Fig 9. Expression of Grnd in cyst cells is elevated by reproduction.

(A-D’) Testes immunostained for Grnd (green in A-D; white in A’-D’), co-stained for Arm (red in A-D). Asterisks mark the hubs. While there were almost no specific anti-Grnd signals in the testes from young males (1-3-day old) (A) and from 3w-old unmated males (C), Grnd was clearly detected in the cyst cell cytoplasm in testes of mated 3w-old males (B). Anti-Grnd signal was absent from the cyst cells in mated 3w-old grnd^minos testis (D). (E) Box-and-whisker plots showing normalized anti-Grnd/anti-Arm intensity in cyst cells. All the relative anti-Grnd intensities were further normalized to the median value for the testes of 3w-old mated males. (F) Fold changes of grnd transcript levels in testes from mated and unmated 3w-old males. Transcript levels were measured by qRT-PCR. The mean grnd/rp49 levels in different samples were normalized to that in the unmated testes. N (biological repeat) = 4. Error bars represent SEM. (G-G”‘) A testis with level-1 ectopic Zfh-1 phenotype from mated 5w-old w¹¹¹⁸ male immunostained for Grnd (green in G, and white in G”‘), co-stained for Zfh-1 (blue in G; white in G’) and Arm (red in G; white in G”). Arrows indicate the anti-Grnd signals in the ectopic Zfh-1-positive cells. (H and I) Suppression of reproduction-induced ectopic Zfh-1 expression (H) and expansion of early germ cells (I) in testes of mated 4w-old males by two independent grnd RNAi lines expressed via eyaA3^ts at 29°C. (J) Knockdown of wgn in cyst cells failed to reduce ectopic Zfh-1 expression in the testes of mated males. Statistic significances were calculated with Mann-Whitney test (E), paired t test (F), and Chi-squared test (H-I). *p<0.05, **p<0.01, and ***p<0.001. Mass-mating scheme were conducted for all experiments.