Identification of bioneutralization epitopes of human follicle stimulating hormone in the regions 31-52 and 66-75 of its beta-subunit.

The crucial role played by follicle stimulating hormone (FSH) in regulating both male and female reproduction and the possibilities of developing contraceptive methods for males by blocking the function of the hormone, makes it important to delineate the hormone-specific bioneutralization epitopes of human follicle stimulating hormone (hFSH) on its beta-subunit. Predictive methods were used to identify the potential surface-oriented regions of hFSH-beta. Peptides corresponding to these regions, i.e. 31-52, 66-75 and 86-95 hFSH-beta, were synthesized, anti-peptide antibodies were elicited in rabbits and the properties of these antisera to bind hFSH and neutralize its biological activity were assessed. Anti-31-52 hFSH-beta antisera bound hFSH specifically, whereas anti-66-75 and anti-86-95 hFSH-beta antisera did not show any detectable binding, proving the region 31-52 hFSH-beta to be a specific antigenic determinant of hFSH. The bioneutralizing abilities of the anti-peptide antibodies were assessed by measuring the hFSH-induced progesterone secretion by rat granulosa cells in vitro. Antibodies to 31-52 and 66-75 hFSH-beta neutralized the bioactivity of hFSH, but anti-86-95 hFSH-beta antibodies did not. Furthermore, the three linear peptides and two disulphide looped peptides of 31-52 hFSH-beta and 86-95 hFSH-beta were also subjected to the in-vitro granulosa cell assay. The linear peptides 31-52 hFSH-beta and 66-75 hFSH-beta and the cyclic 31-52 hFSH-beta disulphide loop peptide significantly inhibited the hFSH-induced progesterone secretion by rat granulosa cells, but the linear 86-95 hFSH-beta peptide and the corresponding cyclic disulphide loop peptide did not. The results clearly show that the regions 31-52 and 66-75 of hFSH-beta harbor bioneutralization epitopes of the hormone. The studies also indicate that cyclization of the linear 31-52 hFSH-beta peptide greatly enhances receptor recognition and that the region 66-75 hFSH-beta may also be involved in hormone-receptor interaction.