Novel Synaptonemal Complex Protein SYCE3 Figure 6. Syce3 mutation results in disruption of meiosis. Histological analysis of testes (day 30) and ovaries (day 32) from Syce3+/+, Syce3+/2 and Syce32/2 mice stained with haematoxylin and eosin. Syce32/2 testis lack postmeiotic stages whereas all stages of the spermatogenic cycle occur in the wild-type testis. The size of mutant ovaries is greatly reduced compared to wild-type littermates and additionally Syce32/2 ovaries are completely depleted of follicles. (Ser) Sertoli cells, (Sc) spermatocytes, (Sp) spermatids, (eSp) elongated spermatids, (aSc) apoptotic spermatocytes, (Sg) spermatogonia, (Oc) oocyte, (Od) oviduct. Long and short bars, 100 mm and 175 mm, respectively. doi:10.1371/journal.pgen.1002088.g006 recombination intermediates [41,43]. In Syce32/2 spermatocytes, RAD51 and RPA form numerous foci, localized to chromosome cores during zygotene (data not shown). This pattern of distribution resembles that of wild-type spermatocytes in early stages of prophase I (data not shown). These observations are consistent with the notion that early DNA-DNA interactions can be mediated by RAD51 and RPA in the absence of SYCE3. However, the following processing steps are likely to be disturbed as RAD51 and RPA remain associated with chromosomes in pachytene-like spermato- cytes (Figure 8). MLH1 marks presumed future crossover sites [43– 45]. Correspondingly, in wild-type pachytene spermatocytes each bivalent displayed one or two MLH1 foci. In contrast, male littermates lacking SYCE3 have no MLH1 foci, pointing to a disruption of crossover formation (Figure 8). To rule out the possibility that the persistence of RPA on chromosome axes and the lack of MLH1 foci in Syce3-deficient spermatocytes is due to their arrest in pachytene and their subsequent elimination by apoptosis rather than reflecting a direct function of SYCE3 in homologous recombination we additionally performed a close examination of recombination in Syce32/2 oocytes at 19.5 dpc (days post coitum). In concordance with earlier reports [46,47] the majority of 19.5 dpc oocytes were staged at late pachytene or diplotene. Consistent with this, SCYP3 labeled AEs were fully synapsed in wild-type pachytene oocytes. As already described above for Syce32/2 spermatocytes synapsis was com- pletely abolished in SYCE3-deficient pachytene-like oocytes. Compared to the situation in the male (see Figure 7A and Figure 8), however, pairing and alignment in 19.5 dpc Syce32/2 oocytes seemed to be more strongly affected (Figure 9A). Whether this finding reflects a female–specific function of SYCE3 in homologous pairing and/or alignment or is rather a secondary effect caused by defective synapsis cannot be judged at present. Careful analysis of recombination markers in 19.5 dpc oocytes strongly supported the notion that SYCE3 is essential for progression of meiotic recombination (Figure 9A). Consistent with our findings in pachytene-like Syce32/2 spermatocytes, in 19.5 dpc oocytes of SYCE3-deficient animals cH2AX stayed associated with chromosome axes, unlike in oocytes of wild-type littermates (Figure 9A). This suggests the persistence of unrepaired DSBs. Moreover, as judged by quantitative analysis of RPA and MLH1 dynamics, processing of TNs to RNs appeared to be considerably affected by the absence of SYCE3: In wild-type oocytes the median of RPA foci per cell was 22 during pachytene and decreased to 4 at diplotene stage. At the same time, the median number of MLH1 foci per cell was 22 at pachytene and 7.5 at diplotene stage in the controls. Thus, both the dynamics of RPA and MLH1 foci as well as their relative abundance found in our wild-type controls were consistent with data reported previously [41]. In clear contrast, the median number of RPA foci in SYCE3- deficient pachytene-like oocytes was significantly higher compared to wild-type controls (67 vs. 22; p,0.001). Additionally, RPA foci persisted on chromosome axes of Syce32/2 oocytes at diplotene- like stage (median, 27 vs. 4; p,0.001) indicating that processing of TNs to RNs was impaired in the absence of SYCE3 (Figure 9B). In line with this notion, MLH1 was virtually completely absent from chromosome axes of Syce32/2 oocytes at both late pachytene- (median, 0) and dilpotene-like stage (median, 0) (Figure 9B). To rule out the possibility that the considerable alterations of RPA and MHL1 dynamics in SYCE3-deficient oocytes could be caused by a delay of meiotic progression per se, we quantified the ratio of pachytene (WT, n = 44; Syce32/2, n = 53) and diplotene (WT, n = 33; Syce32/2, n = 35) stages in 19.5 dpc oocytes of both Syce32/2 and wild-type females. Here, no significant difference could be observed (p = 0,688; Figure 9C). Together, our data strongly argues that while progression through meiotic stages per se remains unaffected by the loss of SYCE3 in females, progression of recombination (i.e. processing of recombination intermediates into MLH1-marked late RNs, which PLoS Genetics | www.plosgenetics.org 7 May 2011 | Volume 7 | Issue 5 | e1002088