Comparison, Characterization, and Identification of Proteases and Protease Inhibitors in Epididymal Fluids of Domestic Mammals. Matrix Metalloproteinases Are Major Fluid Gelatinases1

Abstract

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.

Gelatinases in Boar Seminal Plasma and Their Relation to Semen Indicators

“…Based on previous reports (Metayer et al 2002;Shimokawa et al 2002;McCauley et al 2001;Tentes et al 2007), the bands with 225, 78 and 66 kDa correspond to dimer-MMP-9, proMMP-2 and active MMP-2. The variation in molecular weights (by 10 to 15 kDa) of gelatinases of different origins can be result of different glycosylation (Metayer et al 2002). Bands with lower molecular weights can represent the degradation products of metalloproteases with enzymatically active domain (Shimokawa et al 2002).…”

“…Although gelatinases have been detected in boar epididymal fluids (Metayer et al 2002), this is the first report of gelatinases in boar seminal plasma and their correlation with semen indicators. The influence of boar breed and age was not the aim of this study.…”

“…Seminal plasma contains many proteinases originating either from testicular cells or prostate and other accessory sex glands. Most proteinases described for seminal plasma are serine proteinases, however, proteinases from other classes are present in semen from human and domestic mammals (Metayer et al 2002).…”

“…Gelatinases have been detected in epididymal fluids of ram, stallion and boar (Metayer et al 2002). Metalloproteases, serine proteinases and serine proteinase inhibitors have been detected in turkey seminal plasma (Kotlowska et al 2005).…”

Perfil oxidativo e avaliação funcional de sêmen criopreservado de touros (Bos taurus taurus e Bos taurus indicus) criados em clima tropical

“…A porção fluida dessa suspensão é chamada de plasma seminal (MIES FILHO;BARRETO, 1949 (FOLTZ, 1995;FOUCHÉCOURT et al, 2002;MÉTAYER;GATTI, 2002 …”