Effect of sialylation and complexity of FSH oligosaccharides on inhibin production by granulosa cells

Abstract

Granulosa cell (GC) inhibin A and B production is regulated by FSH and gonadal factors. This gonadotrophin is released as a mixture of glycoforms, which induce different biological responses in vivo and in vitro. Our aim was to determine the effect of recombinant human FSH (rhFSH) glycosylation variants on inhibin A and B production by rat GCs. Preparative isoelectro focusing was used to isolate more acidic/sialylated (pH !4.00) and less acidic/sialylated (pH O5.00) rhFSH charge analogues. Concanavalin A was used to isolate unbound and firmly bound rhFSH glycoforms on the basis of their oligosaccharide complexity. GCs, obtained from oestrogen-primed immature rats, were cultured with either native rhFSH or its glycosylation variants. Inhibin A and B were determined using specific ELISAs. Results were expressed as meanGS.E.M. Under basal conditions, inhibin A was the predominant dimer produced (inhibin A: 673G55; inhibin B: 80G4 pg/ml). More acidic/sialylated charge analogues stimulated inhibin B production when compared to inhibin A at all doses studied; by contrast, less acidic/sialylated charge analogues stimulated inhibin A production and elicited no effect on inhibin B. Glycoforms bearing complex oligosaccharides showed a potent stimulatory effect on inhibin B when compared to inhibin A production (i.e. dose 1 ng/ml: 4.9G0.5 vs 0.9G0.1-fold stimulation, P!0.001). Glycoforms bearing hybrid-type oligosaccharides favoured inhibin A production (i.e. dose 4 ng/ml 2.9G0.1 vs 1.6G0.1-fold stimulation, P!0.05). These results show that the sialylation degree as well as the complexity of oligosaccharides present in the rhFSH molecule may be considered additional factors that differentially regulate dimeric inhibin production by rat GCs.

Comparative pharmacology of a new recombinant FSH expressed by a human cell line

“…In addition to inducing progesterone production to similar levels, both rFSH proteins increased 3B-HSD expression, the enzyme responsible for the conversion of pregnenolone to progesterone (26). The modest induction of CYP19a1 gene expression by both rFSH proteins is consistent with previous reports in granulosa cells (27, 28, 29) as was the induction of both INHA gene expression and inhibin A protein secretion (30). The lack of increase in estrogen and inhibin B in these studies is consistent with the stage at which the granulosa cells were collected, after patients had been through controlled ovarian stimulation and triggering of final oocyte maturation, shifting them from follicular gene expression and hormone production toward luteal phase gene expression (31).…”

A human FSHB transgene encoding the double N-glycosylation mutant (Asn7Δ Asn24Δ) FSHβ subunit fails to rescue Fshb null mice

“…Micro-heterogeneity in glycoprotein hormones contributed by terminal sugar residues has been extensively studied (Creus et al, 2001, 1996; Loreti et al, 2013a,b). Over the past few years our group has identified and biochemically characterized several naturally occurring hypo-glycosylated FSH glycoforms resulting from macro-heterogeneity contributed by presence or absence of individual N-linked glycans on FSHβ subunit (Bousfield et al, 2007; Davis et al, 2014; Walton et al, 2001; Bousfield et al, 2014a,b; Butnev et al, 2015).…”

Influence of gonadotropins on ovarian follicle growth and developmentin vivoandin vitro

“…These isoforms have a smaller half-life period, indicating the existence of regulatory mechanisms that control the intensity and duration of FSH signal exposure (Macklon et al ., 2006). Less sialylated forms of FSH more actively stimulate estradiol and progesterone production by granulosa cells than the acidic isoforms (Loreti et al ., 2013b).…”