Author Summary Meiosis is a special type of cell division that takes place in the germ line of sexually reproducing diploid organisms. Major events during meiosis are the pairing, recombina- tion, and segregation of homologous chromosomes. As a consequence, daughter cells are haploid and genetically diverse. Therefore, meiosis is of utmost importance for the life of sexually reproducing species as it maintains the species-specific chromosome number and generates genetic diversity within a species. Proper segregation of homologous chromosomes during meiosis requires ho- molog pairs to be physically linked. The synaptonemal complex (SC), a meiosis-specific structure conserved in evolution, is essential for this process. Defective assembly of the SC has deleterious effects on germ cells and can cause infertility in mice and humans. Here, we report on a newly identified protein component of the mammalian SC that we have named SYCE3. SYCE3 is strongly conserved among mammals. Using the mouse as a model system, we demonstrate that loss of SYCE3 leads to infertility in both sexes. Infertility is caused by disruption of meiosis due to the inability of Syce32/2 mice to assemble the central element of SCs. Our findings provide new insights into the complexity of SC assembly and its relevance to mamma- lian fertility. proteins [17–19]. Mice deficient for SYCP3 lack AEs and fail to form continuous cohesin cores [20–22]. Interestingly, knockout of the Sycp3 gene results in a sexually dimorphic phenotype: SYCP3- deficient males are sterile due to massive apoptotic cell death during zygotene [20], whereas females are fertile, but exhibit a sharp reduction in litter size caused by aneuploidy resulting in embryo death in utero [21]. AEs become linked by numerous transverse filaments (hence, AEs are referred to as LEs) when synapsis is initiated in zygotene of wild-type mice. Transverse filaments (TFs) are mainly composed of SYCP1, a fibrillar protein with a central coiled-coil domain flanked by two globular N- and C-terminal domains [23]. SYCP1 molecules most likely form homodimers with a parallel orientation with both C-termini anchored in an LE and the N- termini interacting head-to-head in the CE with a SYCP1 dimer of the opposite LE [24–26]. Two facts indicate the prominent function of SYCP1 during SC assembly: (1) disruption of the mouse Sycp1 gene leads to sterility in both sexes, which is caused by massive apoptotic events during spermatogenesis and oogenesis. Detailed analysis of Sycp1 mice revealed a complete breakdown of synapsis of homologous chromosomes, although the mice show normal AE formation and chromosome alignment. Furthermore, SYCP1 is required for crossover formation and the repair of DNA double-strand breaks (DSBs) [27]. (2) When expressed in a heterologous system SYCP1 molecules have the capability to self-assemble and form structures that closely resemble SCs (i.e. polycomplexes; [26]). Therefore, SYCP1 may function as a molecular framework to which other proteins attach to accomplish SC assembly and progression of recombination events ([26,27]; and below). Because of the prominent function of SYCP1 in meiosis and its relevance for fertility, the identification and characterization of its interaction partners has received intense interest over the past years. Of particular interest are three proteins, SYCE1, SYCE2 and Tex12, which exclusively localize to the CE and, interestingly, contain a coiled-coil domain, which constitutes a common protein interaction motif [28,29]. These proteins, together with SYCP1, form a complex in the CE. SYCE1 and SYCE2 were found to Novel Synaptonemal Complex Protein SYCE3 bind to each other and to SYCP1, whereas Tex12 forms a complex with SYCE2 and, therefore, indirectly interacts with SYCP1. The hypothesis that SYCP1 serves as a molecular framework is supported by the fact that disruption of the central region by eliminating Sycp1 results in a mislocalization of all three CE-specific proteins [29]. In addition, knockout models of each of the three known CE proteins revealed that loss of either of these alters higher order polymerization properties and localization of SYCP1 [30–32]. This indicates that the in vivo relationship between SYCP1 and all currently known CE proteins is reciprocal. Correct SC assembly is required for proper meiotic recombi- nation and conversely, recombination is essential for correct SC assembly. This is particularly well documented in mice deficient for Spo11, which are characterized by the lack of Spo11-dependent DSBs. In these mice SC formation is much reduced or SCs form between non-homologous chromosomes [33,34]. Thus SC assem- bly and meiotic recombination are mutually dependent on each other. Here we report on the characterization of SYCE3, a novel protein specifically located in the CE of the mammalian SC. SYCE3 is exclusively expressed during male and female meiosis and colocalizes with SYCP1 and SYCE1. We show that SYCE3 is part of the previously described CE complex [28,29] where it interacts with SYCE1 and SYCE2. To gain more insights into the function of SYCE3 we generated a Syce3 knockout mouse. These mice are characterized by infertility in both sexes as well as complete disruption of synapsis and mislocalization of previously described CE proteins, indicating that SYCE3 is required for synapsis initiation and chromosomal loading of the other CE proteins (i.e. SYCE1, SYCE2 and Tex12). Furthermore, we demonstrate that loss of SYCE3 has no influence on the initiation of meiotic recombination, but is required for its progression. Results Identification and Characterization of SYCE3 We selected a set of genes identified by means of (1) predominant expression in testis and (2) a predicted nuclear localization, as well as a coiled-coil domain, within the encoded protein sequence from a gene expression profile initially used to elucidate the impact of Dazl knockout on gene expression in the developing gonads [35]. Using RT-PCR we demonstrated the selective expression of one of these genes -1700007E06Rik [35] - in adult testes and embryonic ovaries, and its absence in somatic tissues (Figure 1B). 1700007E06Rik encodes a protein (which we have named SYCE3) consisting of 88 amino acids. SYCE3 can be found in all vertebrate classes from fish to human (Figure 1A). It is highly conserved among mammals with an identity of 90% (96% similarity) at the amino acid level between mouse and human. PSORTII and SMART prediction of protein structural motifs revealed that SYCE3 contains a short coiled-coil motif (amino acids 6–39) which is conserved in all analyzed vertebrate sequences except for fish. In order to determine the exact temporal expression pattern of SYCE3 mRNA during spermatogenesis, we performed additional RT-PCR experiments with whole mRNA fractions of pubertal mice testes of different ages. Analysis of the first wave of spermatogenesis revealed that SYCE3 mRNA is first detectable on day 12 (i.e. the onset of the prophase I of meiosis) and persists in older animals. Comparison of the temporal expression profile of SYCE3 mRNA with that of SYCP3 revealed a striking similarity, suggesting that SYCE3 also functions during the prophase I stage of meiosis (Figure 1C). To analyze SYCE3 expression at the protein level, we generated two antibodies against the full-length PLoS Genetics | www.plosgenetics.org 2 May 2011 | Volume 7 | Issue 5 | e1002088