Identification of a heparin-binding protein in bovine seminal fluid as tissue inhibitor of metalloproteinases-2

Presence or absence of three distinct bovine seminal heparin-binding protei ns (21-31 kDa) recognized in sperm extracts by a monoclonal antibody, M1, i s a diagnostic indicator of fertility differences among bulls producing nor mal semen. We recently identified a 31 kDa fertility-associated antigen in bovine seminal fluid as a unique DNase I-like protein. We now report purifi cation and identification of a 24 kDa seminal heparin-binding protein (HBP- 24) recognized by M1. N-terminal microsequence analysis of HBP-24 purified from seminal fluid yielded 20 amino acid residues that displayed 90% identi ty to the N-terminus of a bovine metalloproteinase inhibitor identified as tissue inhibitor of metalloproteinases-2 (TIMP-2). A single immunoreactive band migrating at 24 kDa was detected in Western blots of cauda epididymal sperm extracts following incubation with purified seminal heparin-binding p roteins and subsequent washing in vitro, indicating TIMP-2 bound to sperm m embranes. Expression of TIMP-2 mRNA was detected by RT-PCR in bovine bulbou rethral gland, prostate, and seminal vesicles. Mobility of the 24 kDa hepar in-binding protein increased under nonreducing SDS-PAGE to similar to 21 kD a, characteristic of the reported molecular mass of TIMP-2. To our knowledg e, this is the first report of TIMP-2 binding to spermatozoa and of TIMP-2 mRNA expression in bovine accessory sex glands. These results corroborate p revious reports regarding the site of production of heparin-binding protein s that are related to bull fertility, and suggest that TIMP-2 influences fe rtility of bulls, either through inhibition of metalloprotease activity in semen or via undefined activities independent of matrix metalloproteinase ( MMP) inhibition. (C) 2001 Wiley-Liss, Inc.