PROJECT SUMMARY The menopausal transition represents a complex and dynamic physiological aging phenomenon and often leads to adverse life-style effects. Nearly 85% of post-menopausal women will experience multiple symptoms. Treatment of clinical symptoms in post-menopausal women is difficult without knowing the underlying biological mechanisms and molecular players and hence requires further investigation. The anterior pituitary-derived heterodimeric glycoprotein hormone, follicle-stimulating hormone (FSH) consists of an alpha and a beta subunit. The FSH subunit undergoes significant age-dependent changes in N-glycosylation. Higher ratios of fully glycosylated FSHβ subunit in older, post-menopausal women compared to younger, reproductively active women were identified in human pituitaries and urine. FSH levels rise significantly due to concomitantly declining ovarian estrogen and progesterone during menopause and high levels of old age specific FSH may be deleterious in tissues such as bone and adipose, where FSH acts noncanonically via inflammatory pathways. This observation is of great significance because osteoporosis and weight gain are two of the most common symptoms post-menopausal women experience. This project focuses on the N-glycosylation process within anterior pituitary gonadotrope cells and seeks to identify the molecular players involved in the “glycosylation shift” to a more fully glycosylated phenotype in older age females, as well as the factors that regulate this process. We have recently identified differentially regulated N-glycosylation enzymes expressed in both young (~4- months) and older age (8-month) female mouse gonadotrope cells via RNA-sequencing analysis. Several glycosylation pathway-encoding genes (for example, Man2a1, B4galt5) exhibited a significant upregulation in gonadotropes of older age mice. We hypothesize that these N-glycosylation enzyme- encoding genes may contribute to the age-related “glycosylation shift” in FSHβ seen in older, post-reproductively active female mice. In Specific Aim 1, we will define age-dependent changes in expression of N-glycosylation enzymes MAN2A1 and B4GALT4 in gonadotrope cells of young and old female mice (using a GFP-tagged gonadotrope mouse model) and immunostaining whole pituitary and gonadotrope cells, as well as performing enzyme activity assays. In Specific Aim 2, we will determine the role of estrogen and progesterone receptor signaling in regulation of the N- glycosylation pathway-encoding enzymes in gonadotrope cells of young and old female mice using gonadotrope-specific knockout of either Esr1 or Pgr and performing fluorescence-activated cell sorting (FACS), RNA-sequencing, qPCR, immunostaining, and enzyme activity assays in pituitary gonadotrope cells. Ultimately, the work proposed here represents a novel genetic and biochemical approach and provide new knowledge on the regulation of FSHβ N-glycosylation during reproductive aging and how this may indirectly contribut...