Identification of a structural protein component of rat synaptonemal complexes

Synaptonemal complexes (SCs) are evolutionarily conserved nuclear structures of meiotic cells which form during the zygotene stage of the first meiotic prophase and are responsible for the pairing of homologous chromosomes. Their formation appears to be a prerequisite for crossing-over events and proper chromosome segregation during the first meiotic division. Despite knowledge of their central role in genetic recombination processes very little is known about the molecular composition and the mechanisms governing the assembly of the SCs. In the present study we report on the characterization of a monoclonal antibody (SC14f10) which enabled us to identify a novel SC protein termed SC48. Protein SC48 has a Mr of 48,000 and migrates in two-dimensional gels with a pH value of 6.9. By means of immunogold EM we localized this protein to the central region of the SC. In cell fractionation experiments we recovered protein SC48 together with SC-residual structures in a karyoskeletal fraction of pachytene spermatocytes. Our results indicate that SC48 is a meiosis-specific structural protein component of the SC probably involved in the pairing of homologous chromosomes.     

In vivo analysis of synaptonemal complex formation during yeast meiosis

During meiotic prophase a synaptonemal complex (SC) forms between each pair of homologous chromosomes and is believed to be involved in regulating recombination. Studies on SCs usually destroy nuclear architecture, making it impossible to examine the relationship of these structures to the rest of the nucleus. In Saccharomyces cerevisiae the meiosis-specific Zip1 protein is found throughout the entire length of each SC. To analyze the formation and structure of SCs in living cells, a functional ZIP1::GFP fusion was constructed and introduced into yeast. The ZIP1::GFP fusion produced fluorescent SCs and rescued the spore lethality phenotype of zip1 mutants. Optical sectioning and fluorescence deconvolution light microscopy revealed that, at zygotene, SC assembly was initiated at foci that appeared uniformly distributed throughout the nuclear volume. At early pachytene, the full-length SCs were more likely to be localized to the nuclear periphery while at later stages the SCs appeared to redistribute throughout the nuclear volume. These results suggest that SCs undergo dramatic rearrangements during meiotic prophase and that pachytene can be divided into two morphologically distinct substages: pachytene A, when SCs are perinuclear, and pachytene B, when SCs are uniformly distributed throughout the nucleus. ZIP1::GFP also facilitated the enrichment of fluorescent SC and the identification of meiosis-specific proteins by MALDI-TOF mass spectroscopy.     

Protein SYCP2 provides a link between transverse filaments and lateral elements of mammalian synaptonemal complexes

Synaptonemal complexes (SCs) are evolutionarily conserved meiosis-specific nuclear structures critically involved in synapsis, recombination, and segregation of homologous chromosomes. SCs are proteinaceous structures composed of (a) two lateral elements (LEs), to which the chromatin of the homologs is attached, (b) numerous transverse filaments (TFs) that link the LEs, and (c) a central element (CE). Major protein components of mammalian SCs are the TF protein SYCP1 and the LE proteins SYCP2 and SCYP3. How SCs become assembled is presently poorly understood, in particular, it is not known how TFs assemble at the plane of LEs to interconnect the homologous chromosomes. Therefore, we have investigated possible interactions between SYCP1 and other SC proteins. In immunoprecipitation experiments we could find that SYCP1 and SYCP2 interact in extracts of meiotic cells. Using the yeast two-hybrid system, we were able to demonstrate that the C-terminus of SYCP1 directly interacts with SYCP2. These results were confirmed by different interaction traps. Furthermore, we could narrow down the interacting domain of the SYCP2 molecule to its C-terminal region. We propose that SYCP2 acts as a linker between SYCP1 and SYCP3 and therefore would be the missing connecting link between LEs and TFs essential for proper chromosome synapsis.     

联会复合体——原发无精症发病中的重要角色

联会复合体(synaptonemal complex,SC)是一种减数分裂特异性超分子蛋白质结构,与减数分裂I(改罗文)中同源染色体的凝缩、配对、重组和分离密切相关。近年来,联会复合体的研究取得了一系列重要的进展,包括在其组成成分和功能上的一些新发现。在小鼠不育模型中联会复合体及其编码基因的异常可引起精子发生障碍。更重要的是,联会复合体编码基因之一SCP3单个碱基缺失导致的无精症已在人类原发不育患者中得到证实。对联会复合体基因SCP1的进一步研究也正在进行之中。      

Lateral elements inside synaptonemal complex-like polycomplexes in ndt80 mutants of yeast bind DNA

The synaptonemal complex (SC) keeps the synapsed homologous chromosomes together during pachytene in meiotic prophase I. Structures that resemble stacks of SCs, polycomplexes, are sometimes found before or after pachytene. We have investigated ndt80 mutants of yeast, which arrest in pachytene. SCs appear normal in spread chromosome preparations, but are only occasionally found in intact nuclei examined in the electron microscope. Instead, large polycomplexes occur in almost every ndt80 mutant nucleus. Immunoelectron microscopy using DNA antibodies show strong preferential labeling to the lateral element parts of the polycomplexes. In situ hybridization using chromosome-specific probes confirms that the chromosomes in ndt80 mutants are paired and attached to the SCs. Our results suggest that polycomplexes can be involved in binding of chromosomes and possibly also in synapsis.     

Expression of the Meiosis-Specific Synaptonemal Complex Protein 1 in a Heterologous System Results in the Formation of Large Protein Structures

The synaptonemal complex is a meiosis-specific structure essential for synapsis of homologous chromosomes. The synaptonemal complex protein 1 (SCP1) is a major constituent of the transversal filament, a fibrous structure that connects the central element of the synaptonemal complex with the two lateral elements. The SCP1 protein forms filamentous dimers with the two molecules that have the same polarity, with the C-termini being anchored in the lateral elements and the N-termini reaching into the central element. We investigated whether the SCP1 protein can take part in the formation of higher order protein structures by expressing it in a heterologous system. We find that expression of SCP1 in Swiss-3T3 fibroblast cells results in the formation of large protein structures. These protein structures resemble a higher order protein structure produced by overexpression of a yeast transversal filament protein in meiotic cells. Our results show that SCP1 is a structural protein and that it most likely is directly involved in the assembly of the synaptonemal complex.     

The murine SCP3 gene is required for synaptonemal complex assembly, chromosome synapsis, and male fertility

During meiosis, the homologous chromosomes pair and recombine. An evolutionarily conserved protein structure, the synaptonemal complex (SC), is located along the paired meiotic chromosomes. We have studied the function of a structural component in the axial/lateral element of the SC, the synaptonemal complex protein 3 (SCP3). A null mutation in the SCP3 gene was generated, and we noted that homozygous mutant males were sterile due to massive apoptotic cell death during meiotic prophase. The SCP3-deficient male mice failed to form axial/lateral elements and SCs, and the chromosomes in the mutant spermatocytes did not synapse. While the absence of SCP3 affected the nuclear distribution of DNA repair and recombination proteins (Rad51 and RPA), as well as synaptonemal complex protein 1 (SCP1), a residual chromatin organization remained in the mutant meiotic cells.     

Synaptonemal complex protein SYCP3: Conserved polymerization properties among vertebrates

Synaptonemal complexes (SCs) are meiosis-specific, nuclear structures that are critically involved in synapsis, recombination and segregation of homologous chromosomes. Although the SC structure is conserved in evolution this is not the case for its protein components. To provide information on SC proteins which would be important for our understanding of the conserved SC structure and function, here we compared ortholog SYCP3 proteins of two evolutionary distant vertebrate species, namely rat and medaka fish. To this end we have investigated the polymerization properties of both proteins by immunocytochemistry, electron microscopy and cell fractionation. We found that despite of the sequence differences that have accumulated over the last 450 million years mammalian and fish SYCP3 have similar properties that allow them to co-assemble higher order structures under experimental conditions. We also provide a likely explanation as to how heterozygous mutations in the SYCP3 gene can lead to a defective meiosis.     

A novel mouse synaptonemal complex protein is essential for loading of central element proteins, recombination, and fertility

The synaptonemal complex (SC) is a proteinaceous, meiosis-specific structure that is highly conserved in evolution. During meiosis, the SC mediates synapsis of homologous chromosomes. It is essential for proper recombination and segregation of homologous chromosomes, and therefore for genome haploidization. Mutations in human SC genes can cause infertility. In order to gain a better understanding of the process of SC assembly in a model system that would be relevant for humans, we are investigating meiosis in mice. Here, we report on a newly identified component of the murine SC, which we named SYCE3. SYCE3 is strongly conserved among mammals and localizes to the central element (CE) of the SC. By generating a Syce3 knockout mouse, we found that SYCE3 is required for fertility in both sexes. Loss of SYCE3 blocks synapsis initiation and results in meiotic arrest. In the absence of SYCE3, initiation of meiotic recombination appears to be normal, but its progression is severely impaired resulting in complete absence of MLH1 foci, which are presumed markers of crossovers in wild-type meiocytes. In the process of SC assembly, SYCE3 is required downstream of transverse filament protein SYCP1, but upstream of the other previously described CE-specific proteins. We conclude that SYCE3 enables chromosome loading of the other CE-specific proteins, which in turn would promote synapsis between homologous chromosomes.     

Characterization of all human male synaptonemal complexes by subtelomere multiplex-FISH

During meiotic prophase I, homologous chromosomes synapse and recombine. Both events are of vital importance for the success of meiosis. When homologous chromosomes synapse, a proteinaceous structure called synaptonemal complex (SC) appears along the pairing axis and meiotic recombination takes place. The existence of immunolabeling techniques for SC proteins (SCP1, SCP2 and SCP3) and for DNA mismatch repair proteins present in late recombination nodules (MLH1) allow analyses of both synapsis and meiotic recombination in the gametocyte I. In situ hybridization methods can be applied afterwards because chromatin is preserved during cell fixation for immunoanalysis. The combination of both methodologies allows the analysis of synapsis and the creation of recombination maps for each bivalent. In this work we apply the seven-fluorochrome subtelomere-specific multiplex FISH assay (stM-FISH) to human male meiotic cells previously labeled by immunofluorescence (SCP1, SCP3, MLH1, CENP) to assess its utility for human SC karyotyping. This FISH method consists of microdissected subtelomeric probes labeled combinatorially with seven different fluorochromes. Results prove its usefulness for the identification of all human SCs. Furthermore, by labeling subtelomeric regions this one-single-step method enables the characterization of interstitial and terminal SC fragments and SC delineation even if superposition is present in pachytene spreads.     

SUMO Localizes to the Central Element of Synaptonemal Complex and Is Required for the Full Synapsis of Meiotic Chromosomes in Budding Yeast

The synaptonemal complex (SC) is a widely conserved structure that mediates the intimate alignment of homologous chromosomes during meiotic prophase and is required for proper homolog segregation at meiosis I. However, fundamental details of SC architecture and assembly remain poorly understood. The coiled-coil protein, Zip1, is the only component whose arrangement within the mature SC of budding yeast has been extensively characterized. It has been proposed that the Small Ubiquitin-like MOdifier, SUMO, plays a role in SC assembly by linking chromosome axes with Zip1''s C termini. The role of SUMO in SC structure has not been directly tested, however, because cells lacking SUMO are inviable. Here, we provide direct evidence for SUMO''s function in SC assembly. A meiotic smt3 reduction-of-function strain displays reduced sporulation, abnormal levels of crossover recombination, and diminished SC assembly. SC structures are nearly absent when induced at later meiotic time points in the smt3 reduction-of-function background. Using Structured Illumination Microscopy we furthermore determine the position of SUMO within budding yeast SC structure. In contrast to previous models that positioned SUMO near Zip1''s C termini, we demonstrate that SUMO lies at the midline of SC central region proximal to Zip1''s N termini, within a subdomain called the “central element”. The recently identified SUMOylated SC component, Ecm11, also localizes to the SC central element. Finally, we show that SUMO, Ecm11, and even unSUMOylatable Ecm11 exhibit Zip1-like ongoing incorporation into previously established SCs during meiotic prophase and that the relative abundance of SUMO and Ecm11 correlates with Zip1''s abundance within SCs of varying Zip1 content. We discuss a model in which central element proteins are core building blocks that stabilize the architecture of SC near Zip1''s N termini, and where SUMOylation may occur subsequent to the incorporation of components like Ecm11 into an SC precursor structure.     

A Meiotic Chromosomal Core Consisting of Cohesin Complex Proteins Recruits DNA Recombination Proteins and Promotes Synapsis in the Absence of an Axial Element in Mammalian Meiotic Cells

The behavior of meiotic chromosomes differs in several respects from that of their mitotic counterparts, resulting in the generation of genetically distinct haploid cells. This has been attributed in part to a meiosis-specific chromatin-associated protein structure, the synaptonemal complex. This complex consist of two parallel axial elements, each one associated with a pair of sister chromatids, and a transverse filament located between the synapsed homologous chromosomes. Recently, a different protein structure, the cohesin complex, was shown to be associated with meiotic chromosomes and to be required for chromosome segregation. To explore the functions of the two different protein structures, the synaptonemal complex and the cohesin complex, in mammalian male meiotic cells, we have analyzed how absence of the axial element affects early meiotic chromosome behavior. We find that the synaptonemal complex protein 3 (SCP3) is a main determinant of axial-element assembly and is required for attachment of this structure to meiotic chromosomes, whereas SCP2 helps shape the in vivo structure of the axial element. We also show that formation of a cohesin-containing chromosomal core in meiotic nuclei does not require SCP3 or SCP2. Our results also suggest that the cohesin core recruits recombination proteins and promotes synapsis between homologous chromosomes in the absence of an axial element. A model for early meiotic chromosome pairing and synapsis is proposed.     

The synaptonemal complex is assembled by a polySUMOylation-driven feedback mechanism in yeast

During meiotic prophase I, proteinaceous structures called synaptonemal complexes (SCs) connect homologous chromosomes along their lengths via polymeric arrays of transverse filaments (TFs). Thus, control of TF polymerization is central to SC formation. Using budding yeast, we show that efficiency of TF polymerization closely correlates with the extent of SUMO conjugation to Ecm11, a component of SCs. HyperSUMOylation of Ecm11 leads to highly aggregative TFs, causing frequent assembly of extrachromosomal structures. In contrast, hypoSUMOylation leads to discontinuous, fragmented SCs, indicative of defective TF polymerization. We further show that the N terminus of the yeast TF, Zip1, serves as an activator for Ecm11 SUMOylation. Coexpression of the Zip1 N terminus and Gmc2, a binding partner of Ecm11, is sufficient to induce robust polySUMOylation of Ecm11 in nonmeiotic cells. Because TF assembly is mediated through N-terminal head-to-head associations, our results suggest that mutual activation between TF assembly and Ecm11 polySUMOylation acts as a positive feedback loop that underpins SC assembly.     

Architecture of the nuclear periphery of rat pachytene spermatocytes: distribution of nuclear envelope proteins in relation to synaptonemal complex attachment sites

The nucleus of spermatocytes provides during the first meiotic prophase an interesting model for investigating relationships of the nuclear envelope (NE) with components of the nuclear interior. During the pachytene stage, meiotic chromosomes are synapsed via synaptonemal complexes (SCs) and attached through both ends to the nuclear periphery. This association is dynamic because chromosomes move during the process of synapsis and desynapsis that takes place during meiotic prophase. The NE of spermatocytes possesses some peculiarities (e.g., lower stability than in somatic cells, expression of short meiosis-specific lamin isoforms called C2 and B3) that could be critically involved in this process. For better understanding of the association of chromosomes with the nuclear periphery, in the present study we have investigated the distribution of NE proteins in relation to SC attachment sites. A major outcome was the finding that lamin C2 is distributed in the form of discontinuous domains at the NE of spermatocytes and that SC attachment sites are embedded in these domains. Lamin C2 appears to form part of larger structures as suggested by cell fractionation experiments. According to these results, we propose that the C2-containing domains represent local reinforcements of the NE that are involved in the proper attachment of SCs.     

Synaptonemal complexes in insects

The synaptonemal complex (SC) is the key nuclear element formed in meiotic prophase I to join 2 homologous chromosomes at the pachytene bivalent. It is a highly conserved structure that is universally present in eukaryotes. The SC is presented as a tripartite protein structure, which consists of 2 lateral elements and a central region. In insects, the central region is particularly distinct and highly ordered. This made it possible to describe the fine structure of the central region and propose a model of its architecture. Chromatid DNA is arranged in chromatin loops extending radially from the SC. The loops appear to consist of a basic chromatin fiber with a diameter of 20–30 nm. In many insect species, synaptonemal polycomplexes occur in postpachytene cells. They represent one of the possible ways of SC degradation. Another process, which occurs beyond pachytene, is the formation of proteinaceous chromatid axis, the silver-stained chromatid core. Based on results in insect models, the chromatid cores have been related to the structure and formation of the SC. Research on insect models significantly contributed to understanding individual steps of the SC formation and temporal sequence of chromosome pairing. These include the formation of lateral elements of the SC, pairing initiation, interlocking of chromosomes, and synapsis of homologous chromosomes. Attention is also given to non-homologous pairing, including synaptic adjustment, correction of pairing, and pairing of sex chromosomes. In the next section, chiasmatic and achiasmatic modes of meiosis are compared with respect to the SC formation. In the chiasmatic mode, the SCs display recombination nodules that are believed to mediate the process of recombination. These nodules were discovered in insects, and indirect evidence for their role comes from insects. Two different examples of achiasmatic meiosis, occurring in the heterogametic sex of several insect orders, are given: one involves the SC formation, whereas in the other, SCs are absent. Finally, the potential of SC karyotyping for analysis of the insect genome is discussed.     

Formation of cytoplasmic synaptonemal-like polycomplexes at leptotene and normal synaptonemal complexes at zygotene in Ascaris suum male meiosis

A thread-like (more than 70 cm long) testis of Ascaris suum, when examined under the light and electron microscope, reveals the linear succession of meiotic stages. Beginning from, at least, late leptotene, the spermatocytes are synchronous in their development. Thus within each transverse section of the testis all the spermatocytes are in the same stage. The spermatocytes at each stage of prophase I occupies several (4 to 10) cm of the whole testis length. — At leptotene, synaptonemal-like polycomplexes of lateral and central stacked elements are formed in the cytoplasm of spermatocytes. At late leptotene, the polycomplexes are attached to the external nuclear membrane. The polycomplexes disappear at zygotene. Slightly discernable axial cores are observed in the late leptotene chromosomes. The synaptonemal complexes (SCs) are formed at the zygotene stage, their structure being characteristically tripartite. The SCs disappear from the nuclei at the diffuse stage of prophase I. In other organisms completely developed polycomplexes of stacked lateral and central elements were never found during the presynaptic period of meiosis, although single or two parallel layers of aggregated central regions of SC were found in Neottiella meiocytes at the stage prior to chromosome pairing (Westergaard and von Wettstein, 1970, 1972). — First appearance of the polycomplexes in the cytoplasm insetead of the nucleus is also a novel fact. It is concluded that the polycomplexes at leptotene are formed by a self-assembly of the SC molecular material precociously synthesized in the cytoplasm. Two hypotheses regarding possible function and the further fate for leptotene polycomplexes are discussed.     

Interplay between synaptonemal complex, homologous recombination, and centromeres during mammalian meiosis

The intimate synapsis of homologous chromosome pairs (homologs) by synaptonemal complexes (SCs) is an essential feature of meiosis. In many organisms, synapsis and homologous recombination are interdependent: recombination promotes SC formation and SCs are required for crossing-over. Moreover, several studies indicate that initiation of SC assembly occurs at sites where crossovers will subsequently form. However, recent analyses in budding yeast and fruit fly imply a special role for centromeres in the initiation of SC formation. In addition, in budding yeast, persistent SC–dependent centromere-association facilitates the disjunction of chromosomes that have failed to become connected by crossovers. Here, we examine the interplay between SCs, recombination, and centromeres in a mammal. In mouse spermatocytes, centromeres do not serve as SC initiation sites and are invariably the last regions to synapse. However, centromeres are refractory to de-synapsis during diplonema and remain associated by short SC fragments. Since SC–dependent centromere association is lost before diakinesis, a direct role in homolog segregation seems unlikely. However, post–SC disassembly, we find evidence of inter-centromeric connections that could play a more direct role in promoting homolog biorientation and disjunction. A second class of persistent SC fragments is shown to be crossover-dependent. Super-resolution structured-illumination microscopy (SIM) reveals that these structures initially connect separate homolog axes and progressively diminish as chiasmata form. Thus, DNA crossing-over (which occurs during pachynema) and axis remodeling appear to be temporally distinct aspects of chiasma formation. SIM analysis of the synapsis and crossover-defective mutant Sycp1^−/− implies that SCs prevent unregulated fusion of homolog axes. We propose that SC fragments retained during diplonema stabilize nascent bivalents and help orchestrate local chromosome reorganization that promotes centromere and chiasma function.     

SCP2: a major protein component of the axial elements of synaptonemal complexes of the rat.

In the axial elements of synaptonemal complexes (SCs) of the rat, major protein components have been identified, with relative electrophoretic mobilities (M rs) of 30 000-33 000 and 190 000. Using monoclonal anti-SC antibodies, we isolated cDNA fragments which encode the 190 000 M r component of rat SCs. The translation product predicted from the nucleotide sequence of the cDNA, called SCP2 (for synaptonemal complex protein 2), is a basic protein (pI = 8.0) with a molecular mass of 173 kDa. At the C-terminus, a stretch of approximately 50 amino acid residues is predicted to be capable of forming coiled-coil structures. SCP2 contains two clusters of S/T-P motifs, which are common in DNA-binding proteins. These clusters flank the central, most basic part of the protein (pI = 9.5). Three of the S/T-P motifs are potential target sites for p34(cdc2) protein kinase. In addition, SCP2 has eight potential cAMP/cGMP-dependent protein kinase target sites. The gene encoding SCP2 is transcribed specifically in the testis, in meiotic prophase cells. At the amino acid sequence and secondary structural level, SCP2 shows some similarity to the Red1 protein, which is involved in meiotic recombination and the assembly of axial elements of SCs in yeast. We speculate that SCP2 is a DNA-binding protein involved in the structural organization of meiotic prophase chromosomes.