Biochemical evidence of haploid gene activity in spermatogenesis of the mouse

Mice were exposed to two X-ray doses of 300 and 100 R with 4 days interval in order to deplete the testes of spermatogonia and early meiotic cells. After X-ray treatment, the seminiferous tubules were labelled in culture with radioactive RNA precursors, dispersed into single cells by trypsin treatment and these were fractionated into several cell classes by velocity sedimentation at unit gravity in a Ficoll gradient. With this method quasi-homogeneous populations of middle-late pachytene spermatocytes and round spermatids (steps 1–8 of spermiogenesis) were obtained. RNA was extracted from these two cell types and analysed by linear sucrose gradient fractionation and by affinity chromatography on a poly(U)-Sepharose column. The results showed that round spermatids, as well as pachytene spermatocytes, synthesize both ribosomal RNA (rRNA) and poly(A)⁺ RNA (presumptive messenger RNA) (mRNA). The post-meiotic synthesis of RNA ceases completely in mid-spermiogenesis after nuclear elongation in spermatids has set in.