Meiotic failure in male mice lacking an X-linked factor

Abstract

Meiotic silencing of sex chromosomes may cause their depletion of meiosis-specific genes during evolution. Here, we challenge this hypothesis by reporting the identification of TEX11 as the first X-encoded meiosis-specific factor in mice. TEX11 forms discrete foci on synapsed regions of meiotic chromosomes and appears to be a novel constituent of meiotic nodules involved in recombination. Loss of TEX11 function causes chromosomal asynapsis and reduced crossover formation, leading to elimination of spermatocytes, respectively, at the pachytene and anaphase I stages. Specifically, TEX11-deficient spermatocytes with asynapsed autosomes undergo apoptosis at the pachytene stage, while those with only asynapsed sex chromosomes progress. However, cells that survive the pachytene stage display chromosome nondisjunction at the first meiotic division, resulting in cell death and male infertility. TEX11 interacts with SYCP2, which is an integral component of the synaptonemal complex lateral elements. Thus, TEX11 promotes initiation and/or maintenance of synapsis and formation of crossovers, and may provide a physical link between these two meiotic processes.

Figure 1.

TEX11 forms distinct foci on meiotic chromosomes. (A–D) Distribution of TEX11 foci in spermatocytes. TEX11 foci are absent in leptonema (A), present in zygonema (B) and early pachynema (C), but absent in late pachynema (D). (B) Note that TEX11 foci are present on synapsed regions (arrows) in zygonema. (E–G) TEX11 foci mostly (74%) colocalize with RPA foci in spermatocytes. The same spermatocyte is shown for TEX11 alone (E), RPA alone (F), and a composite image (G). CREST antiserum stains centromeres. Expanded view of the chromosome in the square in G is shown in H and H′ (with offset channels). (I–K) A majority of TEX11 foci (60%) overlap with MSH4 foci in spermatocytes. The same spermatocyte is shown for TEX11 alone (I), MSH4 alone (J), and a composite image (K). Expanded view of the two chromosomes in the square in K is shown in L and L′ (with offset channels). (H′,L′) The direction of deliberate shift of the red channel is indicated by an arrow. (M) TEX11 foci rarely overlap with DMC1 foci in spermatocytes. (N) TEX11 forms arrays of foci in Sycp1^−/− pachynema. (O) TEX11 foci are located between the two aligned but separated lateral elements in Sycp1^−/− pachynema. The same cell is shown in N. (P) Electron microscopy shows immunogold labeling of TEX11 between lateral elements (LEs) in spermatocytes. (CE) Central element.

Figure 2.

Tex11 is essential for male meiosis. (A) Schematic diagram of Tex11^fl and null alleles. Two loxP sites in the same orientation were introduced into introns 2 and 29. Exons are shown as black bars and the exon numbers are indicated. (B) Significant size reduction in 12-wk-old Tex11^−/Y testis. (C) Western blot analysis shows the absence of TEX11 protein in Tex11^−/Y testis. (D–G) Histological analysis of testes from 8-wk-old wild-type and Tex11^−/Y mice. In contrast to wild-type tubules with full spermatogenesis (D), Tex11-deficient tubules (E) exhibit meiotic arrest. (F) Segregation of chromatin in a wild-type anaphase spermatocyte from a Stage XII tubule. Arrows indicate two chromatin masses (i.e., two sets of segregating chromosomes). (G) Abnormal chromosome segregation in Tex11-deficient Stage XII tubules. Arrows and arrowheads indicate abnormally segregating chromatin masses. (Z) Zygotene, (P) pachytene, (ES) elongated spermatids. Bars, 25 μm.

Figure 3.

TEX11 promotes chromosome synapsis. (A) Wild-type pachynema with 19 pairs of fully synapsed autosomes (yellow) and partially synapsed X–Y chromosomes (red). (B) Tex11^−/Y pachynema with asynapsed X–Y. (C) Tex11^−/Y pachynema with eight asynapsed chromosomes: the X–Y and three pairs of autosomes (arrowheads). Notably, each pair of asynapsed homologs (as judged by equal SC length except for XY) is in close proximity and alignment, indicating a possible interaction. (D) Tex11^−/Y diplonema with univalents (arrowheads). (E) An apparently normal Tex11^−/Y pachynema with partially synapsed X–Y in the γH2AX-positive sex body. (F) Tex11^−/Y pachynema. Asynapsed X–Y chromosomes are confined to a single γH2AX-positive domain. (G) Tex1^−/Y pachynema with one pair of asynapsed autosomes (arrowhead) and asynapsed X–Y, all of which are γH2AX-positive. (H) Tex11^−/Y diplonema with a large number of univalents (arrowheads). Some autosomes are bivalents (arrow). X–Y univalents are confined to a single γH2AX-positive domain. In contrast, all autosomal univalents are γH2AX-negative. (I) Analysis of asynapsed chromosomes in early (H1t-negative) and mid-to-late (H1t-positive) Tex11^−/Y pachynema. Testes from four 7.5-wk-old Tex11^−/Y mice were analyzed by triple immunostaining of spread nuclei with anti-SYCP1, anti-SYCP3, and anti-histone H1t antibodies. All pachynema in view were first divided into H1t⁻ and H1t⁺ groups, and asynapsed chromosomes were then examined. More than 200 pachynema from each mouse were analyzed. (J) Two distinct rounds of apoptosis in adult Tex11^−/Y testis. Seminiferous tubules were divided into three groups: H1t-negative (Stages I–IV), H1t-positive (Stages V–XI), and Stage XII.

Figure 4.

TEX11 modulates the formation of meiotic crossovers. (A–C) A dramatic reduction in the number of MLH1 foci (green) in Tex11^−/Y pachynema (∼15 foci) relative to wild type (22 foci). (B) Note the absence of MLH1 foci on four synapsed chromosomes (asterisks) in the mutant. (D) Twenty bivalents (pairs of homologs) are connected via chiasmata in wild-type metaphase I spermatocytes. (E) Presence of univalents in Tex11-deficient metaphase I spermatocytes. Asterisks indicate four univalents. (F) A sharp decrease in the number of bivalents in Tex11-deficient metaphase I spermatocytes. (G) TUNEL analysis of wild-type tubules. A few apoptotic cells (green) are occasionally observed in Stage XII tubules. (H) Massive apoptosis in Tex11^−/Y Stage XII tubules. Apoptotic cells presumably corresponded to anaphase I spermatocytes.

Figure 5.

TEX11 interacts with SYCP2. (A) Five unique overlapping SYCP2 fragments identified in the two-hybrid screen using the full-length TEX11 protein as bait. Numbers indicate the position of terminal residues. The bottom clone with an internal deletion of SYCP2 (1346–1476 amino acids) does not interact with TEX11. (B) Binding of in vitro translated TEX11 to GST-SYCP2 (amino acids 1374–1500). (C) Coimmunoprecipitation of SYCP2 with TEX11 from testis. Immunoprecipitation was performed with rabbit anti-TEX11 antibodies or preimmune control serum and probed with guinea pig anti-SYCP2 antibodies on Western blot. (D) Model illustrating the association of TEX11 foci and the lateral elements at the zygotene stage. A pair of homologous chromosomes and transverse filaments of the SC are also shown.