Urinary follicle-stimulating hormone glycoform analysis by automated western blot
AdvisorBousfield, George R.
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Follicle-stimulating hormone (FSH) is one of the glycoprotein hormones synthesized and secreted by the gonadotropic cells of the anterior pituitary gland. In females, FSH stimulates ovarian follicular development and estradiol synthesis. Human FSH exists as three major forms. The fully-glycosylated FSH24 possesses two α-subunit and two β-subunit oligosaccharides. Two hypo-glycosylated variants, FSH18 and FSH21, possess only one of the two FSHβ glycans and both of the a-subunit oligosaccharides. Human pituitary FSH21 exhibits reduced relative abundance with increasing age in women. FSH glycoform concentrations vary in serum during the human menstrual cycle. As FSH21 exhibits greater FSH biologic activity than does FSH24, ageand cycle-related changes in glycoform abundance may contribute to fertility regulation. The goal of this project was to evaluate the relative abundance of FSH glycoforms in female urine samples. Human FSH and other urinary proteins were ethanol-precipitated and immune-captured using the anti-FSHβ monoclonal antibody, 46.3H6.B7. Automated Western blotting of FSH samples was employed to measure the relative abundance of both FSH glycoforms based on the density of the 21kDa- and 24kDa-FSHb subunit bands. Preliminary studies involved antibody selection and improvements to urinary FSH purification procedures. Evaluation of six post-menopausal urine samples demonstrated consistency with glycoform abundance in previously published Western blot studies (Bousfield, Butnev et al. 2014). Evaluation of FSH in 18 of 30 urine samples obtained from post-menopausal subjects before and after estrogen treatment revealed unexpected variability in glycoform abundance. This variability prevented firm conclusions, however, the estrogen treatment had no effect on FSH secretion and did not appear to reduce FSH24 abundance.
Thesis (M.S.)-- Wichita State University, College of Liberal Arts and Sciences, Dept. of Biological Sciences