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dc.contributor.advisorCampbell, Rebecca
dc.contributor.authorMarshall, Christopher Joseph
dc.identifier.citationMarshall, C. J. (2020). Dissecting alterations to arcuate nucleus neuropeptide Y neural circuitry in a model of polycystic ovary syndrome (Thesis, Doctor of Philosophy). University of Otago. Retrieved from
dc.description.abstractPolycystic ovary syndrome (PCOS) is the most common form of infertility among women of reproductive age. Despite the prevalence of PCOS, the underlying mechanisms behind how PCOS arises and causes infertility remain poorly understood. While primarily considered a disorder of the ovary, a growing body of evidence suggests that PCOS is associated with dysfunctional neuroendocrine circuitry in the hypothalamus that orchestrates gonadotrophin hormone secretion. Women with PCOS frequently exhibit rapid pulsatile luteinizing hormone (LH) secretion. While LH secretion is tightly regulated by ovarian steroid hormonal feedback in fertile women, women with PCOS are unresponsive to these hormones. Elevated LH secretion likely reflects dysfunctions to the gonadotropin-releasing hormone (GnRH) neuronal network in the hypothalamus that gates LH secretion and integrates steroid hormone feedback. As GnRH neurons themselves do not express the receptors necessary for feedback to occur, research has sought to identify alterations to GnRH neural afferents that may arise in PCOS. Prior work within our lab has utilized a prenatal androgen (PNA) treated mouse model of PCOS to identify enhancement of excitatory GABAergic inputs to GnRH neurons. In particular, GABA neurons in the hypothalamic arcuate nucleus (ARN) have been identified with enhanced input to GnRH neurons, and reduced sensitivity to an ovarian steroid hormone, progesterone. Neuropeptide Y (NPY) neurons compose a large proportion of GABA neurons within the ARN, and are known to play an important role in restraint of LH secretion via inhibition of GnRH neurons. Interestingly, plasma NPY is often reduced in women with PCOS, indicating impairment of its neural secretion. The overarching aim of work presented in this thesis was therefore to determine whether NPY neurons within the ARN also exhibit PNA-induced alterations, which may advance our understanding of how unrestrained LH secretion occurs in PCOS. Initially, the proportion of NPY neurons that are GABAergic within the ARN was assessed by pairing immunohistochemical (IHC) labelling of NPY with transgenic reporter identification of GABA neurons, in both PNA-treated mice and fertile controls. Additionally, the molecular identity of ARN GABA neurons was further dissected to assess whether they express other peptides known to regulate GnRH neurons. This work established that ARN NPY neurons compose approximately one-third of ARN GABAergic neurons, a far larger proportion than any other neural phenotype examined, and demonstrated that the vast majority (> 93%) of ARN NPY neurons are GABAergic. While fertile male mice were observed to have a larger population of ARN NPY neurons compared with both fertile (p = 0.0006) and PNA-treated females (p = 0.0192), no differences were found between female groups, indicating the size of this population is not masculinized by PNA-treatment. To investigate remodelling of NPY neurons in the ARN specifically, the validity of an agouti-related peptide (AgRP)-Cre mouse line was characterised as AgRP is highly co-expressed with NPY, and exclusively expressed in the ARN. After crossing AgRP-Cre mice onto a Cre-dependant GFP reporter it was demonstrated that within the ARN, GFP reporter was highly specific and effective at identifying NPY neurons. Additionally, GFP expression was overwhelmingly confined to the ARN only. Whole brain mapping of axon-associated GFP expression revealed that this reporter was highly effective for visualising distal axons. In order to assess whether ARN NPY neurons projecting to GnRH neurons could be targeted more specifically, a fluorescent retrograde tracer was injected into the preoptic area where the majority of GnRH neurons reside. This revealed that approximately 10% of ARN NPY neurons project to the area where GnRH neurons are located, and that these neurons are scattered throughout the ARN rather than composing an anatomically defined population. These experiments demonstrated that AgRP-GFP mice are an appropriate model for further dissection of ARN NPY neurons. Using this model, the hypothesis that PNA exposure leads to remodelling of ARN NPY sensitivity to steroid hormones, and their projections to GnRH neurons was addressed. Interestingly, ARN NPY neurons did not express the progesterone receptor or estrogen receptor . However, a greater proportion of ARN NPY neurons expressed androgen receptor (AR) in PNA mice compared with fertile controls (p = 0.045). When close appositions between ARN NPY axons and GnRH neurons was examined, indicating putative innervation, it was revealed that there were no differences in the close appositions onto GnRH neurons in the PNA-treated mouse. Overall, these results suggest that ARN NPY neurons are not remodelled in the same way as ARN GABA neurons, but are subject to their own distinct alterations in PNA-treated mice in the form of elevated androgen sensitivity. The role of leptin, a major regulator of ARN NPY neurons and permissive cue in fertility, was then examined in PNA-treated mice compared to fertile controls. Plasma leptin was measured in fed and fasted states, and there were no differences in leptin levels in PNA-treated mice. Interestingly, fasting reduced plasma leptin in PNA-treated mice similar to controls, contrasting previous findings indicating derangements to fasted leptin levels. Furthermore, leptin responsiveness indicated by induction of pSTAT3 was not different in PNA-treated mice. These results indicate that leptin signalling in the ARN is normal in PNA-treated mice and leptin acting within this region is not likely to contribute to altered GnRH neuron activity. While ARN NPY neurons and circuitry were found to be largely unaffected by PNA-treatment, a sensitive NanoString assay for gene expression revealed evidence for potential reductions in downstream signalling within hypothalamic regions important for GnRH neural regulation. Expression of the NPY Y1 receptor was downregulated in the rostral periventricular region of the third ventricle, a region important for generating the preovulatory LH surge. Additionally, a sizable reduction in leptin receptor gene expression was observed in the preoptic area in the vicinity of GnRH neurons, an area in which leptin signalling plays a role in regulating LH secretion. Taken together these results do not support the hypothesis that alterations within ARN NPY-to-GnRH neuron circuitry plays a major role underpinning neuroendocrine disruption in PNA-treated mice and PCOS. While ARN NPY neurons do not appear to compose a subset of ARN GABA neurons that are remodelled following PNA-treatment, elevated androgen sensitivity was identified. As androgens disrupt negative feedback in PCOS, this opens up the possibility that androgens act via ARN NPY neurons to disrupt appropriate response by other steroid hormone sensitive afferent populations. Differences in NPY receptor expression were discovered in hypothalamic regions important for regulation of GnRH neurons, and potentially indicates altered NPY signalling within the GnRH afferent network. These findings establish a platform upon which future research will elucidate the importance of these circuit alterations in generating neuroendocrine disruptions in PCOS.
dc.publisherUniversity of Otago
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dc.subjectPolycystic ovary syndrome
dc.titleDissecting alterations to arcuate nucleus neuropeptide Y neural circuitry in a model of polycystic ovary syndrome
dc.language.rfc3066en of Philosophy of Otago
otago.openaccessAbstract Only
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