ADENOSINE 3',5'-CYCLIC-MONOPHOSPHATE DEPENDENT PROTEIN-KINASE ACTIVITY IN DIFFERENTIATING GERM-CELLS OF THE MOUSE TESTIS

Protein kinase activity was studied in middle-late pachytene spermatocytes, round spermatids (steps 1-8 of spermiogenesis) and elongating spermatids (steps 9-13) isolated by velocity sedimentation from the seminiferous tubules of the mouse testis. The 3 cell fractions obtained by this method were 80-90% homogeneous and devoid of somatic cells as judged by light microscopy. cAMP-dependent protein kinase activity was present both in pachytene spermatocytes and in spermatids and was mostly recovered in the soluble fraction of the cell homogenate. The phosphorylative activity expressed per cell was comparable to that described in somatic testicular cells, thus excluding that the activity measured was due to minor somatic contamination undetected with the morphological analysis. Further studies on the phosphokinase activity have shown a stimulation of several fold by cAMP, a preference for protamine as substrate (protamine > total histone > histone F2a) and ATP and Mg2+ requirements similar to those of kinase characterized in other compartments of the testis and in other tissues. Fractionation on DEAE-cellulose of the cytosol prepared from pachytene spermatocytes and round spermatids demonstrated two major peaks of activity eluted with 40 and 180 mM KCl; both enzymes were stimulated by cAMP and specifically bound 3H-cAMP. The same type of analysis performed on elongating spermatids demonstrated that most of the kinase activity eluted with 180 mM KCl. © 1979.