Fig. 1

Localization and quantification of Igf3 and Amh protein in zebrafish testis following Fsh or saline injections. Sertoli cells and type A spermatogonia are Igf3-positive (green) in zebrafish testis 24 h after the second injection, i.e. 48 h after the start of treatment with saline (A-C) or Fsh (D-F). The Igf3 staining shows as a finely dispersed labeling covering larger areas in Sertoli cells contacting type A spermatogonia (as well as in the type A spermatogonia themselves) while a lower number of intensely green, somewhat larger structures were present in Sertoli cells contacting later stages of germ cell development (see higher magnification in M). We observed no major change in the Igf3 pattern in response to Fsh. Sertoli cells are Amh-positive (green) in zebrafish testis 24 h after the second injection with saline (G-I) or Fsh (J-L). Fsh treatment resulted in a more restricted staining pattern: while Sertoli cells contacting type A spermatogonia kept an intense label, the signal became weaker and covered less area in Sertoli cells contacting all later germ cell stages. Fsh treatment also reduced the staining among the spermatozoa in the tubular lumen. M) Higher magnification of testis from Fsh-treated male, showing Sertoli cells contacting type A spermatogonia (asterisks) with a finely dispersed staining, while larger spots (arrowheads) are more typical of Sertoli cells contacting germ cells in more advanced stages of differentiation. N) Quantification of Amh and Igf3 immunoreactive material per surface area of zebrafish testis sections in response to two Fsh (2 × 100 ng/g bw) or saline injections. Testis tissue was fixed 24 h after the second Fsh or saline injection. Data are expressed as mean ± SEM (n = 8) percentage immunoreactive material, determined as indicated in the Supplemental video. Asterisks indicate significant differences between treatments (unpaired t-test, ***p < 0.001; *p < 0.05). 1, type A spermatogonia (including type A_und and A_diff spermatogonia); 2, type B spermatogonia; 3, spermatocytes; 4, spermatids; and 5, spermatozoa. Propidium iodide (red) was used as DNA counterstaining. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)