435 Age-dependent Regulation of Follicle-Stimulating Hormone N-glycosylation in Female Gonadotrope Cells

OBJECTIVES/GOALS: Age-specific N-glycosylation occurs on follicle- stimulating hormone (FSH) in pituitaries of post-menopausal women and results in a higher ratio of fully glycosylated to hypoglycosylated FSH. Our goal is to identify in vivo the N-glycosylation pathway enzymes and the regulatory mechanisms in gonadotropes of young and old female mice. METHODS/STUDY POPULATION: Pituitaries were isolated from female mice (at 4m and 8m; n=5 per group) carrying an Fshb-Cre transgene on a Rosa mT/mG genetic background and the GFP-tagged gonadotropes were purified by FACS. RNA-Seq analysis, and subsequent qPCR assays were performed on GFP+ cells from pituitaries of female mice at 4m (reproductively young), 8m (reproductively mid age) and ≥ 12m (reproductively old) of age. To identify the role of progesterone signaling in age-dependent N-glycosylation in gonadotropes, a gonadotrope-specific knockout of Pgr was achieved. Gonadotropes from these mutant mice at 4-, 8-, and ≥12 months of age (n=5 per group) were isolated for qPCR analysis of N-glycosylation enzyme gene expression. Predicted progesterone receptor (PR) promoter binding sequences was performed using JASPAR. RESULTS/ANTICIPATED RESULTS: RNA-seq identified 28 differentially expressed N-glycosylation enzyme-encoding mRNAs in gonadotropes of female mice at 4- and 8-months. Three genes showed significant differences between ages (Man2a1, Man1c1, and B4galt5.), and further qPCR analyses revealed six out of eight genes analyzed showed age-dependent expression, including Man2a1, Man1c1, and B4galt5. The promoters of all N-glycosylation enzyme genes showed strong predicted binding sequences for PR. Further qPCR analysis showed age-and genotype-dependent differences in N-glycosylation enzyme expression in Pgr cKO females, with the most striking differences observed at 13 months, where B4galt5, Man1a2, Mgat5, and Man2a1 were downregulated in Pgr cKO gonadotropes compared to controls. DISCUSSION/SIGNIFICANCE: We identified changes in the N-glycosylation machinery in female mouse gonadotropes and confirmed the age- and Pr-dependent regulation of the corresponding mRNAs. Our results provide insights into the mechanisms at the level of the pituitary by which old age-specific FSH glycoform regulates osteoporosis and weight gain in post-menopausal women.