Morphological Characterization of Vital Cell Preparations Derived from Maturing Spermatogenic Epithelium
At 8 days of postnatal age. At this time-point, initiation of spermatogenesis could be observed. Large gonocytes exhibiting typical morphology and prominent nucleoli were seen to entering into mitosis (Fig. 1A). These gonocytes were surrounded by possible daughter cells, which demonstrated a size and morphology comparable to those of type A spermatogonia in the mature rat testis (Fig. 1A). Neither intermediate nor type B spermatogonia were present. The Sertoli cells were relatively small and rounded-up, and some were undergoing mitosis. This morphological pattern was similar for each of the samples studied, and no individual stages of spermatogenesis could be identified.
At 18 days of postnatal age. At this point in development, pachytene spermatocytes in stage VIII were the most differentiated type of germ cell observed, although at a frequency lower than that in the 26-day-old rat testis. These samples could be divided into three groups on the basis of the presence of intermediate and type B spermatogonia in stages II-VI (Fig. 1B), preleptotene and midpachytene spermatocytes in stages VII-VIII (Fig. 1C), or type A spermatogonia, leptotene, and zygotene spermatocytes in stages IX-I (Fig. 1D). The exact numbers of samples in each group are presented in Table 1. Stages II-VI formed the shortest group of stages, having an average total length of 5 mm of the consecutive tubular segments. Spermatogonia in stages IX-I of the cycle exhibited few mitoses. The Sertoli cells were elongated, had nucleoli characteristic of mature Sertoli cells (Fig. 1B), and were never seen to be undergoing mitosis.
FIG. 1. Phase-contrast microscopy of vital cell preparations derived from immature rat testis. A) 8-Day-old rat testis contains gonocytes (Go) entering into mitosis (m) and surrounded by type A spermatogonia (A). B-D) 18-Day-old rat testis exhibits (B) intermediate spermatogonia (In) in stages II-VI sharing common cytoplasm, together with a Sertoli cell (Sc) having mature-type nuclear morphology; (C) preleptotene (Pl) and pachytene spermatocytes (Pa) in stages VII-VIII, with three apoptotic pachytene spermatocytes (indicated by arrows); and (D) type A spermatogonia and zygotene spermatocytes (Z) in stages IX-I. E-G) 26-Day-old rat testis shows (E) step 3-4 spermatids (S) and pachytene spermatocytes in stages II-VI, (F) preleptotene and pachytene spermatocytes in stages VII-VIII, and (G) zygotene spermatocytes, live type A spermatogonia, and four apoptotic type A spermatogonia (arrows) in stages IX-I. C’ and G’) TUNEL staining of the cells exhibiting apoptotic morphology in C and G, respectively. Bar = 10 ^m.