Chao Jiang1, Xiaoying Hou1, Cheng Wang1, Jeffrey V May1, Viktor Y Butnev1, George R Bousfield1, John S Davis1. 1. The Olson Center for Women's Health, Department of Obstetrics and Gynecology (C.J., X.H., C.W., J.S.D.), University of Nebraska Medical Center, Omaha, Nebraska 68198; Nebraska-Northwest Iowa VA Medical Center (X.H., J.S.D.), Omaha, Nebraska 68105; Department of Biological Sciences (J.V.M., V.Y.B., G.R.B.), Wichita State University, Wichita, Kansas 67260; and Department of Biochemistry and Molecular Biology (J.S.D.), University of Nebraska Medical Center, Omaha, Nebraska 68198.
Abstract
CONTEXT: Previous studies suggest that aging in women is associated with a reduction in hypoglycosylated forms of FSH. OBJECTIVE: Experiments were performed to determine whether glycosylation of the FSHβ subunit modulates the biological activity of FSH in human granulosa cells. DESIGN AND SETTING: Recombinant human FSH (hFSH) derived from GH3 pituitary cells was purified into fractions containing hypoglycosylated hFSH(21/18) and fully glycosylated hFSH(24). The response to FSH glycoforms was evaluated using the well-characterized, FSH-responsive human granulosa cell line, KGN at an academic medical center. INTERVENTIONS: Granulosa cells were treated with increasing concentrations of fully- or hypoglycosylated FSH glycoforms for periods up to 48 hours. MAIN OUTCOME MEASURE(S): The main outcomes were indices of cAMP-dependent cell signaling and estrogen and progesterone synthesis. RESULTS: We observed that hypoglycosylated FSH(21/18) was significantly more effective than fully glycosylated FSH(24) at stimulating cAMP accumulation, protein kinase A (PKA) activity, and cAMP response element binding protein (CREB) (S133) phosphorylation. FSH(21/18) was also much more effective than hFSH(24) on the stimulation CREB-response element-mediated transcription, expression of aromatase and STAR proteins, and synthesis of estrogen and progesterone. Adenoviral-mediated expression of the endogenous inhibitor of PKA, inhibited FSH(21/18)- and FSH(24)-stimulated CREB phosphorylation, and steroidogenesis. CONCLUSIONS: Hypoglycosylated FSH(21/18) has greater bioactivity than fully glycosylated hFSH(24), suggesting that age-dependent decreases in hypoglycosylated hFSH contribute to reduced ovarian responsiveness. Hypoglycosylated FSH may be useful in follicle stimulation protocols for older patients using assisted reproduction technologies.
CONTEXT: Previous studies suggest that aging in women is associated with a reduction in hypoglycosylated forms of FSH. OBJECTIVE: Experiments were performed to determine whether glycosylation of the FSHβ subunit modulates the biological activity of FSH in human granulosa cells. DESIGN AND SETTING: Recombinant human FSH (hFSH) derived from GH3 pituitary cells was purified into fractions containing hypoglycosylated hFSH(21/18) and fully glycosylated hFSH(24). The response to FSH glycoforms was evaluated using the well-characterized, FSH-responsive human granulosa cell line, KGN at an academic medical center. INTERVENTIONS: Granulosa cells were treated with increasing concentrations of fully- or hypoglycosylated FSH glycoforms for periods up to 48 hours. MAIN OUTCOME MEASURE(S): The main outcomes were indices of cAMP-dependent cell signaling and estrogen and progesterone synthesis. RESULTS: We observed that hypoglycosylated FSH(21/18) was significantly more effective than fully glycosylated FSH(24) at stimulating cAMP accumulation, protein kinase A (PKA) activity, and cAMP response element binding protein (CREB) (S133) phosphorylation. FSH(21/18) was also much more effective than hFSH(24) on the stimulation CREB-response element-mediated transcription, expression of aromatase and STAR proteins, and synthesis of estrogen and progesterone. Adenoviral-mediated expression of the endogenous inhibitor of PKA, inhibited FSH(21/18)- and FSH(24)-stimulated CREB phosphorylation, and steroidogenesis. CONCLUSIONS:Hypoglycosylated FSH(21/18) has greater bioactivity than fully glycosylated hFSH(24), suggesting that age-dependent decreases in hypoglycosylated hFSH contribute to reduced ovarian responsiveness. Hypoglycosylated FSH may be useful in follicle stimulation protocols for older patients using assisted reproduction technologies.
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