Comparison, characterization, and identification of proteases and protease inhibitors in epididymal fluids of domestic mammals. Matrix metalloproteinases are major fluid gelatinases.
The testicular and epididymal fluids of ram, boar, and stallion were analyzed by means of one-dimensional and two-dimensional gelatin gel zymography. Five main gelatinolytic bands were revealed in the ram and at least seven were observed in the boar and stallion. These proteolytic bands showed regionalized distribution throughout the organs. The two main proteolytic activities at around 54-66 kDa retrieved in all three species were inhibited by EDTA and phenanthroline, indicating that they were metallo-dependent enzymes. The activity of some of the low-molecular-weight gelatinases was also decreased by EDTA, whereas others were inhibited by serine protease inhibitors. One of the main proteases at 60-62 kDa from the caput fluid of the stallion and the ram was N-terminal sequenced; in both cases, high sequence homology was found with the N-terminal of the matrix-metalloproteinase-2 pro-form (pro-MMP-2). Antibodies against MMP-2, MMP-3, and MMP-9 gelatinases confirmed the regional distribution in the fluids of pre -, pro-, active, or degraded forms of these metalloproteases in all three species. We also observed the presence of acrosin in epididymal fluids, which was probably released by dead spermatozoa, but this enzyme did not explain all the serine protease activity. Moreover, the majority of this enzyme is bound to the protease inhibitor alpha(2)-macroglobulin, which is present in the fluids of all three species. TIMP-2, a potent inhibitor of MMPs, was present in the fluid of the caput regions in the ram and boar, and in the caput and caudal fluids of the stallion. This study demonstrated that similar types of proteases and inhibitors are regionally distributed in the epididymal fluids of three domestic species, suggesting an identical role in the sperm maturation process, the plasticity of this organ, or both.
Métayer
Sonia
S
Equipe "Spermatozoïdes," UMR 6073 INRA-CNRS, Institut National de la Recherche Agronomique, INRA Nouzilly, 37380 Monnaie, France.
Dacheux
Françoise
F
Dacheux
Jean-Louis
JL
Gatti
Jean-Luc
JL
D000176:Acrosin / Q000032:analysis / Q000276:immunology; D000818:Animals; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D001826:Body Fluids / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000032:analysis*; D004822:Epididymis / Q000737:chemistry*; D018093:Gelatinases / Q000032:analysis*; D006736:Horses; D007120:Immunochemistry; D007525:Isoelectric Focusing; D008297:Male; D020782:Matrix Metalloproteinases / Q000032:analysis* / Q000276:immunology; D011480:Protease Inhibitors / Q000032:analysis*; D011817:Rabbits / Q000276:immunology; D012756:Sheep; D013379:Substrate Specificity; D013552:Swine; D013737:Testis / Q000737:chemistry; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000037:antagonists & inhibitors; D000511:alpha-Macroglobulins / Q000032:analysis
D000906:Antibodies; D011480:Protease Inhibitors; D000511:alpha-Macroglobulins; D019716:Tissue Inhibitor of Metalloproteinase-2; D010450:Endopeptidases; D000176:Acrosin; D018093:Gelatinases; D020782:Matrix Metalloproteinases