The Aging Pituitary/Gonadal Axis

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Project Summary/Abstract - Overall This integrated program project was pursuant to the discovery of naturally occurring, partially glycosylated follicle-stimulating hormone (FSH) glycoforms by the Bousfield laboratory. Two of these glycoforms possessed 3 of 4 N-glycans, exhibited greater biological activity, and were more abundant than fully-glycosylated FSH in young women. Furthermore, hypo-glycosylated FSH variants exhibited an age-dependent decrease concomitant with a well-known decline in fertility in women. Projects are organized to reflect the recognition of the importance of the development of numerous mouse genetic models in previous funding periods inspired by biochemical advances in understanding of the FSH glycoforms, and which now, will inspire in turn, such studies at the signaling, trafficking, and structural biology levels. In Project 1(Kumar), powerful genetic models now make it possible to study each specific glycoform independently, both by injection of purified glycoform preparations into Fshb-null mice as well as in transgenic mice expressing FSH18, FSH21, or FSH24. Advances in single-cell RNA-sequencing coupled with a transgenic line that expresses green fluorescent protein specifically in gonadotropes permit evaluation of the more than 50 enzymes responsible for N-linked oligosaccharide synthesis. Since non-ovarian targets of FSH have raised concerns and confusion for women undergoing IVF, several mouse models were devised to address questions raised by conflicting experimental outcomes, beginning with deletion of the Fshr gene, which would effectively eliminate all forms of putative FSHR proteins, including alternative splicing. In Project 2(Davis), studies involving FSH glycoform signals within primary cultures of granulosa cells, focus on the disparate responses of ovarian target cells to biased agonists FSH18, FSH21, and FSH24. Two major, differentially glycoform-regulated transcription factors (TF), CREB and YAP1, will have their downstream pathways characterized in granulosa cells. Granulosa cells of young and advanced age mice and women will provide basic information relevant to translational efforts. The interface between FSH and FSHR, both monomeric and oligomeric receptor forms, will be studied in Project 3(Jonas). Activation of cAMP accumulation is now known to involve FSHR dynamics that will be evaluated, including internalization. FSH glycoforms are anticipated to alter FSHR conformation to varying degrees, leading to biased agonist signals. Project 4(Bousfield) will incorporate FSHR into lipid nanodiscs to provide a means to evaluate glycoform-FSHR- membrane interactions, using cryogenic electron microscopy (cyro-EM). This has been done with LH and TSH receptors (LHR & TSHR) and those studies have revealed the ligand binding site undergoes a major rotation, lifting it away from the membrane, during receptor activation. The location of FSH oligosaccha-rides is on the back side of the hormone, away from the hormone-receptor interface, which suggests interaction with the cell membrane or membrane proteins. Taken together, the projects integrate FSH glycoform signaling at overlapping levels ranging from the intact organism to the glycosylated hormone itself.