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

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.

Figure 1.

Human FSH glycoforms used in these studies. A, Fully glycosylated, recombinant GH₃-hFSH²⁴, which possesses glycans at all four N-glycosylation sites. B, Hypoglycosylated GH₃-hFSH²¹, which lacks Asn²⁴ N-glycan. C, Hypoglycosylated GH₃-hFSH¹⁸, which lacks Asn⁷ glycan. The glycan diagrams depict abundant glycans found in the nano-electrospray ionization mass spectrometry analysis of GH₃-hFSH glycans using the Oxford Glycobiology Institute system (38, 39). D, Western blot comparing reduced, 1-μg samples of recombinant GH₃-hFSH glycoforms with commercially available recombinant hFSH and pituitary hFSH. The primary antibody was RFSH20, diluted 1:5000. Lane 1, MW markers, as indicated; lane 2, Follistim; lane 3, Gonal f; lane 4, hypoglycosylated GH₃-hFSH^21/18 mixture; lane 5, GH₃-hFSH²⁴; lane 6, pituitary hFSH (AFP7298A); lane 7, MW markers.

Figure 2.

FSH^21/18 is more effective than FSH²⁴ at stimulating cAMP accumulation and PKA activation in human KGN granulosa cells. Granulosa cells were treated for 30 minutes with increasing concentrations of FSH^21/18 or FSH²⁴. A, cAMP levels in the medium were measured using a cAMP chemiluminescent immunoassay kit as described in the Methods. Results are means ± SEM; n = 3 separate experiments. *, P < .05 vs control. **, P < .05, FSH^21/18 vs FSH²⁴. B, Representative Western blot analysis of FSH-induced phosphorylation of PKA-substrates using an antibody directed against the PKA-consensus phosphorylation site. Actin served as a loading control.

Figure 3.

FSH^21/18 is more effective than FSH²⁴ at stimulating CREB phosphorylation in human KGN granulosa cells. A, Granulosa cells were treated for 30 minutes with increasing concentrations of FSH^21/18 or FSH²⁴. Cell lysates were prepared and Western blot analysis performed using phospho-Ser 133-CREB (P-CREB), CREB, and β-actin antibodies. Bars represent means ± SEM; n = 3 separate experiments. *, P < .05 vs control. **, P < .05, FSH^21/18 vs FSH²⁴. B, Granulosa cells were treated for up to 120 minutes with 30 ng/mL FSH^21/18 or FSH²⁴. Shown is a representative Western blot analysis of the time course of FSH-induced phosphorylation of P-CREB and PKA-substrates. Levels of β-actin protein served as a loading control

Figure 4.

FSH^21/18 is more effective than FSH²⁴ at stimulating CRE-mediated transcription. A, Granulosa cells were treated for 30 minutes with 0, 10, and 100 ng/mL of either FSH²¹ or FSH²⁴. The number of phospho-CREB-positive nuclei were quantified and expressed as a percent of the total cells counted in each experiment. Bars represent means ± SEM; n = 3 separate experiments. *, P < .05 vs control. B, Granulosa cells were infected with an adenovirus expressing a CRE-luciferase reporter (CRE-Luc) or green fluorescent protein as a control. After 24 hours, granulosa cells were treated for 6 hours with 0, 10, or 100 ng/mL FSH^21/18 or FSH²⁴. Relative CRE-luciferase activity (rlu) was determined as described in the Methods. Bars represent means ± SEM; n = 3 separate experiments. *, P < .05 vs control. **, P < .05, FSH^21/18 vs FSH²⁴. ns, not significant.

Figure 5.

FSH^21/18 is more effective than FSH²⁴ at stimulating estradiol and progesterone synthesis in human KGN granulosa cells. Granulosa cells were treated for 48 hours with control medium (C) or increasing concentrations (1–100 ng/mL) of FSH^21/18 or FSH²⁴. A and B, Conditioned medium was collected and estradiol and progesterone levels were measured as described in the Methods. Symbols represent means ± SEM; n = 3 experiments. *, P < .05 vs control. **, P < .05, FSH^21/18 vs FSH²⁴. C, Cell lysates were prepared and Western blot analysis of aromatase (CYP19), STAR, and β-actin proteins was performed.