8602 Classical progesterone signaling in kisspeptin neurons is not required for negative feedback regulation of the GnRH pulse generator in mice

Abstract Disclosure: K.M. Dillon: None. D.B. Lohr: None. A.G. Novak: None. A.V. Petriv: None. N.T. Neifert: None. A.M. Moore: None. Mammalian reproductive function depends on the ability of progesterone to suppress pulsatile gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion in a homeostatic negative feedback loop. Impairments in progesterone negative feedback are associated with the development of polycystic ovary syndrome (PCOS), the leading cause of infertility for people with ovaries. Although previous research indicates cells upstream from GnRH neurons expressing the classical progesterone receptor (PR) are required for negative feedback, the identity of these cells and the mechanism by which they reduce GnRH/LH pulsatile secretion remain unclear. In this study, we aimed to address the hypothesis that PR expressed by a neuronal population in the arcuate nucleus expressing Kisspeptin, Neurokinin B, and Dynorphin, recently defined as the GnRH pulse generator, is required for negative feedback regulation of LH pulses. To achieve this, we utilized Cre-lox technology in mice to conditionally delete PR from kisspeptin cells (KPRKO mice) and assessed fecundity, LH pulsatility, and the mRNA expression of KNDy neuropeptides. Female KPRKO mice paired with B6 stud males for 3 months produced significantly fewer litters (p<0.05, n=5) and pups per litter (p<0.05) compared to WT controls (n=5) (students t-tests), recapitulating a previously reported subfertile phenotype in this model. In a separate cohort of animals, serial blood samples were collected every 6 minutes for 2 hours from the tail-tip of habituated WT and KPRKO mice in diestrus (n=6/group) and estrus (n=6/group), and an ultra-sensitive ELISA was used to detect LH pulses. However, compared to WT controls, KPRKO mice did not exhibit any significant changes in total and baseline LH release, pulse frequency or pulse amplitude in either cycle stages (p>0.05, students t-tests). At the completion of serial blood sampling, brains were collected from WT and KPRKO mice and RNAscope in situ hybridization was used to quantify mRNA for kisspeptin (KISS1) and dynorphin (PDYN), which are excitatory and inhibitory to GnRH/LH secretion, respectively. No differences in the number of KISS1- and PDYN-expressing cells or the number of KISS1 and PDYN mRNA transcripts were observed between WT and KPRKO mice (n=4/group, p>0.05, students t-tests). These data suggest that progesterone signaling in kisspeptin cells through PR is not essential for negative feedback regulation of LH pulses and is unlikely to contribute to the infertile phenotype of KPRKO mice. These results suggest that negative feedback may be maintained via non-classical mechanisms or an unidentified PR-positive neuronal population. Presentation: 6/1/2024