DNA Environment of Centromeres and Non-Homologous Chromosomes Interactions in Mouse

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.

DNA Environment of Centromeres and Non-homologous Chromosomes Interactions in Mouse

Pericentromeric regions of chromosomes enriched in tandemly repeated satellite DNA although representing a significant part of eukaryotic genomes are still understudied 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 was refuted. Integration of data about molecular composition, chromosome behaviour and details of in situ organization of pericentromeric regions is of great interest. The objective of this work was a cytogenetic analysis of the interactions of pericentromeric regions non-homologous chromosomes in mouse spermatocytes using immuno-FISH. We analysed two events: the associations between cerntomeric regions of X chromosome and autosomes, and associations between centromeric regions of autosomal bivalents forming chromocenters. We conclude that X chromosome form temporary synaptic associations with different autosomes in early meiotic prophase I which normally can be found at pachytene-diplotene without signs of pachytene arrest. These associations are formed between the satellite DNA-enriched centomeric regions of X chromosome and different autosomes but not involve the satellite-poor centromeric region of Y-chromosome. We suggest the mechanism of X chromosome competitive replacement from such associations during synaptic correction. We showed that centromeric region of the X chromosome remains free of γH2Ax-dependent chromatin inactivation, while Y chromosome is completely inactivated. This findings highlights the predominant role of associations between satellite DNA-enriched regions of different chromosomes including X. We assume that X-autosome temporary associations is a manifestation of an additional synaptic disorders checkpoint. These associations are normally corrected before the late diplotene. We revealed that the intense spreading conditions applied to the spermatocytes I nuclei did not lead to destruction of stretched chromatin fibers i.e. elongated chromocenters enriched in satellite DNA. Revealed by us tight associations between pericentromeric regions of different autosomal bivalents and X chromosome may represent the basis for repeat stability maintenance in autosomes an X chromosome. The consequences of our findings are discussed. We obtained the preparations of mouse spermatocytes nuclei in the meiotic prophase I using two approaches: standard and extremely intense surface spread techniques. Using immuno-FISH we visualized tandemly repeated mouse Major and Minor satellite DNA located in the pericentromeric regions of chromosomes and performed a morphological comparison of the standard- and intensely spreaded meiotic nuclei. Based on our results, we assume the remarkable strength of the chromocenter-mediated associations, “chromatin “bridges”, between different bivalents at the pachytene and diplotene stages. We have demonstrated that the chromocenter “bridges” between the centromeric ends of meiotic bivalents are enriched in both tandemly repeated Major and Minor satellite DNA. Association of centromeric regions of autosomal bivalents and X-chromosome but not with Y-chromosome correlates with the absence of Major and Minor satellites on Y-chromosome. We suggest that revealed tight associations between pericentromeric regions of bivalents may represent the network-like system providing dynamic stability of chromosomal territories, as well as add new data for the hypothesis of ectopic recombination in these regions which supports sequence homogeneity between non-homologous chromosomes and does not contradict the meiotic restrictions imposed by the crossing-over interference near centromeres. We conclude that nuclear architecture in meio-sis may play an essential role in contacts between the non-homologous chromosomes providing the specific characteristics of pericentromeric DNA.

Cytogenetic Studies on Three Species of Genus Burmagomphus of Family Gomphidae (Odonata: Anisoptera) from India

Male germ cell chromosomes of Burmagomphus divaricatus, Burmagomphus pyramidalis and Burmagomphus sivalikensis of family Gomphidae have been investigated by using conventional staining, C-banding, silver nitrate staining and sequence specific staining. The species were collected from Punjab and Himachal Pradesh, India. All the species possess the chromosome number 2n = 23 which is the type number of the family. Terminal C bands and NOR’s are present at the autosomal bivalents and X chromosome is C positive and NOR rich in all the three species, while m bivalents show variation in distribution of C- heterochromatin and NOR’s. In the sequence specific staining, whole complement shows bright DAPI signals in B. divaricatus, bright CMA3 signals in B. pyramidalis and both DAPI and CMA3 signals in B. sivalikensis.

Evolution of the Sex Chromosomes in Beetles. I. The Loss of the Y Chromosome

In the males of Coleoptera, the most frequent sex chromosome constitution is XY. At metaphase I of meiosis, the X and Y are linked by nucleolar proteins, forming the so-called parachute bivalent (Xyp), which is assumed to allow the non-synapsed X and Y to segregate correctly at anaphase I. However, X0 males are not exceptional, and we explored the relationships between the X and nucleolar proteins in the absence of the Y chromosome in 6 species belonging to different families/subfamilies. Using C-banding and silver staining, we show that nucleolar proteins always remain in contact with the X until anaphase I. These proteins are generally more abundant than in the Xyp bivalent, may remain associated with the NOR during diakinesis, and frequently link the X to 1 or 2 autosomal bivalents, which seem to play the same role as the Y. This role may also be played by B chromosomes, which appear to be more frequent in X0 than in XY males. In conclusion, following Y chromosome loss, various strategies using nucleolar proteins have been developed to facilitate the migration of the unique X at meiotic anaphase I.

Alterations in chromosomal synapses and DNA repair in apoptotic spermatocytes of Mus m. domesticus

<p>We investigated whether apoptotic spermatocytes from the mouse <em>Mus m. domesticus</em> presented alterations in chromosomal synapses and DNA repair. To enrich for apoptotic spermatocytes, the scrotum’s temperature was raised by partially exposing animals for 15 min to a 42ºC water bath. Spermatocytes in initial apoptosis were identified <em>in situ</em> by detecting activated Caspase-9. SYCP1 and SYCP3 were markers for evaluating synapses or the structure of synaptonemal complexes and Rad51 and γH2AX for detecting DNA repair and chromatin remodeling. Apoptotic spermatocytes were concentrated in spermatogenic cycle stages III-IV (50.3%), XI-XII (44.1%) and IX-X (4.2%). Among apoptotic spermatocytes, 48% were in middle pachytene, 44% in metaphase and 6% in diplotene. Moreover, apoptotic spermatocytes showed several structural anomalies in autosomal bivalents, including splitting of chromosomal axes and partial asynapses between homologous chromosomes. gH2AX and Rad51 were atypically distributed during pachytene and as late as diplotene and associated with asynaptic chromatin, single chromosome axes or discontinuous chromosome axes. Among apoptotic spermatocytes at pachytene, 70% showed changes in the structure of synapses, 67% showed changes in gH2AX and Rad51 distribution and 50% shared alterations in both synapses and DNA repair. Our results showed that apoptotic spermatocytes from <em>Mus m. domesticus</em> contain a high frequency of alterations in chromosomal synapses and in the recruitment and distribution of DNA repair proteins. Together, these observations suggest that these alterations may have been detected by meiotic checkpoints triggering apoptosis.</p>

Equal frequencies of recombination nodules in both sexes of the pigeon suggest a basic difference with eutherian mammals

The total number of recombination nodules (RNs) in the autosomal synaptonemal complexes (SCs) is statistically equivalent in oocytes and spermatocytes from the domestic pigeon Columba livia. The distribution on RNs along the three longest autosomes is also equivalent in oocytes and spermatocytes. The numbers of RNs show a linear relationship when plotted against SC length both in oocytes and spermatocytes. On the other hand, the ZW pair shows a single and strictly localized RN near the synaptic termini, but the ZZ pair shows unrestricted location of RNs (average 3.8). The ZW and ZZ pairs of the pigeon are euchromatic and do not show specific chromatin packing at pachytene in either sex. The lack of sex-specific differences in the number and location of RNs in the autosomal bivalents of C. livia and previous data on the chicken, suggest that the regulation of crossing-over is basically different in birds and mammals.Key words: meiosis, genetic recombination, recombination nodules, pigeon gametogenesis.

Cytogenetics of chromosome rearrangements in Tribolium castaneum

The karyotype of the red flour beetle, Tribolium castaneum, was reexamined and improved by restriction enzyme banding with HpaII. After this treatment, each of the 10 chromosomes were identified in spermatogonial metaphase cells and 3 of the 8 autosomal bivalents and the XY pair were identified in spermatocyte metaphase I nuclei. Based on centromere position, relative length, and banding pattern, probable correlations between some of the mitotic chromosomes and some of the metaphase I bivalents were ascertained. Thus improved, the karyotypes of beetles harboring genetically defined translocations were investigated. Spermatocyte metaphase I nuclei were most informative, as normal chromosome pairing was visibly disrupted by rearrangements. Bivalents associated with each rearrangement were identified. Results demonstrated that each of the five best defined T. castaneum linkage groups corresponds to a different chromosome and established correspondence between bivalents and linkage groups 1–4. The relevance of these findings is discussed with regard to Tribolium genetics and evolution.Key words: beetles, red flour beetle, Coleoptera, linkage groups, chromosome banding.

Synaptonemal complex analysis of domestic sheep (Ovis aries) with Robertsonian translocations. I. Pachytene karyotype substaging of normal sheep.

Meiotic chromosome pairing behaviour in three normal sheep was surveyed by synaptonemal complex (SC) analysis at the electron microscope level. The SC karyotype of normal rams is composed of a sex pair, three metacentric or submetacentric pairs, and 23 acrocentric autosomal bivalents, with the total autosomal SC length ranging from 309 to 315 μm. Five nucleoli are terminally located on the three metacentric, and one large and one small acrocentric autosomal bivalents. XY morphology was used to divide pachytene into five substages. Although pairing abnormalities (mostly SC interactions) were recorded in an average of 16% of the spermatocytes, bivalent–XY association occurred in only 4.7% of the cells.Key words: sheep, synaptonemal complex, XY pair, pachytene substaging.

Chromosomal synapsis and the meiotic process in male mesquite lizards, Sceloporus grammicus complex

Meiosis in males of the F5 cytotype of Sceloporus grammicus was examined through the analysis of synaptonemal complexes (SCs), diakinetic (metaphase I) nuclei, and secondary spermatocytes (metaphase II configurations). These data allowed the establishment of criteria for substaging of zygonema and pachynema, morphological characterization of the SC complement, and comparison of the orientation and segregation of the autosomes and sex chromosomes. The analysis of nuclei from all stages of meiotic prophase I (leptonema through diakinesis) provided a useful means of partitioning the temporal sequence of early meiotic events. Three substages of zygonema (Z1–Z3) were established, based on the extent of synapsis of the microchromosomal and macrochromosomal elements. Synaptic initiation of the autosomes and sex chromosomes was synchronous. Two patterns of macrochromosomal synapsis were observed. Whereas synapsis of the biarmed elements was biterminal (i.e., progressing from both ends of the homologs), synapsis of the acrocentric elements was uniterminal involving only the distal (noncentromeric) ends of the homologs. Unique sex-chromosomal characteristics were not observed in S. grammicus and, therefore, the substaging of pachynema was based on subjective criteria. Examination of diakinesis – metaphase I and metaphase II configurations indicated low levels of diakinetic irregularities with balanced segregation of the autosomal bivalents and the sex-chromosomal trivalent.Key words: Sceloporus grammicus, meiosis, synaptonemal complex, substaging.