Identification of living spermatogenic cells of the mouse by transillumination-phase contrast microscopic technique for 'in situ' analyses of DNA polymerase activities.

The stages of spermatogenesis can be identified in freshly isolated, unstained adult mouse seminiferous tubules using a transillumination method. Late acrosome- and maturation phase spermatids, arranged in bundles at stages XII-VI give rise to a spotty transillumination pattern. Before spermiation, these cells form a continuous layer on the top of the seminiferous epithelium, recognized by a strong homogeneous central light absorption in the freshly isolated seminiferous tubules at stages VII and VIII. Other stages have a pale light absorption pattern. The accurate determination of the developmental stages of the germ cells was based on the morphology of the developing acrosomic system and of the nuclei of the spermatids, as revealed by phase contrast microscopy. Using this procedure, the activity levels of DNA polymerases alpha and beta have been studied by autoradiography of squash preparations. Using endogenous templates, assay conditions that differentiate between the solubilized DNA polymerases alpha and beta in vitro, were used to distinguish between these activities in situ in different stages of mouse spermatogenesis. Except in very late spermatids shortly before spermiation, DNA polymerases alpha and beta were detectable in all cell types examined. Coinciding with the nuclear protein transitions, elongating spermatids at steps 10-12 and maturation phase spermatids at steps 13-14 showed high DNA polymerase activities. As no replication occurs in these cells, the observations support the view that both DNA polymerases alpha and beta could be involved in repair DNA synthesis.