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.     

Involvement of synaptonemal complex proteins in sex chromosome segregation during marsupial male meiosis

Marsupial sex chromosomes break the rule that recombination during first meiotic prophase is necessary to ensure reductional segregation during first meiotic division. It is widely accepted that in marsupials X and Y chromosomes do not share homologous regions, and during male first meiotic prophase the synaptonemal complex is absent between them. Although these sex chromosomes do not recombine, they segregate reductionally in anaphase I. We have investigated the nature of sex chromosome association in spermatocytes of the marsupial Thylamys elegans, in order to discern the mechanisms involved in ensuring their proper segregation. We focused on the localization of the axial/lateral element protein SCP3 and the cohesin subunit STAG3. Our results show that X and Y chromosomes never appear as univalents in metaphase I, but they remain associated until they orientate and segregate to opposite poles. However, they must not be tied by a chiasma since their separation precedes the release of the sister chromatid cohesion. Instead, we show they are associated by the dense plate, a SCP3-rich structure that is organized during the first meiotic prophase and that is still present at metaphase I. Surprisingly, the dense plate incorporates SCP1, the main protein of the central element of the synaptonemal complex, from diplotene until telophase I. Once sex chromosomes are under spindle tension, they move to opposite poles losing contact with the dense plate and undergoing early segregation. Thus, the segregation of the achiasmatic T. elegans sex chromosomes seems to be ensured by the presence in metaphase I of a synaptonemal complex-derived structure. This feature, unique among vertebrates, indicates that synaptonemal complex elements may play a role in chromosome segregation.     

Meiotic Cohesin Promotes Pairing of Nonhomologous Centromeres in Early Meiotic Prophase

A period of pairing between nonhomologous centromeres occurs early in meiosis in a diverse collection of organisms. This early, homology-independent, centromere pairing, referred to as centromere coupling in budding yeast, gives way to an alignment of homologous centromeres as homologues synapse later in meiotic prophase. The regulation of centromere coupling and its underlying mechanism have not been elucidated. In budding yeast, the protein Zip1p is a major component of the central element of the synaptonemal complex in pachytene of meiosis, and earlier, is essential for centromere coupling. The experiments reported here demonstrate that centromere coupling is mechanistically distinct from synaptonemal complex assembly. Zip2p, Zip3p, and Red1p are all required for the assembly of Zip1 into the synaptonemal complex but are dispensable for centromere coupling. However, the meiotic cohesin Rec8p is required for centromere coupling. Loading of meiotic cohesins to centromeres and cohesin-associated regions is required for the association of Zip1 with these sites, and the association of Zip1 with the centromeres then promotes coupling. These findings reveal a mechanism that promotes associations between centromeres before the assembly of the synaptonemal complex, and they demonstrate that chromosomes are preloaded with Zip1p in a manner that may promote synapsis.     

Pds5 is required for homologue pairing and inhibits synapsis of sister chromatids during yeast meiosis

During meiosis, homologues become juxtaposed and synapsed along their entire length. Mutations in the cohesin complex disrupt not only sister chromatid cohesion but also homologue pairing and synaptonemal complex formation. In this study, we report that Pds5, a cohesin-associated protein known to regulate sister chromatid cohesion, is required for homologue pairing and synapsis in budding yeast. Pds5 colocalizes with cohesin along the length of meiotic chromosomes. In the absence of Pds5, the meiotic cohesin subunit Rec8 remains bound to chromosomes with only minor defects in sister chromatid cohesion, but sister chromatids synapse instead of homologues. Double-strand breaks (DSBs) are formed but are not repaired efficiently. In addition, meiotic chromosomes undergo hypercondensation. When the mitotic cohesin subunit Mcd1 is substituted for Rec8 in Pds5-depleted cells, chromosomes still hypercondense, but synapsis of sister chromatids is abolished. These data suggest that Pds5 modulates the Rec8 activity to facilitate chromosome morphological changes required for homologue synapsis, DSB repair, and meiotic chromosome segregation.     

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.     

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.     

Sex chromosomes, synapsis, and cohesins: a complex affair

During first meiotic prophase, homologous chromosomes are held together by the synaptonemal complex, a tripartite proteinaceous structure that extends along the entire length of meiotic bivalents. While this feature is applicable for autosomes, sex chromosomes often escape from this rule. Many species present sex chromosomes that differ between them in their morphology, length, and gene content. Moreover, in some species, sex chromosomes appear in a single dose in one of the sexes. In all of these cases, the behavior of sex chromosomes during meiosis is conspicuously affected, and this includes the assembly and dynamics of the synaptonemal complex. We review in this study the structure of the synaptonemal complex in the sex chromosomes of three groups of organisms, namely: mammals, orthopterans, and hemipterans, which present different patterns of sex chromosome structure and behavior. Of special interest is the analysis of the organization of the axial/lateral elements of the synaptonemal complex in relation to other axial structures organized along meiotic chromosomes, mainly the cohesin axis. The differences found in the behavior of both axial structures reveal that while the organization of a cohesin axis along sex chromosomes is a conserved feature in most organisms and it shows very little morphological variations, the axial/lateral elements of the synaptonemal complex present a wide range of structural modifications on these chromosomes.Electronic Supplementary Material Supplementary material is available for this article at The synaptonemal complex—50 years     

The Synaptonemal Complex Protein SCP3 Can Form Multistranded, Cross-striated Fibers In Vivo

The synaptonemal complex protein SCP3 is part of the lateral element of the synaptonemal complex, a meiosis-specific protein structure essential for synapsis of homologous chromosomes. We have investigated the fiber-forming properties of SCP3 to elucidate its role in the synaptonemal complex. By synthesis of SCP3 in cultured somatic cells, it has been shown that SCP3 can self-assemble into thick fibers and that this process requires the COOH-terminal coiled coil domain of SCP3, as well as the NH₂-terminal nonhelical domain. We have further analyzed the thick SCP3 fibers by transmission electron microscopy and immunoelectron microscopy. We found that the fibers display a transversal striation with a periodicity of ~20 nm and consist of a large number of closely associated, thin fibers, 5–10 nm in diameter. These features suggest that the SCP3 fibers are structurally related to intermediate filaments. It is known that in some species the lateral elements of the synaptonemal complex show a highly ordered striated structure resembling that of the SCP3 fibers. We propose that SCP3 fibers constitute the core of the lateral elements of the synaptonemal complex and function as a molecular framework to which other proteins attach, regulating DNA binding to the chromatid axis, sister chromatid cohesion, synapsis, and recombination.     

Meiotic cohesin STAG3 is required for chromosome axis formation and sister chromatid cohesion

The cohesin complex is essential for mitosis and meiosis. The specific meiotic roles of individual cohesin proteins are incompletely understood. We report in vivo functions of the only meiosis‐specific STAG component of cohesin, STAG3. Newly generated STAG3‐deficient mice of both sexes are sterile with meiotic arrest. In these mice, meiotic chromosome architecture is severely disrupted as no bona fide axial elements (AE) form and homologous chromosomes do not synapse. Axial element protein SYCP3 forms dot‐like structures, many partially overlapping with centromeres. Asynapsis marker HORMAD1 is diffusely distributed throughout the chromatin, and SYCP1, which normally marks synapsed axes, is largely absent. Centromeric and telomeric sister chromatid cohesion are impaired. Centromere and telomere clustering occurs in the absence of STAG3, and telomere structure is not severely affected. Other cohesin proteins are present, localize throughout the STAG3‐devoid chromatin, and form complexes with cohesin SMC1β. No other deficiency in a single meiosis‐specific cohesin causes a phenotype as drastic as STAG3 deficiency. STAG3 emerges as the key STAG cohesin involved in major functions of meiotic cohesin.     

The Rad9 Gene of Coprinus Cinereus Encodes a Proline-Rich Protein Required for Meiotic Chromosome Condensation and Synapsis

The rad9 gene of Coprinus cinereus is essential for the normal completion of meiosis. We examined surface-spread preparations of wild-type and rad9-1 nuclei from the meiotic stages of karyogamy through metaphase I, and we determined the primary sequence, structure, and meiotic expression of the rad9 gene. In wild-type C. cinereus, karyogamy is followed by condensation and alignment of homologous chromosomes. Condensation and axial core development largely precede synapsis, which often initiates at telomeres. A diffuse diplotene phase coincides with dissolution of the synaptonemal complex, and subsequently chromosomes further condense as the cells progress into metaphase I. In contrast, although karyogamy and nucleolar fusion are apparently normal in rad9-1 basidia, only short stretches of synaptonemal complex form. These correlate with stretches of condensed chromatin, mostly at apparent chromosome ends, and regions of presumptive triple synapsis are numerous. rad9-1 basidia enter the diffuse stage of early diplotene, and then 50% of these cells enter metaphase I by the criteria of nucleolar elimination and at least some chromatin condensation. rad9 gene expression is induced after gamma irradiation and during meiosis. The gene has 27 exons and encodes a predicted protein of 2157 amino acids, with a proline-rich amino terminus.     

The Cohesion Protein SOLO Associates with SMC1 and Is Required for Synapsis,Recombination, Homolog Bias and Cohesion and Pairing of Centromeres in Drosophila Meiosis

Cohesion between sister chromatids is mediated by cohesin and is essential for proper meiotic segregation of both sister chromatids and homologs. solo encodes a Drosophila meiosis-specific cohesion protein with no apparent sequence homology to cohesins that is required in male meiosis for centromere cohesion, proper orientation of sister centromeres and centromere enrichment of the cohesin subunit SMC1. In this study, we show that solo is involved in multiple aspects of meiosis in female Drosophila. Null mutations in solo caused the following phenotypes: 1) high frequencies of homolog and sister chromatid nondisjunction (NDJ) and sharply reduced frequencies of homolog exchange; 2) reduced transmission of a ring-X chromosome, an indicator of elevated frequencies of sister chromatid exchange (SCE); 3) premature loss of centromere pairing and cohesion during prophase I, as indicated by elevated foci counts of the centromere protein CID; 4) instability of the lateral elements (LE)s and central regions of synaptonemal complexes (SCs), as indicated by fragmented and spotty staining of the chromosome core/LE component SMC1 and the transverse filament protein C(3)G, respectively, at all stages of pachytene. SOLO and SMC1 are both enriched on centromeres throughout prophase I, co-align along the lateral elements of SCs and reciprocally co-immunoprecipitate from ovarian protein extracts. Our studies demonstrate that SOLO is closely associated with meiotic cohesin and required both for enrichment of cohesin on centromeres and stable assembly of cohesin into chromosome cores. These events underlie and are required for stable cohesion of centromeres, synapsis of homologous chromosomes, and a recombination mechanism that suppresses SCE to preferentially generate homolog crossovers (homolog bias). We propose that SOLO is a subunit of a specialized meiotic cohesin complex that mediates both centromeric and axial arm cohesion and promotes homolog bias as a component of chromosome cores.     

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.     

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.     

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.     

Meiotic Transverse Filament Proteins: Essential for Crossing Over

Meiosis is a specialized set of two nuclear divisions, meiosis I and II, by which a diploid cell produces four haploid daughters. After premeiotic DNA replication, homologous chromosomes pair and recombine, and then disjoin at meiosis I. Subsequently, at meiosis II, the sister chromatids of each chromosome segregate. In nearly all eukaryotes, meiotic chromosome pairing culminates in the formation of a ladderlike supramolecular protein structure, the synaptonemal complex (SC) (Page and Hawley, 2004). The rungs of the ladder are known as transverse filaments (TFs). Genes encoding TF proteins have been identified in a limited number of organisms, and their function has been studied by mutational analysis. Although TF proteins show little amino acid sequence conservation, their structure and function are largely conserved. In all analyzed species, TF proteins are required for meiotic reciprocal recombination (crossing over).     

Maize meiotic mutants with improper or non-homologous synapsis due to problems in pairing or synaptonemal complex formation

During meiotic prophase homologous chromosomes find each other and pair. Then they synapse, as the linear protein core (axial element or lateral element) of each homologous chromosome is joined together by a transverse central element, forming the tripartite synaptonemal complex (SC). Ten uncloned Zea mays mutants in our collection were surveyed by transmission electron microscopy by making silver-stained spreads of SCs to identify mutants with non-homologous synapsis or improper synapsis. To analyse the mutants further, zyp1, the maize orthologue of the Arabidopsis central element component ZYP1 was cloned and an antibody was made against it. Using antibodies against ZYP1 and the lateral element components AFD1 and ASY1, it was found that most mutants form normal SCs but are defective in pairing. The large number of non-homologous synapsis mutants defective in pairing illustrates that synapsis and pairing can be uncoupled. Of the ten mutants studied, only dsy2 undergoes normal homologous chromosome recognition needed for homologous pairing. The dsy2 mutation fails to maintain the SC. ZYP1 elongation is blocked at zygotene, and only dots of ZYP1 are seen at prophase I. Another mutant, mei*N2415 showed incomplete but homologous synapsis and ASY1 and AFD1 have a normal distribution. Although installation of ZYP1 is initiated at zygotene, its progression is slowed down and not completed by pachytene in some cells and ZYP1 is not retained on pachytene chromosomes. The mutants described here are now available through the Maize Genetics Cooperation Stock Center (http://maizecoop.cropsci.uiuc.edu/).     

Immunofluorescent synaptonemal complex analysis in azoospermic men

The molecular cause of germ cell meiotic defects in azoospermic men is rarely known. During meiotic prophase I, a proteinaceous structure called the synaptonemal complex (SC) appears along the pairing axis of homologous chromosomes and meiotic recombination takes place. Newly-developed immunofluorescence techniques for SC proteins (SCP1 and SCP3) and for a DNA mismatch repair protein (MLH1) present in late recombination nodules allow simultaneous analysis of synapsis, and of meiotic recombination, during the first meiotic prophase in spermatocytes. This immunofluorescent SC analysis enables accurate meiotic prophase substaging and the identification of asynaptic pachytene spermatocytes. Spermatogenic defects were examined in azoospermic men using immunofluorescent SC and MLH1 analysis. Five males with obstructive azoospermia, 18 males with nonobstructive azoospermia and 11 control males with normal spermatogenesis were recruited for the study. In males with obstructive azoospermia, the fidelity of chromosome pairing (determined by the percentage of cells with gaps [discontinuities]/splits [unpaired chromosome regions] in the SCs, and nonexchange SCs [bivalents with 0 MLH1 foci]) was similar to those in normal males. The recombination frequencies (determined by the mean number of MLH1 foci per cell at the pachytene stage) were significantly reduced in obstructive azoospermia compared to that in controls. In men with nonobstructive azoospermia, a marked heterogeneity in spermatogenesis was found: 45% had a complete absence of meiotic cells; 5% had germ cells arrested at the zygotene stage of meiotic prophase; the rest had impaired fidelity of chromosome synapsis and significantly reduced recombination in pachytene. In addition, significantly more cells were in the leptotene and zygotene meiotic prophase stages in nonobstructive azoospermic patients, compared to controls. Defects in chromosome pairing and decreased recombination during meiotic prophase may have led to spermatogenesis arrest and contributed in part to this unexplained infertility.     

Protein Phosphatase 4 Promotes Chromosome Pairing and Synapsis,and Contributes to Maintaining Crossover Competence with Increasing Age

Prior to the meiotic divisions, dynamic chromosome reorganizations including pairing, synapsis, and recombination of maternal and paternal chromosome pairs must occur in a highly regulated fashion during meiotic prophase. How chromosomes identify each other''s homology and exclusively pair and synapse with their homologous partners, while rejecting illegitimate synapsis with non-homologous chromosomes, remains obscure. In addition, how the levels of recombination initiation and crossover formation are regulated so that sufficient, but not deleterious, levels of DNA breaks are made and processed into crossovers is not understood well. We show that in Caenorhabditis elegans, the highly conserved Serine/Threonine protein phosphatase PP4 homolog, PPH-4.1, is required independently to carry out four separate functions involving meiotic chromosome dynamics: (1) synapsis-independent chromosome pairing, (2) restriction of synapsis to homologous chromosomes, (3) programmed DNA double-strand break initiation, and (4) crossover formation. Using quantitative imaging of mutant strains, including super-resolution (3D-SIM) microscopy of chromosomes and the synaptonemal complex, we show that independently-arising defects in each of these processes in the absence of PPH-4.1 activity ultimately lead to meiotic nondisjunction and embryonic lethality. Interestingly, we find that defects in double-strand break initiation and crossover formation, but not pairing or synapsis, become even more severe in the germlines of older mutant animals, indicating an increased dependence on PPH-4.1 with increasing maternal age. Our results demonstrate that PPH-4.1 plays multiple, independent roles in meiotic prophase chromosome dynamics and maintaining meiotic competence in aging germlines. PP4''s high degree of conservation suggests it may be a universal regulator of meiotic prophase chromosome dynamics.     

Expression of Epitope-Tagged SYN3 Cohesin Proteins Can Disrupt Meiosis in Arabidopsis

a-kleisins are core components of meiotic and mitotic cohesin complexes. Arabidopsis contains genes encoding four a-kleisins. SYN1, a REC8 ortholog, is essential for meiosis, while SYN2 and SYN4 appear to be SCCI orthologs and function in mitosis. SYN3 is enriched in the nucleolus of meiotic and mitotic cells and is essential for megagametogenesis. It was recently shown that expression of SYN3-RNAi constructs in buds cause changes in meiotic gene expression that result in meiotic alterations. In this report we show that expression of SYN3 from the 35S promoter with either a c-terminal Myc or FAST tag causes a reduction in SYN1 mRNA levels that results in al- terations in sister chromatid cohesion, homologous chromosome synapsis female meiosis. and synaptonemal complex formation during both male and     

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.