Morphological characteristics of zebrafish type A undifferentiated (A_und) and type A differentiating (A_diff) and type B spermatogonia and Leydig cells and in situ hybridization for insl3 mRNA in adult zebrafish testis. a Type A_und spermatogonia (arrow) are single germ cells showing a large and clear nucleus, with poorly condensed chromatin and one or two compact nucleoli. b A cyst containing two type A_diff spermatogonia (arrowhead) that show smaller nuclei stained more intensely by toluidine-blue. Type B spermatogonia (thin arrow) have still smaller, slightly elongated/ovoid nuclei that moreover show a high amount of heterochromatin. c Interstitial space (dashed line) with a group of Leydig cells. d Adult zebrafish testis section showing the detection of insl3 mRNA by in situ hybridization in Leydig cells cluster (dashed lines). Inset in d non-specific staining obtained with the sense probe. a-c Sections were prepared for morphological analysis according to Leal et al. (2009b). Magnification ×1000 (a, b), ×600 (c, d). Bars 10 µm

a, b Testis tissue sections showing BrdU-positive germ cells: type A_und (arrow), type A_diff (thin arrows) and type B (arrowhead) spermatogonia. Magnification ×1000. Bars 10 µm. c Mitotic indices of type A_und and A_diff spermatogonia after incubation for 7 days in the absence (Basal) and presence (hINSL3) of 100 ng hINSL3/ml. *Significant difference (P < 0.05) between treated and control. Results are presented as means ± SEM (n = 8)

a, b Testis tissue sections showing BrdU-positive Sertoli cells nuclei (arrows) in association with BrdU-positive (white star) and BrdU-negative (black star) type A_und spermatogonia. c BrdU-positive Leydig cell nucleus (thin arrow). d Mitotic indices of Sertoli cells in association with BrdU-negative or BrdU-positive type A_und spermatogonia and of Leydig cells after incubation for 7 days in the absence (Basal) or presence (hINSL3) of 100 ng hINSL3/ml. Different letters indicate significant differences (P < 0.05) between the absence and presence of hINSL3. Magnification ×1000 (a-c). Bars 10 µm. Results are presented as means ± SEM (n = 8)