PARENT SESSION
Epididymis and Sperm Maturation
301
ORIGIN AND CHARACTERIZATION OF RAM EPIDIDYMAL FLUID PROTEASES.
Gatti, Jean-Luc1, Métayer, Sonia1, Dacheux, Françoise1, Guérin, Yvon1, Dacheux, Jean-Louis1, 1
ABSTRACT- The acquisition of sperm fertilizing ability results from changes in the sperm plasma membrane during epididymal transit. Among these changes proteolytic cleavage of surface compounds have been shown, resulting in their redistribution or release in the surrounding media. Because only few studies on the proteases of the epididymal fluid have been performed, we have begun research into gelatinase activity by gel zymography. The fluid from the testis and ten different regions of the epididymis was separated by non-reducing gels containing 0.15 mg/ml gelatin. Five main proteases were revealed with molecular weights of about 35, 40, 50, 67 and 85-90 kDa and different distribution throughout the organ. The most active at 67 kDa was present from the testis to the end of the epididymis, while the 35, 40 and 85-90 kDa proteases appeared in zone 2 and their activity decreased thereafter. The 50 kDa protease was present in zone 4 and 5. The effect of specific inhibitors indicated that the 67 kDa protease was a metalloprotease and the 35 and 40 kDa proteases were serine proteases. Fluids from the mid-caput and cauda epidydimis were analyzed by two dimensional zymograms. The 50, 67 and 85-90 kDa proteases showed series of spots with pIs between 6.0 and 6.5 while the 35 and 40 kDa proteases showed a trailing pattern with pIs from 7 to 9. Use of polyclonal antibodies directed against the gelatinolytic matrix metalloproteases MMP-2 and MMP-9 indicated that they were present in the epididymal fluid of restricted zones but were not the main gelatinases observed by zymography. None of these proteases was a major protein of the ram epididymal fluid. This study demonstrated that different proteases with specific distributions exist in the epididymis and can be characterized by one and two dimensional zymography. This study may lead to the identification of these proteins and allow further analysis of their role in sperm maturation.
KEY WORDS: protease, epididymal maturation, zymography, sheep