Background:Meiotic transcription is a highly regulated process involving stage-specific chromatin remodeling and dynamic RNA polymerase II (RNAPII) activity. Nevertheless, our current understanding is largely based on classical model organisms, while many nonmodel mammals remain unexplored. This study investigates the transcriptional dynamics and chromatin features during meiotic prophase I in Nannospalax xanthodon, a subterranean blind mole rat species with extensive intraspecific karyotype variation. Materials and methods:Using immunofluorescence, we analyzed synapsis and transcriptional activity in spermatocyte nuclei of two geographically distant N. xanthodon males, detecting SYCP3, CREST, γH2AFX, and phosphorylated RNAPII (Ser5, Ser2). Telomere-associated proteins RAP1 and TERT were also examined to assess transcriptional activity at chromosomal ends. Results:Transcriptional activity was detected at all substages of prophase I. Unlike classical models, N. xanthodon exhibited no sharp pachytene-specific transcriptional reactivation; instead, RNAPII-Ser2 and Ser5 signals increased gradually from leptotene to pachytene. Notably, RNAPII-Ser5, but not Ser2, was consistently localized at telomeric regions of autosomes and sex chromosomes. These RNAPII-Ser5 foci coincided with RAP1-positive telomeres, suggesting a poised but transcriptionally inactive polymerase state at chromosome ends. The variability of RNAPII signals around the XY bivalent may also suggest a dynamic regulation of sex chromosome silencing. This is the first analysis of meiotic transcription in mole rats. The gradual transcriptional enhancement in the chromatin of prophase I nuclei and the RNAPII-Ser5 enrichment at telomeres reflect species-specific regulatory features. Conclusions:These findings highlight the importance of investigating nonmodel organisms to uncover novel mechanisms of meiotic regulation and suggest that transcriptional control during meiosis may be more evolutionarily diverse than previously recognized.
We first studied several rare cases of over-diploid spermatocyte emergence using advanced immunocytochemical methods and a cross-species approach in subterranean rodents Ellobius tancrei (Blasius, 1884), E. alaicus Vorontsov et al., 1969, E. talpinus (Pallas, 1770), and Nannospalax leucodon (Nordmann, 1840) (all belong to the order Rodentia). The tetraploid spermatocytes exhibited specific features during meiotic prophase I, including symmetric and asymmetric chromosome quadrivalents with partner-switching, extended asynapsis, altered recombination patterns, and variable chromatin inactivation. These anomalies suggest that meiotic checkpoints, which are potentially triggered by failed synapsis or incomplete sex chromosome silencing, may act to prevent progression of polyploid spermatocytes. However, the quadrivalents assembled shelterin complexes at chromosome ends, as observed in E. talpinus, and these ends were connected to the nuclear envelope through the linker of nucleoskeleton and cytoskeleton (LINC) complex, as observed in E. alaicus, similarly to normal spermatocytes.
Cytogenetic mechanisms of unisexuality in diploid parthenogenetic species of the genus Darevskia have remained debatable until recently. The mechanism that allows the unisexual form to maintain heterozygosity in a number of generations is important for its long-term existence in nature. In this work, for the first time, for parthenogenetic species of the genus Darevskia, in addition to primary oocytes with the usual ploidy (18 + ZW bivalents in meiotic prophase I), oocytes that underwent premeiotic genome endoduplication and carried a doubled number of bivalents (36 + ZZ + WW) were found. Here, we present a detailed comparative analysis of the preparations of synaptonemal complexes in oocyte nuclei without and with genome endoduplication and the behavior of sex Z and W chromosomes. We show the details of the assembly of bivalents in pachytene nuclei, where either homeologs or doubled identical copies of chromosomes compete for synapsis and form multivalents. For the first time, the WW sex bivalent has been visualized in parthenogenetic reptiles. We show the reverse side of meiotic deviations in obligate parthenogenesis—cases of nonviable embryos with specific abnormalities.
Hybrid parthenogenetic animals are an exceptionally interesting model for studying the mechanisms and evolution of sexual and asexual reproduction. A diploid parthenogenetic lizard Darevskia unisexualis is a result of an ancestral cross between a maternal species Darevskia raddei nairensis and a paternal species Darevskia valentini and presents a unique opportunity for a cytogenetic and computational analysis of a hybrid karyotype. Our previous results demonstrated a significant divergence between the pericentromeric DNA sequences of the parental Darevskia species; however, an in-depth comparative study of their pericentromeres is still lacking. Here, using target sequencing of microdissected pericentromeric regions, we reveal and compare the repertoires of the pericentromeric tandem repeats of the parental Darevskia lizards. We found species-specific sequences of the major pericentromeric tandem repeat CLsat, which allowed computational prediction and experimental validation of fluorescent DNA probes discriminating parental chromosomes within the hybrid karyotype of D. unisexualis. Moreover, we have implemented a generalizable computational method, based on the optimization of the Levenshtein distance between tandem repeat monomers, for finding species-specific fluorescent probes for pericentromere staining. In total, we anticipate that our comparative analysis of Darevskia pericentromeric repeats, the species-specific fluorescent probes that we found and the pipeline that we developed will form a basis for the future detailed cytogenomic studies of a wide range of natural and laboratory hybrids.
Under certain conditions, fungi are capable to fast (down to seconds) change the size of their cells. For example, it is known that the cells of many species of yeasts under hyperosmosis instantly and reversibly shrink entirely, without plasmolysis, with a decrease in volume of up to 40% or more. There is limited data that filamentous fungi can also instantly change the diameter of their unspecialized hyphae. This property is fundamental and is a powerful tool for survival and preservation of the physiological functions of the fungus in sharply changing external conditions, but requires detailed study. In this work, which is a large-scale (more than 50,000 cells measured) light microscopic study, the ability of three unrelated basidiomycete species to fast change the diameter of their hyphae under the influence of various factors was analyzed. It was found, for example, that all three fungi respond similarly to moderate hyperosmotic shock and shrink by an average of 12-14% in diameter, but inhibitors of actin assembly can cause both expansion and shrinkage of hyphae, or have no effect on a fungus. Combining the current results with those previously obtained in the study of macroinvagination systems of the fungal plasma membrane, different approaches for basidiomycetes to respond to sudden stress changes were proposed. The previously presented curtain model has also been advanced. The model describes a general mechanism for the rapid regulation of hyphal sizes, which involves the elastic properties of the cell wall, adhesion of the plasma membrane to it, regulation of turgor, a system of actin cables that regulate the tension of the plasma membrane and cell size, as well as a complex system of macroinvaginations of the plasma membrane, ensuring constant integrity and functionality of the latter.### Competing Interest StatementThe authors have declared no competing interest.
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. Natural hybrids between extremely divergent by karyotypes chromosomal races produce a complex meiotic configuration chain-of-eleven or undecavalent. It is expected that they may suffer from reduced fertility, but our results showed that such a multivalent probably does not negatively affect progression of meiosis, since morphologically normal spermatozoa were revealed.image The Moscow-Seliger F1 hybrids of the common shrew produce a longest meiotic configuration, consisting of 11 chromosomes with monobrachial homology (undecavalent or chain-of-eleven: CXI).Chains-of-eleven have been identified in hybrid spermatocytes at the pachytene and diakinesis stages using immunocytochemical, electron and light microscopy methods.In CXI, arms of chromosomes were synapsed and recombined, although recombination nodules may lack in some arms.Unsynapsed regions of the undecavalents undergo meiotic inactivation.A large number of morphologically normal spermatozoa were observed.It is most likely that common-shrew F1 male hybrids were not sterile.
It was demonstrated that in the meiotic prophase I, in heterozygotes for the Robertsonian translocations, different combinations of meiotic configurations (different trivalent numbers and different chromosome chain structures) in the same individual could be identified. In the present study, two types of experimental hybrids of the eastern mole vole Ellobius tancrei , heterozygous for four Robertsonian translocations, were examined. Instead of the expected four trivalents at the pachytene stage, various types of meiotic configurations, up to the chains of ten elements, were identified. It is suggested that the progression of meiosis occurring in different cells depends on the structure of the formed chains and the possibility of their correction. These variations may lead to the decreased gamete production, but not to the arrest of gametogenesis, which is the basis for the maintenance of Robertsonian translocations in the population.
Hybrid zones are “natural laboratories” for studying speciation. In the common shrew Sorex araneus , the hybrid zone between extremely divergent in karyotypes the Moscow and Seliger chromosomal races is unique, because complex heterozygotes (interracial hybrids) form the longest meiotic configuration consisting of 11 chromosomes with monobrachial homology (undecavalent or chain-of-eleven – CXI). Different studies suggested that such a multivalent would negatively affect meiotic progression and, in general, would significantly reduce fertility. In this work, using immunocytochemical and electron microscopy methods, we investigated chromosome synapsis, recombination, and meiotic silencing in pachytene spermatocytes carrying undecavalent. Despite some abnormalities detected in spermatocytes, such as single associations of chromosomes, stretched centromeres in the multivalent, shifted recombinational peak towards distal parts of chromosomal arms of superchain, heterozygous shrews were able to form a large number of morphologically normal and active spermatozoa. Possible low stringency of pachytene checkpoints, proper segregation of homologous chromosomes, and the ability of hybrids to form mature germ cells imply rapid evolutionary fixation and circulation of Rb chromosomes within shrew populations, leading to a variety of chromosomal races.
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.
The basic causes of postzygotic isolation can be elucidated if gametogenesis is studied, which is a drastically different process in males and females. As a step toward clarifying this problem, we obtained an experimental inbred lineage of the eastern mole vole Ellobius tancrei, whose founder animals were animals with identical diploid numbers 2n = 50 but with different Robertsonian translocations (Rb), namely 2Rb4.12 and 2Rb9.13 in the female and 2Rb.2.18 and 2Rb5.9 in the male. Here, we analyzed strictly inbred hybrids (F1, fertile and F10, sterile) using immunocytochemical methods in order to study spermatocytes during the meiotic prophase I. Previously, the presence of trivalents was assumed to have no significant effect on spermatogenesis and fertility in hybrids, but we demonstrated that spermatogenesis might be disturbed due to the cumulative effects of the retarded synapses of Rb bivalents as well as trivalents and their associations with XX sex bivalents. Alterations in the number of gametes due to the described processes led to a decrease in reproductive capacity up to sterility and can be examined as a mechanism for reproductive isolation, thus starting speciation.
Vipera berus is the species with the largest range of snakes on Earth and one of the largest among reptiles in general. It is also the only snake species found in the Arctic Circle. Vipera berus is the most involved species of the genus Vipera in the process of interspecific hybridization in nature. The taxonomy of the genus Vipera is based on molecular markers and morphology and requires clarification using SC-karyotyping. This work is a detailed comparative study of the somatic and meiotic karyotypes of V. berus, with special attention to DNA and protein markers associated with synaptonemal complexes. The karyotype of V. berus is a remarkable example of a bimodal karyotype containing both 16 large macrochromosomes and 20 microchromosomes. We traced the stages of the asynchronous assembly of both types of bivalents. The number of crossing-over sites per pachytene nucleus, the localization of the nucleolar organizer, and the unique heterochromatin block on the autosomal bivalent 6-an important marker-were determined. Our results show that the average number of crossing-over sites per pachytene nucleus is 49.5, and the number of MLH1 sites per bivalent 1 reached 11, which is comparable to several species of agamas.
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.
This paper presents results of the experiments performed on a nonconventional and extremely interesting in regard to evolution, creature, the European river lamprey Lampetra fluviatilis (Petromyzontiformes, Cyclostomata), one of the oldest taxa of vertebrates. We present detailed immunocytochemical and electron microscopy analyses of chromosome synapsis, the transcription process, and chromatin dynamics in lamprey prophase I, which is the first time for science. We found that not all chromosomes complete synapsis. 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 to meiotic chromatin throughout prophase I, and this pattern has not been previously detected in the animals. Thus, the findings made it possible to identify synaptic and epigenetic patterns specific for this group, and to add new pieces of the puzzle to the discussions of the scientific issues under study. The research on lamprey meiotic chromatin and chromosomal dynamics raises many questions leading to new discoveries.
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.
The development of mycological gerontology requires effective methods for assessing the biological age of fungal cells. This assessment is based on the analysis of a complex of aging and oxidative stress markers. One of the most powerful such markers is the protein carbonylation. In this study, the already known method of dry immune dot blotting is adapted for mycological studies of the content of protein carbonyl groups. After testing the method on a number of filamentous fungi species, some features of the accumulation of carbonylated proteins in mycelium were established. Among these features: (i) a weak effect of exogenous oxidative stress on the accumulation of carbonyls in a number of fungi, (ii) reversibility of the carbonyl accumulation, (iii) possibility of arbitrary regulation of carbonyl content by fungus itself and (iv) the influence of hormesis. In addition, two polar strategies for the accumulation of carbonyl modification were revealed, named Id-strategy (Indifferent) and Cn-strategy (Concern). Thus, even the analysis of one marker allows making some preliminary general assumptions and conclusions. For example, the idea that fungi can freely regulate their biological age is confirmed. This feature makes fungi very flexible in terms of responding to environmental influences and promising objects for gerontology.
Introduction. Metabolic syndrome (MS) can cause impaired spermatogenesis and a decrease in sperm counts. However, the details of the effect of MS on developing spermatogenic cells remain unclear. Difficulties in solving this problem, the inconsistency of published clinical data, indicate the advisability of using experimental models to solve this urgent problem of andrology and reproductology. The study objective is to describe to investigate the specifics of the course of meiotic prophase I and the activity of the processes of phagocytosis and autophagy in Sertoli cells of rats with experimentally induced MS and in the course of therapeutic and prophylactic procedures during the development of experimental MS. Materials and methods. The animals were divided into three groups, each of which included four sexually mature male rats: 1st group – males fed a standard diet; 2nd group – males receiving a diet high in fat and fructose for 60 days; 3rd group – males with MS receiving sulphate mineral waters therapy, low-intensity ultrahigh frequency electromagnetic radiation therapy. Testicular cells were examined using light and transmission electron microscopy. For the first time in animals with MS, an immunocytochemical study of the peculiarities of chromosome synapsis in prophase I of meiosis was carried out on the basis of analysis of spread synaptonemal complexes of meiotic chromosomes and immunocytochemical analysis of Sertoli cells and spermatogenic cells in squashed preparations of seminiferous tubules. The parametric Student’s t-test and the nonparametric Mann–Whitney U-test were used for statistical data processing. Results. As a result of a histological study of the structure of the seminiferous tubules of animals of three groups, a statistically significant decrease in the indices of the spermatogenesis index in 2nd and 3rd groups compared to the control was revealed. Immunomorphologically, in the spread nuclei of primary spermatocytes of rats of the 2nd and 3rd groups, violations of the architectonics of nuclei, the formation of synaptonemal complexes fragments and circular synaptonemal complexes, numerous atypical inclusions were found. Signs of pachytene arrest were found in 40–50 % of spermatocyte nuclei. In the study of squashed cells preparations of the seminiferous tubules of rats of the 2nd and 3rd groups, signs of phagocytosed synaptonemal complexes were found in the cytoplasm of Sertoli cells, which were confirmed using antibodies to the SCP3 protein. Thus, evidence for the phagocytosis of degenerating primary spermatocytes by Sertoli cells has been obtained. In Sertoli cells, spermatocytes and spermatids, many autophagosomes are found, using LC3B protein marker. The presence of autophagosomes in Sertoli cells and spermatogenic cells in animals of these two groups was also confirmed by electron microscopy. In male rats of the 2nd group, significant disturbances in the structure of the pachytene nuclei were revealed. In the cytoplasm of Sertoli cells and spermatids of rats of the 2nd group, lipid droplets, numerous phagolysosomes containing cell detritus were revealed. Structural damage and phagocytosis of mitochondria were found in Sertoli cells and spermatocytes. Аutophagy in Sertoli cells were most distinctive in animals of the 3rd group. Conclusion. In male rats with experimental MS, significant disturbances in the structure of the nuclei of meiotic cells, a high content of primary spermatocytes with signs of pachytene arrest were revealed. The results obtained are in good agreement with the data of other authors, who revealed a decrease in the number of spermatozoa in the epididymis of rats and mice when modeling MS. It is assumed that the activation of autophagy is an important factor in supporting the viability of Sertoli cells and supporting the viability of germ cells in stressful situations, including MS. Apparently, autophagy is an adaptive mechanism that removes the remnants of apoptotic spermatogenic cells that are selected as a result of MS development.
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.
Although the pericentromeric regions of chromosomes that are enriched in tandemly repeated satellite DNA represent a significant part of eukaryotic genomes, they remain understudied, which is mainly due to interdisciplinary knowledge gaps. Recent studies suggest their important role in genome regulation, karyotype stability, and evolution. Thus, the idea of satellite DNA as a junk part of the genome has been refuted. The integration of data regarding molecular composition, chromosome behaviour, and the details of the in situ organization of pericentromeric regions is of great interest. The objective of this work was a cytogenetic analysis of the interactions between pericentromeric regions from non-homologous chromosomes in mouse spermatocytes using immuno-FISH. We analysed two events: the associations between centromeric regions of the X chromosome and autosomes and the associations between the centromeric regions of the autosomal bivalents that form chromocenters. We concluded that the X chromosome forms temporary synaptic associations with different autosomes in early meiotic prophase I, which can normally be found until the pachytene–diplotene, without signs of pachytene arrest. These associations are formed between the satellite-DNA-rich centromeric regions of the X chromosome and different autosomes but do not involve the satellite-DNA-poor centromeric region of the Y chromosome. We suggest the hypothetical model of X chromosome competitive replacement from such associations during synaptic correction. We showed that the centromeric region of the X chromosome in association remains free of γH2Ax-dependent chromatin inactivation, while the Y chromosome is completely inactivated. This finding highlights the predominant role of associations between satellite DNA-rich regions of different chromosomes, including the X chromosome. We suppose that X-autosomal transient associations are a manifestation of an additional synaptic disorder checkpoint. These associations are normally corrected before the late diplotene stage. We revealed that the intense spreading conditions that were applied to the spermatocyte I nuclei did not lead to the destruction of stretched chromatin fibers of elongated chromocenters enriched in satellite DNA. The tight associations that we revealed between the pericentromeric regions of different autosomal bivalents and the X chromosome may represent the basis for a mechanism for maintaining the repeats stability in the autosomes and in the X chromosome. The consequences of our findings are discussed.
Introduction. Metabolic syndrome (MS) can cause impaired spermatogenesis and a decrease in sperm counts. However, the details of the effect of MS on developing spermatogenic cells remain unclear. Difficulties in solving this problem, the inconsistency of published clinical data, indicate the advisability of using experimental models to solve this urgent problem of andrology and reproductology.The study objective is to describe to investigate the specifics of the course of meiotic prophase I and the activity of the processes of phagocytosis and autophagy in Sertoli cells of rats with experimentally induced MS and in the course of therapeutic and prophylactic procedures during the development of experimental MS.Materials and methods. The animals were divided into three groups, each of which included four sexually mature male rats: 1st group – males fed a standard diet; 2nd group – males receiving a diet high in fat and fructose for 60 days; 3rd group – males with MS receiving sulphate mineral waters therapy, low-intensity ultrahigh frequency electromagnetic radiation therapy. Testicular cells were examined using light and transmission electron microscopy. For the first time in animals with MS, an immunocytochemical study of the peculiarities of chromosome synapsis in prophase I of meiosis was carried out on the basis of analysis of spread synaptonemal complexes of meiotic chromosomes and immunocytochemical analysis of Sertoli cells and spermatogenic cells in squashed preparations of seminiferous tubules. The parametric Student’s t-test and the nonparametric Mann–Whitney U-test were used for statistical data processing.Results. As a result of a histological study of the structure of the seminiferous tubules of animals of three groups, a statistically significant decrease in the indices of the spermatogenesis index in 2nd and 3rd groups compared to the control was revealed. Immunomorphologically, in the spread nuclei of primary spermatocytes of rats of the 2nd and 3rd groups, violations of the architectonics of nuclei, the formation of synaptonemal complexes fragments and circular synaptonemal complexes, numerous atypical inclusions were found. Signs of pachytene arrest were found in 40–50 % of spermatocyte nuclei. In the study of squashed cells preparations of the seminiferous tubules of rats of the 2nd and 3rd groups, signs of phagocytosed synaptonemal complexes were found in the cytoplasm of Sertoli cells, which were confirmed using antibodies to the SCP3 protein. Thus, evidence for the phagocytosis of degenerating primary spermatocytes by Sertoli cells has been obtained. In Sertoli cells, spermatocytes and spermatids, many autophagosomes are found, using LC3B protein marker. The presence of autophagosomes in Sertoli cells and spermatogenic cells in animals of these two groups was also confirmed by electron microscopy. In male rats of the 2nd group, significant disturbances in the structure of the pachytene nuclei were revealed. In the cytoplasm of Sertoli cells and spermatids of rats of the 2nd group, lipid droplets, numerous phagolysosomes containing cell detritus were revealed. Structural damage and phagocytosis of mitochondria were found in Sertoli cells and spermatocytes. Аutophagy in Sertoli cells were most distinctive in animals of the 3rd group.Conclusion. In male rats with experimental MS, significant disturbances in the structure of the nuclei of meiotic cells, a high content of primary spermatocytes with signs of pachytene arrest were revealed. The results obtained are in good agreement with the data of other authors, who revealed a decrease in the number of spermatozoa in the epididymis of rats and mice when modeling MS. It is assumed that the activation of autophagy is an important factor in supporting the viability of Sertoli cells and supporting the viability of germ cells in stressful situations, including MS. Apparently, autophagy is an adaptive mechanism that removes the remnants of apoptotic spermatogenic cells that are selected as a result of MS development.
Parthenogenetic species of Caucasian rock lizards of the genus Darevksia are important evidence for reticulate evolution and speciation by hybridization in vertebrates. Female-only lineages formed through interspecific hybridization have been discovered in many groups. Nevertheless, critical mechanisms of oogenesis and specifics of meiosis that provide long-term stability of parthenogenetic species are still unknown. Here we report cytogenetic characteristics of somatic karyotypes and meiotic prophase I nuclei in the diploid parthenogenetic species Darevskia unisexualis from the new population "Keti" in Armenia which contains an odd number of chromosomes 2n = 37, instead of the usual 2n = 38. We revealed 36 acrocentric chromosomes and a single metacentric autosomal chromosome, resulting from Robertsonian translocation. Comparative genomic hybridization revealed that chromosome fusion occurred between two chromosomes inherited from the maternal species, similar to another parthenogenetic species D. rostombekowi. To trace the chromosome behaviour in meiosis, we performed an immunocytochemical study of primary oocytes' spread nuclei and studied chromosome synapsis during meiotic prophase I in D. unisexualis based on analysis of synaptonemal complexes (SCs). We found meiotic SC-trivalent composed of one metacentric and two acrocentric chromosomes. We confirmed that the SC was assembled between homeologous chromosomes inherited from two parental species. Immunostaining of the pachytene and diplotene nuclei revealed a mismatch repair protein MLH1 loaded to all autosomal SC bivalents. Possible mechanisms of meiotic recombination between homeologous chromosomes are discussed.