STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF HFSH AND HLH ISOFORMS

Human follicle-stimulating hormone (hFSH) and luteinizing hormone (hLH ) are gonadotropins which are secreted as multiple forms by the pituit ary. Evidence supporting the structural and functional heterogeneity o f 15 purified hFSH isoforms and 20 purified hLH isoforms from pituitar y extracts will be presented. Gonadotropin isoforms were purified by a combination of preparative isoelectric focusing and ion-exhange chrom atography. The protein mass of each isoform was determined by amino ac id analysis, which also correlated (data for hLH) (r = 0.999, P < 0.00 1, n = 15) with the UV area under the curve at 280 nm of the isoforms following gel-filtration HPLC. The alpha and beta subunits of FSH and LH were shown to be intact by SDS-PAGE under reducing conditions, with no evidence of proteolytic nicking or presence of contaminating prote ins. hFSH radioreceptor activity varied over a seven-fold range: and a positive correlation (r = 0.85, P < 0.001, n = 9) was observed betwee n FSH receptor activity and the sialic acid (SA) content (1.5-13.7 mol SA/mol hFSH) of the isoforms, as determined by an HPLC-based microflu orometric assay. FSH in vitro activities varied over a similar range w ith a high correlation (r = 0.82, n = 15) with receptor activities, su ggesting that the initial association of the hormone with the receptor is the key interaction with less differences attributed to subsequent effects in the signaling pathway. A similar result was seen with the hLH isoforms. To explore FSH/LH in vivo, the circulating half-life (LH /FSH) and the in vivo bioactivity (LH) using an acute in vivo assay wa s investigated. The clearance of hLK and hFSH showed a bi-exponential pattern for all isoform preparations with the proportion of the slower dissociating component (t(1/2) 50-60 min) increasing three-fold with increasing sialic acid content of the isoform. The more rapidly cleare d component (t(1/2) approx 10 min) is attributed to hepatically cleare d gonadotropin, rather than gonadotropin equilibration between body co mpartments. The in vivo assay procedure for LH was based on the 24 h i ntegrated plasma testosterone levels in rats following administration of graded doses of hLH isoform or standard. A 16-fold range in in vivo activities between LH isoforms (n = 14) was observed. A comparison be tween hLH in vitro and in vivo activities showed a good correlation (r = 0.75) with the slope of the regression line (1.39) not significantl y different from unity. These results suggest that in this acute in vi vo assay method, the differences in circulating half-lives between hLH isoforms although large is not a key factor in their in vivo activiti y. However, in chronic in vivo assay systems the differences in cleara nce rates between isoforms may be important in their subsequent biolog ical reponse. It is concluded that structural heterogeneity of FSH and LH contributes to functional differences, with a key interaction occu rring at the receptor level. The contribution of sialic acid to these activities was also investigated. Copyright (C) 1996 Elsevier Science Ireland Ltd.