In-vivo analysis of hypothalamic kisspeptin neuron activity patterns
Mammalian reproduction is regulated by the brain, pituitary gland, and gonads. In the brain, gonadotropin-releasing hormone (GnRH) is released to stimulate the pituitary gland. In females, two distinct modes of GnRH secretion are controlled by two populations of hypothalamic kisspeptin neurons located in the arcuate nucleus (ARNKISS), known as the pulse generator, and in the rostral periventricular area of the third ventricle (RP3VKISS) operating as the surge generator. The pituitary gland responds to GnRH by releasing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) which regulate gametogenesis and steroidogenesis in the gonads. This thesis aims to understand the functional activity of these kisspeptin populations in female reproduction using in-vivo GCaMP fibre photometry in mice. Polycystic ovary syndrome (PCOS) affects approximately one in ten women of reproductive age, often presenting with subfertility and hyperandrogenism. Another hallmark of PCOS is increased LH pulsatility, suggesting potential dysfunction in the ARNKISS neuron pulse generator. I examined the activity of the pulse generator in two mouse models of PCOS. In the peripubertal androgen (PPA) model, there is a reduction in the frequency of ARNKISS neuron synchronisation events (SEs) that drive LH pulses. The prenatal androgen (PNA) model exhibited highly variable patterns of pulse generator activity with no significant differences detected in ARNKISS neuron SEs, pulsatile LH secretion, or serum testosterone, estradiol, and progesterone concentrations. However, a machine-learning approach revealed that acyclic PNA mice retained cyclical ARNKISS neuron activity, resembling the normal estrous cycles. The frequency of ARNKISS neuron SEs was significantly increased in algorithm-identified ‘diestrous stage’ PNA mice compared to controls. Further experiments showed reduced feedback suppression of progesterone on ARNKISS neurons in PNA mice and decreased gonadotroph sensitivity to GnRH. These findings highlight the complexity of GnRH pulse generator dysfunction in PCOS and the need for a deeper understanding of deficits across the whole hypothalamic-pituitary-gonadal axis in PCOS models. Ovulation is triggered by a surge of LH release at mid-cycle. Although the RP3VKISS neurons have been proposed as the surge generator, their in-vivo activity has never been recorded in any animal. My second aim is to make the first ever in-vivo recordings of RP3VKISS neurons and investigate the effects of estrogen on their activity. Recordings in freely behaving intact female mice showed that RP3VKISS neurons exhibited elevated levels of activity on proestrus comprised of large, slow baseline oscillations accompanied by frequent fast calcium transients. These activities correlated with the proestrous LH surge. Following ovariectomy, RP3VKISS neurons activity remained low and unvarying, and was restored during the estrogen-induced LH surge, resembling the proestrous surge but with a reduced amplitude. These findings provide the first direct evidence of RP3VKISS neuron activity in vivo that strongly support their role as the key neuronal signal driving ovulation in mammals. Overall, this thesis examined ARNKISS neuron dysfunction in PCOS models and provided the first in-vivo recordings of RP3VKISS neurons. These findings offer new insights into the physiological and pathophysiological roles of hypothalamic kisspeptin neurons in female reproduction.
endocrinology, fertility, GnRH, hypothalamus, kisspeptin, LH, neuroendocrinology, PCOS, reproduction, reproductive neuroendocrinology, women's reproductive health
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