Dmrt7 Is Required for Male but Not Female Gametogenesis
Breeding of Dmrt7 heterozygotes produced homozygous mutant progeny of both sexes at the expected frequency (63/264; 23%). Male and female homozygous mutants were viable, grew to adulthood normally, and exhibited normal sexual behavior. Female homozygotes were fertile, produced litters of normal size, and had no obvious ovarian abnormalities as judged by histological analysis (unpublished data). In contrast, Dmrt7 homozygous mutant males were completely infertile and had testes about one-third the weight of those of heterozygous or wild-type adult littermates (Figure 2). To determine when defective testis development begins in Dmrt7 mutants, we compared the testes of wild-type and mutant littermates during the first wave of spermatogenesis. Prior to postnatal day 14 (P14), mutant testes appeared histologically normal and the testis weights were similar to those of heterozygous and wild-type littermates, indicating that spermatogonia and early meiotic germ cells form normally (Figure 2B; unpublished data). Thereafter, the testes of the Dmrt7 mutant mice ceased to grow and the weight difference was significant. Microscopic examination of P21 and P42 Dmrt7 mutant testes revealed that germ cells arrest in pachynema, and later stages of germ cells are largely missing (Figure 2C and 2D). Dmrt7 mutant mice are deficient in postmeiotic spermatids and lack epididymal spermatozoa, although a few cells develop to the round spermatid stage. These meiotic defects are in agreement with a recent preliminary analysis of another Dmrt7 mutation [42]. While some Dmrt7 mutant tubules are highly vacuolated and contain primarily Sertoli cells and spermatogonia, others have abundant primary spermatocytes. In addition, some tubules contain multinucleated cells and cells with darkly stained nuclei that are typical of apoptotic cells (Figure 2D).
Figure 2  Reduced Testis Size and Germ Cell Apoptosis in Mice with Targeted Deletion in Dmrt7
(A) Testes from a 6-wk-old wild-type (+/+) mouse and a homozygous (−/−) Dmrt7 mutant littermate.
(B–D) Sections of testes from 14-d-old (B), 21-d-old (C), and 42-d-old mice (D) stained with hematoxylin and eosin. Wild-type is in left column and mutant in right. No significant difference is observed at 14 d (B), but by 21 d some tubules are lacking abundant spermatocytes (C, asterisk) or cells with typical apoptotic morphology are present (open arrowhead, C and D). Mutant tubules contain multinucleate cells (closed arrowhead, D).
(E and F) TUNEL labeling of Dmrt7-deficient mouse testes. Testes from wild-type and homozygous mutant littermates were analyzed by TUNEL labeling to detect apoptotic cells. Testis sections from 21-d-old (E) and 6-wk-old mice (F). Apoptotic cells (brown) are much more abundant in seminiferous tubules of homozygous Dmrt7 mutant mice relative to wild-type. Bars in (B–F) represent 100 μm. Since Dmrt7 mutant testes lack most post-pachytene cells, we used TUNEL analysis to test whether the missing cells are eliminated by apoptosis. At 3 wk, Dmrt7 mutant testes contain significantly more apoptotic cells than those of wild-type controls. The percentage of tubule sections with five or more apoptotic nuclei was about three times higher in Dmrt7 mutants compared with wild-type (20% versus 7%; Figure 2E). A similar elevation of apoptosis was apparent in mutant testes at 7 wk (Figure 2F). In mutants, many apoptotic cells were in the middle of the tubules, whereas the apoptotic cells in wild-type occur mainly near the seminiferous tubule periphery. The numbers of Sertoli cells were not significantly different between wild-type and mutant testes, and we observed no difference in somatic cell apoptosis in mutants (unpublished data). From these results, we conclude that loss of Dmrt7 causes a block in meiotic progression, mainly in pachynema, leading to the elimination, by apoptosis, of the arrested cells.