EXPRESSION OF C-KIT RECEPTOR AND ITS AUTOPHOSPHORYLATION IN IMMATURE RAT TYPE-A SPERMATOGONIA

The objective of this study was to examine the expression and activati on of the c-kit receptor, a specific receptor for kit ligand (stem cel l factor, steel factor), in rat type A spermatogonia. Testes were obta ined from 9-day-old rats, decapsulated, and then subjected to sequenti al enzymatic digestion. The mixture of testicular cell types was then separated by sedimentation velocity at unit gravity. The isolated type A spermatogonia were characterized by light and electron microscopy. They exhibited spherical nuclei containing several nucleoli and associ ated chromatin clumps and organelles generally in a perinuclear locati on similar to that found in the in vivo 9-day-old testis. The synthesi s of the c-kit receptor by the spermatogonia was established by hybrid ization of total RNA with a specific cDNA for mouse c-kit receptor. Tw o mRNA transcripts migrating at 4.8 kb and 12 kb were observed. Locali zation of the c-Lit receptor in the isolated cells was determined by i mmunocytochemistry using an antibody to c-kit protein. Specific staini ng for c-kit receptor was observed in the cytoplasm of the isolated ty pe A spermatogonia. Furthermore, the presence of the c-kit receptor pr otein in the spermatogonia was confirmed by Western blot analysis usin g the same antibody. The antibody recognized the c-kit receptor at sim ilar to 160 kDa. In an attempt to determine whether this receptor has a functional significance, we examined the effect of kit ligand on the phosphorylation of the c-kit receptor. The c-rt ir receptor appeared to be constitutively autophosphorylated on tyrosine at low basal level s, and upon stimulation with kit ligand, the amount of phosphorylated protein increased significantly. These observations indicate that kit ligand induces autophosphorylation of the c-kit receptor, which may le ad to the activation of other cellular target proteins responsible for spermatogonial proliferation and/or differentiation.