Natural male hybrid common shrews with a very long chromosomal multivalent at meiosis appear not to be completely sterile.

Among 36 known chromosomal hybrid zones of the common shrew Sorex araneus, the Moscow-Seliger hybrid zone is of special interest because inter-racial complex heterozygotes (F1 hybrids) produce the longest meiotic configuration, consisting of 11 chromosomes with monobrachial homology (undecavalent or chain-of-eleven: CXI). Different studies suggest that such a multivalent may negatively affect meiotic progression and in general should significantly reduce fertility of hybrids. In this work, by immunocytochemical and electron microscopy methods, we investigated for the first time chromosome synapsis, recombination and meiotic silencing in pachytene spermatocytes of natural inter-racial heterozygous shrew males carrying CXI configurations. Despite some abnormalities detected in spermatocytes, such as associations of chromosomes, stretched centromeres, and the absence of recombination nodules in some arms of the multivalent, a large number of morphologically normal spermatozoa were observed. Possible low stringency of pachytene checkpoints may mean that even very long meiotic configurations do not cause complete sterility of such complex inter-racial heterozygotes.

Speciation on the Roof of the World: Parallel Fast Evolution of Cryptic Mole Vole Species in the Pamir-Alay-Tien Shan Region.

Speciation is not always accompanied by morphological changes; numerous cryptic closely related species were revealed using genetic methods. In natural populations of Ellobius tancrei (2n = 54-30) and E. alaicus (2n = 52-48) of the Pamir-Alay and Tien Shan, the chromosomal variability due to Robertsonian translocations has been revealed. Here, by comprehensive genetic analysis (karyological analyses as well as sequencing of mitochondrial genes, cytb and COI, and nuclear genes, XIST and IRBP) of E. alaicus and E. tancrei samples from the Inner Tien Shan, the Alay Valley, and the Pamir-Alay, we demonstrated fast and independent diversification of these species. We described an incompletely consistent polymorphism of the mitochondrial and nuclear markers, which arose presumably because of habitat fragmentation in the highlands, rapid karyotype changes, and hybridization of different intraspecific varieties and species. The most intriguing results are a low level of genetic distances calculated from mitochondrial and nuclear genes between some phylogenetic lines of E. tancrei and E. alaicus, as well significant species-specific chromosome variability in both species. The chromosomal rearrangements are what most clearly define species specificity and provide further diversification. The "mosaicism" and inconsistency in polymorphism patterns are evidence of rapid speciation in these mammals.

The First Analysis of Synaptonemal Complexes in Jawless Vertebrates: Chromosome Synapsis and Transcription Reactivation at Meiotic Prophase I in the Lamprey Lampetra fluviatilis (Petromyzontiformes, Cyclostomata).

Transcription is known to be substage-specific in meiotic prophase I. If transcription is reactivated in the mid pachytene stage in mammals when synapsis is completed, then this process is observed in the zygotene stage in insects. The process of transcriptional reactivation has been studied in a small number of different taxa of invertebrates and vertebrates. Here, for the first time, we investigate synapsis and transcription in prophase I in the European river lamprey Lampetra fluviatilis (Petromyzontiformes, Cyclostomata), which is representative of jawless vertebrates that diverged from the main branch of vertebrates between 535 and 462 million years ago. We found that not all chromosomes complete synapsis in telomeric regions. Rounded structures were detected in chromatin and in some synaptonemal complexes, but their nature could not be determined conclusively. An analysis of RNA polymerase II distribution led to the conclusion that transcriptional reactivation in lamprey prophase I is not associated with the completion of chromosome synapsis. Monomethylated histone H3K4 is localized in meiotic chromatin throughout prophase I, and this pattern has not been previously detected in animals. Thus, the findings made it possible to identify synaptic and epigenetic patterns specific to this group and to expand knowledge about chromatin epigenetics in prophase I.

Nonhomologous Chromosome Interactions in Prophase I: Dynamics of Bizarre Meiotic Contacts in the Alay Mole Vole Ellobius alaicus (Mammalia, Rodentia).

Nonhomologous chromosome interactions take place in both somatic and meiotic cells. Prior to this study, we had discovered special contacts through the SYCP3 (synaptonemal complex protein 3) filament between the short arms of nonhomologous acrocentrics at the pachytene stage in the Alay mole vole, and these contacts demonstrate several patterns from proximity to the complete fusion stage. Here, we investigated the nonhomologous chromosome contacts in meiotic prophase I. It turned out that such contacts do not introduce changes into the classic distribution of DNA double-strand breaks. It is noteworthy that not all meiotic contacts were localized in the H3k9me3-positive heterochromatic environment. Both in the mid zygotene and in the early-mid diplotene, three types of contacts (proximity, touching, and anchoring/tethering) were observed, whereas fusion seems to be characteristic only for pachytene. The number of contacts in the mid pachytene is significantly higher than that in the zygotene, and the distance between centromeres in nonhomologous contacts is also the smallest in mid pachytene for all types of contacts. Thus, this work provides a new insight into the behavior of meiotic contacts during prophase I and points to avenues of further research.

Geographic Mosaic of Extensive Genetic Variations in Subterranean Mole Voles Ellobius alaicus as a Consequence of Habitat Fragmentation and Hybridization.

Restricted mobility, sociality, and high inbreeding-characteristic for subterranean mammals-lead to rapid changes in their genome structure. Up to now, the Alay mole vole Ellobius alaicus was a data-deficient species; its spatial and phylogenetic relationships with a sibling species, E. tancrei, were not clarified. We carried out a genetic analysis including differential G-banding of chromosomes and mitochondrial (cytb) and nuclear gene (XIST and IRBP) sequencing. The phylogenetic reconstruction based on cytb represented the expected phylogenetic relationships of two species. Using the XIST, we revealed two new lineages among E. alaicus from the Alay Valley (Southern Kyrgyzstan). Analysis of IRBP demonstrated presence of the specific genotype in most of E. alaicus specimens, but also revealed the haplotype, typical for E. tancrei, in some Alay mole voles. The results may be explained as persistence of ancestral gene polymorphism in E. alaicus or limited interspecific hybridization with E. tancrei. Several chromosomal forms were revealed in E. alaicus in the Alay Valley. We propose that 'mosaic' genetic polymorphism might appear in E. alaicus due to fragmentation of their habitats in highlands of the Alay Valley, Tien Shan, and Pamir-Alay as well as due to hybridization with E. tancrei or persistence of ancestral polymorphisms.

Heterogeneity in conservation of multifunctional partner enzymes with meiotic importance, CDK2 kinase and BRCA1 ubiquitin ligase.

The evolution of proteins can be accompanied by changes not only to their amino acid sequences, but also their structural and spatial molecular organization. Comparison of the protein conservation within different taxonomic groups (multifunctional, or highly specific) allows to clarify their specificity and the direction of evolution. Two multifunctional enzymes, cyclin-dependent kinase 2 (CDK2) and BRCA1 ubiquitin ligase, that are partners in some mitotic and meiotic processes were investigated in the present work. Two research methods, bioinformatics and immunocytochemical, were combined to examine the conservation levels of the two enzymes. It has been established that CDK2 is a highly conserved protein in different taxonomic lineages of the eukaryotic tree. Immunocytochemically, a conserved CDK2 pattern was revealed in the meiotic autosomes of five rodent species and partially in domestic turkey and clawed frog. Nevertheless, variable CDK2 distribution was detected at the unsynapsed segments of the rodent X chromosomes. BRCA1 was shown to be highly conserved only within certain mammalian taxa. It was also noted that in those rodent nuclei, where BRCA1 specifically binds to antigens, asynaptic regions of sex chromosomes were positive. BRCA1 staining was not always accompanied by specific binding, and a high nonspecificity in the nucleoplasm was observed. Thus, the studies revealed different conservation of the two enzymes at the level of protein structure as well as at the level of chromosome behavior. This suggests variable rates of evolution due to both size and configuration of the protein molecules and their multifunctionality.

Sex differences in the meiotic behavior of an XX sex chromosome pair in males and females of the mole vole Ellobius tancrei: turning an X into a Y chromosome?

Sex determination in mammals is usually provided by a pair of chromosomes, XX in females and XY in males. Mole voles of the genus Ellobius are exceptions to this rule. In Ellobius tancrei, both males and females have a pair of XX chromosomes that are indistinguishable from each other in somatic cells. Nevertheless, several studies on Ellobius have reported that the two X chromosomes may have a differential organization and behavior during male meiosis. It has not yet been demonstrated if these differences also appear in female meiosis. To test this hypothesis, we have performed a comparative study of chromosome synapsis, recombination, and histone modifications during male and female meiosis in E. tancrei. We observed that synapsis between the two X chromosomes is limited to the short distal (telomeric) regions of the chromosomes in males, leaving the central region completely unsynapsed. This uneven behavior of sex chromosomes during male meiosis is accompanied by structural modifications of one of the X chromosomes, whose axial element tends to appear fragmented, accumulates the heterochromatin mark H3K9me3, and is associated with a specific nuclear body that accumulates epigenetic marks and proteins such as SUMO-1 and centromeric proteins but excludes others such as H3K4me, ubiH2A, and γH2AX. Unexpectedly, sex chromosome synapsis is delayed in female meiosis, leaving the central region unsynapsed during early pachytene. This region accumulates γH2AX up to the stage in which synapsis is completed. However, there are no structural or epigenetic differences similar to those found in males in either of the two X chromosomes. Finally, we observed that recombination in the sex chromosomes is restricted in both sexes. In males, crossover-associated MLH1 foci are located exclusively in the distal regions, indicating incipient differentiation of one of the sex chromosomes into a neo-Y. Notably, in female meiosis, the central region of the X chromosome is also devoid of MLH1 foci, revealing a lack of recombination, possibly due to insufficient homology. Overall, these results reveal new clues about the origin and evolution of sex chromosomes.

Kinase CDK2 in Mammalian Meiotic Prophase I: Screening for Hetero- and Homomorphic Sex Chromosomes.

Cyclin-dependent kinases (CDKs) are crucial regulators of the eukaryotic cell cycle. The critical role of CDK2 in the progression of meiosis was demonstrated in a single mammalian species, the mouse. We used immunocytochemistry to study the localization of CDK2 during meiosis in seven rodent species that possess hetero- and homomorphic male sex chromosomes. To compare the distribution of CDK2 in XY and XX male sex chromosomes, we performed multi-round immunostaining of a number of marker proteins in meiotic chromosomes of the rat and subterranean mole voles. Antibodies to the following proteins were used: RAD51, a member of the double-stranded DNA break repair machinery; MLH1, a component of the DNA mismatch repair system; and SUN1, which is involved in the connection between the meiotic telomeres and nuclear envelope, alongside the synaptic protein SYCP3 and kinetochore marker CREST. Using an enhanced protocol, we were able to assess the distribution of as many as four separate proteins in the same meiotic cell. We showed that during prophase I, CDK2 localizes to telomeric and interstitial regions of autosomes in all species investigated (rat, vole, hamster, subterranean mole voles, and mole rats). In sex bivalents following synaptic specificity, the CDK2 signals were distributed in three different modes. In the XY bivalent in the rat and mole rat, we detected numerous CDK2 signals in asynaptic regions and a single CDK2 focus on synaptic segments, similar to the mouse sex chromosomes. In the mole voles, which have unique XX sex chromosomes in males, CDK2 signals were nevertheless distributed similarly to the rat XY sex chromosomes. In the vole, sex chromosomes did not synapse, but demonstrated CDK2 signals of varying intensity, similar to the rat X and Y chromosomes. In female mole voles, the XX bivalent had CDK2 pattern similar to autosomes of all species. In the hamster, CDK2 signals were revealed in telomeric regions in the short synaptic segment of the sex bivalent. We found that CDK2 signals colocalize with SUN1 and MLH1 signals in meiotic chromosomes in rats and mole voles, similar to the mouse. The difference in CDK2 manifestation at the prophase I sex chromosomes can be considered an example of the rapid chromosome evolution in mammals.

Meiotic Nuclear Architecture in Distinct Mole Vole Hybrids with Robertsonian Translocations: Chromosome Chains, Stretched Centromeres, and Distorted Recombination.

Genome functioning in hybrids faces inconsistency. This mismatch is manifested clearly in meiosis during chromosome synapsis and recombination. Species with chromosomal variability can be a model for exploring genomic battles with high visibility due to the use of advanced immunocytochemical methods. We studied synaptonemal complexes (SC) and prophase I processes in 44-chromosome intraspecific (Ellobius tancrei × E. tancrei) and interspecific (Ellobius talpinus × E. tancrei) hybrid mole voles heterozygous for 10 Robertsonian translocations. The same pachytene failures were found for both types of hybrids. In the intraspecific hybrid, the chains were visible in the pachytene stage, then 10 closed SC trivalents formed in the late pachytene and diplotene stage. In the interspecific hybrid, as a rule, SC trivalents composed the SC chains and rarely could form closed configurations. Metacentrics involved with SC trivalents had stretched centromeres in interspecific hybrids. Linkage between neighboring SC trivalents was maintained by stretched centromeric regions of acrocentrics. This centromeric plasticity in structure and dynamics of SC trivalents was found for the first time. We assume that stretched centromeres were a marker of altered nuclear architecture in heterozygotes due to differences in the ancestral chromosomal territories of the parental species. Restructuring of the intranuclear organization and meiotic disturbances can contribute to the sterility of interspecific hybrids, and lead to the reproductive isolation of studied species.

Meiotic Chromosome Contacts as a Plausible Prelude for Robertsonian Translocations.

Robertsonian translocations are common chromosomal alterations. Chromosome variability affects human health and natural evolution. Despite the significance of such mutations, no mechanisms explaining the emergence of such translocations have yet been demonstrated. Several models have explored possible changes in interphase nuclei. Evidence for non-homologous chromosomes end joining in meiosis is scarce, and is often limited to uncovering mechanisms in damaged cells only. This study presents a primarily qualitative analysis of contacts of non-homologous chromosomes by short arms, during meiotic prophase I in the mole vole, Ellobius alaicus, a species with a variable karyotype, due to Robertsonian translocations. Immunocytochemical staining of spermatocytes demonstrated the presence of four contact types for non-homologous chromosomes in meiotic prophase I: (1) proximity, (2) touching, (3) anchoring/tethering, and (4) fusion. Our results suggest distinct mechanisms for chromosomal interactions in meiosis. Thus, we propose to change the translocation mechanism model from 'contact first' to 'contact first in meiosis'.

Rapid chromosomal evolution in enigmatic mammal with XX in both sexes, the Alay mole vole Ellobiusalaicus Vorontsov et al., 1969 (Mammalia, Rodentia).

Evolutionary history and taxonomic position for cryptic species may be clarified by using molecular and cytogenetic methods. The subterranean rodent, the Alay mole vole Ellobiusalaicus Vorontsov et al., 1969 is one of three sibling species constituting the subgenus Ellobius Fischer, 1814, all of which lost the Y chromosome and obtained isomorphic XX sex chromosomes in both males and females. E.alaicus is evaluated by IUCN as a data deficient species because their distribution, biology, and genetics are almost unknown. We revealed specific karyotypic variability (2n = 52-48) in E.alaicus due to different Robertsonian translocations (Rbs). Two variants of hybrids (2n = 53, different Rbs) with E.tancrei Blasius, 1884 were found at the Northern slopes of the Alay Ridge and in the Naryn district, Kyrgyzstan. We described the sudden change in chromosome numbers from 2n = 50 to 48 and specific karyotype structure for mole voles, which inhabit the entrance to the Alay Valley (Tajikistan), and revealed their affiliation as E.alaicus by cytochrome b and fragments of nuclear XIST and Rspo1 genes sequencing. To date, it is possible to expand the range of E.alaicus from the Alay Valley (South Kyrgyzstan) up to the Ferghana Ridge and the Naryn Basin, Tien Shan at the north-east and to the Pamir-Alay Mountains (Tajikistan) at the west. The closeness of E.tancrei and E.alaicus is supported, whereas specific chromosome and molecular changes, as well as geographic distribution, verified the species status for E.alaicus. The case of Ellobius species accented an unevenness in rates of chromosome and nucleotide changes along with morphological similarity, which is emblematic for cryptic species.

Reorganization of the Y Chromosomes Enhances Divergence in Israeli Mole Rats Nannospalax ehrenbergi (Spalacidae, Rodentia): Comparative Analysis of Meiotic and Mitotic Chromosomes.

The Y chromosome in mammals is variable, even in closely related species. Middle East blind mole rats Nannospalax ehrenbergi demonstrate autosomal variability, which probably leads to speciation. Here, we compare the mitotic and meiotic chromosomes of mole rats. For the first time, we studied the behavior of their sex chromosomes in the meiotic prophase I using electron microscopy and immunocytochemical analysis. Unexpectedly, the sex chromosomes of the 52- and 60-chromosome forms of mole rats showed different synaptic and recombination patterns due to distinct locations of the centromeres on the Y chromosomes. The absence of recombination in the 60-chromosome form, the asymmetric synapsis, and the short-term disturbance in the synaptic co-orientation of the telomeric regions of the X and Y chromosomes were revealed as specific features of mole rat sex bivalents. We suggest several scenarios of Y chromosome alteration in connection with species differentiation in mole rats.

Chromosomal Evolution in Mole Voles Ellobius (Cricetidae, Rodentia): Bizarre Sex Chromosomes, Variable Autosomes and Meiosis.

This study reports on extensive experimental material covering more than 30 years of studying the genetics of mole voles. Sex chromosomes of Ellobius demonstrate an extraordinary case of mammalian sex chromosomes evolution. Five species of mole voles own three types of sex chromosomes; typical for placentals: XY♂/XX♀; and atypical X0♂/X0♀; or XX♂/XX♀. Mechanisms of sex determination in all Ellobius species remain enigmatic. It was supposed that the Y chromosome was lost twice and independently in subgenera Bramus and Ellobius. Previous to the Y being lost, the X chromosome in distinct species obtained some parts of the Y chromosome, with or without Sry, and accumulated one or several copies of the Eif2s3y gene. Along with enormous variations of sex chromosomes, genes of sex determination pathway and autosomes, and five mole vole species demonstrate ability to establish different meiotic mechanisms, which stabilize their genetic systems and make it possible to overcome the evolutionary deadlocks.

Dual mechanism of chromatin remodeling in the common shrew sex trivalent (XY 1Y 2).

Here we focus on the XY1Y2 condition in male common shrew Sorex araneus Linnaeus, 1758, applying electron microscopy and immunocytochemistry for a comprehensive analysis of structure, synapsis and behaviour of the sex trivalent in pachytene spermatocytes. The pachytene sex trivalent consists of three distinct parts: short and long synaptic SC fragments (between the X and Y1 and between the X and Y2, respectively) and a long asynaptic region of the X in-between. Chromatin inactivation was revealed in the XY1 synaptic region, the asynaptic region of the X and a very small asynaptic part of the Y2. This inactive part of the sex trivalent, that we named the 'head', forms a typical sex body and is located at the periphery of the meiotic nucleus at mid pachytene. The second part or 'tail', a long region of synapsis between the X and Y2 chromosomes, is directed from the periphery into the nucleus. Based on the distribution patterns of four proteins involved in chromatin inactivation, we propose a model of meiotic silencing in shrew sex chromosomes. Thus, we conclude that pachytene sex chromosomes are structurally and functionally two different chromatin domains with specific nuclear topology: the peripheral inactivated 'true' sex chromosome regions (part of the X and the Y1) and more centrally located transcriptionally active autosomal segments (part of the X and the Y2).

Reticulate Evolution of the Rock Lizards: Meiotic Chromosome Dynamics and Spermatogenesis in Diploid and Triploid Males of the Genus Darevskia.

Knowing whether triploid hybrids resulting from natural hybridization of parthenogenetic and bisexual species are fertile is crucial for understanding the mechanisms of reticulate evolution in rock lizards. Here, using males of the bisexual diploid rock lizard species Darevskia raddei nairensis and Darevskia valentini and a triploid hybrid male Darevskia unisexualis × Darevskia valentini, we performed karyotyping and comparative immunocytochemistry of chromosome synapsis and investigated the distribution of RAD51 and MLH1 foci in spread spermatocyte nuclei in meiotic prophase I. Three chromosome sets were found to occur in cell nuclei in the D. unisexualis × D. valentini hybrid, two originating from a parthenogenetic D. unisexualis female and one from the D. valentini male. Despite this distorted chromosome synapsis and incomplete double-strand breaks repair in meiotic prophase I, the number of mismatch repair foci in the triploid hybrid was enough to pass through both meiotic divisions. The defects in synapsis and repair did not arrest meiosis or spermatogenesis. Numerous abnormal mature spermatids were observed in the testes of the studied hybrid.

Unique sex chromosome systems in Ellobius: How do male XX chromosomes recombine and undergo pachytene chromatin inactivation?

Most mammalian species have heteromorphic sex chromosomes in males, except for a few enigmatic groups such as the mole voles Ellobius, which do not have the Y chromosome and Sry gene. The Ellobius (XX ♀♂) system of sex chromosomes has no analogues among other animals. The structure and meiotic behaviour of the two X chromosomes were investigated for males of the sibling species Ellobius talpinus and Ellobius tancrei. Their sex chromosomes, despite their identical G-structure, demonstrate short synaptic fragments and crossover-associated MLH1 foci in both telomeric regions only. The chromatin undergoes modifications in the meiotic sex chromosomes. SUMO-1 marks a small nucleolus-like body of the meiotic XX. ATR and ubiH2A are localized in the asynaptic area and the histone γH2AFX covers the entire XX bivalent. The distribution of some markers of chromatin inactivation differentiates sex chromosomes of mole voles from those of other mammals. Sex chromosomes of both studied species have identical recombination and meiotic inactivation patterns. In Ellobius, similar chromosome morphology masks the functional heteromorphism of the male sex chromosomes, which can be seen at meiosis.

Analysis of meiotic chromosome structure and behavior in Robertsonian heterozygotes of Ellobius tancrei (Rodentia, Cricetidae): a case of monobrachial homology.

Synaptonemal complex (SC) chains were revealed in semisterile intraspecific F1 hybrids of Ellobius tancrei Blasius, 1884 (2n = 49, NF=56 and 2n=50, NF=56), heterozygous for Robertsonian (Rb) translocations. Chains were formed by Rb submetacentrics with monobrachial homology. Chromosome synapsis in spermatocytes of these hybrids was disturbed, apparently because of the problematic release of the chromosomes from the SC chains. These hybrids suffer from low fertility, and our data support the opinion that this is because a formation of Rb metacentrics with monobrachial homology within different races of the same species might be an initial event for the divergence of chromosomal forms.

Synaptonemal complex analysis of interracial hybrids between the Moscow and Neroosa chromosomal races of the common shrew Sorex araneus showing regular formation of a complex meiotic configuration (ring-of-four).

Immunocytochemical and electron microscopic analysis of synaptonemal complexes (SCs) was carried out for the first time in homozygotes and complex Robertsonian heterozygotes (hybrids) of the common shrew, Sorex araneus Linnaeus, 1758, from a newly discovered hybrid zone between the Moscow and the Neroosa chromosomal races. These races differ in four monobrachial homologous metacentrics, and closed SC tetravalent is expected to be formed in meiosis of a hybrid. Indeed, such a multivalent was found at meiotic prophase I in hybrids. Interactions between multivalent and both autosomes and/or the sex chromosomes were observed. For the first time we have used immunocytochemical techniques to analyse asynapsis in Sorex araneus and show that the multivalent pairs in an orderly fashion with complete synapsis. Despite some signs of spermatocytes arrested in the meiotic prophase I, hybrids had large number of active sperm. Thus, Moscow - Neroosa hybrid males that form a ring-of-four meiotic configuration are most likely not sterile. Our results support previous demonstrations that monobrachial homology of metacentrics of the common shrew does not lead to complete reproductive isolation between parapatric chromosomal races of the species.