Purification of Rat Spermatogenic Cells and Preliminary Biochemical Analysis of These Cells

Abstract

A method for obtaining highly purified fractions of rat testicular cells is described. Single cell suspensions from adult rat testes were separated by centrifugal elutriation. Fractions enriched in pachytene primary spermatocytes, early spermatids, and cytoplasts detached from late spermatids were obtained. These fractions were further separated by equilibrium density centrifugation on gradients of Percoll. In this manner fractions of 3 x 10⁷ pachytene spermatocytes (98% purity), 1.1 x 10⁸ early spermatids (93% purity), and 1.1 x 10⁸ cytoplasts (98% purity) were obtained within 6 h after sacrificing the rats. The cells appeared to be morphologically intact and to have retained their biochemical integrity.

Analysis of acid-soluble nuclear proteins by polyacrylamide gel electrophoresis showed that histone 4 is synthesized during the pachytene stage, and confirmed that testis-specific histones are also synthesized during this stage. Analysis of a microsomal RNA preparation from purified pachytene spermatocytes and purified early spermatids by sucrose gradients indicated that intact ribosomal RNA (rRNA) can be obtained from purified cells. Both cell types are active in synthesizing presumptive messenger RNA (mRNA) with a wide range of sedimentation values, but no appreciable rRNA synthesis was detected.