ISOLATION OF THE SYNCHRONIZED A SPERMATOGONIA FROM ADULT VITAMIN-A-DEFICIENT RAT TESTES

A method for isolating A spermatogonia from the adult vitamin A-defici ent (VAD) rat testis is described. After removal, the testes were deca psulated and tubules were dissected. An enzymatic digestion with colla genase, hyaluronidase, and trypsin was performed first to eliminate mo st of the interstitial cells. A second digestion with collagenase and hyaluronidase was performed to obtain a cell suspension with a high nu mber of A spermatogonia. The cell suspension was further enriched with A spermatogonia by preplating on peanut agglutinin and separating on a discontinuous Percoll gradient. By this procedure, purification of t he suspension to 70-90% A spermatogonia was obtained. In the seminifer ous tubules of the VAD rats, only Sertoli cells, A spermatogonia, and some preleptotene spermatocytes are present, In our rats, the A sperma togonia are almost all arrested in the G(1) phase of the cell cycle be fore the S phase of A(1) spermatogonia, and presumably before their di fferentiation into A(1) spermatogonia. After administration of vitamin A, spermatogenesis starts synchronously from these A spermatogonia. T he isolation of these synchronized A spermatogonia opens ways to inves tigate the regulation of differentiation and proliferation of A sperma togonia and the biochemical characteristics of the subsequent types of A spermatogonia.