The beginning of the menopausal transition lacks suitable diagnostic markers. The transition is attended by significant morbidity including irregular reproductive cycles, dysfunctional uterine bleeding, urogenital changes, impaired fertility, declining bone mass, vasomotor symptoms, and psychological impairment. The long-term objective of this project is to understand the role of follicle-stimulating hormone (FSH) glycosylation variants hFSH21 and hFSH24 in aging. Young women possess more hypo-glycosylated hFSH21 than fully-glycosylated hFSH24, while older women possess less hFSH21. As both glycoforms are found in urine, both enter the circulation where they affect gonadal and non-gonadal targets. In the previous funding period we determined that hFSH21 is much more active than hFSH24 in binding FSHR, activating gonadal target cells in vitro, and stimulating the ovary in vivo. In contrast, hFSH24 is much more active than hFSH21 in activating osteoclast differentiation. Thus, our overall hypothesis is, in the face of a senescing ovary, the additional switch from hFSH21 to hFSH24 further compromises fertility and may contribute to bone loss. Each project will test various aspects of this hypothesis via the following specific aims: 1. Determine the mechanism(s) by which partial glycosylation of hFSH leads to significantly increased FSHR binding. Our working hypothesis is that hFSH21 and small molecule binding alter FSHR conformation, making more FSH binding sites available to both glycoforms, thus increasing bioactivity. An exciting element is collaborative super resolution microscopy studies that will monitor FSHR oligomerization directly. 2. Define the different signaling pathways activated by FSH glycoforms in traditional gonadal targets as well as in bone, a nontraditional target cell. Under this aim, Project 2 will compare FSH glycoform activation of murine or human granulosa cells or cell lines, and osteoclast precursors. Our working hypothesis is that hFSH21 is more active than hFSH24 in gonadal target cells, while the reverse is true for non-gonadal target cells. The age-related shift in glycoform ratio thus has important physiological consequences. 3. Use genetic models to study the role of FSH glycoforms in the aging ovary and age-related bone loss. Under this aim, FSH hypo-glycosylated glycoforms will be evaluated by Project 3 for their ability to rescue Fshb null female mice from infertility and their effects on bone loss. Our working hypothesis is the age-related shift from FSH21 to FSH24 has deleterious effects on ovarian function, yet promotes osteoclast activation. These projects will be supported by two scientific cores. Core B will provide critical FSH glycoform preparations to all projects. Core C will assist in analyzing mass spectrometry data, provide next generation sequencing (NGS) and analyze NGS data. Together, the projects will provide new knowledge about the role of FSH glycoforms in reproductive aging that can be used to diagnose the onset of the menopausal transition, and evidence to support or refute the controversial role of FSH in bone loss in aging women.
Loss of hypo-glycosylated hFSH21, as a function of age reduces reproductive function and corresponding increase in fully-glycosylated FSH24 may accelerate bone loss. These will be studied for effects on FSH receptor binding, cellular signaling, and target tissue function in both ovary and bone. The results will address the controversial issue of the role of FSH in bone loss.
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