OR17-3 Immortalized AVPV Kisspeptin Neurons Transiently Express GnRH Receptors under Specific Sex Steroid Conditions in vitro
Authors: Lee Blake , Rayzman Noa , Chappell Patrick 
Published: November 2022

In female animals, ovarian estradiol (E2) acts as negative feedback inhibition of GnRH secretion and positive feedback stimulation at ovulation. Both E2-regulated mechanisms work via stimulation or repression of distinct neuronal populations of Kisspeptin (KP)-synthesizing neurons. While E2 clearly stimulates AVPV KP neurons to increase kiss1 expression during the preovulatory surge, mechanisms required for optimal potentiation of maximal GnRH surge release remain incompletely characterized. We used two immortalized KP cell lines demonstrating increased kiss1 expression under high E2 (KTaV-3), or kiss1 suppression under low E2 (KTaR-1), to probe GnRH receptor (GnRHR) expression levels under E2 conditions of varying doses and times of exposure. Additionally, we have begun to investigate transcriptional mechanisms underlying gnrhr expression in KP neurons in vitro.

AVPV-derived KTaV-3 cells were treated with either E2 alone (5-100pM) or E2 with progesterone (4-40nM) for varying durations (4-96h), either constitutively or under modulating levels approximating changes found during the murine estrous cycle. All treatments were undertaken in serum-free (SF) media (DMEM) following a 50% serum synchronization. After total RNA isolation, cDNAs were probed with mouse gnrhr primers. Results reveal that gnrhr expression, not observed in typical growth media conditions, is induced in KTaV-3 cells to varying degrees dependent on prior E2/P4 exposure. Following shorter treatment times (12-24h), proestrus levels (100pM E2; 4nM P4) of sex steroids noticeably induced gnrhr, while exposure of KTaV-3 cells to a 96h estrous cycle revealed maximal gnrhr expression at the diestrus II→proestrus transition, with exposure cycles concluding with declining E2/rising P4 exhibiting no to minimal gnrhr expression. These results suggest that maximal KP neuronal expression of gnrhr may occur prior to the E2 peak on proestrus, potentially preparing AVPV KP neurons for GnRH surge release 12-16h later. Similar steroid conditions also increased dlx3 expression, a transcription factor required for GnRHR in pituitary, only in KTaV-3 cells. In KTaR-1 cells, gnrhr expression was observed only following E2 decreases, while dlx3 was constitutively elevated. Since cFos/JunB complex formation on the gnrhr promoter has been implicated in gonadotrope GnRHR levels, we explored how transfection of these transcription factors might affect gnrhr expression in KTaV-3 cells. Preliminary results reveal that fos/jun co-transfection stimulates gnrhr expression maximally at intermediate (DII) E2 levels, while higher E2 levels are required with fos or jun expression alone. While reciprocal GnRH-Kisspeptin connections have not yet been observed in vivo, these results suggest KP neurons may possess the capacity to respond to intrahypothalamic GnRH only in certain estrous cycle stages, by differentially modulating GnRH receptivity in the AVPV. These findings supplement the prevailing model of feed-forward KP regulation of GnRH with potential short-loop KP-GnRH positive feedback regulation, providing an increased understanding of feedback mechanisms required for normal neuroendocrine regulation of reproduction.

Presentation: Sunday, June 12, 2022 11:30 a.m. - 11:45 a.m.