Re-examination of spermatogonial renewal in the rat by means of seminiferous tubules mounted “in toto”

Observations on dissected tubules, fixed in Carnoy, stained with hematoxylin and mounted “in toto” revealed that there were five distinct classes of type A spermatogonia. The type A₁ found in stages II–VIII of the cycle of the seminiferous epithelium had round, pale-stained nuclei, typically arranged in linear clusters of four or eight along the tubular wall. They all divided at stage IX to produce type A₂ cells. These in turn divided at stage XII to produce type A₃ spermatogonia. The type A₂ and A₃ cells had large ovoid nuclei containing globular masses of deeply stained chromatin and were randomly distributed in the space between Sertoli nuclei. The type A₃ spermatogonia divided at stage XIV to produce type A₄ cells. These had smaller nuclei, sometimes lobulated, containing more deeply stained chromatin granulation, free in the nucleus or adhering to the nuclear membrane. They divided in stage I of the cycle to yield two classes of spermatogonia: intermediate type and new type A₁. Hence, type A₁–type A₄ spermatogonia were considered as “renewing stem cells.” The fifth class of type A spermatogonia (A₀) was found at all stages of the cycle. Rare, isolated or in pairs, they had oval nuclei with deeply stained chromatin granulations. Seldom seen to divide, they did not appear to be actively involved in cell renewal and were tentatively considered as “reserve stem cells”.