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

• Published in: Molecular reproduction and development Vol. 58; no. 3; pp. 336 - 341
• Main Authors: McCauley, T.C., Zhang, H.M., Bellin, M.E., Ax, R.L.
• Format: Journal Article
• Language: English
• Published: New York John Wiley & Sons, Inc 01.03.2001; Wiley-Liss
• Subjects: accessory sex gland; Amino Acid Sequence; Animals; Biological and medical sciences; Blotting, Western; Cattle; Fertility; Fundamental and applied biological sciences. Psychology; Heparin - metabolism; Male; Mammalian male genital system; matrix metalloproteinase; Molecular Sequence Data; Morphology. Physiology; Protease Inhibitors - chemistry; Protease Inhibitors - metabolism; Reverse Transcriptase Polymerase Chain Reaction; Semen - chemistry; Sequence Alignment; Spermatozoa - metabolism; Tissue Inhibitor of Metalloproteinase-2 - chemistry; Tissue Inhibitor of Metalloproteinase-2 - genetics; Tissue Inhibitor of Metalloproteinase-2 - metabolism; Vertebrates: reproduction; Spermatozoa; Enzyme; Enzyme inhibitor; Metalloendopeptidases; Ox; Male genital duct; Germinal cell; Gene expression; Male genital system; Gelatinase A; Peptidases; Binding protein; Vertebrata; Epididymis; Biological fixation; Mammalia; Fertility; Accessory sex organ; Hydrolases; Artiodactyla; Heparin; Seminal plasma; Ungulata
• ISSN: 1040-452X; 1098-2795
• DOI: 10.1002/1098-2795(200103)58:3<336::AID-MRD12>3.0.CO;2-Z

Presence or absence of three distinct bovine seminal heparin‐binding proteins (21–31 kDa) recognized in sperm extracts by a monoclonal antibody, M1, is a diagnostic indicator of fertility differences among bulls producing normal semen. We recently identified a 31 kDa fertility‐associated antigenin bovine seminal fluid as a unique DNase I‐like protein. We now report purification 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% identity 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 proteins and subsequent washing in vitro, indicating TIMP‐2 bound to sperm membranes. Expression of TIMP‐2 mRNA was detected by RT‐PCR in bovine bulbourethral gland, prostate, and seminal vesicles. Mobility of the 24 kDa heparin‐binding protein increased under nonreducing SDS‐PAGE to ∼ 21 kDa, characteristic of the reported molecular mass of TIMP‐2. To our knowledge, 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 previous reports regarding the site of production of heparin‐binding proteins that are related to bull fertility, and suggest that TIMP‐2 influences fertility of bulls, either through inhibition of metalloprotease activity in semen or via undefined activities independent of matrix metalloproteinase (MMP) inhibition. Mol. Reprod. Dev. 58:336–341, 2001. © 2001 Wiley‐Liss, Inc.