Histochemical demonstration of zinc ions in ejaculated human semen

A revised in-vitro technique for autometallographic demonstration of chelatable zinc in the human ejaculate is presented, and the localization of the loosely bound pool of zinc ions is described in semen smears and at the ultrastructural level. In semen smears, black autometallographic (AMG) grains indicated the presence of zinc ions dispersed between the spermatozoa. These AMG grains have the same size as grains associated with the sperm tail and may have the same origin. EM analysis of AMG-developed smears fixed in osmium suggested that the detected zinc ions might be related to huge protein molecules present in semen and adhering to the surface of the spermatozoa. Spermatozoa in AMG-stained smears exhibited zinc ions in the midpiece and head, and also joined to the membrane of the tail. Washed spermatozoa exhibited zinc ions only within the midpiece. Ultrastructurally, they were found located in the helecine mitochondria. A few grains were found in the acrosome of the washed spermatozoa. Treatment with the chelating agent DEDTC resulted in complete bleaching of the zinc staining. These findings and the fact that calcium EDTA acid blocks the plasma and surface staining, but not the acrosomal and mitochondrial staining, suggest that chelatable zinc ions exist in two separate pools in human semen.