PMON55 Calcium Imaging in the Intact Mouse Pituitary: A Novel Design for Understanding the Formation and Modulation of the Gonadotrope Network

Abstract

Abstract The reproductive cells of the pituitary, the gonadotropes, develop after e12.75 in mice and continue to differentiate and migrate throughout puberty. In response to physiological signals, these cells change in morphology to alter connections with other cells. Previous studies have indicated that gonadotropes have functional relationships in an interconnected, organized network. However, little work has been done to determine (1) what developmental cues are needed to organize this network, or (2) how this network functions to promote gonadotropin secretion. Activation of gonadotropin-releasing hormone receptors (GnRHRs) leads to an increase in intracellular calcium; therefore, this pathway can be used for visualizing the activation of gonadotropes. We have developed a novel system to analyze the gonadotrope network which will allow us to study the influence of physiological signals to this interconnected population. To visualize calcium signals specifically in gonadotropes, we created a line of Gonadotrope-GCaMP mice using Gnrhr-driven Cre to express the GCaMP calcium indicator. For present studies, intact pituitary glands were taken from adult Gonadotrope-GCaMP mice (males and proestrous females). Whole pituitaries were mounted in a chamber, and gonadotrope network responses to GnRH (1 or 5nM, at least 4 pituitaries/dose) were recorded using confocal microscopy. Calcium signals from an average of 177 ± 8 gonadotropes per pituitary were analyzed. MATLAB software was used to quantify network connections of gonadotropes for each treatment. Correlation coefficients were determined by their patterns of calcium signaling. Gonadotrope connectivity increases following GnRH stimulation in a dose-dependent manner in female pituitaries (17% following 1nM GnRH, p<0.05; 44% following 5nM GnRH, p<0.05). While male gonadotropes also increase connectivity following stimulation with GnRH, the effect is not enhanced by the higher dose (27% following 1nM GnRH, p<0.05; 20% following 5nM GnRH, NS). Although the strength of gonadotrope correlations is not changed by 1nM GnRH in females, higher dosage of GnRH doubles the strength of correlations over pre-stimulation levels (Pre: 0.08, Post: 0.16, p<0.05). The strength of correlations in males was not significantly altered by GnRH at either dose. In conclusion, the data from whole mouse pituitaries confirm that gonadotropes are capable of high levels of coordinated activity. All pituitaries showed baseline levels of coordination among gonadotropes, confirming the presence of the network. Network activation is significantly increased by GnRH stimulation in both males and females. In females, coordination of gonadotropes is also strengthened by GnRH in a dose-dependent manner. Using a novel gonadotrope-specific calcium indicator mouse model, we have shown GnRH-mediated changes in gonadotrope network connectivity in the intact mouse pituitary. Future studies in our lab will determine how other physiological signals are involved in development and/or maintenance of this coordinated activation. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.