Figure 4. SYCE3 mirrors SYCE1 localization. Co-labeling of zygotene and pachytene spermatocyte spread preparations with SYCE3 (red) and SYCE1 or SYCE2 (green) acquired using a confocal microscope. Co-staining for SYCE3 and SYCE1 in zygotene and pachytene spermatocytes shows that these two proteins co-localize along the synapsed chromosomes (see inset). In contrast, SYCE3 and SYCE2 do not necessarily co-localize in zygotene and pachytene cells (see inset). Here, SYCE3 is distributed in a more continuous pattern whereas SYCE2 appears more punctuated. Bar, 10 mm. doi:10.1371/journal.pgen.1002088.g004 offspring with the mutated locus in Mendelian ratio. Correct deletion of Syce3 in heterozygote and homozygote animals was confirmed by Southern blot analysis (Figure S2D). Syce32/2 mice displayed no overt somatic phenotype, but repeated mating attempts of wild-type with both Syce32/2 male and female mice did not produce offspring implying that both male and female Syce32/2 mice were infertile. Consistent with this, Syce32/2 testes of adult mice have a clearly reduced size compared to their wild- type littermates as previously reported for other infertile mice (Figure S2E; i.e. [27]). In addition, many TUNEL-positive meiotic cells (Figure S3A) and no postmeiotic cells were found in histological sections of Syce32/2 testes, indicating a defect during meiosis resulting in programmed cell death at stage IV (Figure 6 and Figure S3B). Compared to wild type females, ovaries of Syce32/2 littermates showed a sharp size reduction and lacked mature follicles suggesting a disruption of oogenesis (Figure 6). Initiation of Synapsis Is Dependent on SYCE3 To address the question why Syce3 knockout leads to disruption of meiosis, we performed immunofluorescence analysis on spread mouse spermatocytes in which AEs were labeled with SYCP3 and TFs with SYCP1 as a marker for synapsis. As expected, in wild- type spermatocytes homologs were aligned in close juxtaposition Novel Synaptonemal Complex Protein SYCE3 during zygotene (data not shown) and full synapsis was achieved during pachytene (Figure 7A). In Syce32/2 pachytene-like spermatocytes the vast majority of AEs of the homologs were paired and aligned along their entire lengths. Incorrect alignment of autosomes was very rare (Figure 7A) and was most probably due to harsh chromosome spreading. Sex chromosomes on the other hand were frequently unpaired and appeared as univalents mimicking the phenotypes previously described for other CE proteins [27,30–32]. In clear contrast to the wild-type situation, AEs completely failed to synapse in pachytene-like Syce32/2 spermatocytes (Figure 7A). To further investigate the effects of SYCE3 depletion on synapsis, we compared SYCP1 localization in Syce32/2, Syce12/2 and Syce22/2 spermatocytes. As previously described, in Syce22/2 cells SYCP1-staining is confined to regions where homologous chromosomes are in closer association (see Figure S4B and [31]). In contrast, in Syce12/2 cells SYCP1 localizes to the chromosome axes in a weak discontinuous pattern regardless of whether they are closely aligned or not (see Figure S4A and [30]). A similar distribution of SYCP1 was observed in cells deficient for SYCE3: here, too, SYCP1 was distributed in a weak, discontinuous pattern along the AEs, irrespective of whether they were closely aligned or not (Figure 7A). This is in strong contrast to wild-type spermatocytes, where SYCP1 localizes only to the synapsed areas of homologous chromosomes, but not to aligned AEs (Figure 7A). This suggests that SYCP1 is able to bind to the AEs via its C- terminus, and that the N-terminal interactions are impaired in the absence of SYCE3. Furthermore, these data clearly show that immunofluorescence analysis obtained under our experimental conditions can reproduce previously obtained weak immunofluo- rescence signals (compare Figure S4 and [30,31]) and thus allow precise comparison of the different CE-mutant phenotypes. We also investigated the localization of other CE proteins in Syce32/2 mice. To this end, we labeled spread Syce32/2 spermatocytes with SYCP3 as an AE marker in combination with either SYCE1 or SYCE2. Interestingly, both SYCE1 and SYCE2 were completely absent from the axes in cells lacking SYCE3 (Figure 7A). These results provide clear evidence that SYCE3 is required for loading of the other CE proteins. To obtain more detailed information about synapsis defects in the Syce3 knockout mice, we performed electron microscopic analysis on Syce32/2 testis. In wild-type spermatocytes, normal SCs composed of LEs with attached chromatin and a CE were observed. In contrast, we found partially aligned AEs in Syce32/2 spermatocytes, but no CE or CE-like structures at all (Figure 7B). This phenotype resembles the situation found in Sycp12/2 and Syce12/2 mice, but differs from that of Syce22/2 and Tex122/2 mice in which synapsis appears to initiate due to the assembly of short CE-like structures [30–32]. SYCE3 Is Essential for Normal Progression of Meiotic Recombination During leptotene, meiotic recombination is initiated by the introduction of DNA DSBs. These sites become marked by histone cH2AX. During leptotene and zygotene, cH2AX is located in large domains around the DNA breaks, but as meiotic prophase I progresses, it becomes restricted to the sex chromosomes [38]. In Syce32/2 spermatocytes, however, immunostaining of cH2AX revealed altered dynamics. While distribution of cH2AX in early (leptotene, zygotene) mutant spermatocytes resembles that of early wild-type cells (data not shown), cH2AX is not restricted to the sex chromosomes during the pachytene-like stage (Figure 8). Instead, cH2AX remains associated with most of the chromosomes in a cloud-like manner. The persistence of cH2AX-staining up to the PLoS Genetics | www.plosgenetics.org 5 May 2011 | Volume 7 | Issue 5 | e1002088