Loss of TEX12 results in infertility

TEX12 has been shown to localize to the central element of the synaptonemal complex (Hamer et al., 2006). To gain more insight in the function of this protein during meiosis, we generated Tex12 knockout mice. To inactivate the Tex12 gene, a targeting vector, in which a NEO cassette replaced exon 2 to exon 5 of the gene, was electroporated into embryonic stem (ES) cells. Successfully targeted ES cells were used for generation of chimeric mice that transmitted the Tex12^–/– allele to the germline. The Tex12^–/– allele transmitted in a mendelian fashion in Tex12^+/– intercrosses and the wild-type Tex12 gene, gene transcript or encoded protein could not be detected in the Tex12^–/– mice (Fig. 1).

Interruption of Tex12 leads to complete elimination of spermatocytes at epithelial stage IV of spermatogenesis and subsequent infertility (Fig. 2). Analysis of Tex12^–/– females showed that, in comparison to the wild type, only ∼30% of the oocytes are present at embryonic stage E16.5 in Tex12^–/– ovaries (Fig. 2). Notably, as the Tex12^–/– ovary size is proportionally smaller than comparable wild-type ovaries, the density of oocytes in Tex12^–/– ovaries is similar to the wild-type situation. The surviving Tex12^–/– oocytes reach the dictyate arrest stage and 1 day after birth ∼30% of the oocytes are still present (Fig. 2). However, Tex12^–/– oocytes are not able to form healthy primordial follicles and eventually degenerate. As a consequence, hardly any follicles could be observed in Tex12^–/– ovaries 1 week after birth (Fig. 2).

TEX12 is essential for elongation of synapsis between the homologous chromosomes

To study the role of TEX12 in synaptonemal complex formation and synapsis, we analyzed meiotic cells from Tex12^–/– and wild-type testes and ovaries using cell spread preparations and immunofluorescence microscopy. Because staining with antibodies against SYCP2 or SYCP3 interfered more with co-staining of other proteins, we used an antibody against the protein STAG3 to visualize the axial elements. STAG3 is part of the meiotic cohesin core, which keeps the sister chromatids together during meiosis, and colocalizes perfectly with the axial elements in both Tex12^–/– and wild-type cells. By studying staining of SYCP3 (data not shown) and STAG3, we observed that the axial elements form normally and that the meiotic chromosomes align in cells without TEX12 (Fig. 3A). However, progression of synapsis was affected in Tex12^–/– cells, resulting in only partially synapsed meiotic chromosomes (Fig. 3A). In wild-type cells, synapsis of the homologous chromosomes is marked by staining of the transversal filament protein SYCP1 and the central element protein SYCE1 along the axial elements. Tex12^–/– meiotic cells also showed staining for SYCP1 and SYCE1, which overlapped with STAG3 on the axial elements (Fig. 3A). However, instead of covering the complete meiotic axes as observed in the wild type, SYCP1 and SYCE1 only formed foci and small stretches in Tex12^–/– meiotic cells (Fig. 3A). Importantly, the central element protein SYCE2 was completely absent from the Tex12^–/– chromosome cores (Fig. 3B).

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Fig. 3.

Initiation of synapsis without an intact central element. Meiotic chromosomes in wild-type and Tex12^–/– spermatocytes and oocytes immunolabeled for SYCP1 (transverse filaments, green), STAG3 (axial elements, blue) and SYCE1 (central elements, red) (A) or STAG3 (axial elements, green) and SYCE2 (central elements, red) (B). Centromeres are labeled with CREST (white) (B). Bar: 5 μm.

We also studied the structure of the meiotic chromosomes using electron microscopy (EM) and compared wild-type (full synapsis), Tex12^–/– (partial synapsis) and Sycp1^–/– (no synapsis) testes and ovaries (Fig. 4). In line with the immunofluorescence experiments, short synapsed axial element structures in Tex12^–/– cells could be confirmed at the EM level in both spermatocytes and oocytes. Although partially synapsed, the Tex12^–/– synaptonemal complex showed a weakly stained and disrupted central-element-like structure. We also observed very small areas of chromosome convergence in Sycp1^–/– spermatocytes. However, these areas of convergence completely lacked any obvious central-element-like structure (Fig. 4).

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Fig. 4.

Initiation of synapsis without an intact central element. Electron microscopy of the synaptonemal complex in wild-type, Tex12^–/– and Sycp1^–/– oocytes and spermatocytes. AE, axial elements (lateral elements after synapsis); TF, transverse filaments; CE, central element; CE^*, central-element-like structure. Bar: 100 nm. A schematic representation of these results is shown in the cartoon at the bottom left.

DNA double-strand breaks fail to develop into meiotic crossovers

The absence of TEX12 results in a disrupted central element and only partial synapsis of the meiotic chromosomes, which could have consequences for the progression of meiotic recombination. Because the Tex12^–/– spermatocytes are eliminated from the testis as early as epithelial stage IV, we monitored the effects of these structural deficiencies on the progression of meiotic recombination in Tex12^–/– and wild-type oocytes, which survive until the dictyate arrest stage. As described for the Syce2^–/– spermatocytes (Bolcun-Filas et al., 2007), the Tex12^–/– spermatocytes show normal loading of early recombination markers but do not form an XY body marked by BRCA1 or γ-H2AX as observed in wild-type spermatocytes (supplementary material Fig. S1). We stained Tex12^–/– and wild-type oocytes for proteins that mark defined stages of meiotic DNA double-strand break (DSB) processing: presence of DSBs and asynapsis (γ-H2AX, BRCA1), homologous recombination (DMC1, RPA) and sites of crossing-over (MLH1) (Mahadevaiah et al., 2001; Moens et al., 2002; Turner et al., 2004).

At the leptotene stage, meiotic DSBs are formed by SPO11 and marked by phosphorylation of H2AX (then referred to as γ-H2AX) (Mahadevaiah et al., 2001). These meiotic DSBs will be resolved by homologous recombination, which can be visualized as early recombination nodules (DMC1 foci), transformed recombination nodules (RPA foci) and sites of crossovers (MLH1 foci) (Moens et al., 2002).

In early oocytes from stage E16.5 embryos, we observed wild-type levels of DMC1 foci in both wild-type and Tex12^–/– oocytes (Fig. 5). However, at a later stage (E18.5), high levels of DMC1 foci were still present in the Tex12^–/– oocytes, whereas these foci were lost in wild-type oocytes (Fig. 5). Later during meiosis, the DMC1 foci are gradually replaced by RPA (Moens et al., 2002) and, in accordance, we observed high levels of RPA in both wild-type and Tex12^–/– oocytes at stage E17.5 (Fig. 5). However, these RPA foci failed to be removed in the Tex12^–/– oocytes and high RPA levels were still present at stage E18.5 in the Tex12^–/– oocytes (Fig. 5). Hence, the Tex12^–/– oocytes never complete the transition from transformed recombination nodules to meiotic crossovers. In accordance with the impaired processing of DMC1 and RPA foci, we could not observe any MLH1 foci in Tex12^–/– oocytes (Fig. 5).

When the DSBs become properly resolved at the pachytene stage of meiosis, γ-H2AX is only retained on unsynapsed chromosomal regions, a process dependent on BRCA1 (Baart et al., 2000; Mahadevaiah et al., 2001; Turner et al., 2004; Turner et al., 2005). In accordance, we found γ-H2AX and BRCA1 to persist on the unsynapsed chromosomal regions in Tex12^–/– oocytes (Fig. 6).

Progression of synapsis requires an intact central element structure

The transverse filament protein SYCP1 is required for recruitment of the central element proteins to the homologous chromosomes (Hamer et al., 2006). In cultured mammalian cells, initiation of SYCP1 fiber formation completely depends on the presence of the C-terminus (axial region) of SYCP1, whereas absence of the N-terminus (central region) only decreases the efficiency of fiber formation and elongation (Ollinger et al., 2005). Together, these results indicate that initiation of synapsis starts with the interaction of the C-terminus of SYCP1 with the axial elements. It has been proposed that the central element protein SYCE1 stabilizes the interaction between the two opposing N-termini of SYCP1 in the center of the synaptonemal complex, whereas SYCE2 would longitudinally connect these SYCP1 dimers to shape a complete central element (Bolcun-Filas et al., 2007). The central element consists of multiple layers of columns and pillars that shape an almost crystalline 3D structure in the center of the synaptonemal complex (Schmekel and Daneholt, 1995). We found that TEX12 is essential for the inclusion of SYCE2 in the central element and confirm the notion that these proteins form an independent complex (Hamer et al., 2006), possibly corresponding to the columns and pillars observed in the structural studies of the central element (Schmekel and Daneholt, 1995). Without TEX12, this structure is not properly assembled. As a consequence, the synaptonemal process does not proceed in Tex12^–/– meiotic cells and the synapsed stretches containing SYCP1 are not elongated along the axial elements.

Progression of meiotic recombination requires structural maturation of the central element of the synaptonemal complex

In Saccharomyces cerevisiae, initiation and elongation of the synaptonemal complex depend on a protein complex called the synapsis initiation complex, consisting of the ZMM proteins (Zip1, Zip2, Zip3, Zip4, Mer3 and Msh4) (Fung et al., 2004; Lynn et al., 2007). Of these proteins Zip2, Zip3 and Zip4 appear functionally related to the mammalian central element proteins: they are essential for synaptonemal complex formation, they are located at the center of the synaptonemal complex and without them 80% of all crossovers (Class I crossovers) do not occur (Agarwal and Roeder, 2000; Chua and Roeder, 1998; Lynn et al., 2007; Tsubouchi et al., 2006). Binding of Zip3 to synapsis initiation sites recruits Zip2 and Zip4, which in turn are responsible for polymerization of Zip1 (Agarwal and Roeder, 2000; Tsubouchi et al., 2006). The fact that these proteins are necessary for class I crossovers and that the number of synapsis initiation sites corresponds with the number of these crossovers has led to the proposal that initiation of synapsis determines the sites of future crossovers during meiosis (Henderson and Keeney, 2004; Henderson and Keeney, 2005; Zickler, 2006).

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Fig. 5.

The central element is required for the development of meiotic crossovers. (A) Meiotic chromosomes in wild-type and Tex12^–/– oocytes immunolabeled for STAG3 (axial elements, green) and DMC1 (red, E16.5 and E18.5), RPA (red, E17.5 and E18.5) or MLH1 (red, E18.5). Centromeres are labeled with CREST (white). Bar: 5 μm. (B) Average number of DMC1 and RPA foci (± s.e.m.) at E16.5 (wt, n=7; –/–, n=4) and E18.5 (wt, n=12; –/–, n=9) and E17.5 (wt, n=6; –/–, n=10) and E18.5 (wt, n=10; –/–, n=9).

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Fig. 6.

Repair of DNA double-strand breaks is impaired without an intact central element. Meiotic chromosomes in wild-type and Tex12^–/– E17.5 oocytes immunolabeled for STAG3 (axial elements, green) and γ-H2AX (red) or BRCA1 (red). Centromeres are labeled with CREST (white). Bar: 5 μm.

Also in mammals, synapsis and meiotic recombination are two highly intertwined events. In the mouse, although meiotic recombination initiates prior to and independently of synapsis (Baudat et al., 2000; Mahadevaiah et al., 2001), synapsis is required for recombination sites to develop into meiotic crossovers (de Vries et al., 2005). Furthermore, synapsis of the homologous chromosomes is completely dependent on the initiation of meiotic recombination (Baudat et al., 2000). However, in Tex12^–/– oocytes, even though synapsis is initiated, crossing-over still does not occur. Hence, merely initiating synapsis is not sufficient to generate meiotic crossovers in the mouse.

How synapsis, including a fully functional central element, could promote the formation of meiotic crossovers remains unclear. The lack of TEX12 or incomplete synapsis might trigger an unknown checkpoint in the Tex12^–/– oocytes. Moreover, TEX12 could even be part of the recombination or checkpoint machinery itself. Activation of a checkpoint in the Tex12^–/– oocytes could possibly lead to a cell cycle arrest that would prevent the meiotic prophase to progress to the pachytene stage during which crossovers would normally occur. It has also been postulated that the mechanical properties (such as robustness or flexibility) of the synaptonemal complex affect how meiotic DSBs are resolved (Blat et al., 2002; Borner et al., 2004; Moens, 1978). The lack of crossovers in Tex12^–/– mice could then be explained by a structural defect caused by the absence of central element proteins from the synaptonemal complex. Either way, without full synapsis along the meiotic cores and a functional central element, the limited synapsed areas on the Tex12^–/– chromosomes fail to support the development of meiotic crossovers.

Animals

The Tex12 mutant mouse line was established at the MCI/ICS (Mouse Clinical Institute/Institut Clinique de la Souris, Illkirch, France; http://www-mci.u-strasbg.fr). The targeting vector was constructed as follows. A 4.4 kb fragment encompassing Tex12 exon 1 was amplified by PCR (129S2/SvPas) and subcloned in an MCI proprietary vector, resulting in a step 1 plasmid. This MCI vector has a floxed neomycin-resistance cassette. A 4.1 kb fragment was amplified by PCR and subcloned in the step1 plasmid to generate the final targeting construct (Fig. 1). The linearized construct was electroporated in 129S2/SvPas mouse embryonic stem cells. After selection, targeted clones were identified by PCR using external primers and further confirmed by Southern blot with neomycin and external probes. Two positive ES cell clones were injected into C57BL/6J blastocysts, and male chimaeras derived gave germline transmission. The Tex12 mice were further analyzed by PCR, RT-PCR and western blot using guinea pig anti-TEX12 (Hamer et al., 2006) and mouse anti-α-tubulin (SIGMA).

Wild-type, Tex12 and Sycp1 (de Vries et al., 2005) mice were used and maintained according to regulations provided by the animal ethical committee of the Karolinska Institute who also approved of the experiments.

Immunohistochemistry, immunocytochemistry and electron microscopy

Histology and immunohistochemistry were performed as described (Hamer et al., 2001; Wang and Hoog, 2006). Immunocytochemistry was performed as described (Kouznetsova et al., 2005) using a `drying-down' technique (Peters et al., 1997) and the following antibodies: rabbit anti-SYCP1 (1:50) (Liu et al., 1996), mouse anti-SYCP1 (1:200) (gift from C. Heyting, Wageningen, The Netherlands,), rabbit anti-SYCP3 (1:200) (Liu et al., 1996), human anti-CREST (1:1500) (Hadlaczky et al., 1986), rabbit anti-STAG3 (1:400) (Pezzi et al., 2000), guinea pig anti-STAG3 (1:200) (Kouznetsova et al., 2005), guinea pig anti-SYCE1 (1:1500) (Hamer et al., 2006), guinea pig anti-SYCE2 (1:400) (Hamer et al., 2006), rabbit anti-γ-H2AX (Upstate Biotechnology) (1:100), rabbit anti-BRCA1 (gift from J. M. A. Turner, National Institute for Medical Research, London, UK) (1:1000), rabbit anti-DMC1 (1:100) and rabbit anti-RPA (1:500) (gifts from P. Moens, York University, Toronto, Canada) and mouse anti-MLH1 (1:50) (BD Biosciences). Secondary antibodies were applied as described (Kouznetsova et al., 2005; Wang and Hoog, 2006). Electron microscopy was performed on ultra thin sections of testis or ovary tissue fixed in 2.5% glutaraldehyde and 1% OsO₄ as described previously and according to standard protocols (Liebe et al., 2004).

Microscopy and imaging

Slides were viewed at room temperature using Leica DMRA2 and DMRXA microscopes and 100× objectives with epifluorescence. Images were captured with a Hamamatsu digital charge-coupled device camera C4742-95 and Openlab™ software version 3.1.4. Images were processed using Adobe Photoshop version 9.0.