ISOLATION OF HIGHLY PURIFIED TYPE-A SPERMATOGONIA FROM PREPUBERTAL RAT TESTIS

We have developed a new method that allows isolation of highly purifie d type A spermatogonia from prepubertal rats. The procedure is based o n the maximal release of spermatogania from the seminiferous epitheilu m obtained by the complete enzymatic digestion of the tubular basal la mina, followed by removal of contaminating somatic cells through adhes ion to plastic dishes coated with the lectin Datura stramonium aggluti nin and fractionation on a discontinuous Percoll gradient. The cell su spension obtained contains up to 85% type A spermatogonia. Besides mor phological criteria, the identification of germ cells and somatic cell s has been performed by means of immunocytochemical markers, such as c -kit receptor, which is present only in germ cells, and vimentin, whic h is present only in somatic cells. All type A spermatogonia isolated were c-kit positive, thus suggesting that c-kit receptor is present in both undifferentiated and differentiating type A spermatogonia, Preli minary culture experiments demonstrate that spermatogonia survival in vitro was significantly improved by the addition of 10% fetal calf ser um or horse serum to the culture medium; however, optimal culture cond itions remain to be established. in vitro studies on isolated spermato gonia may provide a significant contribution toward elucidation of the mechanisms regulating spermatogonial proliferation and differentiatio n.