INSULIN-LIKE GROWTH-FACTOR (IGF)-I AND (IGF)-II, IGF-BINDING PROTEINS, AND IGF-BINDING PROTEIN PROTEASES ARE PRODUCED BY THECA AND STROMA OF NORMAL AND POLYCYSTIC HUMAN OVARIES

There is increasing evidence for an important regulatory role for the insulin-like growth factor (IGF) system in the human ovary. IGF-I and -II and IGF-binding proteins (IGFBPs)-1 to -4 have been identified by analysis of follicular fluid and granulosa cell-conditioned medium and by in situ hybridization and Northern and dot blot analyses of ovaria n tissues. It has been suggested that abnormalities of intraovarian IG F-I or IGFBPs may play a part in the pathogenesis of polycystic ovary syndrome. The aim of this study was to identify production of IGF-I an d II and IGFBP-1 to -4 by unstimulated normal and polycystic ovaries. IGF-I and -II were measured by RIA after acid-gel exclusion chromatogr aphy in medium conditioned by incubation for 48 h with granulosa cells or explants of theca or stroma. Both IGF-I and -II were present in th e low nanograms per mt range in theca- and stroma-conditioned medium ( T+SCM). IGFBPs in T+SCM were initially analyzed by Western Ligand blot ting, which revealed that low mol wt IGFBPs were predominant, especial ly IGFBP-2 (35 kDa). There was a band corresponding to 26 kDa with sma ller amounts of a 31-kDa band, but only a trace of IGFBP-3 (44 and 40 kDa, confirmed by immunoblot). We found no consistent differences betw een normal and polycystic ovary syndrome ovaries, and although there w as a trend toward increased IGFBP accumulation in response to LH, this was not consistent. We were unable to detect IGFs or IGFBPs by Wester n ligand blotting in granulosa cell-conditioned medium. In further stu dies we attempted to measure IGFBP-3 by RIA using two different antise ra (alpha-BP-3gl and 1287-2-14) that detect different epitopes of IGFB P-3 and allow the presence of proteolytic activity to be demonstrated. Results obtained using alpha-BP-3gl were lower than those using 1287- 2-14, suggesting proteolysis of IGFBP-3 in the medium. There was no ev idence of proteolysis of serum IGFBP-3 after incubation with condition ed medium, but in contrast, radiolabeled [I-125]IGFBP-3 was cleaved af ter incubation with T+SCM. Immunoblotting revealed intact IGFBP-2 (35 kDa) and bands of various sizes between 16-33 kDa. Immunoreactive frag ments of IGFBP-3 between 13-40 kDa were seen. In conclusion, T+SCM con tained IGF-I and -II. IGFBP-2 and -4 were the predominant species of I GFBP in T+SCM. T+SCM also contained significant protease activity dire cted toward IGFBP-2 and -3. Proteolytic activity may be an important m echanism by which bioactive IGFs are made available to these tissues.