Separation of Specific Stages of Spermatids from Vitamin A-Synchronized Rat Testes for Assessment of Nucleoprotein Changes during Spermiogenesis1

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Abstract
Studies of the precise steps of spermiogenesis at which dramatic changes occur in the nuclear proteins have been limited by the inability to obtain sufficient quantities of these cells in narrowly defined developmental stages, especially those between steps 1 and 12. This limitation can now be overcome by vitamin A-induced synchronization of rat testes into a few stages of the seminiferous epithelial cycle. Cell suspensions from stage-synchronized rat testes were separated by centrifugal elutriation, and selected fractions were further purified on Percoll gradients. Fractions enriched in spermatids in steps 1–3, 7–8, 9–10, and 11–12 were obtained. Analysis of the basic nucleoproteins from these cells by PAGE revealed the following changes. Between steps 3 and 7, histone (H) 2A variants, H2A.1, H2A.2, and TH2A, became post-translationally modified; and during steps 9–11, Hit became modified. H4, which was monoacetylated in steps 1–3, showed maximal levels of hyperacetylation in steps 11–12. The histones were the major basic nuclear proteins in spermatids through step 12. The low levels of transition proteins 1 and 2 observed in a fraction enriched in steps 11–12 could be largely accounted for by contamination from step 13–15 spermatids. All results were consistent with those obtained from normal, unsynchronized rats. The technique of vitamin A synchronization is therefore useful in more precisely defining biochemical changes during spermiogenesis.

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