Role of arcuate nucleus kisspeptin neurons in mediating oestrogen negative feedback on gonadotrophin-releasing hormone neurons in adult mouse

Abstract

Reproduction and fertility in mammals is critically dependent on the neurosecretion of gonadotrophin-releasing hormone (GnRH) released by the hypothalamic GnRH neurons into the hypophyseal portal system of the anterior pituitary. GnRH binds to GnRH receptors on the pituitary gonadotrophs and directs gonadotrophin secretion into the peripheral circulation. The oestrogen negative feedback mechanism is important in controlling the fluctuating profiles of pulsatile GnRH and luteinizing hormone (LH) release. For the major part of the oestrous cycle, oestrogen exerts negative feedback actions on both GnRH neurons and pituitary gonadotrophs to suppress GnRH release and ultimately restrain LH secretion at its nadir. The precise neurobiological mechanisms underlying oestrogen’s inhibitory effects on GnRH/LH release are largely unknown. It is hypothesised that kisspeptin neurons are required to mediate oestrogen negative feedback actions to suppress pulsatile GnRH/LH secretion. This thesis focused on investigating the role of arcuate nucleus (ARN) kisspeptin neurons in mediating oestrogen negative feedback actions on GnRH neurons in adult female mice.

Anterograde tract tracing was conducted to determine the projections of both ARN and rostral periventricular area of the third ventricle (RP3V) kisspeptin neurons. Retrograde tracing was performed to verify the axonal projections of ARN and RP3V kisspeptin neurons into the rostral preoptic area and ARN, respectively. The ARN kisspeptin neurons were found to project widely within both the medial and lateral aspects of the hypothalamus and associated limbic structures, and possibly innervate GnRH neuron cell bodies or dendrites in the rostral preoptic area. The RP3V kisspeptin neurons exhibited a more medial projection pattern throughout the brain and were found to provide substantial inputs into the ARN. The diversity of brain regions targeted by kisspeptin neurons suggests their involvement in regulating multiple circuits in addition to the GnRH neuronal network. Despite the wide projections of kisspeptin neurons into various regions, none of the kisspeptin neuron populations project to the external zone of the median eminence (ME). Together, these data indicate that ARN and RP3V kisspeptin neurons do not innervate GnRH nerve terminals in the external zone of the ME. It is possible that ARN kisspeptin neurons regulate GnRH nerve terminals through a nonsynaptic interaction, whereby the kisspeptin may be released from terminals in the internal zone of the ME and act through volume transmission to eventually reach and control GnRH release in the external zone.

Transgenic mouse models harbouring global and neuron-specific deletions of Gpr54 gene were examined for oestrogen negative feedback dynamics to assess the role of kisspeptin-Gpr54 signalling in mediating oestrogen negative feedback. Both animal models exhibited abnormal oestrogen negative feedback profiles with the absence of postovariectomy LH elevations. This implied that kisspeptin neuron input may be required for the generation of high-amplitude GnRH pulses to stimulate rapid LH secretion from the pituitary after ovariectomy. Finally, the role of ARN oestrogen receptor alpha (ERα)-expressing neurons in mediating oestrogen negative feedback was assessed by using adeno-associated virus (AAV)-mediated Cre/loxP recombination to induce ERα deletion specifically in the ARN. The results indicate that ARN ERα-expressing neurons are essential for oestrous cyclicity and appear to be important components of the multimodal negative feedback pathways. Overall, the ARN kisspeptin neurons are unlikely the key neurons conveying oestrogen negative feedback on GnRH pulses/LH secretion. Instead, they are part of the network modulating high amplitude GnRH pulses stimulating LH secretion in the absence of oestrogen inhibitory effects.