Fig. 4

Spermatogonia cell survival requires Chd4. A Histological sections of wild type and ddx4-chd4^−/− testis cord from 9 days old mice stained with H&E (a, b) and Hematoxylin and immunostained with Stra8 antibodies (marking differentiating spermatogonia) (c, d). Arrows mark Stra8 positive cells present in wild type but reduced in number or absent in the mutant. Experiments were done in at least three different mice showing similar results. B Immunostaining of sections of developing testis reveals severe loss of germ cells (Tra98) in chd4^−/− testis cords. C Quantitation of number of cell positive for Tra98, Plzf, and Sox9. 4 dpp testis (Plzf, wild type, 9.9 ± 4.1, n = 47 seminiferous tubules; chd4^−/− 2.2 ± 1.4, n = 56, P < 0.0001, t test. Tra98, wild type 12.6 ± 5.3, n = 35; chd4^−/− 3.0 ± 1.9, n = 37, P < 0.0001, t test). 7 dpp testes (Plzf, wild type, 14.6 ± 5.0, n = 28; chd4^−/− 3.4 ± 2.4, n = 45, P < 0.0001, t test. Tra98, wild type 19.6 ± 5.9, n = 48; chd4^−/− 3.0 ± 2.1, n = 24, P < 0.0001, t test). Experiments were done in three different mice showing similar results combined in our quantification. D Higher cell death in chd4^−/− knockout testes than in wild-type controls at 4 days post-partum. Apoptotic cells were visualized using TUNEL staining in three mice of each genotype. Series of images (tiles) were used to construct the lower magnification (top panel) and the magnification showed a single tile in the bottom panel. The percentage of apoptotic cells was counted only in the tubule cross sections that contained apoptotic cells and was normalized to total number of germ cells. 19.05 ± 11.17, n = 468 (mean ± SD, n = number of seminiferous tubules counted) wild type and 38.25 ± 13.72 chd4^−/−, n = 344; P < 0.0001 (two-tailed Student t test). The arrows indicate apoptotic cells and arrowhead non-apoptotic germ cells