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 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 approximately 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.
McCauley
T C
TC
Department of Animal Sciences, University of Arizona, Tucson, Arizona 85721-0038, USA.
Zhang
H M
HM
Bellin
M E
ME
Ax
R L
RL
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D005298:Fertility; D006493:Heparin / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000737:chemistry* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012661:Semen / Q000737:chemistry*; D016415:Sequence Alignment; D013094:Spermatozoa / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D011480:Protease Inhibitors; D019716:Tissue Inhibitor of Metalloproteinase-2; D006493:Heparin