Fig 2. Reproduction disrupts cyst cell differentiation.

(A-D) 5w-old w¹¹¹⁸ testes immunostained for Zfh-1 (green), co-stained for Vasa (blue), and DNA (red). Asterisks mark the hubs. Arrows indicate ectopic Zfh-1-positive cells. (A) Normal testis from an unmated male (30 vs. 0). Zfh-1-positive cells and early germ cells were located near the hub. (B-D) Testes from mated w¹¹¹⁸ males (10 vs.20), exhibiting ectopic Zfh-1expression at level 1 (B), level 2 (C), and level 3 (D). (E-H) Percentages of testes with ectopic Zfh-1-positive cells from the males at different ages. Numbers of the testes scored are shown in the parentheses. (E-H) Testes from mass-mated (10 vs. 20) or unmated (30 vs. 0) males of w¹¹¹⁸ (E), Canton-S (F), yw (G), and Oregon R (H) at different ages. (I) Testes from 5w-old w¹¹¹⁸ males mated with females (10 vs. 20) for five weeks (week 1–5), the first three weeks (week 1–3), or the last three weeks (week 3–5). (J) Testes from 4w-old w¹¹¹⁸ and Canton-S males mated to females with the ratios of 30:0, 10:20, and 25:5. P-values in I and J were calculated with Chi-squared test. ns: p>0.05, *p<0.05, **p<0.01, and ***p<0.001. (K and K’) 3-day-old Dad-lacZ testis immunostained for β-galactosidase (red in K), co-stained for FasIII and Eya (green in K, and white in K’) and Vasa (blue in K). Arrows indicate the LacZ-positive and Eya-negative cyst cells. Arrowheads mark the cells double positive for LacZ and Eya. Asterisks indicate hubs. (L and M) Graphs showing the distance of Zfh-1-positive cells to the hub in the mated testes from Dad-lacZ (L) and w¹¹¹⁸ males (M). Each dot indicates a single Zfh-1-positive cell. Ectopic Zfh-1-positive cells are defined as cells 200 μm away from the hub (dash line). Percentages of the double positive cells among the total ectopic Zfh-1-positive cells are shown at the top of each column. (N) A testis from mated 5w-old w¹¹¹⁸ exhibiting ectopic Zfh-1 expression in differentiated cyst cells. Arrowheads mark the ectopically expressed Zfh-1 (green) in Eya (red)-positive cells away from the hub (large, Vasa-positive cells). (O) EdU labeling of a testis displaying level 3 ectopic Zfh-1 expression from mated 5w-old w¹¹¹⁸ males. The arrows indicate the double-positive cells for EdU (red) and Zfh-1 (green) away from the hub. Mass-mating schemes were conducted for all experiments.