THIOL-DISULFIDE STATUS OF HUMAN SPERM PROTEINS

The thiol-disulfide status in proteins of human spermatozoa categorize d as normozoo-spermic, teratozoospermic and asthenozoospermic was exam ined. Washed spermatozoa were incubated with or without dithiothreitol (DTT) to reduce disulfides (SS) to thiols (SH), and then labelled wit h the specific fluorescence thiol labelling agent monobromobimane (mBB r). The SH and SS in intact labelled spermatozoa were evaluated by flu orescence microscopy and by flow cytometry analysis; mBBr-labelled spe rmatozoa were solubilized and sperm proteins analysed by gel electroph oresis (SDS-PAGE for non-basic, whole sperm proteins and acid urea-PAG E for sperm nuclear basic proteins). Microscopy and flow cytometry sho wed that normozoospermic samples (having normal sperm count, morpholog y and motility) contained both SH and SS, with more SS than SH. Hetero geneity in the proportion of SH/(SH plus SS) was observed among sperma tozoa within the ejaculates. The total SH plus SS was similar among th e ejaculates, with some variability in SH/(SH plus SS) noted among the m. SDS-PAGE of solubilized normozoospermic cells showed differences in the SH and SS content of the protein bands. Acid urea-PAGE of basic p roteins 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 sample s. Teratozoospermic samples (in which > 90% of the spermatozoa exhibit ed abnormal morphology) were similar in thiol-disulfide status to norm ozoospermic samples, but contained non-protamine basic proteins in add ition to protamines. Spermatozoa in asthenozoospermic samples (in whic h > 90% of the spermatozoa were immotile) contained lower amounts of S H than did those of normozoospermic samples; the total (SH plus SS) in the asthenozoospermic samples was similar to that in the normozoosper mic samples, as shown by microscopy, flow cytometry and gel electropho resis. The significantly lower SH/(SH plus SS) was evident in most spe rm proteins, including the protamines, of the asthenozoospermic sample s. This 'over oxidation' of sperm thiols may result from an abnormal m aturation in the epididymis or from an effect of the seminal plasma.