Unexpected silicon localization in calcium carbonate exoskeleton of cultured and fossil coccolithophores.

Coccolithophores, marine calcifying phytoplankton, are important primary producers impacting the global carbon cycle at different timescales. Their biomineral structures, the calcite containing coccoliths, are among the most elaborate hard parts of any organism. Understanding the morphogenesis of coccoliths is not only relevant in the context of coccolithophore eco-physiology but will also inform biomineralization and crystal design research more generally. The recent discovery of a silicon (Si) requirement for crystal shaping in some coccolithophores has opened up a new avenue of biomineralization research. In order to develop a mechanistic understanding of the role of Si, the presence and localization of this chemical element in coccoliths needs to be known. Here, we document for the first time the uneven Si distribution in Helicosphaera carteri coccoliths through three synchrotron-based techniques employing X-ray Fluorescence and Infrared Spectromicroscopy. The enrichment of Si in specific areas of the coccoliths point to a targeted role of this element in the coccolith formation. Our findings mark a key step in biomineralization research because it opens the door for a detailed mechanistic understanding of the role Si plays in shaping coccolith crystals.

PGRMC1 participates in late events of bovine granulosa cells mitosis and oocyte meiosis.

Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in both oocyte and ovarian somatic cells, where it is found in multiple cellular sub-compartments including the mitotic spindle apparatus. PGRMC1 localization in the maturing bovine oocytes mirrors its localization in mitotic cells, suggesting a possible common action in mitosis and meiosis. To test the hypothesis that altering PGRMC1 activity leads to similar defects in mitosis and meiosis, PGRMC1 function was perturbed in cultured bovine granulosa cells (bGC) and maturing oocytes and the effect on mitotic and meiotic progression assessed. RNA interference-mediated PGRMC1 silencing in bGC significantly reduced cell proliferation, with a concomitant increase in the percentage of cells arrested at G2/M phase, which is consistent with an arrested or prolonged M-phase. This observation was confirmed by time-lapse imaging that revealed defects in late karyokinesis. In agreement with a role during late mitotic events, a direct interaction between PGRMC1 and Aurora Kinase B (AURKB) was observed in the central spindle at of dividing cells. Similarly, treatment with the PGRMC1 inhibitor AG205 or PGRMC1 silencing in the oocyte impaired completion of meiosis I. Specifically the ability of the oocyte to extrude the first polar body was significantly impaired while meiotic figures aberration and chromatin scattering within the ooplasm increased. Finally, analysis of PGRMC1 and AURKB localization in AG205-treated oocytes confirmed an altered localization of both proteins when meiotic errors occur. The present findings demonstrate that PGRMC1 participates in late events of both mammalian mitosis and oocyte meiosis, consistent with PGRMC1's localization at the mid-zone and mid-body of the mitotic and meiotic spindle.

Mathematical modeling of growth and death dynamics of mouse embryonic stem cells irradiated with γ-rays.

Following ionizing radiation, mouse embryonic stem cells (mESCs) undergo both apoptosis and block at G2/M phase of the cell cycle. The dynamics of cell growth and the transition through the apoptotic phases cannot be directly inferred from experimental data, limiting the understanding of the biological response to the treatment. Here, we propose a semi-mechanistic mathematical model, defined by five compartments, able to describe the time curves of untreated and γ-rays irradiated mESCs and to extract the information therein embedded. To this end, mESCs were irradiated with 2 or 5 Gy γ-rays, collected over a period of 48 h and, at each time point, analyzed for apoptosis by using the Annexin V assay. When compared to unirradiated mESCs, the model estimates an additional 0.2 probability to undergo apoptosis for the 5 Gy-treated cells, and only a 0.07 (not statistically significantly different from zero) when a 2 Gy-irradiation dose is administered. Moreover, the model allows us to estimate the duration of the overall apoptotic process and also the time length of its early, intermediate, and late apoptotic phase.

The genomic and proteomic blueprint of mouse megakaryocytes derived from embryonic stem cells.

BACKGROUND: Platelets are specialized cells, produced by megakaryocytes (MKs) in the bone marrow, which represent the first defense against hemorrhage. There are many diseases where platelet production or function is impaired, with severe consequences for patients. Therefore, new insights into the process of MK differentiation and platelet formation would have a major impact on both basic and clinical research. OBJECTIVES: Embryonic stem (ES) cells represent a good in vitro model to study the differentiation of MKs, with the possibility of being genetically engineered and constituting an unlimited source of MKs. However, lack of knowledge about the molecular identity of ES-derived MKs (ES-MKs) may prevent any further development and application of this model. METHODS: This paper presents the first comprehensive transcriptional and proteome profile analyses of mouse ES-MKs in comparison with MKs derived from mouse fetal liver progenitors (FL-MKs). RESULTS: In ES-MKs we found a down-regulation of cytoskeleton proteins, specific transcription factors and membrane receptors at both transcriptional and protein levels. At the phenotypic level, this molecular blueprint was displayed by ES-MKs' lower polyploidy, lower nuclear/cytoplasm ratio and reduced capacity to form proplatelets and releasing platelets. CONCLUSIONS: Overall our data demonstrate that ES-MKs represent a useful model to clarify many aspects of both MK physiology and pathological conditions where impaired MK functions are related to defective MK development, as in inherited thrombocytopenias and primary myelofibrosis.

Karyotype analysis of the euploid cell population of a mouse embryonic stem cell line revealed a high incidence of chromosome abnormalities that varied during culture.

It is common knowledge that mouse embryonic stem cell (mESC) lines accumulate chromosomal changes during culture. Despite the wide use of mESCs as a model of early mammalian development and cell differentiation, there is a lack of systematic studies aimed at characterizing their karyological changes during culture. We cultured an mESC line, derived in our laboratory, for a period of 3 months investigating its chromosome complement at different times. About 60% of the metaphases analysed were euploid throughout the culture period but, from passage 13, only 50% of the euploid metaphases had a proper chromosome complement. The remaining 50% showed chromosome abnormalities, mainly gain or loss of entire chromosomes, both within the same passage and among different passages analysed. The very heterogeneous spectrum of abnormalities indicates a high frequency of chromosome mutations that arise continuously during culture. The heterogeneity of the aberrant chromosome constitution of 2n = 40 metaphases, observed at different passages of culture, might be due either to their elimination or to a shift towards the hypoeu- or hypereuploid population of those metaphases that accumulate further chromosome abnormalities. The stability of the frequency of eu-, hypoeu- and hypereuploid populations during culture might, however, be due to the elimination of those cells that carry a high mutational burden. Based on our results, we suggest that karyotype analysis of the euploid cell population of mESC lines is necessary when such lines are used in the production of chimeric mice, for their contribution to the germ line, or when they are differentiated into specific cell types.

Genome sizes in afrotheria, xenarthra, euarchontoglires, and laurasiatheria.

Topical literature and Web site databases provide genome sizes for approximately 4,000 animal species, invertebrates and vertebrates, 330 of which are mammals. We provide the genome size for 67 mammalian species, including 51 never reported before. Knowledge of genome size facilitates sequencing projects. The data presented here encompassed 5 Metatheria (order Didelphimorphia) and 62 Eutheria: 15 Xenarthra, 24 Euarchontoglires (Rodentia), as well as 23 Laurasiatheria (22 Chiroptera and 1 species from Perissodactyla). Already available karyotypes supplement the haploid nuclear DNA contents of the respective species. Thus, we established the first comprehensive set of genome size measurements for 15 Xenarthra species (armadillos) and for 12 house-mouse species; each group was previously represented by only one species. The Xenarthra exhibited much larger genomes than the modal 3 pg DNA known for mammals. Within the genus Mus, genome sizes varied between 2.98 pg and 3.68 pg. The 22 bat species we measured support the low 2.63 pg modal value for Chiroptera. In general, the genomes of Euarchontoglires and Laurasiatheria were found being smaller than those of (Afrotheria and) Xenarthra. Interspecific variation in genome sizes is discussed with particular attention to repetitive elements, which probably promoted the adaptation of extant mammals to their environment.

Single-cell quantitative RT-PCR analysis of Cpt1b and Cpt2 gene expression in mouse antral oocytes and in preimplantation embryos.

Fatty acids represent an important energy source for preimplantation embryos. Fatty acids oxidation is correlated with the embryo oxygen consumption which remains relatively constant up to the 8-cell stage, but suddenly increases between the 8-cell and morula stages. The degradation of fatty acids occurs in mitochondria and is catalyzed by several carnitine acyl transferases, including two carnitine palmitoyl transferases, CPT-I and CPT-II. We have carried out a study to determine the relative number of transcripts of Cpt1b and Cpt2 genes encoding for m-CPT-I and CPT-II enzymes, during mouse preimplantation development. Here we show that Cpt1b transcripts are first and temporally detected at the 2-cell stage and reappear at the morula and blastocyst stage. Cpt2 transcripts decrease following fertilization to undetectable levels and are present again later at the morula stage. These results show that transcription of both Cpt1b and Cpt2 is triggered at the morula stage, concomitantly with known increasing profiles of oxygen uptake and fatty acids oxidation. Based on the number of Cpt2 transcripts detected, we could discriminate the presence of two groups of embryos with high and low number of transcripts, from the zygote throughout preimplantation development. To further investigate if the establishment of these two groups of embryos occurs prior to fertilization, we have analyzed the relative number of transcripts of both genes in antral and ovulated MII oocytes. As for preimplantation embryos, MII oocytes show two groups of Cpt2 expression. Antral oocytes, classified according to their chromatin configuration in SN (surrounded nucleolus, in which the nucleolus is surrounded by a rim of Hoechst-positive chromatin) and NSN (not surrounded nucleolus, in which this rim is absent), show three groups with different numbers of Cpt2 transcripts. All NSN oocytes have a number of Cpt2 transcripts doubled compared to that of the group of MII oocytes with high expression. Instead, SN oocytes could be singled out into two groups with high and low numbers of Cpt2 transcripts, similar to those found in MII oocytes. The results of this study point out a correlation between the timing of fatty acids oxidation during preimplantation development and the expression of two genes encoding two enzymes involved in the oxidative pathway. Furthermore, although the biological meaning for the presence of two groups of oocytes/embryos with different levels of Cpt2 transcripts remains unclear, the data obtained suggest a possible correlation between the levels of Cpt2 expression and embryo developmental competence.

High-resolution organization of mouse telomeric and pericentromeric DNA.

We studied the organization of telomeric, major and minor satellite DNA sequences located in the pericentromeric regions of mouse telocentric and Robertsonian metacentric chromosomes by high-resolution fluorescence in situ hybridization. Molecular data have already proved that in telocentrics, from the physical chromosome end, telomeric sequences are followed by minor and then by major satellite DNA. We showed that the three families of repetitive DNA are organized as uninterrupted long-range cluster repeats and that there is no intermingling between telomeric and minor satellite DNA or between the major and the minor tandem repeats or with non-satellite DNA. The pericentromeric region of metacentric chromosomes consists of a small block of minor satellite DNA sandwiched between two blocks of major satellite DNA.

The other chromatin.

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.

Human-dominated ecosystems and restoration ecology: Seveso today.

Seveso is a town (40,000 inhabitants) 16 km north of Milan, which from 10 July 1976 became synonymous with the chemically induced ecological catastrophe because of the large number of people affected by dioxin exposure and of the large area involved. The most polluted area (about 43 ha) was artificially reconstructed and transformed into a wood composed mainly of oaks with some scattered green fields and some bushy areas, the Bosco delle Querce urban park. A four-year survey monitoring the present ecological and biological risk parameters of the artificially reconstructed ecosystem shows its full ecological recovery as an urban park. Plant and animal coenoses are well composed and the park has been colonized by annelids, insects, amphibians, reptiles, birds and mammals. All these animals are useful biological reagents for risk-assessment because of their potential long-term exposure to TCDD. When some of the endpoints of the xenoestrogen-like molecules' action were studied (i.e., gametogenesis and the gross morphology of genital organs in rabbits and house mice), no signs of TCDD effects were detected. Mutagenicity tests and the house mouse sperm DNA COMET assay do not reveal the presence of any biological risk. The study of the carabidocoenosis and the housefly cytogenetics corroborates this last indication, thus guaranteeing the successful ecological recovery of the formerly most polluted Seveso area.

Pericentromeric organization at the fusion point of mouse Robertsonian translocation chromosomes.

In mammals, Robertsonian (Rb) translocation (the joining of two telo/acrocentric chromosomes at their centromere to form a metacentric) is the most effective process in chromosomal evolution leading to speciation; its occurrence also affects human health (through the induction of trisomies) and the fertility of farm animals. To understand the mechanism of Rb translocation, we used the house mouse as a model system and studied the organization of pericentromeric satellite DNAs (satDNA) of telocentrics and Rb chromosomes, both minor and major satDNA. The chromosome-orientation fluorescence in situ hybridization (CO-FISH) technique was used to analyze the major satDNA. To detect the very small amount of minor satDNA, a procedure was developed that combines CO-FISH with primed in situ labeling and conventional FISH and is five times more sensitive than the CO-FISH procedure alone. It was found that both the major and the minor satDNA tandem repeats are oriented head-to-tail in telocentric and Rb chromosomes, and their polarity is always the same relative to the centromere. We suggest that all tandemly repetitive satDNAs in a species probably are locked into such a symmetry constraint as a universal consequence of chromosomal evolution. Rb translocation breakpoints were found localized within the minor satDNA of telocentrics, and these sequences contributed symmetrically to the formation of the centromeric region of the Rb chromosomes. These results are important for an understanding of the geometry of Rb translocations and suggest the study of DNA orientation as a new tool for investigating these rearrangements.

Alteration of nuclear architecture in male germ cells of chromosomally derived subfertile mice.

The mammalian cell nucleus consists of numerous compartments involved in the regular unfolding of processes such as DNA replication and transcription, RNA maturation, protein synthesis and cell division. Knowledge is increasing of the relationships between high-order levels of chromatin organization and its spatial organization, and of how these relationships contribute to the various functions carried out in the nucleus. We have studied the spatial arrangement of mouse telocentric chromosomes 5, 11, 13, 15, 16 and 17, some of their metacentric Robertsonian derivatives, and X and Y chromosomes by whole chromosome painting in male germ (spermatogonia, pachytene spermatocytes and spermatids) and Sertoli cells of homozygous and heterozygous individuals. Using dual-colour fluorescence in situ hybridization we found that these chromosomes occupy specific nuclear territories in each cell type analysed. When chromosomes are present as Robertsonian metacentrics in the heterozygous state, that is, as Robertsonian metacentrics and their homologous telocentrics, differences in their nuclear positions are detectable: heterozygosity regularly produces a change in the nuclear position of one of the two homologous telocentrics in all the cell types studied. In the Robertsonian heterozygotes, the vast majority of the Sertoli cells show the sex chromosomes in a condensed state, whereas they appear decondensed in the Robertsonian homozygotes. As the Robertsonian heterozygosities we studied produce a chromosomally derived impairment of male germ-cell differentiation, we discuss the possibility that changes in chromosome spatial territories may alter some nuclear machinery (e.g., synapsis, differential gene expression) important for the correct unfolding of the meiotic process and for the proper functioning of Sertoli cells.

Nuclear transfer, genome reprogramming and novel opportunities in cell therapy.

The knowledge of the molecular mechanism involved in cell differentiation during embryonic development is central for the understanding of differentiative processes including those involved in the progression of genetic diseases. This knowledge would permit the development of new strategies for cell and gene therapies. It has recently been shown that mice can develop to term enucleated oocytes injected with the nuclei of somatic cells. These experiments demonstrate the capacity of the mouse oocyte to remodel the genetic programme of a somatic cells nucleus in order to make it capable of initiating and continuing embryonic development. The activation of zygotic genes occurs in the mouse by the 2-cell stage and it is a crucial event in the life of the newly formed mouse embryo as lack or wrong timing of zygotic gene expression leads to the death of the embryo. For these reasons the gentic modifications (reprogramming) induced by the oocyte over the newly injected somatic nucleus must be completed before zygotic genome activation occurs. The understanding of the mechanisms that intervene in the processes of cell differentiation and in those that make it a reversible process, would allow to repeat the process of nucleus reprogramming in an in vitro system, without the use of the female gamete. Here we will describe some of the genome modifications that might be involved in the reprogramming process following the transfer of a terminally differentiated somatic nucleus into the cytoplasm of an enucleated oocyte.

Fetal erythroblast isolation up to purity from cord blood and their culture in vitro.

BACKGROUND: Erythroblasts have been the most encouraging candidate cell type for noninvasive prenatal genetic investigation. We previously showed that human erythroblasts can be recovered from bone marrow and blood bank buffy coats by a physical cell separation. In the present study, we modified our previous methodology, taking into account the peculiar behavior of erythroblasts in response to modifications of pH and osmolality of the separation medium. METHODS: Twenty to forty milliters of cord blood were initially centrifuged on Ficoll/diatrizoate (1.085 g/ml). The interphase cells were further separated on a continuous density gradient (1.040-1.085 g/ml). Two different gradients were initially compared: the first was iso-osmolar and neutral, whereas the second also contained an ionic strength gradient and a pH gradient (triple gradient). A subsequent monocyte depletion was performed by using magnetic microbeads coated with anti-CD14 monoclonal antibody (mAb), and erythroblasts were purified by sedimentation velocity. Purified cells were investigated by analyses with fluorescence-activated cell sorting (FACS) and fluorescence in situ hybridization (FISH) and immunocytochemistry with mAb against fetal hemoglobin and were cultured in vitro. RESULTS: When nucleated cells were spun on an iso-osmolar and neutral continuous density gradient, two separated bands of nucleated red blood cells (NRBCs) were obtained: a light fraction banding at 1.062 g/ml and an heavy fraction banding at 1.078 g/ml. Conversely, when cells were spun in the triple gradient, NRBCs were shifted to the low-density region. Monocyte depletion by immunomagnetic microbeads and velocity sedimentation provided a pure erythroblast population. FACS and FISH analyses and immunocytochemistry substantiated the purity of the isolated cell fraction, which was successfully cultured in vitro. CONCLUSIONS: We have shown that fetal erythroblasts can be purified up to homogeneity from cord blood, but further refinements of the isolation procedure are necessary before the same results can be obtained from maternal peripheral blood.

Gene expression and chromatin organization during mouse oocyte growth.

Mouse oocytes can be classified according to their chromatin organization and the presence [surrounded nucleolus (SN) oocytes] or absence [nonsurrounded nucleolus (NSN) oocytes] of a ring of Hoechst-positive chromatin around the nucleolus. Following fertilization only SN oocytes are able to develop beyond the two-cell stage. These studies indicate a correlation between SN and NSN chromatin organization and the developmental competence of the female gamete, which may depend on gene expression. In the present study, we have used the HSP70.1Luc transgene (murine HSP70.1 promoter + reporter gene firefly luciferase) to analyze gene expression in oocytes isolated from ovaries of 2-day- to 13-week-old females. Luciferase was assayed on oocytes after classification as SN or NSN type. Our data show that SN oocytes always exhibit a higher level of luciferase activity, demonstrating a higher gene expression in this category. Only after meiotic resumption, metaphase II oocytes derived from NSN or SN oocytes acquire the same level of transgene expression. We suggest that the limited availability of transcripts and corresponding proteins, excluded from the cytoplasm until GVBD in NSN oocytes, could explain why these oocytes have a lower ability to sustain embryonic development beyond the two-cell stage at which major zygotic transcription occurs. With this study we have furthered our knowledge of epigenetic regulation of gene expression in oogenesis.

Six-year-old archival chromosome preparations are still good biological reagents for repeated primed in situ labelling (rPRINS).

Six-year-old chromosome preparations of Eulemur coronatus were used for in situ localization of highly repetitive DNA sequences. The application of the repeated primed in situ labelling (rPRINS) technique allowed the detection of positive signals whereas conventional fluorescence in situ hybridization gave negative results on the same archival preparations. This paper reveals the possibility of rescuing archival chromosome preparations for both clinical and comparative cytogenetics. Moreover, the chromosomal localization of the ECO-SAT 503 bp highly repetitive DNA sequences were found to localize in the pericentromeric region of every chromosome, with the exception of chromosome 9.

Timing of gene expression and oolemma localization of mouse alpha6 and beta1 integrin subunits during oogenesis.

The sperm antigen fertilin alpha/beta and the integrin complex alpha6beta1 present on the oolemma are two of the most promising candidates to mediate gamete interaction. During growth, the plasma membrane of both hamster and mouse zona-free oocytes acquires the capacity to fuse with acrosome-reacted sperm when oocytes reach the size of 25-30 microm in diameter, suggesting changes in the membrane molecular composition. The present study has two aims: to determine the timing of (1) gene expression of alpha6 and beta1 integrins and (2) localization of these integrin subunits on the plasma membrane in primordial germ cells and in oocytes during oogenesis. We found that both alpha6 and beta1 genes are expressed in female germ cells during all the stages of development analyzed, from 10.5 to 18.5 d.p. c., during oocyte growth, and in ovulated eggs. The alternatively spliced isoform alpha6B is expressed from 10.5 d.p.c., whereas alpha6A begins to be expressed at 12.5 d.p.c., suggesting a different role for the two variants. In situ immunodetection of alpha6 or beta1 shows a ring of fluorescence on the female germ cell plasma membrane for both integrins at 10.5 d.p.c., then the fluorescent signal becomes undetectable at 12.5 d.p.c. to reappear again, this time with a patchy distribution, at 18.5 d.p.c. This pattern of localization is maintained in oocytes isolated from newborn individuals and only when oocytes during growth reach the size of about 25-30 microm in diameter does the fluorescence become homogenous all around the whole oocyte surface. These data, although not conclusive, support the hypothesis of an involvement of alpha6 and beta1 integrins in sperm-egg fusion.

Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei.

Until recently, fertilization was the only way to produce viable mammalian offspring, a process implicitly involving male and female gametes. However, techniques involving fusion of embryonic or fetal somatic cells with enucleated oocytes have become steadily more successful in generating cloned young. Dolly the sheep was produced by electrofusion of sheep mammary-derived cells with enucleated sheep oocytes. Here we investigate the factors governing embryonic development by introducing nuclei from somatic cells (Sertoli, neuronal and cumulus cells) taken from adult mice into enucleated mouse oocytes. We found that some enucleated oocytes receiving Sertoli or neuronal nuclei developed in vitro and implanted following transfer, but none developed beyond 8.5 days post coitum; however, a high percentage of enucleated oocytes receiving cumulus nuclei developed in vitro. Once transferred, many of these embryos implanted and, although most were subsequently resorbed, a significant proportion (2 to 2.8%) developed to term. These experiments show that for mammals, nuclei from terminally differentiated, adult somatic cells of known phenotype introduced into enucleated oocytes are capable of supporting full development.