Ultrastructural description and cytochemical study of the spermatozoon of Crotallus durissus (Squamata, Serpentes)

The present study was undertaken to elucidate some aspects about the nature of the spermatozoon ultrastructure of Crotallus durissus using cytochemical methods. We also provide for the first time the ultrastructural description of this species spermatozoon. Cytochemical studies of spermatozoa have not been performed so far in the Serpentes, and species spermatozoon may prove helpful to better understand the reproductive biology of this group. Besides the synapomorphies of the Squamata and Serpentes, the C. durissus spermatozoon possess the following: circular acrosome tip; rounded perforatorium tip with a stopper-like basal modification; bilateral stratified laminar structures; central electron-dense structure within the proximal centriole; fibrous sheath extending until the level of the second mitochondrial ring; rounded mitochondria in cross-section, but with variable shape and organization in longitudinal and oblique sections, respectively; linear annulus; developed multilaminar membranes in the nuclear region and the midpiece. The formation of membrane filipin–sterol complexes occur sparsely along the head region, specially around the nucleus; the complexes were also present in the midpiece membrane and scarcely lining the flagellum. The complexes were present in the different layers of the multilaminar membranes. The ethanol-phosphotungstic acid (E-PTA) treatment releaved the presence of basic proteins in acrosome vesicle, pericentriolar material, peripheral fibers of the axoneme and fibrous sheath. The tannic acid technique revealed the microtubules of the centrioles and the axoneme; the extracellular tubules encircling the spermatozoa and those spread in the epididymal lumen were also observed. However, the immunocytochemistry assay using antibodies against alpha-tubulin and beta-tubulin, the primary microtubule monomers, does not support the existence of composition similarity between these tubular structures, since the extracellular tubules were not labeled by the antibodies. The results obtained in this work demonstrate that the utilization of electron microscopic techniques may provide relevant information to the study of ophidian reproductive biology, particularly in analyses concerning spermatozoal ultrastructure.