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The thiol–disulfide status in proteins of human spermatozoa categorized as normozoospermic, teratozoospermic and asthenozoospermic was examined. Washed spermatozoawere incubated with or without dithiothreitol (DTT) to reduce disulfides (SS) to thiols (SH), and then labelled with the specific fluorescence thiol labelling agent monobromobimane (mBBr). The SH and SS in intact labelled spermatozoa were evaluated by fluorescence microscopy and by flow cytometry analysis; mBBr-labelled spermatozoa were solubilized and sperm proteins analysed by gel electrophoresis (SDS-PAGE for non-basic, whole sperm proteins and acid urea-PAGE for sperm nuclear basic proteins). Microscopy and flow cytometry showed that normozoospermic samples (having normal sperm count, morphology and motility) contained both SH and SS, with more SS than SH. Heterogeneity in the proportion of SH/(SH plus SS) was observed among spermatozoa within the ejaculates. The total SH plus SS was similar among the ejaculates, with some variability in SH/(SH plus SS) noted among them. SDS-PAGE of solubilized normozoospermic cells showed differences in the SH and SS content of the protein bands. Acid urea-PAGE of basic proteins isolated from normozoospermic samples showed protamines P1 and P2 and traces of non-protamine basic proteins. P1 and P2 contained SH and SS, with variability in SH/(SH plus SS) observed among the samples. Teratozoospermic samples (in which > 90% of the spermatozoa exhibited abnormal morphology) were similar in thiol–disulfide status to normozoospermic samples, but contained non-protamine basic proteins in addition to protamines. Spermatozoa in asthenozoospermic samples (in which > 90% of the spermatozoa were immotile) contained lower amounts of SH than did those of normozoospermic samples; the total (SH plus SS) in the asthenozoospermic samples was similar to that in the normozoospermic samples, as shown by microscopy, flow cytometry and gel electrophoresis. The significantly lower SH/(SH plus SS) was evident in most sperm proteins, including the protamines, of the asthenozoospermic samples. This 'over oxidation' of sperm thiols may result from an abnormal maturation in the epididymis or from an effect of the seminal plasma.
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