Reactivated movement of decondensed rat sperm models and a description of their ultrastructure

This study aimed at finding optimal conditions to decondense rat sperm nuclear chromatin with minimal damage. This was judged by the ability of the sperm-tail axoneme in the partially decondensed sperm models to be reactivated. Decondensation was assessed by phase contrast microscopy. Partial decondensation was judged to occur when the bright refractive appearance of the sperm nucleus turned black, and full decondensation when the nucleus turned pale and increased in volume. Demembranation was shown to have occurred by electron microscopy. With 0.03% Triton X-100 rat caudal epididymal sperm were partially demembranated to produce sperm models. Demembranation using a 0.1% solution of Triton X-100 was complete, but as with the solution of 0.05% Triton X-100, resulted in poorer reactivation of the partially decondensed sperm models. Reactivated movement of decondensed sperm models was used to assess the effect of the decondensing agents DTT and heparin. We were only able to achieve reactivation of sperm models that had undergone partial decondensation. Optimal reactivation was obtained after rat sperm models had decondensed in the decondensation solution containing 5 mM DTT, 6 mM EDTA, and 27.3 or 34.1 USP/ml heparin. Concentrations of heparin above or below these values resulted in a decrease in the number of sperm models reactivated. Ultrastructurally, sperm partially decondensed with 5 mM DTT, 6 mM EDTA, and 34.1 USP/ml heparin had their plasma membrane further extracted compared with sperm treated with 0.03% Triton X-100 alone. Decondensation was greatest in the peripheral regions of the nucleus with extraction of the acrosome but not of the perforatorium. The decondensed regions had a filamentous appearance. This procedure will allow access to sperm nuclear chromatin for experimental manipulation in rat sperm models.