Project 1: Functional Analysis of Age-Specific FSH Analogs Using Genetically Altered Mice

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PROJECT SUMMARY/ABSTRACT – Project 1(Kumar) The long-term goal of Project 1 is to study the mechanisms of follicle-stimulating hormone (FSH) actions in gonads and extra-gonadal tissues in the aging female. Both the FSH subunits are glycosylated with two N-linked sugar chains on each subunit. It is known that glycosylation of FSH is estrous/menstrual cycle- and age-specific. Previous studies identified FSH variants consisting of 2 sugar chains on the α but either one or none on the β subunit. These variants are known as hypoglycosylated FSH glycoforms and designated as FSH21, and FSH18, in contrast to the fully glycosylated FSH24. Most importantly, the ratio of hypo- (FSH21 or FSH18) to fully- glycosylated FSH forms (FSH24) is age-dependent, with high levels of FSH21/18 glycoforms predominant in young age for optimal ovarian function and high levels of FSH24 predominantly present in peri/post-menopausal women and may contribute to the aging-associated bone loss. However, the distinct in vivo biological functions of these changing ratios of FSH glycoforms in vivo are unknown in ovarian and bone physiology in aging. Moreover, whether the documented FSH actions on bone are mediated via the bona-fide ovarian FSH receptors has never been tested. The central hypothesis is that estrogen regulates an age-dependent glycosylation switch on FSH and the resulting shift in higher abundance of fully-glycosylated FSH24 mediates bone-specific actions via FSH receptors on osteoclasts. This hypothesis will be tested using genetically engineered novel mouse models. In Aim 1, we will purify GFP-tagged gonadotropes from young and old age female mouse pituitaries by flow sorting and perform RNA-Seq analysis to identify age-specific and estrogen-dependent glycosylation pathway enzymes. Additionally, we will introduce an HFSHB24 transgene onto gonadotrope-specific Esr1 conditional null background and determine whether loss of estrogen signaling leads to changes in abundance of FSH glycoforms. In Aim 2, we will engineer Fshb null mice expressing either a FSH18 or a FSH21 hypo- glycosylated form and a Tet-inducible HFSHB24 transgene. This genetic strategy will allow us to temporally regulate at desired times, the age-specific changing ratios of FSH glycoforms and test systematically the in vivo biological actions of changing ratios of FSH glycoforms in ovarian physiology in the absence of endogenous mouse FSH. In Aim 3, we will selectively delete Fshr in osteoclasts by a Cre-lox genetic approach to unequivocally test the direct cell-autonomous actions of FSH in osteoclasts in the bone. Successful completion of Project 1 studies may unravel a novel phenomenon of age-dependent N-glycosylation switch on FSH resulting in alterations in target tissue specificity (ovary versus bone) and may potentially lead to new therapeutic options for preserving ovarian function and intervention of bone loss in aging women. The three Aims of Project 1 are well integrated into the overall P01 which tests the central hypothesis that in the face a senescing ovary, the switch from hypo-glycosylated to fully-glycosylated FSH, compromises reproductive potential and contributes to other physiological changes, such as bone loss.