Examining the Networking and Behavior of Gonadotropes in Intact Mouse Pituitaries using GCamp6 Imaging

Asman, Daniel
Pituitary gonadotropes are an integral part of the Hypothalamo-pituitary-gonadal axis. Gonadotropes synthesize and secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH) when acted upon by gonadotropin-releasing hormone (GnRH) from the hypothalamus. Activation of gonadotropes by GnRH induces a rapid biphasic elevation of intracellular calcium. The importance of calcium in gonadotropes are largely from cell culture experiments that lack any spatial resolution to address calcium activity at the population level. To address this gap in knowledge, we have taken an innovative approach of using a genetically encoded calcium indicator (GCaMP6) to detect fluctuating calcium signals within gonadotropes in vivo. Utilizing CRE/Lox technology, we have created a gonadotrope specific GCaMP6 expressing mouse that is highly responsive to GnRH. First, the pituitary was explanted from our GCamp6-positive mice and placed in oxygenated CSF where it was exposed to a pulse of GnRH to assess calcium kinetics. We analyzed the amplitude and frequency of Ca2+ transients from gonadotropes. Several gonadotropes displayed regular oscillations upon GnRH agonist application (57%), while others showed irregular oscillatory behavior (43%). Additionally, synchronization of GnRH-induced calcium transients was observed in several clusters of gonadotropes that were in proximity to one another. Gender differences in GnRH-induced gonadotrope responses were identified. The proportion of gonadotropes exhibiting increases in amplitude was significantly higher in females than males (p<0.05, R2). This study, although still preliminary, shows promising steps forward in the exploration and understanding of calcium signaling within gonadotropes in response to GnRH.
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