Mouse TEX15 is essential for DNA double-strand break repair and chromosomal synapsis during male meiosis

Abstract

During meiosis, homologous chromosomes undergo synapsis and recombination. We identify TEX15 as a novel protein that is required for chromosomal synapsis and meiotic recombination. Loss of TEX15 function in mice causes early meiotic arrest in males but not in females. Specifically, TEX15-deficient spermatocytes exhibit a failure in chromosomal synapsis. In mutant spermatocytes, DNA double-strand breaks (DSBs) are formed, but localization of the recombination proteins RAD51 and DMC1 to meiotic chromosomes is severely impaired. Based on these data, we propose that TEX15 regulates the loading of DNA repair proteins onto sites of DSBs and, thus, its absence causes a failure in meiotic recombination.

Figure 1.

Targeted inactivation of the Tex15 gene. (A) Schematic diagram of the Tex15 targeting strategy. The four exons of Tex15 are drawn in scale as rectangles and are designated by the numbers shown above. The neomycin selection marker is flanked by loxP sites and the orientation of loxP sites is indicated by arrowheads. The LacZ coding sequence is preceded by an IRES sequence and followed by SV40 polyadenylation signal sequence (not depicted). (B) Absence of TEX15 protein in Tex15 ^−/− testes. Western blot analysis was performed on 20 μg each of adult wild-type, Tex15 ^+/−, Tex15 ^−/−, and Sycp2 ^−/− testicular protein extracts. SYCP2 served as a nuclear protein control. (C) TEX15 is present in both the cytoplasm and nucleus in testes. Cytoplasmic (Cyt) and nuclear (Nuc) fractions were prepared from adult wild-type and Sycp2 ^−/− testes. MVH, predominantly expressed in spermatocytes and spermatids, served as a cytoplasmic protein control (Toyooka et al., 2000). SYCP3 served as a nuclear protein control. (D) Dramatic size reduction in Tex15 ^−/− testis. (E) Absence of TEX15 in germ cell–deficient XX^Y* testis. 30 μg of total testicular protein extracts from adult wild-type (WT) or germ cell–deficient (XX^Y*) testes were used for Western blot analysis with antibodies against indicated proteins (Hunt and Eicher, 1991). Protein molecular mass standards are shown in kilodaltons (B, C, and E).

Figure 2.

Meiotic arrest in Tex15-deficient mice. Testes and epididymides from 7-wk wild-type and Tex15 ^−/− mice were subject to histological analysis. (A) A wild-type seminiferous tubule contains a full spectrum of germ cells: zygotene spermatocytes (Z), pachytene spermatocytes (P), and elongating spermatids (ES). (B) In some Tex15 ^−/− seminiferous tubules, two to three layers of zygotene-like spermatocytes (arrows) are present. (C) Tex15 ^−/− tubules with large but lightly stained degenerating spermatocytes (arrows). (D) Tex15 ^−/− tubules with only a single layer of early spermatogenic and Sertoli cells (arrow). (E) Wild-type epididymal tubules were full of spermatozoa. (F) Epididymal tubules from Tex15 ^−/− mice were devoid of germ cells. Bar, 50 μm.

Figure 3.

Failure of chromosomal synapsis in Tex15^−/− spermatocytes. Spread nuclei of wild-type (A, C, and E) and Tex15-deficient (B, D, and F) spermatocytes were stained with anti-SYCP1, 2, and 3 antibodies and CREST antiserum. (A and B) Formation of axial elements in both wild-type and mutant spermatocytes. (C and D) SYCP1 formed long fibers (C, arrows) along synapsed chromosomes in wild-type zygotene spermatocytes, whereas no such long SYCP1 fibers were observed in Tex15-deficient spermatocytes (D). (E) Wild-type zygotene spermatocytes. Note the presence of ∼30 CREST foci. (F) Advanced Tex15-deficient spermatocytes (as judged by formation of axial elements) contained 40 CREST foci. +/+, wild type; −/−, Tex15-deficient. Bar, 10 μm.

Figure 4.

Meiotic DSBs are generated, but not repaired, in Tex15^−/− spermatocytes. Surface-spread nuclei of wild-type and Tex15 ^−/− spermatocytes were immunostained with anti-gH2AX, -RAD51, -DMC1, and -RPA antibodies. Axial elements of SCs were labeled with either anti-SYCP2 or anti-SYCP3 antibodies. (A and B) gH2AX is abundant in both wild-type and Tex15^−/− leptotene spermatocytes. (C–E) Absence or reduction of RAD51 foci in Tex15 ^−/− spermatocytes. (E) A dramatic reduction in the number of RAD51 foci per leptotene/zygotene spermatocyte in Tex15 ^−/− testes. Values shown represent the mean ± SEM. (F and G) Absence of DMC1 foci in Tex15 ^−/− spermatocytes. (H and I) Tex15 ^−/− spermatocytes exhibit abundant RPA foci. (C, F, and H) wild-type zygotene spermatocytes; (D, G, and I) advanced Tex15 ^−/− spermatocytes. Bar, 10 μm.

Figure 5.

A model for the role of TEX15 in early meiotic recombination. Both BRCA1 and 2 interact with RAD51. TEX15, like BRCA1 and 2, apparently functions downstream of SPO11 but upstream of the formation of RAD51 and DMC1 foci at sites of DSBs.