Mouse SYCP2 is required for synaptonemal complex assembly and chromosomal synapsis during male meiosis

During meiosis, the arrangement of homologous chromosomes is tightly regulated by the synaptonemal complex (SC). Each SC consists of two axial/lateral elements (AEs/LEs), and numerous transverse filaments. SC protein 2 (SYCP2) and SYCP3 are integral components of AEs/LEs in mammals. We find that SYCP2 forms heterodimers with SYCP3 both in vitro and in vivo. An evolutionarily conserved coiled coil domain in SYCP2 is required for binding to SYCP3. We generated a mutant Sycp2 allele in mice that lacks the coiled coil domain. The fertility of homozygous Sycp2 mutant mice is sexually dimorphic; males are sterile because of a block in meiosis, whereas females are subfertile with sharply reduced litter size. Sycp2 mutant spermatocytes exhibit failure in the formation of AEs and chromosomal synapsis. Strikingly, the mutant SYCP2 protein localizes to axial chromosomal cores in both spermatocytes and fetal oocytes, but SYCP3 does not, demonstrating that SYCP2 is a primary determinant of AEs/LEs and, thus, is required for the incorporation of SYCP3 into SCs.     

Corona is required for higher-order assembly of transverse filaments into full-length synaptonemal complex in Drosophila oocytes

The synaptonemal complex (SC) is an intricate structure that forms between homologous chromosomes early during the meiotic prophase, where it mediates homolog pairing interactions and promotes the formation of genetic exchanges. In Drosophila melanogaster, C(3)G protein forms the transverse filaments (TFs) of the SC. The N termini of C(3)G homodimers localize to the Central Element (CE) of the SC, while the C-termini of C(3)G connect the TFs to the chromosomes via associations with the axial elements/lateral elements (AEs/LEs) of the SC. Here, we show that the Drosophila protein Corona (CONA) co-localizes with C(3)G in a mutually dependent fashion and is required for the polymerization of C(3)G into mature thread-like structures, in the context both of paired homologous chromosomes and of C(3)G polycomplexes that lack AEs/LEs. Although AEs assemble in cona oocytes, they exhibit defects that are characteristic of c(3)G mutant oocytes, including failure of AE alignment and synapsis. These results demonstrate that CONA, which does not contain a coiled coil domain, is required for the stable ‘zippering’ of TFs to form the central region of the Drosophila SC. We speculate that CONA's role in SC formation may be similar to that of the mammalian CE proteins SYCE2 and TEX12. However, the observation that AE alignment and pairing occurs in Tex12 and Syce2 mutant meiocytes but not in cona oocytes suggests that the SC plays a more critical role in the stable association of homologs in Drosophila than it does in mammalian cells.     

OsSPO11-1 is essential for both homologous chromosome pairing and crossover formation in rice

Spo11 is a homolog of a subunit of archaebacterial topoisomerase, which catalyzes DNA double-strand breaks and initiates homologous chromosome recombination. In the present study, we silenced the SPO11-1 gene in rice (Oryza sativa) using RNAi. Rice plants with loss-of-function of OsSPO11-1 have no apparent growth defects during vegetative development, but homologous chromosome pairing and recombination are significantly obstructed. Telomeres can be assembled as bouquet during the zygotene stage of the OsSPO11-1-deficient plants, just as that in wild type. Although the two axial-associated proteins, REC8 and PAIR2, are loaded onto the chromosomes, the depletion of PAIR2 from the chromosomes is much later than in wild type. The central element of the synaptonemal complex (SC), ZEP1, does not load onto the chromosomes normally, implying that SC formation is disturbed severely. The crossover protein, MER3, isn't efficiently assembled onto chromosomes and the lack of bivalent suggests that crossovers are also affected in the absence of OsSPO11-1. Thus, OsSPO11-1 is essential for both homologous chromosomes pairing and crossover formation during meiosis in rice.     

Peculiarities of chromosome organization in meiosis

Meiotic and mitotic chromosomes have a complex of differences. (1) At the early prophase I of meiosis, chromosomes acquire protein axial elements (AEs) that were absent in mitosis; in addition to somatic cohesins, AEs contain the meiosis-specific cohesins REC8, SMC1β, and STAG3. (2) At the middle prophase I, protein lateral elements (LEs) of synaptonemal complexes (SCs) are formed on the basis of AEs. The LE proteins are not conserved, but in Saccharomyces cerevisiae and Arabidopsis thaliana they contain functional domains with conserved secondary structures. Among the almost 679 thousand proteins of primitive eukaryotes that we studied by bioinformatics methods, in green and brown algae, some lower fungi, and Coelenterata, we revealed proteins or functional domains similar to SC proteins. (3) During the pachytene and diplotene stages of meiosis, chromosomes of spermatocytes and mother pollen cells acquire a general structure resembling the structure of amphibian and avian lampbrush chromosomes in miniature. Lateral chromatin loops with sizes of 90, 160, and even over 480 Kb were observed in human spermatocytes during the diplotene stage. In combination, all these observations confirm the considerable conservation of the scheme of molecular and ultrastructural organization of meiotic chromosomes in a large variety of eukaryotic organisms.     

OsREC8 is essential for chromatid cohesion and metaphase I monopolar orientation in rice meiosis

The successful transmission of chromosomes during mitosis and meiosis relies on the establishment and subsequent release of cohesion between replicated chromatids. Cohesion is mediated by a four-subunit structural maintenance of chromosome complex, called cohesins. REC8 is a key component of this meiotic cohesion complex in most model organisms studied to date. Here, we isolated and dissected the functions of OsREC8, a rice (Oryza sativa) REC8 homolog, using two null Osrec8 mutants. We showed that OsREC8 encodes a protein that localized to meiotic chromosomes from approximately meiotic interphase to metaphase I. Homologous pairing and telomere bouquet formation were abnormal in Osrec8 meiocytes. Furthermore, fluorescent in situ hybridization experiments on Osrec8 meiocytes demonstrated that the mutation eliminated meiotic centromeric cohesion completely during prophase I and also led to the bipolar orientation of the kinetochores during the first meiotic division and accordingly resulted in premature separation of sister chromatid during meiosis I. Immunolocalization analyses revealed that the loading of PAIR2, PAIR3, OsMER3, and ZEP1 all depended on OsREC8. By contrast, the presence of the OsREC8 signal in pair2, pair3, Osmer3, and zep1 mutants indicated that the loading of OsREC8 did not rely on these four proteins. These results suggest that OsREC8 has several essential roles in the meiotic processes.     

Replication Protein A2c Coupled with Replication Protein A1c Regulates Crossover Formation during Meiosis in Rice

Replication protein A (RPA) is a conserved heterotrimeric protein complex comprising RPA1, RPA2, and RPA3 subunits involved in multiple DNA metabolism pathways attributable to its single-stranded DNA binding property. Unlike other species possessing a single RPA2 gene, rice (Oryza sativa) possesses three RPA2 paralogs, but their functions remain unclear. In this study, we identified RPA2c, a rice gene preferentially expressed during meiosis. A T-DNA insertional mutant (rpa2c) exhibited reduced bivalent formation, leading to chromosome nondisjunction. In rpa2c, chiasma frequency is reduced by ∼78% compared with the wild type and is accompanied by loss of the obligate chiasma. The residual ∼22% chiasmata fit a Poisson distribution, suggesting loss of crossover control. RPA2c colocalized with the meiotic cohesion subunit REC8 and the axis-associated protein PAIR2. Localization of REC8 was necessary for loading of RPA2c to the chromosomes. In addition, RPA2c partially colocalized with MER3 during late leptotene, thus indicating that RPA2c is required for class I crossover formation at a late stage of homologous recombination. Furthermore, we identified RPA1c, an RPA1 subunit with nearly overlapping distribution to RPA2c, required for ∼79% of chiasmata formation. Our results demonstrate that an RPA complex comprising RPA2c and RPA1c is required to promote meiotic crossovers in rice.     

The cohesin REC8 prevents illegitimate inter‐sister synaptonemal complex assembly

During meiosis, a specialized chromosome structure is assembled to promote pairing/synapsis of homologous chromosomes and meiotic recombination, a process yielding chiasmata between homologs to ensure accurate segregation. Meiosis‐specific cohesin complexes mediating sister chromatid cohesion play pivotal roles in almost all these events, including synaptonemal complex (SC) formation. In this issue of EMBO Reports, Agostinho and colleagues have examined chromosome axes and SC structures by taking advantage of a hypomorphic Stag3 mutant in which the levels of the cohesin subunit REC8 are partly reduced 6 . Using super‐resolution microscopy, the authors illuminate previously unforeseen chromosome axis structures, showing locally separated axes in regions where REC8 is absent, regardless of RAD21L or RAD21 cohesin localization. Furthermore, they assessed the relationship between sister chromatid cohesion and inter‐sister SC formation, demonstrating that “axial opening” in the REC8‐free region is accompanied by illegitimate SC formation between sister chromatids. This study highlights the physiological importance of REC8 in sister chromatid cohesion and proper SC formation during meiosis, suggesting a new model in which a high density of REC8 deposition along the chromosome prevents illegitimate inter‐sister SC formation.     

Differential distribution and association of repeat DNA sequences in the lateral element of the synaptonemal complex in rat spermatocytes

The synaptonemal complex (SC) is an evolutionarily conserved structure that mediates synapsis of homologous chromosomes during meiotic prophase I. Previous studies have established that the chromatin of homologous chromosomes is organized in loops that are attached to the lateral elements (LEs) of the SC. The characterization of the genomic sequences associated with LEs of the SC represents an important step toward understanding meiotic chromosome organization and function. To isolate these genomic sequences, we performed chromatin immunoprecipitation assays in rat spermatocytes using an antibody against SYCP3, a major structural component of the LEs of the SC. Our results demonstrated the reproducible and exclusive isolation of repeat deoxyribonucleic acid (DNA) sequences, in particular long interspersed elements, short interspersed elements, long terminal direct repeats, satellite, and simple repeats. The association of these repeat sequences to the LEs of the SC was confirmed by in situ hybridization of meiotic nuclei shown by both light and electron microscopy. Signals were also detected over the chromatin surrounding SCs and in small loops protruding from the lateral elements into the SC central region. We propose that genomic repeat DNA sequences play a key role in anchoring the chromosome to the protein scaffold of the SC. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Meiosis-Specific Cohesin Component,Stag3 Is Essential for Maintaining Centromere Chromatid Cohesion,and Required for DNA Repair and Synapsis between Homologous Chromosomes

Cohesins are important for chromosome structure and chromosome segregation during mitosis and meiosis. Cohesins are composed of two structural maintenance of chromosomes (SMC1-SMC3) proteins that form a V-shaped heterodimer structure, which is bridged by a α-kleisin protein and a stromal antigen (STAG) protein. Previous studies in mouse have shown that there is one SMC1 protein (SMC1β), two α-kleisins (RAD21L and REC8) and one STAG protein (STAG3) that are meiosis-specific. During meiosis, homologous chromosomes must recombine with one another in the context of a tripartite structure known as the synaptonemal complex (SC). From interaction studies, it has been shown that there are at least four meiosis-specific forms of cohesin, which together with the mitotic cohesin complex, are lateral components of the SC. STAG3 is the only meiosis-specific subunit that is represented within all four meiosis-specific cohesin complexes. In Stag3 mutant germ cells, the protein level of other meiosis-specific cohesin subunits (SMC1β, RAD21L and REC8) is reduced, and their localization to chromosome axes is disrupted. In contrast, the mitotic cohesin complex remains intact and localizes robustly to the meiotic chromosome axes. The instability of meiosis-specific cohesins observed in Stag3 mutants results in aberrant DNA repair processes, and disruption of synapsis between homologous chromosomes. Furthermore, mutation of Stag3 results in perturbation of pericentromeric heterochromatin clustering, and disruption of centromere cohesion between sister chromatids during meiotic prophase. These defects result in early prophase I arrest and apoptosis in both male and female germ cells. The meiotic defects observed in Stag3 mutants are more severe when compared to single mutants for Smc1β, Rec8 and Rad21l, however they are not as severe as the Rec8, Rad21l double mutants. Taken together, our study demonstrates that STAG3 is required for the stability of all meiosis-specific cohesin complexes. Furthermore, our data suggests that STAG3 is required for structural changes of chromosomes that mediate chromosome pairing and synapsis, DNA repair and progression of meiosis.     

The role of OsCOM1 in homologous chromosome synapsis and recombination in rice meiosis

COM1/SAE2 is a highly conserved gene from yeast to higher eukaryotes. Its orthologs, known to cooperate with the MRX complex (Mre11/Rad50/Xrs2), are required for meiotic DNA double‐strand break (DSB) ends resection and specific mitotic DSB repair events. Here, the rice (Oryza sativa, 2n = 2x = 24) COM1/SAE2 homolog was identified through positional cloning, termed OsCOM1. Four independent mutants of OsCOM1 were isolated and characterized. In Oscom1 mutants, synaptonemal complex (SC) formation, homologous pairing and recombination were severely inhibited, whereas aberrant non‐homologous chromosome entanglements occurred constantly. Several key meiotic proteins, including ZEP1 and OsMER3, were not loaded normally onto chromosomes in Oscom1 mutants, whereas the localization of OsREC8, PAIR2 and PAIR3 seemed to be normal. Moreover, OsCOM1 was loaded normally onto meiotic chromosomes in Osrec8, zep1 and Osmer3 mutants, but could not be properly loaded in Osam1, pair2 and OsSPO11‐1^RNAi plants. These results provide direct evidence for the functions of OsCOM1 in promoting homologous synapsis and recombination in rice meiosis.     

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.     

Absence of mouse REC8 cohesin promotes synapsis of sister chromatids in meiosis

REC8 is a key component of the meiotic cohesin complex. During meiosis, cohesin is required for the establishment and maintenance of sister-chromatid cohesion, for the formation of the synaptonemal complex, and for recombination between homologous chromosomes. We show that REC8 has an essential role in mammalian meiosis, in that Rec8 null mice of both sexes have germ cell failure and are sterile. In the absence of REC8, early chromosome pairing events appear normal, but synapsis occurs in a novel fashion: between sister chromatids. This implies that a major role for REC8 in mammalian meiosis is to limit synapsis to between homologous chromosomes. In all other eukaryotic species studied to date, REC8 phenotypes have been restricted to meiosis. Unexpectedly, Rec8 null mice are born in sub-Mendelian frequencies and fail to thrive. These findings illuminate hitherto unknown REC8 functions in chromosome dynamics during mammalian meiosis and possibly in somatic development.     

Altered distribution of MLH1 foci is associated with changes in cohesins and chromosome axis compaction in an asynaptic mutant of tomato

In most multicellular eukaryotes, synapsis [synaptonemal complex (SC) formation] between pairs of homologous chromosomes during prophase I of meiosis is closely linked with crossing over. Asynaptic mutants in plants have reduced synapsis and increased univalent frequency, often resulting in genetically unbalanced gametes and reduced fertility. Surprisingly, some asynaptic mutants (like as1 in tomato) have wild-type or increased levels of crossing over. To investigate, we examined SC spreads from as1/as1 microsporocytes using both light and electron microscopic immunolocalization. We observed increased numbers of MLH1 foci (a crossover marker) per unit length of SC in as1 mutants compared to wild-type. These changes are associated with reduced levels of detectable cohesin proteins in the axial and lateral elements (AE/LEs) of SCs, and the AE/LEs of as1 mutants are also significantly longer than those of wild-type or another asynaptic mutant. These results indicate that chromosome axis structure, synapsis, and crossover control are all closely linked in plants.     

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.     

Meiotic cohesin REC8 marks the axial elements of rat synaptonemal complexes before cohesins SMC1beta and SMC3

In meiotic prophase, the sister chromatids of each chromosome develop a common axial element (AE) that is integrated into the synaptonemal complex (SC). We analyzed the incorporation of sister chromatid cohesion proteins (cohesins) and other AE components into AEs. Meiotic cohesin REC8 appeared shortly before premeiotic S phase in the nucleus and formed AE-like structures (REC8-AEs) from premeiotic S phase on. Subsequently, meiotic cohesin SMC1beta, cohesin SMC3, and AE proteins SCP2 and SCP3 formed dots along REC8-AEs, which extended and fused until they lined REC8-AEs along their length. In metaphase I, SMC1beta, SMC3, SCP2, and SCP3 disappeared from the chromosome arms and accumulated around the centromeres, where they stayed until anaphase II. In striking contrast, REC8 persisted along the chromosome arms until anaphase I and near the centromeres until anaphase II. We propose that REC8 provides a basis for AE formation and that the first steps in AE assembly do not require SMC1beta, SMC3, SCP2, and SCP3. Furthermore, SMC1beta, SMC3, SCP2, and SCP3 cannot provide arm cohesion during metaphase I. We propose that REC8 then provides cohesion. RAD51 and/or DMC1 coimmunoprecipitates with REC8, suggesting that REC8 may also provide a basis for assembly of recombination complexes.     

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.     

Mutation of the rice gene PAIR3 results in lack of bivalent formation in meiosis

Meiosis is essential for eukaryotic sexual reproduction and important for genetic diversity among individuals. Although a number of genes regulating homologous chromosome pairing and synapsis have been identified in the plant kingdom, their molecular basis remains poorly understood. In this study, we identified a novel gene, PAIR3 ( HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS 3 ), required for homologous chromosome pairing and synapsis in rice. Two independent alleles, designated pair3-1 and pair3-2 , were identified in our T-DNA insertional mutant library which could not form bivalents due to failure of homologous chromosome pairing and synapsis at diakinesis, resulting in sterility in both male and female gametes. Suppression of PAIR3 by RNAi produced similar results to the T-DNA insertion lines. PAIR3 encodes a protein that contains putative coiled-coil motifs, but does not have any close homologs in other organisms. PAIR3 is preferentially expressed in reproductive organs, especially in pollen mother cells and the ovule tissues during meiosis. Our results suggest that PAIR3 plays a crucial role in homologous chromosome pairing and synapsis in meiosis.     

The Ecm11-Gmc2 Complex Promotes Synaptonemal Complex Formation through Assembly of Transverse Filaments in Budding Yeast

During meiosis, homologous chromosomes pair at close proximity to form the synaptonemal complex (SC). This association is mediated by transverse filament proteins that hold the axes of homologous chromosomes together along their entire length. Transverse filament proteins are highly aggregative and can form an aberrant aggregate called the polycomplex that is unassociated with chromosomes. Here, we show that the Ecm11-Gmc2 complex is a novel SC component, functioning to facilitate assembly of the yeast transverse filament protein, Zip1. Ecm11 and Gmc2 initially localize to the synapsis initiation sites, then throughout the synapsed regions of paired homologous chromosomes. The absence of either Ecm11 or Gmc2 substantially compromises the chromosomal assembly of Zip1 as well as polycomplex formation, indicating that the complex is required for extensive Zip1 polymerization. We also show that Ecm11 is SUMOylated in a Gmc2-dependent manner. Remarkably, in the unSUMOylatable ecm11 mutant, assembly of chromosomal Zip1 remained compromised while polycomplex formation became frequent. We propose that the Ecm11-Gmc2 complex facilitates the assembly of Zip1 and that SUMOylation of Ecm11 is critical for ensuring chromosomal assembly of Zip1, thus suppressing polycomplex formation.     

Meiosis in mice without a synaptonemal complex

The synaptonemal complex (SC) promotes fusion of the homologous chromosomes (synapsis) and crossover recombination events during meiosis. The SC displays an extensive structural conservation between species; however, a few organisms lack SC and execute meiotic process in a SC-independent manner. To clarify the SC function in mammals, we have generated a mutant mouse strain (Sycp1(-/-)Sycp3(-/-), here called SC-null) in which all known SC proteins have been displaced from meiotic chromosomes. While transmission electron microscopy failed to identify any remnants of the SC in SC-null spermatocytes, neither formation of the cohesion axes nor attachment of the chromosomes to the nuclear membrane was perturbed. Furthermore, the meiotic chromosomes in SC-null meiocytes achieved pre-synaptic pairing, underwent early homologous recombination events and sustained a residual crossover formation. In contrast, in SC-null meiocytes synapsis and MLH1-MLH3-dependent crossovers maturation were abolished, whereas the structural integrity of chromosomes was drastically impaired. The variable consequences that SC inactivation has on the meiotic process in different organisms, together with the absence of SC in some unrelated species, imply that the SC could have originated independently in different taxonomic groups.     

Meiotic cohesin complexes are essential for the formation of the axial element in mice

Cohesin is a conserved multisubunit protein complex that participates in chromosome segregation, DNA damage repair, chromatin regulation, and synaptonemal complex (SC) formation. Yeast, but not mice, depleted of the cohesin subunit Rec8 are defective in the formation of the axial elements (AEs) of the SC, suggesting that, in mammals, this function is not conserved. In this paper, we show that spermatocytes from mice lacking the two meiosis-specific cohesin subunits RAD21L and REC8 were unable to initiate RAD51- but not DMC1-mediated double-strand break repair, were not able to assemble their AEs, and arrested as early as the leptotene stage of prophase I, demonstrating that cohesin plays an essential role in AE assembly that is conserved from yeast to mammals.