Carlo Alberto Redi

Robertsonian metacentrics of the house mouse lose telomeric sequences but retain some minor satellite DNA in the pericentromeric area

A combination of cytogenetic and molecular biology techniques were used to study the molecular composition and organisation of the pericentromeric regions of house mouse metacentric chromosomes, the products of Robertsonian (Rb) translocations between telocentrics. Regardless of whether mitotic or meiotic preparations were used, in situ hybridisation failed to reveal pericentromeric telomeric sequences on any of the Rb chromosomes, while all metacentrics retained detectable, although reduced (average 50 kb), amounts of minor satellite DNA in the vicinity of their centromeres. These results were supported by slot blot hybridisation which indicated that mice with 2n=22 Rb chromosomes have 65% of telomeric sequences (which are allocated to the distal telomeres of both Rb and telocentric chromosomes and to the proximal telomeres of telocentrics) and 15% the amount of minor satellite, compared with mice with 2n=40 all-telocentric chromosomes. Pulsed field gel electrophoresis and Southern analysis of DNA from Rb mice showed that the size of the telomeric arrays is similar to that of mice with all-telocentric chromosomes and that the minor satellite sequences were hybridising to larger fragments incorporating major satellite DNA. Since the telomeric sequences are closer to the physical end of the chromosome than the minor satellite sequences, the absence of telomeric sequences and the reduced amount of minor satellite sequences at the pericentromeric region of the Rb metacentrics suggest that the breakpoints for the Rb translocation occur very close to the minor satellite-major satellite border. Moreover, it is likely that the minor satellite is required for centromeric function, 50–67 kb being enough DNA to organise one centromere with a functionally active kinetochore.

The analysis of chromatin organisation allows selection of mouse antral oocytes competent for development to blastocyst.

Mouse antral oocytes can be classified in two different types termed SN or NSN oocytes, depending on the presence or absence, respectively, of a ring of Hoechst 33342-positive chromatin surrounding the nucleolus. The aim of the present study was to test the developmental competence to blastocyst of the two types of oocytes. Here we show that following isolation, classification and culture of cumulus-free antral oocytes, 14.7% and 74.5% of NSN and SN oocytes, respectively, reached the metaphase II stage. When fertilised and further cultured none of the metaphase II NSN oocytes developed beyond the 2-cell stage whilst 47.4% of the metaphase II SN oocytes reached the 4-cell stage and 18.4% developed to blastocyst. The findings reported in this paper may contribute to improved procedures of female gamete selection for in vitro fertilisation of humans and farm animals. Furthermore, the selection of oocytes with better developmental potential may be of interest for studies on nuclear/cytoplasm interaction, particularly in nuclear-transfer experiments.

The other chromatin

Abstract. Current understanding of heterochromatin, thanks to molecular data, focuses on its performing several functions in evolution and development. Heterochromatin shows characteristic distribution patterns in karyotypes and contributes to the broad scattering of genome sizes through biological taxa. Heterochromatin remains compacted and thus different from properly stained euchromatin during somatic interphase. A minimum amount of heterochromatin, however, is required for it to be visible in light microscopy. It may further escape notice during the dynamic processes of embryogenesis and gametogenesis. Present-day biology is in search of specific proteins and DNA sequences that comprise heterochromatin. The data that result from overcoming the threshold of visibility will support understanding of interference by heterochromatin in ontogeny and evolution. The contributions of Sigrid and Wolfgang Beermann to the study of heterochromation diminution (DNA elimination) are recalled, and we also discuss the functions and effects of heterochromatin on differential DNA endoreplication and in speciation.

Variable Reprogramming of the Pluripotent Stem Cell Marker Oct4 in Mouse Clones: Distinct Developmental Potentials in Different Culture Environments

A prevailing view of cloning by somatic‐cell nuclear transfer is that reprogramming of gene expression occurs during the first few hours after injection of the nucleus into an oocyte, that the process is stochastic, and that the type of reprogramming needed for cloning success is foreign and unlikely to be readily achieved in the ooplasm. Here, we present evidence that the release of reprogramming capacity is contingent on the culture environment of the clone while the contribution of aneuploidy to altered gene expression is marginal. In particular, the rate of blastocyst formation in clones and the regional distribution of mRNA for the pluripotent stem cell marker Oct4 in clonal blastocysts was highly dependent on the culture environment after cumulus cell nuclear transfer, unlike that in genetically equivalent zygotes. Epigenetic modifications of genetically identical somatic nuclei continue after the first cell division of the clones and are amenable to a degree of experimental control, and their development to the blastocyst stage and appropriate expression of Oct4 predict further outcome, such as derivation of embryonic stem (ES) cells, but not fetal development. This observation indicates that development to the blastocyst stage is not equivalent to full reprogramming and lends support to the novel concept that ES cells are not the equivalent of the inner cell mass, hence the discrepancy between ES cell derivability and fetal development of clones.

Karyotype and genetic evolution in speciation of subterranean mole rats of the genus Spalax in Turkey

Karyotype (2n) and allozyme diversity at 37 gene loci were determined in 69 subterranean mole rats in Turkey belonging to the two superspecies: the ancestor Spalax leucodon (n = 55; 20 populations) and the descendant S. ehrenbergi (n = 14: four populations. We identified remarkable variation of diploid chromosome numbers in the S. leucodon superspecies: 2n = 38, 40, 50, 54, 60 and 62; and in the S. ehrenbergi superspecies: 2n = 52, 56 and 58. Genetic diversity indices were low on average in both S. leucodon and S. ehrenbergi superspecies: Allele diversity, A = 1.081 and 1.074; polymorphism, P-50 0 = 0.077 and 0.068; heterozygosity, H = 0.038 and 0.027; and gene diversity, H = 0.038 and 0.034, respectively. H ranged from 0 in mesic or semimesic regions to 0.088 in arid Anatolia. We consider the populations with different diploid chromosome numbers, 2n, as good biological species. Karyotypic diversity may mark extensive ecological speciation. Nees genetic distances, D average 0.174, range 0.002 0.422) and ecogeographical criteria suggest that almost each population may represent a different biological species, but critical future testing is necessary to support this claim. Karyotypes and allozymes are nonrandomly distributed across Turkey, displaying remarkable correlations with climatic and biotic factors. Both 2n and H are significantly correlated with aridity stress (2n/rainfall. r = −0.74; P < 0.001), and in our region also with climatic unpredictability. These results support the niche-width genetic variation hypothesis in space and time. Climatic selection in Turkey appears to be a major architect of karyotype and genetic (allozyme) diversity and divergence in mole rat evolution, in both speciation and adaptation.

Maternal Oct-4 is a potential key regulator of the developmental competence of mouse oocytes

BackgroundThe maternal contribution of transcripts and proteins supplied to the zygote is crucial for the progression from a gametic to an embryonic control of preimplantation development. Here we compared the transcriptional profiles of two types of mouse MII oocytes, one which is developmentally competent (MIISN oocyte), the other that ceases development at the 2-cell stage (MIINSN oocyte), with the aim of identifying genes and gene expression networks whose misregulated expression would contribute to a reduced developmental competence.ResultsWe report that: 1) the transcription factor Oct-4 is absent in MIINSN oocytes, accounting for 2) the down-regulation of Stella, a maternal-effect factor required for the oocyte-to-embryo transition and of which Oct-4 is a positive regulator; 3) eighteen Oct-4-regulated genes are up-regulated in MIINSN oocytes and are part of gene expression networks implicated in the activation of adverse biochemical pathways such as oxidative phosphorylation, mitochondrial dysfunction and apoptosis.ConclusionThe down-regulation of Oct-4 plays a crucial function in a sequence of molecular processes that leads to the developmental arrest of MIINSN oocytes. The use of a model study in which the MII oocyte ceases development consistently at the 2-cell stage has allowed to attribute a role to the maternal Oct-4 that has never been described before. Oct-4 emerges as a key regulator of the molecular events that govern the establishment of the developmental competence of mouse oocytes.

Organisation of complex nuclear domains in somatic mouse cells

The number and associations of heterochromatin chromocenters, nucleoli, centromeres and telomeres were studied in the nucleus of different somatic cells of Mus domesticus. Fibroblasts of the cell line 3T3, kidney cells (primary culture), and bone marrow cells were used. The above mentioned nuclear and chromosome markers were identified by DAPI/actinomycin D, indirect immunofluorescence with anti-centromere antibodies, silver impregnation for nucleolar proteins and fluorescence in situ hybridisation (FISH) with telomeric probes. The quantitative analysis of the nuclei showed that the pericentromeric heterochromatin is organised in about 18 chromocenters per nucleus in the 3T3 cells, and about seven in kidney and bone marrow cells, having generally a peripheral distribution in the nucleus of all the studied cells. Several aggregated centromeres were participating in each of the chromocenters, about four centromeres per 3T3 cell and about six centromeres per kidney and bone marrow cells. Some of the chromocenters were also in close association with nucleoli. The number of telomeric labels per nucleus was as expected for each chromosome set (2n = 68-70 and 2n = 40). About half of the telomeric signals were loosely aggregated within the heterochromatic blocks while the rest were distributed in the nucleus as unrelated units not bound with chromocenters. The three cell types have complex nuclear territories formed by different chromosomal domains: the pericentromeric heterochromatin, centromeres, proximal telomeres and nucleoli. With the exception of some bone marrow cells, we have not found a nuclear polarisation of the analysed chromosomal markers compatible with the Rabl configuration. However, Rabl anaphasic polarisation allows the contact of centromeric regions making possible that centromeric associations arise. If in addition, associative elements such as constitutive heterochromatin or nucleoli are close to the centromeric regions, like in Mus domesticus chromosomes, then the associations might be consolidated and persist until the interphase. These associations may be the origin of the nuclear domains described here for Mus domesticus somatic cells.

Chromatin organisation and nuclear architecture in growing mouse oocytes

Although the female gamete is blocked at the dictyate stage of the first meiotic prophase during the whole folliculogenesis, many important epigenetic changes occur to organise the genome to attend early embryonic development. In this paper, we will describe the results of a number of studies aimed to improve our understanding of the nuclear organization of the mouse oocyte during folliculogenesis. Using silver methods that stain NOR, centromeres and heterochromatin, as well as, the use of specific antibodies for the demonstration of centromeres, we have described the changes to the chromatin organisation and to the spatial localisation of chromocenters and centromeres during oocyte growth; these changes have been correlated to the developmental competence of the resulting antral and metaphase II (MII) oocyte.

Epigenomic differentiation in mouse preimplantation nuclei of biparental, parthenote and cloned embryos

Chromosomes, sub-chromosomal regions and genes are repositioned during cell differentiation to acquire a cell-type-specific spatial organization. The constraints that are responsible for this cell-type-specific spatial genome positioning are unknown. In this study we addressed the question of whether epigenetic genome modifications may represent constraints to the acquisition of a specific nuclear organization. The organization of kinetochores, pericentric heterochromatin and the nucleolus was analysed in pre-implantation mouse embryos obtained by in-vitro fertilization (IVF), parthenogenetic activation (P) and nuclear transfer (NT) of differentiated somatic nuclei, which possess different epigenomes. Each stage of pre-implantation embryonic development is characterized by a stage-specific spatial organization of nucleoli, kinetochores and pericentric heterochromatin. Despite differences in the frequencies and the time-course of nuclear architecture reprogramming events, by the eight-cell stage P and NT embryos achieved the same distinct nuclear organization in the majority of embryos as observed for IVF embryos. At this stage the gametic or somatic nuclear architecture of IVF or P and NT embryos, respectively, is replaced by a common embryonic nuclear architecture. This finding suggests that the epigenome of the three types of embryos partially acts as a constraint of the nuclear organization of the three nuclear subcompartments analysed.

Differences in the organization and chromosomal allocation of satellite DNA between the European long tailed house mice Mus domesticus and Mus musculus

We compared the organization of satellite DNA (stDNA) and its chromosomal allocation inMus domesticus and inMus musculus. The two stDNAs show similar restriction fragment profiles after digestion (probed withM. domesticus stDNA) with some endonucleases of which restriction sequences are present in the 230–240 bp repetitive unit of theM. domesticus stDNA. In contrast, EcoRI digestion reveals thatM. musculus stDNA lacks most of the GAATTC restriction sites, particularly at the level of the half-monomer. The chromosome distribution of stDNA (revealed by anM. domesticus stDNA probe) shows different patterns in theM. domesticus andM. musculus karyotypes, with about 60% ofM. domesticus stDNA retained in theM. musculus genome. It is particularly noteworthy that the pericentromeric regions ofM. musculus chromosomes 1 and X are totally devoid ofM. domesticus stDNA sequences. In both groups, the differences in energy transfer between the stDNA-bound fluorochromes Hoechst 33258 and propidium iodide suggest that AT-rich repeated sequences have a much more clustered array in theM. domesticus stDNA, as if they are organized in tandem repeats longer than those ofM. musculus. Considering the data as a whole, it seems likely that the evolutionary paths of the two stDNAs diverged after the generation of the ancestral 230–240 bp stDNA repetitive unit through the amplification, in theM. domesticus genome, of a family repeat which included the EcoRI GAATTC restriction sequence.