Synaptonemal complex formation in spermatocytes of the autotriploid rainbow trout, Oncorhynchus mykiss (Pisces, Salmonidae)

Meiotic cells of triploid male rainbow trouts were analyzed by a surface-spreading SC technique in order to show the process of chromosome synapsis. At zygotene the formation of SCs involved, almost exclusively, two sets of lateral elements (LEs), and the remaining set of LEs presented different synaptic configurations involving one, two, three, or four LEs. The absence of SCs involving more than two LEs from mid- to late pachytene indicates that the multivalents produced by synapsis involving more than two LEs, are eliminated before the end of pachytene; thus, the mechanism of chromosome pairing in triploid male rainbow trouts produces, almost exclusively, bivalents with a probable extensive nonhomologous synapsis involving the extra set of chromosomes.

Cytogenetic analysis of Epicauta atomaria (Meloidae) and Palembus dermestoides (Tenebrionidae) with Xyp sex determination system using standard staining, C-bands, NOR and synaptonemal complex microspreading techniques

The mitotic and meiotic chromosomes of the beetles Epicauta atomaria (Meloidae) and Palembus dermestoides (Tenebrionidae) were analysed using standard staining, C-banding and silver impregnation techniques. We determine the diploid and haploid chromosome numbers, the sex determination system and describe the chromosomal morphology, the C-banding pattern and the chromosome(s) bearing NORs (nucleolar organizer regions). Both species shown 2n = 20 chromosomes, the chromosomal meioformula 9 + Xyp, and regular chromosome segregation during anaphases I and II. The chromosomes of E. atomaria are basically metacentric or submetacentric and P. dermestoides chromosomes are submetacentric or subtelocentric. In both beetles the constitutive heterochromatin is located in the pericentromeric region in all autosomes and in the Xp chromosome; additional C-bands were observed in telomeric region of the short arm in some autosomes in P. dermestoides. The yp chromosome did not show typical C-bands in these species. As for the synaptonemal complex, the nucleolar material is associated to the 7th bivalent in E. atomaria and 3rd and 7th bivalents in P. dermestoides. Strong silver impregnated material was observed in association with Xyp in light and electron microscopy preparations in these species and this material was interpreted to be related to nucleolar material.

Synaptonemal complex analysis in the fish species Piaractus mesopotamicus and Colossoma macropomum, and in their interspecific hybrid

The surface-spreading technique for visualization of whole cell synaptonemal complex (SC) complements was employed to study the chromosome synapsis in spermatocytes of P. mesopotamicus, C. macropomum and in their interspecific hybrid (tambacu) with the main objective to analyze possible errors in chromosome pairing that could result in hybrid sterility. SC analysis showed that the parental species P. mesopotamicus and C. macropomum have 27 bivalents homogeneously synapsed. The SC in spermatocytes from the hybrid tambacu showed gross meiotic configurations in all cells analyzed. The spermatocytes exhibited a few chromosomes or well synapsed chromosome segments, while many chromosome segments did not have any synapsis. This result, allied to other genetical and cytogenetical evidence, reinforces the hypothesis that the hybrid tambacu is sterile. Further studies involving other aspects, such as behavior and physiology, should be conduced before the introduction of these hybrids in rivers and lakes.

Surface-spreading technique of meiotic cells and immunodetection of synaptonemal complex proteins in teleostean fishes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Organization and behavior of the synaptonemal complex during achiasmatic meiosis of four buthid scorpions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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 complex (SC) component Zip1 plays a role in meiotic recombination independent of SC polymerization along the chromosomes.

Zip1 is a yeast synaptonemal complex (SC) central region component and is required for normal meiotic recombination and crossover interference. Physical analysis of meiotic recombination in a zip1 mutant reveals the following: Crossovers appear later than normal and at a reduced level. Noncrossover recombinants, in contrast, seem to appear in two phases: (i) a normal number appear with normal timing and (ii) then additional products appear late, at the same time as crossovers. Also, Holliday junctions are present at unusually late times, presumably as precursors to late-appearing products. Red1 is an axial structure component required for formation of cytologically discernible axial elements and SC and maximal levels of recombination. In a red1 mutant, crossovers and noncrossovers occur at coordinately reduced levels but with normal timing. If Zip1 affected recombination exclusively via SC polymerization, a zip1 mutation should confer no recombination defect in a red1 strain background. But a red1 zip1 double mutant exhibits the sum of the two single mutant phenotypes, including the specific deficit of crossovers seen in a zip1 strain. We infer that Zip1 plays at least one role in recombination that does not involve SC polymerization along the chromosomes. Perhaps some Zip1 molecules act first in or around the sites of recombinational interactions to influence the recombination process and thence nucleate SC formation. We propose that a Zip1-dependent, pre-SC transition early in the recombination reaction is an essential component of meiotic crossover control. A molecular basis for crossover/noncrossover differentiation is also suggested.

Chromosome synapsis and recombination in simple and complex chromosomal heterozygotes of tuco-tuco (Ctenomys talarum: Rodentia: Ctenomyidae):Rodentia: Ctenomyidae)

The chromosomal speciation hypothesis suggests that irregularities in synapsis, recombination, and segregation in heterozygotes for chromosome rearrangements may restrict gene flow between karyotypically distinct populations and promote speciation. Ctenomys talarum is a South American subterranean rodent inhabiting the coastal regions of Argentina, whose populations polymorphic for Robertsonian and tandem translocations seem to have a very restricted gene flow. To test if chromosomal differences are involved in isolation among its populations, we examined chromosome pairing, recombination, and meiotic silencing of unsynapsed chromatin in male meiosis of simple and complex translocation heterozygotes using immunolocalization of the MLH1 marking mature recombination nodules and phosphorylated histone γH2A.X marking unrepaired double-strand breaks. We observed small asynaptic areas labeled by γH2A.X in pericentromeric regions of the chromosomes involved in the trivalents and quadrivalents. We also observed a decrease of recombination frequency and a distalization of the crossover distribution in the heterozygotes and metacentric homozygotes compared to acrocentric homozygotes. We suggest that the asynapsis of the pericentromeric regions are unlikely to induce germ cell death and decrease fertility of the heterozygotes; however, suppressed recombination in pericentromeric areas of the multivalents may reduce gene flow between chromosomally different populations of the Talas tuco-tuco.

Complex meiotic configuration of the holocentric chromosomes: the intriguing case of the scorpion Tityus bahiensis

Mitotic and meiotic chromosomes of Tityus bahiensis were investigated using light (LM) and transmission electron microscopy (TEM) to determine the chromosomal characteristics and disclose the mechanisms responsible for intraspecific variability in chromosome number and for the presence of complex chromosome association during meiosis. This species is endemic to Brazilian fauna and belongs to the family Buthidae, which is considered phylogenetically basal within the order Scorpiones. In the sample examined, four sympatric and distinct diploid numbers were observed: 2n = 5, 2n = 6, 2n = 9, and 2 = 10. The origin of this remarkable chromosome variability was attributed to chromosome fissions and/or fusions, considering that the decrease in chromosome number was concomitant with the increase in chromosome size and vice versa. The LM and TEM analyses showed the presence of chromosomes without localised centromere, the lack of chiasmata and recombination nodules in male meiosis, and two nucleolar organiser regions carrier chromosomes. Furthermore, male prophase I cells revealed multivalent chromosome associations and/or unsynapsed or distinctly associated chromosome regions (gaps, less-condensed chromatin, or loop-like structure) that were continuous with synapsed chromosome segments. All these data permitted us to suggest that the chromosomal rearrangements of T. bahiensis occurred in a heterozygous state. A combination of various factors, such as correct disjunction and balanced segregation of the chromosomes involved in complex meiotic pairing, system of achiasmate meiosis, holocentric nature of the chromosomes, population structure, and species dispersion patterns, could have contributed to the high level of chromosome rearrangements present in T. bahiensis.

Complex meiotic configuration of the holocentric chromosomes: the intriguing case of the scorpion Tityus bahiensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Synaptomenal complex analysis of four breeds of Bos taurus taurus x B. taurus indicus hybrids

The synaptonemal complex (SC) was analyzed in four F1 hybrids of Bos taurus taurus and B. taurus indicus including Gyr-Simmental (G-S), Nelore Simmental (N-S), Gyr-Holstein-Friesian (G-H) and Nelore-Piemontese (N-P). We analysed the frequency of various types of SC abnormalities and the frequency of cells with SC abnormalities. The results were compared with similar observations made on purebred animals. All the animals studied possessed 29 autosomal and one sex bivalent. The frequency of cells with abnormalities in the hybrids were 28.0% in the N-P, 29.1% in the G-S, 33.3% in the N-S and 40.0% in the G-H. The frequency of cells with abnormalities in the four hybrids was 31.5%; 57.9% of these abnormalities occurred in zygotene and 42.0% occurred in pachytene. The comparisons among the hybrids and among the hybrids and their parental breeds showed that the only significant difference was between Gyr and Gyr-Holstein-Friesian animals. Some aspects of the relationship between the frequency of cells with anomalies and the fertility of hybrids are discussed.

Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility.

In addition to the five 70-kDa heat shock proteins (HSP70) common to germ cells and somatic tissues of mammals, spermatogenic cells synthesize HSP70-2 during meiosis. To determine if this unique stress protein has a critical role in meiosis, we used gene-targeting techniques to disrupt Hsp70-2 in mice. Male mice homozygous for the mutant allele (Hsp70-2 -/-) did not synthesize HSP70-2, lacked postmeiotic spermatids and mature sperm, and were infertile. However, neither meiosis nor fertility was affected in female Hsp70-2 -/- mice. We previously found that HSP70-2 is associated with synaptonemal complexes in the nucleus of meiotic spermatocytes from mice and hamsters. While synaptonemal complexes assembled in Hsp70-2 -/- spermatocytes, structural abnormalities became apparent in these cells by late prophase, and development rarely progressed to the meiotic divisions. Furthermore, analysis of nuclei and genomic DNA indicated that the failure of meiosis in Hsp70-2 -/- mice was coincident with a dramatic increase in spermatocyte apoptosis. These results suggest that HSP70-2 participates in synaptonemal complex function during meiosis in male germ cells and is linked to mechanisms that inhibit apoptosis.

Cloning,overexpression and purification of functionally active Saccharomyces cerevisiae Hop1 protein from scherichia coli

One of the major limitations to the application of high-resolution biophysical techniques such as X-crystallography and spectroscopic analyses to structure-function studies of Saccharomyces cerevisiae Hop1 protein has been the non-availability of sufficient quantities of functionally active pure protein. This has, indeed, been the case of many proteins, including yeast synaptonemal complex proteins. In this study, we have performed expression screening in Escherichia coli host strains, capable of high-level expression of soluble S. cerevisiae Hop1 protein. A new protocol has been developed for expression and purification of S. cerevisiae Hop1 protein, based on the presence of hexa-histidine tag and double-stranded DNA-Cellulose chromatography. Recombinant S. cerevisiae Hop1 protein was >98% pure and exhibited DNA-binding activity with high-affinity to the Holliday junction. The availability of the recombinant HOP1 expression vector and active Hop1 protein would facilitate structure-function investigations as well as the generation of appropriate truncated and site-directed mutant proteins, respectively. (C) 2010 Elsevier Inc. All rights reserved.