Progesterone Regulation of the GnRH Pulse Generator

Abstract

Kisspeptin neurons in the arcuate nucleus (ARN) represent the gonadotrophin- releasing hormone (GnRH) pulse generator responsible for driving the pulsatile release of GnRH and luteinising hormone (LH) secretion. In a healthy menstruating woman, following the mid-cycle ovulation, progesterone (P4) levels dramatically increase and exert a negative feedback action upon pulsatile LH secretion necessary for maintaining normal fertility. In mice, the ARN kisspeptin neurons display synchronised patterns of firing approximately every hour in diestrus and, similar to humans, this slows dramatically during the luteal phase of the cycle. However, the exact mechanism by which P4 negatively feeds back to regulate pulsatile LH secretion remains unknown. Given that ARN kisspeptin neurons express the progesterone receptor (PR), I hypothesised that this may be the site at which P4 negative feedback occurs. I first assessed the profile of circulating P4 levels in mice at 8-hour intervals throughout the estrous cycle using liquid chromatography-mass spectrometry (LC-MS). The concentration of P4 was found to peak at 6 PM on proestrus (21.94 ng/mL) and was at its lowest at diestrus 10 AM (0.42 ng/mL). I next assessed the changes in Pgr mRNA expression within ARN kisspeptin neurons throughout the cycle using RNAscope. This revealed that Pgr mRNA levels in kisspeptin neurons remain unchanged across the cycle. Building on these findings, I next assessed the impact of circulating P4 on the activity of the GnRH pulse generator using ARN kisspeptin neuron GCaMP fibre photometry in freely behaving mice. Diestrous mice equipped for monitoring the activity of the GnRH pulse generator were injected with either 4 or 8 mg/kg P4 and kisspeptin neuron synchronisation episodes (SEs) were monitored for 24 hours. Both doses resulted in a significantly decreased frequency of SEs compared to the vehicle, which persisted for 6 hours. To establish the site of action of P4, I then undertook a GCaMP fibre photometry study in which 25 or 50 ng/mL of P4 was infused directly into the ARN. Surprisingly, this had no effect on the GnRH pulse generator activity. My next approach to establish the role of PR in kisspeptin neurons involved using CRISPR-Cas9 gene editing to selectively knockdown PR in ARN kisspeptin neurons in adult mice. This achieved a selective knockdown of approximately 42% of PR in ARN kisspeptin neurons. This was not found to have any substantial effect on the GnRH pulse generator activity, pulsatile LH secretion, or estrous cyclicity. However, it partially mitigated the inhibitory actions of peripheral P4 treatment on kisspeptin neuron SEs. Unexpectedly, the major phenotype of mice with ARN kisspeptin neuron PR knockdown was that of substantial obesity due to increased appetite. In summary, my findings suggest that P4 exerts its inhibitory actions on ARN kisspeptin neurons through temporally regulated PR-dependent and PR-independent pathways. The partial mitigation of P4’s inhibitory actions on the GnRH pulse generator activity following PR knockdown in the ARN kisspeptin neurons, along with the observed obesity phenotype, highlights the multifaceted role of PR in this neuronal population. These insights further our knowledge of the specific roles of ARN kisspeptin PR and could be pivotal for the development of new therapeutic strategies for reproductive and metabolic disorders, such as polycystic ovarian syndrome (PCOS).