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 (IG F) peptides, IGF-binding proteins (IGFBPs), and IGFBP-3 proteolytic ac tivity, are present in human seminal plasma (SP). In this study, we ha ve further characterized the IGFBPs in SP using immunoprecipitation an d Western ligand blotting, Western immunoblotting, affinity cross-link ing and immunoprecipitation, and RIA of IGFBP-3 using two different as says and have identified additional proteolytic activities for IGFBP-4 and IGFBP-5 in SP. Immunoprecipitation with antibodies to IGFBP-2, IG FBP-3, and IGFBP-4 before and after affinity cross-linking, demonstrat ed 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 I GF-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 d ifferent RIAs. Proteolytic activities for IGFBP-4 and IGFBP-5 were dem onstrated in SP by incubation with the respective iodinated IGFBPs. On comparing the proteolytic activity for IGFBP-4 by purified prostate-s pecific antigen (PSA; a known IGFBP-3 protease in SP) or by SP with me asured equivalent concentrations of PSA, the dose response and fragmen t patterns were identical. With IGFBP-4 however, proteolysis by purifi ed PSA was different from that by SP with measured equivalent concentr ations of PSA: 1) proteolysis by pure PSA was less efficient than matc hed concentrations of SP; 2) the pattern of fragments after proteolysi s by pure PSA was different from that after proteolysis by matched con centrations of SP; and 3) proteolysis by purified PSA was significantl y inhibited by phenylmethylsulfonylfluoride and aprotinin, but proteol ysis by SP was not. We conclude that human SP contains intact IGFBP-5 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 pres ent in SP. There was no significant difference in any of these finding s in SP from normal volunteers, vasectomized patients, or patients wit h idiopathic azoospermia. The roles of IGFBPs and IGFBP proteases in t he male reproductive system and male infertility remain to be further elucidated.