Hypoglycosylated hFSH Has Greater Bioactivity Than Fully Glycosylated Recombinant hFSH in Human Granulosa Cells.

Abstract

Previous studies suggest that aging in women is associated with a reduction in hypoglycosylated forms of FSH. Experiments were performed to determine whether glycosylation of the FSHβ subunit modulates the biological activity of FSH in human granulosa cells. 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. Granulosa cells were treated with increasing concentrations of fully- or hypoglycosylated FSH glycoforms for periods up to 48 hours. The main outcomes were indices of cAMP-dependent cell signaling and estrogen and progesterone synthesis. 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. 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.