POLYMORPHISM OF HUMAN PITUITARY FSH - ANALYSIS OF IMMUNOREACTIVITY AND IN-VITRO BIOACTIVITY OF DIFFERENT MOLECULAR-SPECIES

Follicle-stimulating hormone is known to be highly heterogeneous in se rum and in the pituitary. In the present study, we have partially sepa rated different molecular species of human pituitary FSH and character ized their immunoreactivity and in vitro bioactivity. Pooled extracts of male (n=15) and female (n=9) human pituitary glands were chromatogr aphed on a column of Sephacryl S-200 and FSH-containing fractions were fractionated by chromatofocusing in the pH range 4-6. FSH was measure d in the individual fractions by an in vitro bioassay, based on the FS H-dependent aromatase activity of immature rat Sertoli cells, and by t he following methods based on commercial kits: radioimmunoassay (RIA), immunofluorimetric assay (IFMA), immunoradiometric assay (IRMA), immu noenzymometric assay (IEMA). In each assay, the kit standard, calibrat ed against the 2nd International Reference Preparation (IRP) 78/549, a nd the International Standard (IS) 83/575 were run in parallel. The re lative potencies of the kit standards in terms of IS 83/575 were: IFMA 3.08, IRMA 1.62, RIA 2.42, IEMA 1.45 and bioassay 1.14. After chromat ofocusing, pituitary FSH eluted mostly in fractions with pH approximat e to 4.5, without sex-related differences. In both sexes approximate t o 25% of bioactive material showed a pI<4 and eluted with 1 M NaCl. Al though the same IS 83/575 was used in the various assays, the profiles of immunoreactive FSH were significantly different. The highest inter method variability was observed in the case of male pituitary FSH. The relative biopotency of the different molecular species of FSH did not appear to change according to their pI but, rather, varied with the a ssay method and the standard. In terms of 2nd IRP 78/549 the activity profiles were similar but not identical to those obtained in terms of IS 83/575 and the ratios between the two values (IS:IRP ratios) were s ignificantly different among the methods. No significant correlation w as found between IS:IRP ratios and FSH concentrations or pH. These dat a suggest that: (1) all the methods have different affinities for stan dard and unknown and/or for different molecular isoforms of FSH; (2) o verall, they perform more accurately when assaying female pituitary FS H, perhaps because of a closer resemblance between standard and unknow n; (3) the variability of the IS:IRP ratios indicates a different affi nity of the antibodies for IS 83/575 and the kit standard, highlightin g the importance of the molecular composition of the reference prepara tions for the final result; and (4) the results do not support the com monly accepted concept of a higher in vitro biopotency of less acidic species of pituitary FSH.