Regional differences in composition of the perforatorium and outer periacrosomal layer of the rat spermatozoon as revealed by immunocytochemistry.

The rat perforatorium is the part of the perinuclear theca that underlies the acrosomic system. It appears to be composed of several polypeptides. The main objective of this study was to determine the distribution of seven of these perforatorial polypeptides in the head of the rat spermatozoon. For this purpose, polyclonal antibodies were affinity purified from these polypeptides and tested 1) for their distribution on electron-microscope sections of late spermatids and spermatozoa by immunogold labeling and 2) for their specificity on Western blots of denatured perforatorial polypeptides by immunoblotting. Immunoblotting showed that all seven of the prominent perforatorial polypeptides had epitopes in common. Immunogold labeling of spermatozoa showed that antibodies against the 13, 13.4, and 16 kDa polypeptides were restricted in their localization to the thicker apical portion of the perforatorium and to the inner zone of the ventral spur. However, antibodies against the 34, 43, 57, and 63 kDa polypeptides reacted with the entire perforatorium but, in addition, reacted with the inner part of the ventral spur and with a portion of the "outer periacrosomal layer" lying between the plasma membrane and the outer acrosomal membrane. These results suggest 1) that there are regional differences in protein composition of the perforatorium, of the outer periacrosomal layer, and of the postacrosomal dense lamina; and 2) that perforatorial polypeptides may not necessarily be restricted to the subacrosomal region, but may also compose portions of the outer periacrosomal layer and postacrosomal dense lamina. Based on both immunoblotting and immunocytochemical results, using an antiactin monoclonal antibody that recognizes all known isoforms of actin, actin was not detected in the perforatorium of step 19 spermatids or spermatozoa. Actin, however, together with the seven perforatorial polypeptides tested, was present in the subacrosomal space of elongating spermatids before the process of condensation of the perforatorium takes place.