Fig 1. Reproduction induces ectopic Zfh-1 expression away from the hub.

(A and B) Basal ends of 5w-old w¹¹¹⁸ testes stained for DNA to visualize the bundles of spermatid nuclei (Hoescht 33342). (A) Testis from males kept in solitude. Sperm bundles are indicated by arrows. (B) Testis from a mated male (1 male vs. 6 females). There was no sperm bundle at the basal end. (C) Percentages of the testes containing at least one sperm bundle. (D-H) Testes from w¹¹¹⁸ males immunostained for Zfh-1 (CySC/early cyst cell, green), co-stained for Vasa (germ cells, blue) and DNA (nuclei, red). Asterisks indicate testis hubs. (D) Apical region of a 1-3-day-old testis. Zfh-1-positive cells and small, DNA-bright Vasa-positive early germ cells were only present in the apical region. Inset shows the Zfh-1 expression in the tip. (E-H) 5w-old testes from the males kept in solitude (unmated) (E), or from single males mated with six females (1 vs. 6) (F-H). Ectopic Zfh-1-positive cells are indicated by arrows in the testes exhibiting level 1 (F), level 2 (G) and level 3 (H) ectopic Zfh-1 expression. In level 2 (G) and level 3 (H) testes, ectopic Zfh-1-positive cells were usually associated with small germ cells. (I and J) Percentages of w¹¹¹⁸ (I) and Canton-S (J) testes with ectopic Zfh-1 expression. Extra Zfh-1-positive cells (levels 1–3) were observed in testes from single male mated with 6 females (1 vs. 6) and 1–2 males (1 vs. 1–2) but rarely seen in the testes from unmated males. The asterisks indicate the significance comparing total testes from week-5 and -6 males in 1 vs. 6 to 1 vs. 1–2 mating ratio. N = number of testes scored. P-values shown in C, I, and J were calculated with Chi-squared test. *p<0.05, **p<0.01, and ***p<0.001.