Search PubMed⌕ Search

Biomedical subjects

P Debey

Publications and source records attributed to P Debey.

At least 37 records · Page 2Linked to original sources
PubMed1998

Three distinct sub-nuclear populations of HMG-I protein of different properties revealed by co-localization image analysis.

We have studied the nuclear distribution of the non-histone HMG-I protein by indirect immunofluorescence in several human and murine somatic cell lines and in growing mouse oocytes. We show that HMG-I, a high mobility-group protein which interacts in vitro with the minor groove of AT-rich B-DNA, is found exclusively in the nucleus and that this localization corresponds to a complex distribution. By comparing the HMG-I-dependent fluorescence signal with the chromatin density determined by Hoechst 33342 or propidium iodide staining, we present evidence for the existence of three HMG-I sub-populations whose contribution to the total fluorescence can be determined using a newly developed quantitative co-localization image analysis program: foci that correspond to regions of heterochromatin, intense dots located within decondensed chromatin, and a more diffuse component extending throughout the nucleoplasm. In addition, we show that these sub-populations differ in their sensitivity to nuclease digestion and in vivo displacement by the minor-groove binder Hoechst 33342. Finally, double immunolabeling of RNA polymerase II-dependent transcription and HMG-I shows that the intense dots are not correlated with sites of high transcriptional activity. We discuss the possibility that these three sub-populations reflect distinct and separable biological functions of the HMG-I protein.

3T3 Cells↗
PubMed1997

Dynamic organization of DNA replication in one-cell mouse embryos: relationship to transcriptional activation.

We have analyzed the spatial and temporal relationship between transcription and replication sites during the first cell cycle in mouse embryos. Embryos were microinjected with both 5-bromouridine-5'-triphosphate and digoxygenin-11-deoxyuridine-5'-triphosphate to visualize transcription and replication sites respectively. We detected six different phases of replication during S phase and dated the onset of zygotic transcription at the end of the S phase. Using confocal microscopy, we showed that there is essentially no colocalization of replication and transcription sites at this stage of development. Moreover, studies on aphidicolin-treated embryos demonstrated that inhibition of DNA replication does not hinder transcriptional activation at the 1-cell stage.

Animals↗
PubMed1996

Conditions favoring RNA polymerase I transcription in permeabilized cells.

RNA synthesis can be detected in nuclei using modified RNA precursors (Br-UTP) introduced in permeabilized cells. Surprisingly, RNA pol I transcripts are detected only after inhibition of RNA pol II or salt enhancement of RNA pol I activity. By modifying a previously reported protocol, we found that RNA pol I transcripts can be detected selectively or simultaneously with RNA pol II transcripts without any drug treatment. Removing glycerol from the permeabilization and transcription buffers and improving the permeabilization using Triton X-100 revealed RNA pol I transcription in two cell lines (mammalian and Xenopus) and in isolated mouse oocytes. The transcripts were most probably rRNA because they were detected in the nucleoli, digested by RNase, sensitive to actinomycin D, and resistant to alpha-amanitin. We found by microinjection of the Br-UTP precursors in living cells that low ionic strength allows the detection of RNA pol I transcription. Electron microscopy of mouse oocytes showed that the "looseness" of the nucleolar organization is associated with the detection of the RNA pol I transcription; this detection does not necessarily need nucleolar disorganization. The data obtained with both permeabilized cells and microinjections of RNA precursors in the absence of glycerol support the hypothesis that the degree of hydration of the cell plays a role in RNA pol I transcription.

Animals↗
PubMed1996

Fusion with activated mouse oocytes modulates the transcriptional activity of introduced somatic cell nuclei.

We have analyzed the transcriptional activity of somatic cell nuclei fused with artificially activated mouse oocytes. Two types of somatic cells have been used: transcriptionally silent thymocytes, obtained from the thymus of newborn mice, and transcriptionally active murine erythroleukemia cells (MEL) from in vitro culture. Cells were fused with activated oocytes, either less than 1 h, or 3 h post-ethanol-treatment. When the fusion occurred early after activation (1 h or less), the transferred somatic cell nuclei reacted by nuclear envelope breakdown (NEBD), which exposed their chromatin to the cytoplasmic environment of the oocytes, and the reconstituted nuclei underwent significant ultrastructural remodeling. No transcriptional activity was ever detected in these reconstituted nuclei during the subsequent 3-4 h of culture of the resulting hybrid cells. In the case of MEL, this means that transcriptional activity ceased as soon as they entered the cytoplasm. In contrast, somatic nuclei which entered the cytoplasm of activated oocytes 3 h postactivation did not undergo NEBD and their remodeling was less pronounced. In contrast to the first group, these nuclei were transcriptionally active during the following 3-4 h of culture. In both cases, the female pronucleus remained transcriptionally silent.

Animals↗
PubMed1996

Development-dependent localization of nuclear antigens in growing mouse oocytes.

We have analyzed the distribution of nuclear and nucleolar proteins during the period of oocyte's growth. Oocytes were isolated mechanically or enzymatically from ovaries of juvenile mice of various ages (from 1 to 28 days after birth). Small nuclear ribonucleoproteins (snRNPs), the splicing factor SC-35, and a protein linked to cell proliferation (p-120) were detected by indirect immunofluorescence. snRNP distribution is consistent with the prophase state of oocyte's nuclei, while SC-35 (and p-120) exhibit a "speckled" distribution throughout the entire period of growth. The number of speckles (or foci) appears maximal around 10 days after birth, i.e., in the period of maximal transcriptional activity, and is sensitive to alpha-amanitin treatment. On the other hand, the immunofluorescent distribution of of nucleolin and p-103 (a nucleolar marker of the granular component) is compared to the ultrastructural distribution of the granular component analyzed by electron microscopy on oocytes of the same age.

Amanitins↗
PubMed1995

Endogenous transcription occurs at the 1-cell stage in the mouse embryo.

In most animal species, the earliest stages of embryogenesis are regulated by maternally inherited components, at least until the activation of the zygotic genome. Although the first proteins derived from the activation of the embryonic genome have been detected long ago at the 2-cell stage in the mouse, the exact timing of transcriptional activity resumption after fertilization is still a matter of debate. Any new information about this critical event is relevant to the practice of transgenesis and cloning of embryos as well as to the general understanding of the regulation of nuclear processes following fertilization. Using a new fluorescent method allowing the detection of in vivo RNA synthesis, we show that endogenous transcription by RNA polymerase II takes place unambiguously as early as at the late 1-cell stage. Furthermore, we demonstrate that transcription is first initiated in the paternal pronucleus.

Animals↗
PubMed1995

Photofrin-induced fluorescence in progressive and regressive murine colonic cancer cells: correlation with cell photosensitivity.

Microspectrofluorometry and fluorescence imaging were used to investigate the intracellular fluorescence of two murine colonic cancer cell lines--a progressive cell line (PROb) and a regressive cell line (REGb)--incubated with Photofrin. These two cell lines, which were initially cloned from the same chemically induced colonic murine cancer, differ in their metastatic properties and have been considered as models to mimic the tumoral cell heterogeneity. The fluorescence from cytoplasmic area of cells incubated with Photofrin appeared as a complex emission, with two maxima at 632 and 695 nm assigned to monomer species, and a poorly resolved band around 665 nm assigned to aggregates. The spectral distribution was shown to depend on the incubation time, with an aggregate contribution increasing for extended periods. The amount of Photofrin uptake, as determined from the total fluorescence intensity, was found for PROb to be twice that for REGb. However, the phototoxicities were quite similar for both cell lines, suggesting that drug concentration may not be the only determining factor in photobiological efficiency.

Animals↗
PubMed1994

Redistribution of nuclear antigens linked to cell proliferation and RNA processing in mouse oocytes and early embryos.

We have systematically analyzed by indirect immunofluorescence the subcellular distribution of nuclear antigens in relation to developmental stages of maturing mouse oocytes and developing embryos. Antigens were of two types: (1) a protein whose nuclear localization in interphase somatic cells depends on their proliferative state protein recognized by a monoclonal antibody 43B1N, and (2) snRNP polypeptides recognized by autoimmune sera of anti-Sm and anti-RNP type. The protein recognized by 43B1N was present in the germinal vesicle of oocytes from antral follicles, but absent from the nuclei during the first hours of embryonic life up to the middle to late 2-cell stage. Starting from this stage, it was always found in nuclei of interphase blastomeres, where its "speckles" co-localized with the speckles containing high concentrations of snRNP polypeptides. SnRNP polypeptides recognized by anti-Sm and anti-RNP sera were in turn found in nuclei of all developmental stages. When embryos were treated with aphidicolin or cytochalasin D to arrest cell division, the 43B1N reacting protein was again localized in the pronuclei at 42 hr post-hCG, i.e., slightly later than the onset of transcriptional activity. These results suggest a progressive building up of nuclei during embryonic development, which could influence gene expression.

Animals↗
PubMed1994

Asymmetrical DNA and AT/GC base content of differential sector of Pleurodeles waltl sexual bivalent: a quantitative fluorescence imaging analysis in lampbrush chromosomes.

The mitotic Z and W sex chromosomes in Pleurodeles seem to be identical. Earlier morphological and molecular analyses of lampbrush paired chromosomes in the female meiosis showed clearly that 20% of the chromosomal length located in the middle part of the sex bivalent (bivalent IV) is heteromorphic. We investigated here the base content and composition of the DNA axes in the heteromorphic region by quantitative fluorescence imaging using various base-specific (DAPI, Hoechst 33342 and chromo-mycin A3) or base-nonspecific (ethidium bromide) fluorescent DNA probes. Our results show a significantly higher percentage of AT bases in Z than in W differential sectors. In addition the entire base content of Z appears slightly higher than that of W.

Animals↗
PubMed1993

Competent mouse oocytes isolated from antral follicles exhibit different chromatin organization and follow different maturation dynamics.

After labelling DNA with the specific vital fluorophore Hoechst 33342, oocytes, isolated by puncture from antral follicles in adult mice, have two essentially different configurations of their nuclear fluorescence images. These have been called SN (where the nucleolus is surrounded by chromatin) and NSN (where the nucleolus is not surrounded by chromatin). Intermediate configurations are also found, although with a lower frequency. The proportion of each class is on the average equal and depends neither on the presence of cumulus cells nor on the age of the mouse. Electron microscopy confirms several ultrastructural differences between these two nuclear configurations, namely, the structure of the nucleolus, which is vacuolated in NSN-type and compact in SN-type oocytes. Using video-enhanced fluorescence microscopy at low level of excitation light, we could follow directly in vitro the meiotic maturation of both classes, without impairing their viability. We show that in germinal vessicle (GV) state, the chromatin does not change from one configuration into the other and that both classes are able to mature to metaphase II, although the maturation has slightly different characteristics.

Animals↗
PubMed1993

Inhibition of protein kinases by 6-dimethylaminopurine accelerates the transition to interphase in activated mouse oocytes.

Mouse oocyte activation is followed by a peculiar period during which the interphase network of microtubules does not form and the chromosomes remain condensed despite the inactivation of MPF. To evaluate the role of protein phosphorylation during this period, we studied the effects of the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP) on fertilization and/or parthenogenetic activation of metaphase II-arrested mouse oocytes. 6-DMAP by itself does not induce the inactivation of histone H1 kinase in metaphase II-arrested oocytes, and does not influence the dynamics of histone H1 kinase inactivation during oocyte activation. However, 6-DMAP inhibits protein phosphorylation after oocyte activation. In addition, the phosphorylated form of some proteins disappear earlier in oocytes activated in the presence of 6-DMAP than in the activated control oocytes. This is correlated with the acceleration of some post-fertilization morphological events, such as sperm chromatin decondensation and its transient recondensation, formation of the interphase network of microtubules and pronuclear formation. In addition, numerous abnormalities could be observed: (1) the spindle rotation and polar body extrusion are inhibited; (2) the exchange of protamines into histones seems to be impaired, as judged by the morphology of DNA fibrils by electron microscopy; (3) the formation of a new nuclear envelope around the sperm chromatin proceeds prematurely, while recondensation is not yet completed. These observations suggest that the 6-DMAP-sensitive kinase(s) is (are) involved in the control of post-fertilization events such as the formation of the interphase network of microtubules, the remodelling of sperm chromatin and pronucleus formation.

Adenine↗
PubMed1992

In vitro DNA fluorescence after in vitro fertilization (IVF) failure.

PURPOSE: A pilot study was performed to test the diagnostic value of in vitro DNA fluorescence in oocytes that failed to fertilize after IVF. Ten patients with a cleavage rate less than 20% after IVF were included. RESULTS: Uncleaved oocytes were observed by fluorescence microscopy after incubation with the DNA fluorescent dye Hoeschst 33342. Four main causes which may have contributed to the low cleavage rate were found: (1) sperm incapacity to penetrate the oocyte despite the absence of the usual criteria for male infertility, (2) oocyte immaturity, (3) delayed fertilization, and (4) oocyte abnormalities revealed by aberrations in the morphology of the female chromatin. CONCLUSIONS: The possibility of a rapid and detailed analysis of the maturational status of unfertilized oocytes, the morphology of the female chromatin, the presence and quantity of spermatozoa tightly bound to the zona pellucida, and sperm penetration into the oocyte without subsequent pronucleus formation, using DNA fluorescence, allows us to clarify further the cause of fertilization failure and to orient infertility treatment toward the male, the female, or both partners.

Adult↗
PubMed1991

Dynamics of paternal chromatin changes in live one-cell mouse embryo after natural fertilization.

Using video-enhanced fluorescence microscopy, we describe in live mouse zygotes the paternal chromatin changes undergone after fertilization. We focus on the sperm recondensation process and the formation of the paternal pronucleus, in relationship with the progression of maternal chromatin. Chromatin is labeled with the vital fluorophore Hoechst 33342. Our conditions of dye concentration and irradiation allow a continuous following of the dynamics of changes without major perturbation. We combine these observations with ultrastructural analysis performed by electron microscopy of the same eggs fixed at chosen stages. We show that the highly recondensed state corresponds to the appearance of the nuclear envelope and therefore the beginning of the pronuclear stage.

Animals↗
PubMed1991

Chromatin behaviour under influence of puromycin and 6-DMAP at different stages of mouse oocyte maturation.

Preovulatory mouse oocytes were cultured in vitro up to each subsequent stages of maturation: germinal vesicle (GV), germinal vesicle breakdown (GVBD), groups of not yet individualized bivalents, circular bivalents, late prometaphase I, metaphase I, anaphase I and telophase I. The stages were identified in living oocytes by fluorescence microscopy using Hoechst 33342 as a specific vital dye. Oocytes from each stage of development developed in vitro and ovulated metaphase II oocytes were subsequently cultured in the presence of puromycin or 6-dimethylaminopurine (6-DMAP), an inhibitor of protein phosphorylation. The effects on chromatin of these drugs were studied during and at the end of culture by fluorescence and electron microscopy. We found that puromycin and 6-DMAP stop meiosis when applied at all stages of oocyte maturation, except for metaphase II. Oocytes at this stage are activated by puromycin. Reaction of the oocytes to the two drugs is different at GV and at metaphase II. All of the other stages react to the drugs by chromatin compaction, which can be followed by chromatin decondensation to form a nucleus. Our results suggest that late prophase chromatin condensation, bivalent individualization and retention of their individuality, as well as individualization of monovalents from telophase and retention of their individuality at metaphase II, are dependent on protein phosphorylation. The events occurring between metaphase I and telophase I are independent of protein synthesis and phosphorylation. The events occurring between metaphase II and formation of the nucleus are independent of protein synthesis.

Adenine↗
PubMed1991

Fertilization and ageing processes in non-divided human oocytes after GnRHa treatment: an analysis of individual oocytes.

Some human oocytes cultured together with spermatozoa for in-vitro fertilization (IVF) do not subsequently divide. The arrest of the fertilization process at different moments during development may provide information about the cause of fertilization failure. Oocytes which subsequently divide are transferred 48 h after insemination; when oocytes do not divide, ageing processes can be observed. Therefore these oocytes are interesting material in which to observe both fertilization and ageing. Our study concerns 72 undivided human oocytes 0, 48 or 72 h post-insemination. DNA of the oocyte and spermatozoa was visualized by the DNA fluorescent dye Hoechst 33342. Living oocytes were observed in toto by fluorescence and bright field microscopy which allowed nuclear and pronuclear membranes to be discerned. Oocytes were subsequently fixed and sectioned for bright field microscopy. Both techniques allowed parallel observations. Oocytes at various stages of fertilization are described: sperm penetration in both mature and immature oocytes, decondensation of sperm-heads, premature condensation of male chromatin, polyspermy and pronucleus formation. Typical ageing processes such as the centripetal migration of the metaphase II chromosomes, the formation of a restitution nucleus and the lagging of chromosomes within a metaphase spindle are observed. DNA fluorescence appears to be a quick, easy and valuable means to analyse fertilization and its failure.

Adult↗
PubMed1989

Dynamics of chromatin changes in live one-cell mouse embryos: a continuous follow-up by fluorescence microscopy.

Conditions of minimal dye concentration and minimal irradiation which allow the continuous observation of pronuclei in live unicellular mouse eggs by fluorescence microscopy have been found with the use of Hoechst 33342 as fluorophore and a camera of high sensitivity coupled with an image processing system allowing true integration of weak fluorescent signals and further treatment and analysis. Under these conditions the developmental potential of the embryos is not affected. Using such an approach, which avoids eventual artifacts due to fixation procedure, we describe the changes in the nuclear organization and chromatin structure, from formation of pronuclei to mitosis, with particular attention to the chromatin associated with nucleoli and the timing process of chromatin condensation.

Animals↗
PubMed1989

[Use of very low density light fluorescence microscopy: study of real-time of the development of chromatin of unicellular mouse embryos].

Video enhanced fluorescence microscopy coupled to digitized image processing was used to follow the dynamics of chromatin changes in live one cell mouse embryos. The experimentation on live material was made possible through the use of very low concentrations of the DNA specific fluorophore, Hoechst 33342, and very low irradiation intensities, which maintain a good viability of embryos.

Animals↗