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Flow cytometric analysis of enzymes in live spermatozoa before and after cryostorage
Author(s) -
Schaller J.,
Glander H.J.
Publication year - 2000
Publication title -
andrologia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.633
H-Index - 59
eISSN - 1439-0272
pISSN - 0303-4569
DOI - 10.1046/j.1439-0272.2000.00362.x
Subject(s) - esterase , biochemistry , collagenase , subtilisin , enzyme , biology , cathepsin g , microbiology and biotechnology , aminopeptidase , fluorescein , elastase , fluorescence , amino acid , leucine , physics , quantum mechanics
Synthetic fluorogenic substrates, like the CellProbe TM reagents, can determine enzymes in vital human spermatozoa. These substrates will enter the cells without previous cell permeabilization and exhibit fluorescence after cleavage depending on enzyme activity. They consist of different peptide sequences, specific for the enzymes, and a fluorescein‐ or rhodamine 110‐dye moiety. The number of positive cells and the intensity of the fluorescence can be determined by flow cytometric analysis. We investigated several enzymes (peptidases, proteinases, esterases, elastases and collagenases) in intact spermatozoa before and after cryoprotection. Semen samples with normal spermiogram parameters were cryoprotected using the freezing medium TEST yolk buffer (TYB). Fresh spermatozoa showed a marked fluorescence after incubation with the synthetic substrates for the aminopeptidase M, butyryl esterase, fluorescein diacetate (FDA)‐and FDA/sodium fluoride (NAF)‐esterase, ala‐ala‐pro‐val (AAPV)‐elastase, gly pro‐leu‐gly pro‐(GPLGP)‐collagenase, gly gly leu‐(GGL)‐subtilisin as well as lys‐ala‐(LA)‐dipeptidyl peptidase (DPP) II. After cryopreservation the spermatozoal fluorescence increased applying substrates for butyryl esterase ( P <0.05), prolyl‐aminopeptidase ( P <0.001) and val‐lys‐(VK)‐cathepsin ( P <0.001) most probably due to elevated enzyme activities. The activities of FDA‐esterase ( P <0.05) and FDA/NAF‐esterase ( P <0.05), AAPV‐elastase ( P <0.01), GPLGP‐collagenase ( P <0.05) and GGL‐subtilisin ( P <0.001) decreased after cryopreservation. The substrates for arg‐gly glut‐ser‐(RGES)‐elastase, gly phenyl‐gly ala‐(GFGA)‐collagenase and threo‐pro‐(TP)‐cathepsin were not cleaved before as well as after cryostorage. The substrates for subtilisin and DPP may play a prominent role in andrology, because subtilisin is a serine protease like acrosin and DPP mediates cell–cell‐interactions. The present results demonstrate for the first time alterations of enzyme patterns in spermatozoa after cryopreservation without disrupting cellular integrity, which is commonly performed by conventional enzyme analysis.