Anna A. Torgasheva

Multiple independent evolutionary losses of XY pairing at meiosis in the grey voles

In many eutherian mammals, X–Y chromosome pairing and recombination is required for meiotic progression and correct sex chromosome disjunction. Arvicoline rodents present a notable exception to this meiotic rule, with multiple species possessing asynaptic sex chromosomes. Most asynaptic vole species belong to the genus Microtus sensu lato. However, many of the species both inside and outside the genus Microtus display normal X–Y synapsis at meiosis. These observations suggest that the synaptic condition was present in the common ancestor of all voles, but gaps in current taxonomic sampling across the arvicoline phylogeny prevent identification of the lineage(s) along which the asynaptic state arose. In this study, we use electron and immunofluorescent microscopy to assess heterogametic sex chromosome pairing in 12 additional arvicoline species. Our sample includes ten species of the tribe Microtini and two species of the tribe Lagurini. This increased breadth of sampling allowed us to identify asynaptic species in each major Microtine lineage. Evidently, the ability of the sex chromosomes to pair and recombine in male meiosis has been independently lost at least three times during the evolution of Microtine rodents. These results suggest a lack of evolutionary constraint on X–Y synapsis in Microtini, hinting at the presence of alternative molecular mechanisms for sex chromosome segregation in this large mammalian tribe.

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

A- and B-chromosome pairing and recombination in male meiosis of the silver fox (Vulpes vulpes L., 1758, Carnivora, Canidae)

We examined A- and B-chromosome pairing and recombination in 12 males from the farm-bred population of the silver fox (2n = 34 + 0–10 Bs) by means of electron and immunofluorescent microscopy. To detect recombination at A and B chromosomes, we used immunolocalisation of MLH1, a mismatch repair protein of mature recombination nodules, at synaptonemal complexes. The mean total number of MLH1 foci at A-autosomes was 29.6 foci per cell. The XY bivalent had one MLH1 focus at the pairing region. Total recombination length of the male fox genome map was estimated as 1,530 centimorgans. We detected single MLH1 foci at 61% of linear synaptic configurations involving B chromosomes. The distribution of the foci along B- and A-bivalents was the same. This may be considered as a first molecular evidence that meiotic recombination does occur in mammalian B chromosomes. There was no correlation between the number of synaptic configurations involving B chromosomes per cell and the recombination rate of the A-genome.

Synapsis and recombination in inversion heterozygotes

Inversion heterozygotes are expected to suffer from reduced fertility and a high incidence of chromosomally unbalanced gametes due to recombination within the inverted region. Non-homologous synapsis of the inverted regions can prevent recombination there and diminish the deleterious effects of inversion heterozygosity. The choice between non-homologous and homologous synapsis depends on the size of inversion, its genetic content, its location in relation to the centromere and telomere, and genetic background. In addition, there is a class of inversions in which homologous synapsis is gradually replaced by non-homologous synapsis during meiotic progression. This process is called synaptic adjustment. The degree of synaptic adjustment depends critically on the presence and location of the COs (crossovers) within the inversion loop. Only bivalents without COs within the loop and those with COs in the middle of the inversion can be completely adjusted and became linear.

Immunocytological Analysis of Meiotic Recombination in the Gray Goose (Anser anser)

Studies on mammals demonstrate wide interspecific variation in the number and distribution of recombination events along chromosomes. Birds represent an interesting model group for comparative analysis of cytological and ecological drivers of recombination rate evolution. Yet, data on variation in recombination rates in birds are limited to a dozen of species. In this study, we used immunolocalization of MLH1, a mismatch repair protein marking mature recombination nodules, to estimate the overall recombination rate and distribution of crossovers along macrochromosomes in female and male meiosis of the gray goose (Anser anser). The average number of MLH1 foci was significantly higher in oocytes than in spermatocytes (73.6 ± 7.8 and 58.9 ± 7.6, respectively). MLH1 foci distribution along individual macrobivalents showed subtelomeric peaks, which were more pronounced in males. Analysis of distances between neighboring MLH1 foci on macrobivalents revealed stronger crossover interference in male meiosis. These data create a framework for future genetic and physical mapping of the gray goose.

Cytological basis of sterility in male and female hybrids between sibling species of grey voles Microtus arvalis and M. levis

To make insight into the cytological basis of reproductive isolation, we examined chromosome synapsis and recombination in sterile male and female hybrids between Microtus arvalis and M. levis. These sibling species differ by a series of chromosomal rearrangements (fusions, inversions, centromere shifts and heterochromatin insertions). We found that meiosis in male hybrids was arrested at leptotene with complete failure of chromosome pairing and DNA double-strand breaks repair. In the female hybrids meiosis proceeded to pachytene; however, the oocytes varied in the degree of pairing errors. Some of them demonstrated almost correct chromosome pairing, while most of them contained a varying number of univalents and multivalents with extensive regions of asynapsis and non-homologous synapsis. Variation between oocytes was probably caused by stochasticity in the ratio of homologous to non-homologous pairing initiations. We suggest that substantial chromosomal and genetic divergence between the parental species affects preliminary alignment of homologues, homology search and elimination of ectopic interhomologue interactions that are required for correct homologous pairing. Apparently, pairing failure in male and aberrant synapsis in female vole hybrids followed by meiotic silencing of unsynapsed chromatin cause apoptosis of gametocytes and sterility.

Effects of sex and gene order on the recombination frequency and distribution in the chromosome 1 of the house mouse

559 Recombination makes a substantial contribution into generation of genetic variability. It mediates homologous chromosome paring and segregation at meiosis [1]. Studies on various species by various methods demonstrated non random distribution of recombination sites along the chromosomes. Mam malian chromosomes show uneven pattern of distribu tion with pronounced recombination peaks near to telomeres and suppression near to centromeres [2]. Detailed analysis of human, mouse, rat and other mammal genomes revealed a series of local hot and cold spots of recombination [3]. Chiasma interfer ence, i.e. a suppression of recombination in the vicin ity of already occurred recombination sites, also plays a substantial role in determination of the uneven dis tribution [4]. Sex differences in recombination pat terns have been observed in many species of mammals. Females usually have higher frequency and more even distribution of recombination along the chromosomes than males [5]. The causes and biological reasons of these differences remain unclear. It is also unclear what does determine the recombi nation rate in certain regions of a chromosome: recombination features of the DNA sequences located in these regions or their position related to centromere and telomere. A comparison of recombination pat terns of the same chromosomes in the wild type homozygotes and homozygotes for a paracentric inversion may help to solve this problem [6]. We carried out immunofluorescent analysis of recombination frequency and distribution in the chro mosome 1 in spermatocytes and oocytes of the mice homozygous for the wild type gene order and those homozygous for the paracentric inversion In(1)1Rk. This inversion is localized in the medial part of the 1 st chromosome and cover 60% of its length. We visualized the recombination sites at the surface spreads of synaptonemal complexes (SC) using anti bodies to MLH1 (mismatch repair protein) and SCP3 (SC axial element protein) as described earlier [7]. Several studies demonstrated that MLH1 is a reliable marker of the recombination sites. The number and distribution of MLH1 foci in the pachytene cells cor respond precisely to chiasma number and distribution at diakinesis [8–10]. The bivalents of the chromosome 1 were identified by fluorescent in situ hybridization (FISH) with microdissection generated DNA probe to this chro mosome as described earlier [11]. The preparations were visualized with an Axioplan 2 Imaging micro scope (Carl Zeiss, Germany) equipped with a CCD camera (CV M300, JAI Corporation, Japan), CHROMA filter sets and ISIS4 image processing package (MetaSystems GmbH, Germany). The length of the SC of the chromosome 1 and the dis tances of MLH1 foci from to the centromere was mea sured using MicroMeasure 3.3 software [12]. The table shows recombination features of the chromosomes studied. Within each karyotypic group the chromosome 1 bivalents in females formed significantly longer SCs and had more MLH1 foci than in males. Average rela tive distances between the neighboring foci was smaller in females. This indicates that chiasma inter ference (suppression of recombination near previously formed recombination sites [10]) in females is weaker than in males. Similar conclusions were drawn from the genetic analysis of recombination in the chromo some 1 of the house mouse [13]. Several studies dem onstrated a high positive correlation between the SC length and recombination frequency [7, 8, 14]. Appar ently, higher recombination frequency in females is due to longer SC and weaker interference. High level of interference in males determines more uneven dis tribution of the recombination sites along the chromo some. Telomeric peaks of recombination are more pronounced in males than in females (figure). Relatively higher recombination frequency and more even distribution of the recombination sites Effects of Sex and Gene Order on the Recombination Frequency and Distribution in the Chromosome 1 of the House Mouse*

Spatial organization of fibroblast and spermatocyte nuclei with different B-chromosome content in Korean field mouse, Apodemus peninsulae (Rodentia, Muridae)

Korean field mouse (Apodemus peninsulae) shows a wide variation in the number of B chromosomes composed of constitutive heterochromatin. For this reason, it provides a good model to study the influence of the number of centromeres and amount of heterochromatin on spatial organization of interphase nuclei. We analyzed the three-dimensional organization of fibroblast and spermatocyte nuclei of the field mice carrying a different number of B chromosomes using laser scanning microscopy and 3D fluorescence in situ hybridization. We detected a co-localization of the B chromosomes with constitutive heterochromatin of the chromosomes of the basic set. We showed a non-random distribution of B chromosomes in the spermatocyte nuclei. Unpaired B chromosomes showed a tendency to occur in the compartment formed by the unpaired part of the XY bivalent.

Chromosome synapsis and recombination in the hybrids between chromosome races of the common vole Microtus aravalis: “arvalis” and “obscurus”

206 Chromosome races “arvalis” and “obscurus” of the common voles (Microtus aravalis) have the same chromosome number 2n = 46, but differ for the num ber of autosome arms (FNa = 80 and 68, correspond ingly). They also differ for nuclear and mitochondrial genomes [1–3]. This difference is due to alternative fixation of pericentric inversions and centromere transpositions [4]. These races are distributed parapat rically and form 5–10 km wide hybrid zone in Vladimir Province where the pure karyotypes, their F1 hybrids and recombinants with variable FNa are detected [2]. To estimate a role of the chromosomal rearrangements in the restriction of the gene flow we examined chromosome pairing and recombination in the males captured in the hybrid zone. Some of them were heterozygotes for the centromere position in one or two chromosome pairs. We detected a normal chro mosome synapsis in the heteromorphic bivalents. Crossingover was completely suppressed between the centromeres in the heteromorphic SCs. Recombina tion block in these regions leads to a local suppression of the gene flow between the chromosome races of the common vole. This maintains genetic divergence between them and may lead to speciation.

Germline-Restricted Chromosome (GRC) is Widespread among Songbirds

The genome of flying birds, the smallest among amniotes, reflects overweight of the extensive DNA loss over the unrestricted proliferation of selfish genetic elements, resulted in a shortage of repeated sequences and lack of B-chromosomes. The only exception of this rule has been described in zebra finch, which possesses a large germ-line restricted chromosome (GRC), transmitted via oocytes, eliminated from male postmeiotic cells and absent in somatic cell. It is considered as a rarity and its origin, content and function remain unclear. We discovered that all songbirds possess GRC: in various size and genetic content it is present in all fifteen songbird species investigated and absent from germ-line genomes of all eight species of other bird orders examined. Our data based on fluorescent in situ hybridization of DNA probes derived from GRCs of four different Passeri species and their sequencing indicate that the GRCs show low homology between avian species. They contain fragments of the somatic genomes, which include various unique and repetitive sequences. We propose that the GRC has formed in the common ancestor of the extant songbirds and undergone subsequent divergence. GRC presence in the germ line of every songbird studied indicate that it could contain genetic element(s) indispensable for gametogenesis, which are yet to be discovered.