Effect of FSH and its Glycosylation Variants on Proliferation, Steroid and Inhibin Production in a Human Ovarian Granulosa-Like Tumor Cell Line (KGN).

KGN is a steroidogenic human ovarian granulosa-like tumor cell line that expresses functional FSH receptor; in these cells steroid production is stimulated by FSH. This glycoprotein is released from the pituitary gland as a mixture of glycosylation variants that act on the target cells inducing different biological responses in vitro and in vivo. The aim of the present study was to determine the effect of recombinant human FSH (rhFSH) and its glycosylation variants on proliferation and estradiol (E2), progesterone (P4), monomeric (Pro-alphaC) and dimeric inhibin (inhibin A and B) production. Preparative isoelectrofocusing was used to isolate rhFSH charge analogues in a pH range of 2.6-7.5. Two preparations were obtained by combining pH 3 to 4 (more acidic/sialylated, AC) and pH 5 to 7 (less acidic/sialylated, BA) fractions. Concanavalin-A chromatography was used to isolate rhFSH glycosylation variants on the basis of glycan complexity. Two preparations were obtained: unbound rhFSH isoforms (UB) bearing complex, highly branched carbohydrate chains and firmly bound rhFSH isoforms (FB) bearing hybrid type oligosaccharides. Cells were cultured on 24-well plate treated with or without rhFSH or its glycosylation variants (20 ng/mL) for 24h or 72h; E2 and P4 (24h) were determined by RIA and inhibins (72h) by specific ELISAs. [Methyl-3H]- thymidine incorporation was used as a measure of proliferative activity (24h). Under basal conditions KGN cells were able to produce E2 in the presence of androstenedione (100nM), P4 in the absence of a steroidogenic substrate (25-OH-Cholesterol), Pro-alphaC and inhibin A. Inhibin B was not detected. There was a direct correlation between basal P4 production and cell density. This condition did not affect either E2 or inhibin production. rhFSH significantly stimulated the production of E2, P4, Pro-alphaC, inhibin A and the incorporation of thymidine. Less acidic/sialylated rhFSH was more biopotent to stimulate the production of E2 and inhibins than its more sialylated counterpart. Progesterone production was not significantly affected by the degree of sialylation of the hormone. UB isoforms showed lower biopotency to stimulate steroids and inhibin production when compared to FB isoforms. These glycosylation variants, bearing hybrid type oligosaccharides, exerted a potent stimulatory effect on E2, P4, Pro-alphaC and inhibin A production. These results suggest that the degree of sialylation as well as the complexity of carbohydrate chains present in rhFSH molecules may be considered as additional factors that differentially regulate KGN cells function. (Supported by CONICET, PIP 5479 and FONCYT, BID-1728 OC/AR PICT 2004, N° 25365, Argentina) (poster)