47). Gonadal steroid hormones in turn provide classic homeostatic feedback signals from the gonads to the GnRH neurons (48). For the majority of the female ovarian cycle, the gonadal steroid hormones estrogen and progesterone suppress GnRH neuron activity through negative feedback (49). During the mid-follicular phase in females, rising levels of estradiol drive a switch from negative to positive central feedback (50). This leads to a massive and continuous release of GnRH (5153) to drive the preovulatory LH surge necessary for ovulation to occur (reviewed in 54,55). Although estradiol is sufficient to drive the GnRH/LH surge (56), progesterone is essential to achieve the full magnitude of the GnRH/LH surge (57,58). Following ovulation, the ovulated follicle is remodelled into a corpus luteum that 5

secretes large quantities of progesterone and estrogen. These gonadal hormones once again provide negative feedback control to slow GnRH/LH pulse frequency during the luteal phase (59,60).

2.2 Increased activity of the GnRH/LH pulse generator in PCOS Elevated LH levels or an elevated LH to FSH ratio is a common clinical feature in PCOS (5,61,62), and a key indicator of neuroendocrine dysfunction. Serial blood sampling in women with PCOS indicates that elevated LH levels result from an increase in LH pulse frequency (63), which is most likely to reflect an increase in GnRH pulse frequency (1,64) (Figure 1). An elevated LH to FSH ratio disrupts normal follicular development and favours androgen synthesis from ovarian follicles (reviewed by 65,66). In female monkeys, excess androgens increase the number of immature ovarian follicles and stimulate proliferation of the thecal cell layer (67), traits which are emulated in the ovaries of PCOS women. The high number of anovulatory follicles additionally become fluid filled to produce the characteristic ‘cyst-like’ follicles from which the syndrome is named (reviewed by 65,66). Androgens also act peripherally to induce the visible symptoms of PCOS, including hirsutism and acne. The mechanisms underlying the persistent increase in LH pulse frequency and amplitude in PCOS are not well understood. There are, however, three current hypotheses implicating the actions of peripheral hormones in the brain. One hypothesis is that hyperinsulinemia enhances GnRH neuron activity or pituitary responsiveness to GnRH. This is supported by work demonstrating that the administration of Metformin, an anti-diabetic drug that suppresses glucose production is able to reduce plasma LH in both lean and obese women with PCOS (68-71). Interestingly, animal studies indicate that metformin’s ability to reduce GnRH neuron activity may be due to direct central actions via activation of the AMP-activated protein kinase second messenger pathway in GnRH neurons (72). A second hypothesis is that low levels of progesterone in the absence of ovulation and follicular remodelling result in the loss of progesterone negative feedback to GnRH secretion that would otherwise occur normally. Progesterone is considered a critical regulator of GnRH pulse frequency. LH pulse frequency inversely correlates with progesterone levels during the menstrual cycle, and the administration of progesterone during the 6

follicular phase slows LH pulse frequency (73). However, LH hypersecretion has been documented in pre-pubertal girls with hyperandrogenism (74). At this stage of maturation, cyclic ovarian function has not been established and therefore elevated LH cannot simply be a direct result of the absence of progesterone signalling. This idea is further discounted by a growing body of evidence gathered from human and animal studies that substantiates a third hypothesis suggesting that hyperandrogenism alters neuronal circuitry critical for relaying steroid hormone negative feedback to GnRH neurons. The specific role of androgens in mediating the impaired steroid hormone feedback of PCOS is discussed in detail below. 2.3 Impaired steroid hormone negative feedback in PCOS Compared to control women, PCOS patients require higher concentrations of estradiol and progesterone to lower pulsatile LH release (75,76), demonstrating an impairment in the ability of steroid hormones to suppress the activity of the GnRH/LH pulse generator. Data from PCOS patients suggests that inappropriately high levels of androgens indirectly establish this impaired negative feedback. In healthy controls, testosterone administration does not increase pulsatile LH levels (77), and in women with PCOS the administration of flutamide, an androgen receptor antagonist, does not restore normal LH secretion (78). However, long-term flutamide treatment is able to re-establish the ability of estradiol and progesterone to suppress LH pulse frequency (78). This suggests that high levels of androgens in adulthood do not directly drive LH hypersecretion, but instead interfere with the ability of estradiol and progesterone to regulate LH release. Direct cellular evidence for this comes from studies in the adult mouse in which testosterone treatment interferes with the ability of progesterone to lower GnRH neuron activity (79). It is hypothesised that HA is established by early pubertal development and precedes the development of the PCOS phenotype that becomes apparent shortly after puberty (80,81). In adolescent girls, elevated testosterone correlates with increased LH pulse frequency compared to developmentally matched subjects with normal testosterone levels (82-85), even before menarche and the establishment of ovulatory cycles (74). The delivery of exogenous progesterone is unable to lower the elevated LH pulse frequency in approximately half of the adolescent patients that present with HA (76,86), and this mirrors a similar proportion of PCOS patients that display impaired progesterone negative feedback in adulthood (75). These findings suggest

7

that the augmented rise in androgens during pubertal development leads to a progressive decline in hypothalamic sensitivity to progesterone negative feedback which in turn perpetuates the downstream consequences of the PCOS phenotype (87). In contrast, HA girls that maintain progesterone sensitivity despite similar degrees of HA may not go on to develop the full PCOS phenotype. A clearer understanding of the definitive pathways through which progesterone alters GnRH neuron activity, and the specific elements that are affected by HA is critical for progressing our understanding of the development and treatment of PCOS.

3. Pathways mediating progesterone negative feedback 3.1 Rapid versus genomic pathways Progesterone can rapidly suppress LH secretion in humans (59) and GnRH/LH pulse frequency in the non-human primate (88). These rapid actions of progesterone on LH suppression are maintained in ovariectomised nuclear progesterone receptor (PR) knockout mice (89), suggesting that the acute effects of progesterone occur via non-nuclear transmembrane receptors that do not require time-consuming gene transcription before a physiological outcome (89-92). Several transmembrane progesterone receptors that rapidly activate signalling cascades are present in the hypothalamus (93). Perhaps best described is the progesterone receptor membrane component 1 (PgRMC1). In a mouse brain slice preparation progesterone has been found to rapidly reduce the activity of GnRH neurons through binding to PgRMC1 (92). In addition, work in immortalised cell lines that express GnRH revealed the inhibition of GnRH release by progesterone may be partly mediated by an inhibitory membrane-bound G-protein-coupled receptor (mPR) (89). Rapid actions of progesterone also occur downstream from the metabolism of progesterone by glial cells into neurosteroids such as 3α-hydroxy-5α-pregnan-20-one (allopregnanolone), a potent modulator of the GABAA receptor (94,95) (Figure 2A). Although allopregnanolone signalling through GABAA has no noted effect on LH release in the ewe (96,97), the reported effects of allopregnanolone on GnRH and LH release in rodents is inconsistent. Both an increase and reduction in GnRH/LH release has been reported following allopregnanolone treatment in the rat (98-100), and similar allopregnanolone treatment in female mice does not alter LH release (89). However, allopregnanolone increases the response of all GnRH neurons to GABA via direct allosteric modulation of the GABAA receptor in the mouse brain slice 8

(101,102). Overall, although progesterone has a clear role in inhibiting GnRH neuron activity, further work is required to understand the rapid modulation of GnRH neuron activity by transmembrane progestin receptors and neurosteroids. In contrast, there is strong support that the ovarian regulation of GnRH release is dependent upon signalling through the classical PR. The classical actions of progesterone occur when progesterone binds to the nuclear PR to form a complex that translocates to the nucleus and regulates the transcription of target genes. The physiological consequences of PR activation usually occur over the time course of several hours following the translation of proteins. Evidence from animal studies suggests that classical PR signalling is required for progesterone negative feedback effects on GnRH/LH activity. The PR antagonist RU486 blocks the ability of progesterone to suppress LH release in ovariectomised ewes (96). PR knockout in mice disrupts normal cycling and fertility, and leads to a significant increase in circulating LH (103,104). Although the negative feedback actions of exogenous progesterone in PR-knockout animals have not been assessed, these studies suggest that PR is an important contributor to progesterone negative feedback. As GnRH neurons do not express PR, progesterone modulation of GnRH neuron activity by classical PR must be via trans-synaptic input (96,105) (Figure 2A).

3.2 Locating the brain circuits mediating progesterone negative feedback Viral tract tracing studies in the mouse have defined areas in the brain that contain hormone-sensitive neurons in synaptic contact with GnRH neurons (106). Several of the identified areas also express PR, such as the rostral periventricular nucleus of the third ventricle, the medial preoptic area and the arcuate nucleus (ARN) (107). The medial basal hypothalamus (MBH), and more specifically the ARN, has generated the best evidence as important regions for progesterone control of GnRH neuron pulse frequency. In the rat, deafferentation of the MBH with the rostral hypothalamus has been used to demonstrate that GnRH pulsatile secretion is maintained, suggesting that synaptic contact to the distal regions of GnRH neurons is maintained within the MBH (108,109). Furthermore, deafferentation combined with the ablation of the ARN completely eliminates pulsatile LH (110). In the ewe, the exogenous administration of progesterone reduces GnRH/LH pulse frequency following deafferentation (111). This indicates that the ARN contains cells that are

9

critical for progesterone regulation of GnRH/LH pulse frequency. It is likely this occurs through PR, as implantation of the PR antagonist RU486 into the ARN of the OVX ewe disrupts progesterone inhibition of GnRH/LH release (112). Supporting that the control of progesterone negative feedback by the ARN cells is unique, the implantation of RU486 into the rostral preoptic area (POA) of the OVX ewe does not disrupt GnRH/LH pulse frequency. Interestingly, microimplants of progesterone alone into the POA or ARN were unable to inhibit LH release in the same study, suggesting that progesterone actions at more than one site within the ARN or steroid actions elsewhere within the brain may be required to elicit full negative feedback effects. There are several neuropeptides within the medial basal hypothalamus that may relay progesterone negative feedback. The neuropeptide kisspeptin is colocalised with PR (113) and is a potent activator of GnRH neurons (114). Direct kisspeptin signalling to GnRH neurons through the G-protein coupled receptor 54 is essential for normal puberty onset, fecundity and estrous cyclicity (115). Kisspeptin neurons specifically located within the ARN, termed KNDy neurons due to their colocalisation with the neuropeptides neurokinin B and dynorphin (116,117), exhibit a high degree of co-expression with PR (118). The majority of studies supporting progesterone negative feedback via KNDy neurons have been performed in the ewe, where the inhibitory neuropeptide dynorphin is implicated to play a major role in mediating this feedback (97). Exogenous progesterone administration increases dynorphin concentrations in cerebrospinal fluid and hypothalamic mRNA (119), and blockade of dynorphin-kappa receptor signalling in the ARN increases LH pulse frequency (120). Information regarding the neuropeptides involved in progesterone negative feedback in other species remains less well understood. In addition to the neuropeptides, classical neurotransmitters are postulated to play a key role in progesterone negative feedback. PR expression has been identified

in

hypothalamic

glutamatergic

(121)

and

GABAergic

neurons

(105,122,123). Electrophysiological studies in the mouse have demonstrated that progesterone treatment of OVX mice reduces the GABA mediated postsynaptic current frequency recorded in GnRH neurons, supporting that progesterone negative feedback is mediated via GABA (124). Although GABA typically acts as an inhibitory neurotransmitter in the adult brain, studies in the mouse have identified that activation of the GABAA receptor has a predominantly excitatory effect on GnRH neuron activity due to elevated cellular chloride levels (125). Therefore, a reduction in 10

GABA-mediated postsynaptic currents would remove excitation of GnRH neuron activity, consistent with a negative feedback effect. Although studies aiming to identify the location of GABAergic afferents to GnRH neurons have implicated the POA (126-130), the anteroventral periventricular nucleus (AVPV) (131,132) and the suprachiasmatic nucleus (SCN) (133), there has been no clear identification of the GABA neurons involved in progesterone negative feedback until recently. Using a viral-mediated tract tracing approach, PR-positive GABA neurons originating specifically within the ARN have been shown to provide robust innervation of the GnRH cell body and proximal dendrite (123), as well as to the distal dendrite and dendron (134) near the median eminence (Moore et al., unpublished data). The discovery that this specific pathway is modified in a PCOSlike animal model (123) lends additional weight to the importance of this ARN GABA pathway in mediating appropriate progesterone mediated steroid hormone feedback as discussed below. 4. Modelling PCOS with prenatal androgen treatment In order to investigate specific changes in the neuronal circuits that regulate negative feedback, numerous animal models of PCOS have been developed through fetal androgen excess (reviewed in 35). Prenatal testosterone or dihydrotestosterone exposure in the female primate (135-137), sheep (138-141) and rat (142-144) leads to many of the reproductive and metabolic symptoms that are present in women with PCOS. The prenatal androgen-treated mouse model displays reproductive abnormalities that closely mimic the syndrome but only minor metabolic disturbances (145-148). This provides a useful model of the “lean PCOS phenotype” observed in ~50% of all PCOS cases in the clinic (149) and conveniently avoids any confounding metabolic disturbances that may impact on reproductive function. PNA-treated animals also develop the key neuroendocrine abnormalities associated with PCOS (reviewed in 150,151). Elevated LH pulse frequency has been measured in adult PNA-treated primates (152). Elevated LH levels are also seen in foetal plasma on PNA-treated primates (135), suggesting that it may be the earliest indicator of the developing PCOS phenotype. An increase in LH pulse frequency has also been detected in PNA-treated sheep (153), rats (142) and mice (123,145). As LH release mirrors that of GnRH, elevated LH pulse frequency in these models is most likely reflective of an increase in GnRH neuron activity in the hypothalamus. This is

11

supported by electrophysiological recordings of GnRH neurons in PNA-treated mice that show an increased GnRH neuron firing rate (72) (Figure 3). As in women with PCOS, the increase in GnRH/LH pulse frequency identified in prenatally androgen-treated animal models most likely occurs due to impaired progesterone negative feedback. Progesterone negative feedback impairment is apparent in the non-human primate model of PCOS (154) and LH pulse frequency is higher than controls during the progesterone dominated luteal phase in T-treated ewes (155). The implantation of a progesterone-releasing device in ovariectomised PNA ewes and mice is also unable to inhibit LH pulse frequency to the extent of control females (123,153). Therefore, PNA-treated animal models provide a useful tool for studying changes in neuroendocrine circuits that relay progesterone feedback information and for determining the specific role that early androgen exposure plays in the genesis of the syndrome. 5. Mechanism of androgen interference with progesterone negative feedback Androgens have well known organisational effects in the brain during critical periods of prenatal development (156,157). Exposure to androgens early in development may result in modified hypothalamic circuitry that is permanently reprogrammed during early development. Alternatively, PNA treatment may programme changes within peripheral organs, such as the ovary, to induce a state of hyperandrogenism that manifests from puberty onset (158). The resulting high levels of androgens may then act in the brain to drive plastic changes within the GnRH neuronal network that, importantly, may be reversible by the blockade of androgen signalling. Potential mechanisms of androgen action in disrupting progesterone negative feedback include both androgen receptor dependent and independent mechanisms (Figure 2).

5.1 Androgen receptor-independent mechanisms High circulating levels of androgens in PCOS may impair progesterone negative feedback indirectly by the metabolism of androgens to neurosteroids that directly alter GnRH neuron activity. Dihydrotestosterone (DHT), a non-aromatisable androgen, and its metabolite 5α-androstan-3α-17β diol (3α-diol) can allosterically modulate GABAA receptors to promote the opening frequency of the channel and increase chloride flux (159-162) (Figure 2). As GABAA receptor activation can excite

12

GnRH neurons and potentially blunt their responsiveness to indirect progesterone negative feedback actions, this may ultimately lead to increased GnRH/LH release in the PCOS state. Alternatively, DHT can be metabolised to 5α-androstan-3β 17β-diol (3β-diol). Although this metabolite cannot allosterically modulate the GABAA receptor (163), it is able to preferentially bind to and activate transcription through ERβ, the only isoform of ER expressed by GnRH neurons (162) (Figure 2). This pathway has a noted role in the regulation of the hypothalamic-pituitary-adrenal axis by reducing stress-induced secretion of corticosterone and adrenocorticotrophic hormone from the anterior pituitary gland (164). ERβ is suggested to play a small role in the rapid negative feedback actions of estradiol (165), however, the downstream consequences of 5α-androstan-3β 17β-diol interaction with ERβ in GnRH neurons remains unknown. In addition to DHT, the progesterone metabolite allopregnanolone may play a role in directly increasing GnRH neuron activity. Cohorts of amenorrheic (166) and both normal and obese PCOS patients possess elevated serum allopregnanolone levels (167,168). Recently, it was shown that the GABAA receptor has a heightened sensitivity to allopregnanolone in PCOS patients, as demonstrated by a saccadic eye velocity test following allopregnanolone administration (167). Although the effect of allopregnanolone on GnRH neuron activity is currently unclear, it is possible that activation of the GABAA receptor by allopregnanolone is also increased to drive GnRH neuron activity in PCOS.

5.2 Androgen receptor-dependent mechanisms Testosterone and DHT can bind to the androgen receptor (AR) in the brain or be aromatised locally to estradiol and bind to receptors for estrogen, whereas the nonaromatizable DHT exclusively binds to AR (169). The AR then binds to androgenresponse elements in the promoter region of target genes to stimulate or inhibit gene transcription (170). There is strong evidence to suggest that elevated androgen levels in PCOS drive impaired progesterone negative feedback through activation of the AR. AR antagonists such as spironolactone and flutamide are routinely prescribed to treat the peripheral symptoms of hyperandrogenaemia (171). In support of a role for AR mediating the brain mechanisms involved in PCOS, flutamide treatment for 5 weeks has been found to restore progesterone negative feedback in women with PCOS and 13

normalise cyclicity and GABA signalling to GnRH neurons in PNA mice (78,145). Although the mechanisms through which the activation of AR impairs progesterone negative feedback is unclear in PCOS patients, excellent progress has been made in animals exposed to androgens and in models of the syndrome. Direct androgen action through AR can interfere with the transcription of PR (172,173) and this could result in the reduced progesterone sensitivity of the hyperandrogenic state. In the rat, prenatal treatment with either testosterone or DHT attenuates the ability of estrogen to induce PR messenger RNA (mRNA) expression in the hypothalamus (142,174,175). In the ewe, prenatal treatment with testosterone masculinises PR expression within the ARN, leading to a reduced number of PRpositive cells (175). The PNA-treated mouse model of PCOS has a dramatic reduction in hypothalamic PR within the ARN (123). These data again highlight the ARN as region where androgen actions on PR expression might lead to impairments in specific neurons mediating negative feedback actions.

6. Arcuate GABA neurons mediate impaired progesterone negative feedback in PCOS Coming full circle, there is growing evidence that GABA neurons, specifically located within the arcuate nucleus, are involved in impaired progesterone negative feedback due to hyperandrogenism typical of the PCOS phenotype. GABAergic drive to GnRH neurons in the PNA-treated mouse is elevated, as shown by an increase in postsynaptic current frequency and amplitude from recordings in the mouse brain slice (145). These functional data correspond with anatomical evidence for increased vesicular GABA transporter appositions with GnRH neurons, representative of GABAergic inputs, in the same model (123). The origin of this increased input was determined with a viral-mediated Cre-lox approach to trace the projections from specific GABA neuron subpopulations. This revealed that GABA neurons within the ARN provide enhanced input to GnRH neuron dendrites in PNA-treated mice, and concomitantly, display reduced co-expression with PR (Figure 4). This is consistent with identifying the specific circuit involved in androgen mediated interference of progesterone negative feedback as DHT has been shown to blunt progesterone negative feedback via GABA inputs to GnRH neurons (124). Together, these findings suggest that reduced PR expression in ARN GABA neurons, most likely resulting from high androgen levels, leads to a state of impaired steroid hormone feedback as 14

progesterone-insensitive ARN GABA neurons increase their connections with the GnRH neuron (Figure 5). Increased excitatory GABAergic input may in turn drive high GnRH neuron firing and episodic release into the median eminence, leading to elevated LH pulsatile release from the pituitary gland that drives the hyperandrogenism and disrupted estrous cyclicity of PCOS. These findings highlight a novel neuronal pathway that is potentially critical for the steroid hormone feedback control of normal fertility, Of course, while these findings implicate GABA neurons in particular, other progesterone-sensitive arcuate subpopulations might also play a role in PCOS. For instance, numerous studies in the human, primate and ewe report that the suppression of GnRH release by progesterone is mediated by inhibitory endogenous opioids (48,118,119,176-181), suggesting a role for ARN dynorphin neurons. Accordingly, the prenatal testosterone-treated sheep exhibits a reduced number of dynorphin neurons in the arcuate nucleus (175). As dynorphin is known to inhibit pulsatile GnRH neuron activity (181-183), a decrease in dynorphin expression or activity in PCOS could provide a mechanism through which GnRH neuron activity is increased. While the role of this specific pathway is currently limited to work in a preclinical mouse model of PCOS, the translation of these findings to the clinical manifestation of PCOS in women is supported by literature reporting on the effects of GABA-acting anti-epileptic drugs (AEDs) on reproductive function.

The anti-

convulsant and anti-manic effects of AEDs such as valporate are attributed to enhanced GABA activity in the brain. The endocrine and reproductive symptoms that manifest in epileptic women treated with AEDs are strikingly typical of PCOS, including hirsutism, polycystic ovaries, hyperandrogenaemia, disrupted menstrual cyclicity and difficulty conceiving (184-187). Although there have been limited studies investigating changes within the GnRH neuronal network induced by AEDs, valproic acid significantly alters GnRH and GABA expression within the preoptic area of rats (188), and increases GABA associations with GnRH neurons in mice (189). This implies that AED treatment in women may induce the PCOS phenotype in a similar manner to PNA treatment in mice, by increasing GABAergic synaptic connectivity with GnRH neurons. Of additional interest, GABA enhancing antiepileptic drugs interfere with the activity of progestogen containing oral contraceptives (190). Although there is evidence that this occurs peripherally through the increased metabolism of contraceptive steroids by the liver (191), the data 15

discussed here raises the possibility that interference with the activity of GABAergic neurons that convey progesterone feedback information to GnRH neurons may also be involved. Dissecting the mechanisms of impaired steroid hormone feedback in PCOS not only extends our understanding of how the brain normally responds to steroid hormone signaling, it also has the potential of identifying future therapeutic targets for the treatment of PCOS. It is appealing to imagine targeting specific brain circuits with novel therapeutics to restore steroid hormone feedback sensitivity and normal GnRH/LH pulsatility. However, the utility of such an approach remains uncertain given the intrinsic pathophysiology of the ovary reported in PCOS. Isolated theca cells from PCOS women are known to be hyperandrogenic (192) and express higher levels of the enzymes critical for androgen biosynthesis (193). Recent findings point toward a genetic basis for these ovarian abnormalities (194) and would suggest that drugs ameliorating the central steroid hormone sensitivity defects would fail to completely restore normal reproductive function. Although, given the heterogeneous nature of PCOS and the most likely varied etiology of the syndrome, the restoration of steroid hormone feedback could be more effective in some patient groups. It will be important to determine whether long-term modification of central steroid hormone feedback can cause sustained changes in ovarian function.

7. Conclusion: PCOS is a prevalent disorder without a clearly defined origin that presents with a spectrum of reproductive and metabolic dysfunction. It is imperative that the causes and consequences of the syndrome be investigated from every possible angle, from peripheral tissues and circulating factors to central brain circuits that both respond to and drive peripheral hormone secretion. Developing a better understanding of the neuroendocrine pathology of PCOS not only enhances our ability to potentially treat or avoid the development of PCOS, it also provides a basis for understanding the essential components of normal steroid hormone feedback and fertility. In response to early clinical observations of impaired central steroid hormone negative feedback in PCOS, several animal models of the syndrome have begun to advance our understanding of where and how this might develop within the brain. The critical questions are: when, where and how do androgens promote the neuroendocrine impairments of PCOS including a loss of progesterone sensitivity; 16

also, where and how does progesterone mediate negative feedback control of GnRH neuron activity? Currently, there is strong evidence for the involvement of GABA neurons in mediating impaired progesterone negative feedback downstream from androgen actions on progesterone signalling. Recently, anatomical work has identified that the GABA neurons residing specifically within the arcuate nucleus of the hypothalamus provide a novel circuit to GnRH neurons through which impaired progesterone negative feedback and increased GnRH/LH pulse frequency may be mediated in the PCOS-state. This work highlights a specific anatomical pathway that is potentially crucial for gonadal steroid hormone negative feedback to GnRH neurons. Future studies are required to explore the functional relevance of this specific arcuate GABA input on GnRH neuron activity in both fertile and PCOS-like states to continue to advance our understanding of the neuroendocrine genesis of PCOS. Acknowledgements: This work was supported by a University of Otago PhD Scholarship and the New Zealand Health Research Council.

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2.

3. 4. 5.

6.

7.

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Figure 1. LH pulse frequency is increased in PCOS. Pulsatile LH secretion over 24 hours in a 17 year old girl with normal fertility (A) and a 17 year old girl with PCOS (B). Stars are representative of a pulse. (Adapted from 1).

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Figure 2. Modes of progesterone and androgen signalling to GnRH neurons. A. Progesterone is hypothesised to act through multiple pathways to modulate GnRH neuron activity. (1) Progesterone may bind to the progesterone receptor expressed by afferents to GnRH neurons, such as neurons expressing kisspeptin, neurokinin B and dynorphin (KNDy) or GABA. (2) Progesterone can act directly through membrane receptors expressed by GnRH neurons, such as PgRMC1. (3) Glial cells can convert progesterone to neurosteroids such as allopregnanolone, which can directly bind to and modulate the GABAA receptor. B. Androgens, including testosterone and dihydrotestosterone (DHT) can (1) act through the androgen receptor in afferents to GnRH neurons and alter the expression of the progesterone receptor. (2) Androgens or the DHT metabolite 5α-androstan-3 α 17β-diol (3α-diol) may directly bind to and modulate the GABAA receptor. (3) The DHT metabolite 5α-androstan-3β 17β-diol (3β-diol) may act through ERβ in GnRH neurons to initiate downstream changes in gene expression.

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Figure 3. Evidence for increased activity of the GnRH/LH pulse generator in prenatal androgen-treated mice. A. Graphs displaying representative traces of GnRH neuron firing rates from control (i) and PNA (ii) mice over a 60-minute period. iii) The firing rate of GnRH neurons was significantly higher in PNA mice (n=17 cells from 10 mice) compared to controls (n=17 cells from 8 mice). Adapted from Roland and Moenter, 2010. B. Representative measurements of LH from serial blood samples taken from a control (i) and PNA (ii) mouse in 10-minute intervals for 120 minutes, stars represent a pulse of LH. iii) LH pulse frequency is significantly increased in PNA mice (n=19) compared to controls (n=14). Adapted from Moore et al. 2015. *, p