Protein composition of the perinuclear theca of bull spermatozoa

The perinuclear theca is a cytoskeletal structure that covers the nucleus of mammalian spermatozoa except for a narrow zone around the insertion of the tail. It shows two distinct regions, a subacrosomal layer or perforatorium and, continuing caudally beyond the acrosomic system, the postacrosomal sheath. The main objective of this study was to analyze the protein composition of the bull perinuclear theca and the distribution of some of its proteins. The perinuclear theca was extracted from demembranated and acrosome-depleted sperm heads by alkaline treatment. Gel electrophoresis revealed that the extract was composed of over 20 polypeptide bands, of which the 15.5-, 25-, 28-, 32-, 36-, and 60-kDa bands were most prominent. Polyclonal antibodies prepared against these major polypeptides were screened on Western blots of the theca extracts and were immunolocalized on sections of spermatozoa prepared for electron microscopy. Antibodies against the 15.5- and 60-kDa polypeptides reacted monospecifically with their respective bands on Western blots, and immunogold labeled the perforatorium and the entire perinuclear theca, respectively. Antibodies against the 25-, 28-, 32-, and 36-kDa polypeptides showed distinctive patterns of cross-reactivity with other polypeptides on Western blots, but immunogold labeled exclusively the entire perinuclear theca. In conclusion, this study provides evidence that the protein composition of the bull perinuclear theca is complex: at least six major polypeptides make up this cytoskeletal element. As to their distribution, the 15.5-kDa polypeptide is localized exclusively to the subacrosomal layer, and the other five major polypeptides analyzed in this study appear to be shared between the subacrosomal and post-acrosomal regions of the perinuclear theca.