Studies on Spermatogenesis in Rats. I. Application of the Sedimentation Velocity Technique to an Investigation of Spermatogenesis

Abstract

A sedimentation velocity chamber at unit gravity (Staput fractionation) was employed to separate cells obtained from suspensions of testes from adult rats, and the differential cell count was determined to estimate the sedimentation profiles of cell populations in various fractions. The incorporation of thymidine-3H into acid-insoluble portions of cell fractions was measured at varying times following the intratesticular administration of thymidine. The kinetic profiles of thymidine-3H incorporation into spermatogonia, primary spermatocytes, spermatids, and spermatozoa were compared with radioautographs prepared from histological sections of testes obtained from the same animals. Good correlations were observed between radioactivity peaks in Staput fractions and grain localization in cells of the spermatogenesis cycle at varying time periods after thymidine-3H injection. Data obtained with the Staput technique can be rapidly quantitated. The limits of the Staput fractionation technique for the separation of cells from the rat testis were evaluated with respect to the possible isolation of homogeneous populations for biochemical studies, and to the currently more amenable separation of heterogeneous classes of cells for kinetic studies. The usefulness of the method was validated for application to subsequent investigations.