Project Summary/Abstract – Project 4(Bousfield) Our long-term goal is to understand how carbohydrate modulates the biological activity of follicle-stimulating hormone (FSH). The human ovary becomes resistant to FSH stimulation with age and circulating FSH concentrations rise, keeping serum estrogen concentrations normal until 2 years before the final menstrual cycle. Around the same time pituitary FSH changes from predominantly hypo-glycosylated hFSH21 and hFSH18 (due to loss of FSHβ subunit N-glycan at Asn24 or Asn7, respectively) to a predominantly fully-glycosylated hFSH24, which possesses all 4 N-glycans. Hypo-glycosylated hFSH18 and hFSH21 exhibit greater apparent affinity for the FSHR, can occupy more FSHR sites, and associate with FSHR more quickly than fully-glycosylated hFSH24. Our collaborators have demonstrated hFSH18 and hFSH21 exhibit greater biological activity in vitro, rescue fertility in transgenic Fshb-null mice, and exhibit distinct patterns of gene expression in granulosa cells. The overall hypothesis of this proposal is that in the face of a senescing ovary the additional switch from hypo- glycosylated FSH18 and FSH21 to fully-glycosylated FSH24 further compromises fertility and contributes to bone loss. The objective of this proposal is to study the mechanisms for these differences in FSHR binding via the following specific aims: 1: Develop FSH glycoform immunoassays with multi-species antibodies. Our working hypothesis is that fully- and partially glycosylated FSH glycoforms secretion patterns diverge in women. The fact that hFSH18 and hFSH21 induce unique gene expression profiles makes it imperative to develop specific immunoassays for all three physiologically relevant glycoforms, hFSH18, hFSH21, and hFSH24. 2: Determine FSHR/FSH-glycoform structures. Our working hypothesis is that FSH18, FSH21, and FSH24 induce different FSHR conformations that result in biased signals. The Project will purify FSHR and FSH/FSHR complexes. Cryogenic electron microscopy (cryo-EM) will be conducted in collaboration with Dr. Zhao Wang at Baylor College of Medicine. Highly purified FSH glycoforms, hFSH18, hFSH21, and hFSH24 are available for both aims. Glycoform-specific assays are expected to provide novel information regarding FSH glycoform changes during the normal human menstrual cycle and as women age. FSH glycoform/FSHR structures are expected to reveal the role of FSH N-glycans in fully activating the FSHR. The potential translational outcomes of this project are unique immunoassays for measuring FSH glycoforms and knowledge of FSHR function to facilitate development of drugs to enhance or inhibit FSHR function.