Figure 4
Inactivation of Huwe1 in the differentiating spermatogonia leads to an activation of DDR and apoptotic cell death. (a) Loss of Huwe1 resulted in elevated proportion of Stra8 + cells showing intense γH2AX foci in unsynchronized 10 dpp testis. Representative immunofluorescence images from unsynchronized 10 dpp WT and Stra8-Cre KO testes stained for γH2AX (green) and Stra8 (red) (left panel). γH2AX+ Stra8- cells in the tubules are germ cells in early meiosis (leptotene/zygotene). The yellow arrowheads indicate Stra8 + differentiating spermatogonia/pre-leptotene spermatocytes and the white arrow indicates a Stra8+ cell that is also γH2AX+ . Quantification of the proportion of Stra8+ cells that are γH2AX + from the images (right panel) WT, n = 4 (89 tubules), KO, n = 4 (128 tubules). Scale bar = 50 μm. (b) Inactivation of Huwe1 leads to the recruitment of pCHK2 to the intense γH2AX foci in unsynchronized 10 dpp testis. Representative images from immunofluorescence staining of 10 dpp WT and Stra8-Cre KO testes stained for γH2AX (green) and pCHK2 (red). Scale bar = 20 μm. (c) Loss of Huwe1 in the differentiating spermatogonia resulted in increased levels of apoptosis in the synchronized 8 dpRA testes. Shown are typical images from TUNEL assay performed on testes sections at 8 dpRA (left panel). The yellow arrowheads indicate apoptotic cells. Scale bar = 90 μm. Quantification from the images (right panel). WT, n = 5 (783 tubules) Stra8-Cre KO, n = 7 (1099 tubules). (d) Loss of Huwe1 resulted in an elevated percentage of cleaved caspase 3 positive tubules in the synchronized 8 dpRA testes. Shown are typical IHC images for cleaved caspase 3 in 8 dpRA WT and Stra8-Cre KO testes (left panel). The yellow arrowhead indicates a cleaved caspase 3 positive cell. Scale bar = 90 μm. Quantification from the images (right panel) WT, n = 4 (269 tubules), KO, n = 4 (286 tubules). Data are shown as mean ± SEM. Student’s t-test. *p < 0.05.
