Y-chromosome haplotypes in azoospermic Israeli men

Among azoospermic and severely oligozoospermic men, 7-15% present microdeletions of a region on the long arm of the Y chromosome that has been called AZF (azoospermia factor). Because these deletions present varying relative frequencies in different populations, we decided to ascertain whether their presence was correlated with specific Y-chromosome haplotypes. For that, we evaluated 51 infertile Israeli men, 9 of whom had microdeletions in AZF. Haplotypes were identified using a hierarchical system with eight biallelic DNA markers. We also checked for the presence of the deletion marker 50f2/C, which was absent in all seven patients with isolated AZFc deletion and also in the one patient with isolated AZFb deletion, suggesting that these microdeletions overlap. As expected, haplogroup J was the most common (47%), followed by equal frequencies of haplogroups Y* (xDE, J, K), P* (xR1a, R1b8), K* (xP), and E. In six patients with AZFc deficiencies of comparable size, three belonged to haplogroup J, two belonged to haplogroup P* (xR1a, R1b8), and one belonged to haplogroup R1a. Also, there were no significant differences in the haplotype frequencies between the groups with and without microdeletions. Thus we did not identify any association of a specific haplogroup with predisposition to de novo deletion of the AZF region in the Israeli population.     

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.     

Cryogenic preservation of mammalian testicular material for synaptonemal complex analysis

Synaptonemal complex (SC) analyses have been used in mammalian cytogenetics to investigate the effects of chromosome heterozygosity on meiotic processes. To date, these analyses have been limited largely to the study of humans, livestock, or species which are easily maintained in the laboratory. The development of a method for the cryogenic preservation of testicular tissue for SC analyses allows for the application of this approach to field-oriented biological problems. This new method is described and data are presented which demonstrate its utility to mammalian cytogenetic studies.     

Protein-protein interactions in the synaptonemal complex.

In mammalian systems, an approximately M(r) 30,000 Cor1 protein has been identified as a major component of the meiotic prophase chromosome cores, and a M(r) 125,000 Syn1 protein is present between homologue cores where they are synapsed and form the synaptonemal complex (SC). Immunolocalization of these proteins during meiosis suggests possible homo- and heterotypic interactions between the two as well as possible interactions with yet unrecognized proteins. We used the two-hybrid system in the yeast Saccharomyces cerevisiae to detect possible protein-protein associations. Segments of hamsters Cor1 and Syn1 proteins were tested in various combinations for homo- and heterotypic interactions. In the cause of Cor1, homotypic interactions involve regions capable of coiled-coil formation, observation confirmed by in vitro affinity coprecipitation experiments. The two-hybrid assay detects no interaction of Cor1 protein with central and C-terminal fragments of Syn1 protein and no homotypic interactions involving these fragments of Syn1. Hamster Cor1 and Syn1 proteins both associate with the human ubiquitin-conjugation enzyme Hsubc9 as well as with the hamster Ubc9 homologue. The interactions between SC proteins and the Ubc9 protein may be significant for SC disassembly, which coincides with the repulsion of homologs by late prophase I, and also for the termination of sister centromere cohesiveness at anaphase II.     

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.     

Physical mapping of translocation breakpoints in rye by means of synaptonemal complex analysis

A physical map including 40 translocation breakpoints has been constructed in rye by means of synaptonemal complex (SC) analysis of well-paired pachytene quadrivalents. The chromosome arms involved in such translocations were previously identified either from mitotic C-banding analysis or from the meiotic configurations observed in the progenies of crosses with a rye line having multiple chromosome rearrangements. The synaptonemal complexes formed by some translocation homozygotes were also analyzed, the relative pachytene SC length of their translocated chromosomes being compared to that observed in the corresponding translocation heterozygotes. In the translocations in which the position of the breakpoint could be well defined from mitotic C-banding analysis, a good correspondence between the relative position of the point showing partner exchange in the pachytene quadrivalents and the actual location of the breakpoint was established. It is concluded that the mapping of translocation breakpoints by SC analysis of pachytene quadrivalents provides a more accurate estimate of the position of the breakpoints than that obtained from mitotic C-banding analysis, due to the lack of evenly-distributed interstitial C-bands in most rye chromosomes. The distribution of the breakpoints along the chromosomes in relation to their spontaneous or induced origin is also discussed.     

Spreading the synaptonemal complex of Neurospora crassa

A protocol was developed to spread the synaptonemal complex (SC) of the fungus Neurospora crassa. It involves direct mechanical breakage of meiotic cells before spreading. This technique makes it possible to examine the SC of the same nucleus with both light and electron microscopy. This protocol is potentially applicable for other Pyrenomycetes. The SCs were examined at zygotene, pachytene and diplotene. The central elements and the recombination nodules (RN) were well revealed by silver staining. Ten to 13 RNs were counted at pachytene. The total genomic SC length varied with the stage. This whole mount electron microscopy of the SC is particularly useful for studying chromosomal rearrangements.     

Meiotic and synaptonemal complex studies in 45 subfertile males

Summary Somatic interphase cells from males and females with normal karyotypes and with variants of the heterochromatic regions on chromosomes 9 and Y were stained with the fluorochrome D287/170. The results showed that only 9h retained the ability to stain with D 287/170 in the interphase state, whereas 15ph and Yqh lost the specific staining pattern seen in metaphase. The number and size of the specific stained interphase bodies correlated with the ploidy of the cell population and the size of the 9h as seen in metaphase.     

Banding studies and synaptonemal complex analysis of an X-autosome translocation in the domestic pig

In a litter of nine domestic pigs, a translocation between the X-chromosome and chromosome 13 was found in six individuals: four males and two females. The translocation was presumed to have originated in the dam. Banding studies indicated that the breaks preceding the translocation had occurred in a distal GTG-negative band of the long arm of the X, 15-30% of the length of Xq from the telomere, and proximally in chromosome 13, 15-25% from the centromere. The normal X of the females invariably replicated its DNA late. Synaptonemal complex analysis of spermatocytes demonstrated a quadrivalent in 75 of 85 analyzable cells (88.2%), and in 10 cells (11.8%) one trivalent and one univalent were found. Extensive nonhomologous pairings were visualized in the pachytene stage by applying an 'overlap' test measuring the sex chromosomes and collating their pairings. An arrest in male meiosis was verified histologically; no meiotic stages later than pachytene developed. This resulted in sterility, with considerable testicular hypoplasia. The records of female fertility were available only for the dam and did not show any deviations from the average of the herd.     

Structural analysis of the human SYCE2-TEX12 complex provides molecular insights into synaptonemal complex assembly

The successful completion of meiosis is essential for all sexually reproducing organisms. The synaptonemal complex (SC) is a large proteinaceous structure that holds together homologous chromosomes during meiosis, providing the structural framework for meiotic recombination and crossover formation. Errors in SC formation are associated with infertility, recurrent miscarriage and aneuploidy. The current lack of molecular information about the dynamic process of SC assembly severely restricts our understanding of its function in meiosis. Here, we provide the first biochemical and structural analysis of an SC protein component and propose a structural basis for its function in SC assembly. We show that human SC proteins SYCE2 and TEX12 form a highly stable, constitutive complex, and define the regions responsible for their homotypic and heterotypic interactions. Biophysical analysis reveals that the SYCE2-TEX12 complex is an equimolar hetero-octamer, formed from the association of an SYCE2 tetramer and two TEX12 dimers. Electron microscopy shows that biochemically reconstituted SYCE2-TEX12 complexes assemble spontaneously into filamentous structures that resemble the known physical features of the SC central element (CE). Our findings can be combined with existing biological data in a model of chromosome synapsis driven by growth of SYCE2-TEX12 higher-order structures within the CE of the SC.     

Evolution of the synaptonemal complex in Helix aspersa spermatocytes

In spermatocytes of Helix aspersa, the structure of the synaptonemal complexes undergoes changes in the course of the pachytene, the lateral elements being transformed into wide bands of lesser density than the chromatin. By using the uranyl-EDTA-lead sequence, which preferentially stains RNA, the lateral elements can be made to appear positive in the early pachytene while the corresponding areas, which become wider and more diffuse, are positive during late pachytene. — Apparently, the lateral elements do not persist in the diplotene and remnants of the central element can occasionally be observed. Using the uranyl-EDTA-lead method reveals some positively stained material surrounding the chromatin, mostly granular in appearance, which is observed in late pachytene and attains its maximum amount during diplotene. Several aspects of these observations are here discussed.     

Searching for synaptonemal complex proteins and their genes

As an alternative to the production and use of monoclonal antisynaptonemal complex (SC) antibodies to isolate SC genes, we have explored the use of polyclonal anti-SC antibodies to identify SC genes from a cDNA expression library. The method proved relatively simple, reliable, and fast and has yielded two SC genes. A homologue of one of these genes from a different species has previously been isolated in another laboratory. © 1993 Wiley-Liss, Inc.     

The pachytene synaptonemal complex complement of the cat

Surface spreads of pachytene spermatocyte nuclei from two cats were used to construct a synaptonemal complex karyotype for the cat. It was possible to recognise the 18 autosomal synaptonemal complexes by reference to a published light microscopic banded somatic karyotype. Some variation from the somatic karyotype was noted, presumably as a result of differential contraction during prophase I. The X and Y chromosome axes were joined by a synaptonemal complex in many of the nuclei, but the structure of the unpaired portion of the X axis was quite variable. In some nuclei it was highly contracted, while in others it was extended and often was split into two or more axes. In most nuclei the autosomal synaptonemal complexes had numerous axial twists.     

DNA repeated sequences may be involved in synaptonemal complex formation

Synaptonemal complexes (SCs) are intranuclear structures that facilitate the reversible lateral synapsis of homologous chromosomes in the course of meiosis. It is still unclear which DNA nucleotide sequences are responsible for the attachment of chromatin to SC lateral elements. Considering the features of the dispersed repeated sequences (RSs), it is possible to assume that they participate in the structure and functional organization of the meiotic chromosomes. Using numerical analysis, we have investigated the relationship between the RS and the distribution of meiotic recombination events in mouse chromosome 1. Using in situ hybridization on spread mouse spermatocytes, we have examined the arrangement of different types of RSs relative to SCs. Hybridization signals of B1(Alu), B2, and minisatellite probes were localized predominantly in SCs regions. Based on the results, we proposed a model of meiotic chromosome organization. According to the model, RSs participate in the attachment of chromatin loops to SCs.     

A possible role for the synaptonemal complex in chiasma maintenance

It is suggested that in addition to whatever function the synaptonemal complex may serve with respect to crossing over, it may serve a previously unsuspected function, i.e. one which normally assures regular meiotic chromosome disjunction in most organisms. This is the provision of the sister chromatid cohesiveness which is probably required for chiasma maintenance until anaphase I and may also be required in the centromere regions for maintenance of dyad integrity until anaphase II. Attention is directed to suggestive correlations of occurrence of normal synapsis through pachytene (homologous or non-homologous) with instances of sister chromatid cohesiveness at later meiotic stages and conversely of lack of normal synapsis with failure of sister chromatid cohesiveness. Chromosome behavior is compared in trisomies and in material homozygous for several meiotic mutants.     

From early homologue recognition to synaptonemal complex formation

This review focuses on various aspects of chromosome homology searching and their relationship to meiotic and vegetative pairing and to the silencing of unpaired copies of genes. Chromosome recognition and pairing is a prominent characteristic of meiosis; however, for some organisms, this association (complete or partial) is also a normal part of nuclear organization. The multiple mechanisms suggested to contribute to homologous pairing are analyzed. Recognition of DNA/DNA homology also plays an important role in detecting DNA segments that are present in inappropriate number of copies before and during meiosis. In this context, the mechanisms of methylation induced premeiotically, repeat-induced point mutation, meiotic silencing by unpaired DNA, and meiotic sex chromosome inactivation will be discussed. Homologue juxtaposition during meiotic prophase can be divided into three mechanistically distinct steps, namely, recognition, presynaptic alignment, and synapsis by the synaptonemal complex (SC). In most organisms, these three steps are distinguished by their dependence on DNA double-strand breaks (DSBs). The coupling of SC initiation to (and downstream effects of) DSB formation and the exceptions to this dependency are discussed. Finally, this review addresses the specific factors that appear to promote chromosome movement at various stages of meiotic prophase, most particularly at the bouquet stage, and on their significance for homologue pairing and/or achieving a final pachytene configuration.The synaptonemal complex - 50 years