Separation of mouse spermatogenic cells by velocity sedimentation

Improvements have been made in methods of preparation of single cell suspensions from mouse testes for separation by sedimentation at unit gravity. Mechanical means of preparation specifically damage spermatogonia, young primary spermatocytes, and spermatids at various stages of development, resulting in apparent sedimentation velocities which correspond closely to those of free nuclei. Trypsin has been used to release these cells from the seminiferous tubules allowing measurement of the sedimentation properties of the intact cell. The exclusion of trypan blue from the cells, resistance to trypsin, and the persistence in vitro of the highly differentiated forms of macromolecular synthesis demonstrate the integrity of the cells in the suspensions prepared with trypsin. Differentiation pathways in terms of sedimentation velocities of the cells and nuclei have been constructed and are in agreement with morphological changes.