Identification of insulin-like growth factor-binding protein-3 (IGFBP-3) fragments and IGFBP-5 proteolytic activity in human seminal plasma: a comparison of normal and vasectomized patients.

Previous studies have demonstrated that insulin-like growth factor (IGF) peptides, IGF-binding proteins (IGFBPs), and IGFBP-3 proteolytic activity, are present in human seminal plasma (SP). In this study, we have further characterized the IGFBPs in SP using immunoprecipitation and Western ligand blotting, Western immunoblotting, affinity cross-linking and immunoprecipitation, and RIA of IGFBP-3 using two different assays and have identified additional proteolytic activities for IGFBP-4 and IGFBP-5 in SP. Immunoprecipitation with antibodies to IGFBP-2, IGFBP-3, and IGFBP-4, before and after affinity cross-linking, demonstrated that intact IGFBP-2 and IGFBP-4 are present in SP, but intact IGFBP-3 is absent. Low mol wt fragments of IGFBP-3, which did not bind to IGF-I or IGF-II on Western ligand blot and did not cross-link to IGF-II, were demonstrated on Western immunoblot and were measurable by two different RIAs. Proteolytic activities for IGFBP-4 and IGFBP-5 were demonstrated in SP by incubation with the respective iodinated IGFBPs. On comparing the proteolytic activity for IGFBP-4 by purified prostate-specific antigen (PSA; a known IGFBP-3 protease in SP) or by SP with measured equivalent concentrations of PSA, the dose response and fragment patterns were identical. With IGFBP-5, however, proteolysis by purified PSA was different from that by SP with measured equivalent concentrations of PSA: 1) proteolysis by pure PSA was less efficient than matched concentrations of SP; 2) the pattern of fragments after proteolysis by pure PSA was different from that after proteolysis by matched concentrations of SP; and 3) proteolysis by purified PSA was significantly inhibited by phenylmethylsulfonylfluoride and aprotinin, but proteolysis by SP was not. We conclude that human SP contains intact IGFBP-2 and IGFBP-4, but has only IGFBP-3 fragments with low affinity for IGF peptides; that PSA is able to proteolyze IGFBP-4 and IGFBP-5 (as well as IGFBP-3); and that an additional IGFBP-5 protease is probably present in SP. There was no significant difference in any of these findings in SP from normal volunteers, vasectomized patients, or patients with idiopathic azoospermia. The roles of IGFBPs and IGFBP proteases in the male reproductive system and male infertility remain to be further elucidated.