Novel Synaptonemal Complex Protein SYCE3 Figure 9. Complete repair of DSBs and crossover formation is not observed in Syce32/2 oocytes. (A) Immunofluorescence analysis on mouse oocytes collected from 19.5 dpc littermates. Wild-type and Syce32/2 oocytes double stained for cH2AX (first row), RPA (second row) or MLH1 (third row) and SYCP3 are shown. Images were acquired using a confocal laser scanning microscope. Bars, 10 mm. (B) Numbers of RPA foci (left) in pachytene (WT, n = 21; Syce32/2, n = 27) and diplotene (WT, n = 15; Syce32/2, n = 15) oocytes depicted in a box-and-whisker plot. Box-and-whisker plot of MLH1 foci (right) counted in pachytene (WT, n = 23; Syce32/2, n = 26) and diplotene (WT, n = 18; Syce32/2, n = 20) oocytes. (C) Pachytene/ diplotene ratio of oocytes collected from control (n = 77) or Syce32/2 (n = 88) 19.5 dpc littermates. doi:10.1371/journal.pgen.1002088.g009 SYCE3 is required for initiating synapsis. Furthermore, we have shown that SYCE1 and the SYCE2/Tex12 complex do not localize to chromosomes in Syce32/2 spermatocytes (Figure 7A). The lack of CE-like structures and CE-specific proteins indicates that SYCE3 is required upstream of SYCE1, SYCE2 and Tex12 and downstream of SYCP1 during the initiation of CE assembly. A possible function of SYCE3 could be to enable recruitment of SYCE1 and the SYCE2/Tex12 complex to SYCP1 N-termini. Loading of SYCE1 and SYCE2 to SYCP1 in turn would stabilize SYCP1 N-termini as proposed by Costa et al. [28]. This hypothesis is consistent with our results obtained from co-immunoprecipita- tion assays with transfected COS-7 cells, revealing that SYCE3 is capable of binding SYCE1 as well as SYCE2 (Figure 10). However, whether the role of SYCE3 in CE assembly is of a structural or regulatory character still needs to be clarified. Taken together, we conclude that SYCE3 is essential for initiating synapsis and for chromosomal loading of SYCE1 and the SYCE2/Tex12 complex. According to our present knowledge the behavior of SYCP1 and SYCE3 in meiotic cells lacking certain CE-specific proteins remains somewhat puzzling. In wild-type cells SYCP1 localization is restricted to synapsed chromosome axes. By contrast, in the absence of SYCE1, SYCP1 is observed in a weak, discontinuous pattern at the unsynapsed chromosome axes, no matter whether they were closely aligned or not. However, this is not the case in the absence of SYCE2 or Tex12, as under these conditions SYCP1 exclusively localizes to small foci at the sites of synapsis initiation [31]. These observations led to the hypothesis that SYCE1 is required to restrict SYCP1 to synapsed areas [30]. Our finding that SYCE3 also localizes to unsynapsed chromosome axes in SYCE1 spermatocytes suggests that SYCE3 restriction to synapsed areas also depends on SYCE1 (Figure 5C). Nevertheless, we presently have no satisfactory explanation as to how this restricted localization can be accomplished. Another aspect that would require further investigation is the relationship between SYCP1 and SYCE3. As mentioned above, chromosomal loading PLoS Genetics | www.plosgenetics.org 10 May 2011 | Volume 7 | Issue 5 | e1002088