| Abstrakt: |
Sperm maturation, taking place during the transit of spermatozoa through the epididymis, represents a key step in the reproduction process. Spermatozoa, particularly the plasma membrane, are exposed to epididymal fluid components representing the natural environment essential for their post-testicular maturation. Changes in the sperm membrane proteins are influenced by proteolytic and glycosidic enzymes present in the epididymal fluid. Accordingly, the occurrence of inhibitors in this reproductive organ is very important for the regulation of sperm membrane protein processing. In the present study, we characterized the proteolytic and inhibiting activity in epididymal fluid of the pig.Boar epididymal fluid was separated by size exclusion chromatography and by high-performance liquid chromatography with reverse phase (RP HPLC). The protein fractions were subjected to SDS-PAGE. The separated proteins were investigated for enzymatic and proteinase inhibiting activities by substrate and reverse substrate zymographies, respectively. Additionally, we monitored gene expression and protein changes of inhibitor to acrosin along the epididymis. The occurrence and changes of acrosin inhibitor in epididymal fluid and spermatozoa was investigated using specific polyclonal antibody.By zymographic methods, we detected serine proteinases and metalloproteinases of 30, 40, 63 and 65 kDa, trypsin-like proteinase inhibitors of 6, 9, 18 and 21 kDa, and serine proteinase and metalloproteinase inhibitors with molecular masses of 12, 14, 17 and 60 kDa. The presence of acrosin inhibitor of 12 kDa and 8 kDa in epididymal fluid and spermatozoa, respectively, was proved using specific antibody. Gene expression of acrosin inhibitor increased gradually throughout the epididymal duct, from its entry to the tail. Proteinases and their inhibitors in reproductive fluids play a key role in reproduction processes and their mutual interaction helps to maintain homeostasis. Acrosin inhibitor in the reproductive tract inactivates prematurely released sperm acrosin and protects spermatozoa and reproductive epithelium against proteolytic degradation. (This work was supported by grants 303/09/1285 and 523/08/HD64 GACR, 1M0601 MSMT and 50520701 AVOZ.)(poster) |
