Activity, substrate detection and immunolocalization of glutathione peroxidase (GPx) in bovine reproductive organs and semen.
The purpose of the current study was to further investigate the role of the antioxidant selenium-dependent enzyme glutathione peroxidase (GPx) in reproductive organs and semen from bulls. To this end a fast and convenient combined method for immune detection and substrate localization was adapted, which allows the assessment of both molecular weight and peroxidase activity of proteins on one and the same SDS-PAGE gel plate. After routine semen analysis of ejaculates, a spectrophotometrical assay of GPx activity in bovine semen was performed. For the immunological analyses performed, a rabbit polyclonal monospecific antibody against GPx was raised. Substrate detection and immunolocalization of GPx in lysates from bovine testis, epididymis, spermatozoa, and seminal plasma was performed. In order to determine the localization of GPx in spermatozoa, immunofluorescence analysis was performed. A positive correlation was established between GPx activity in semen and the number of motile spermatozoa. A negative correlation was observed between GPx activity and the number of immotile spermatozoa. The combined method for immunodetection and substrate localization was tested and proved reliable. Both tetramer and monomer forms of GPx were detected in lysates from testis, epididymis, and spermatozoa. We found no GPx activity in seminal plasma. Immunofluorescence shows the presence of GPx chiefly in the mitochondrial and in the acrosome regions of spermatozoa. GPx activity remained stable under the extreme experimental conditions.
Vaisberg
Chaika Niuma
CN
Cryobiology of Gametes Department, Institute of Biology and Immunology of Reproduction Acad. Kiril Bratanov, Bulgarian Academy of Sciences, 73 Tsarigradsko Shose Bvd., 1113 Sofia, Bulgaria. chaikavaisberg@hotmail.com
Jelezarsky
Lachezar Vasilev
LV
Dishlianova
Blagovesta
B
Chaushev
Todor Angelov
TA
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004822:Epididymis / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D005979:Glutathione Peroxidase / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D008856:Microscopy, Fluorescence; D012661:Semen / Q000201:enzymology*; D013081:Sperm Motility; D013094:Spermatozoa / Q000201:enzymology; D013737:Testis / Q000201:enzymology*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D005979:Glutathione Peroxidase