[Embryonic genome activation]. / Activation du génome embryonnaire.

After fertilization in mammals, the genome of the newly formed embryo is first transcriptionally inactive. Development is then strictly dependent on the maternally inherited RNA and proteins present in the oocyte that were accumulated before ovulation during oocyte growth and maturation. The onset of transcription specific to the embryo, referred to as "embryonic genome activation (EGA)", is initiated later during development at various preimplantation stages according to species. Transcriptional activity can be underlined thanks to several approaches such as precursors incorporation in newly synthesized RNA and expression of reporter genes. These studies show that EGA is established in two phases: a "minor" one, first with reduced transcriptional activity and that does not require any specific transcription factor; second, a "major" phase with rapidly increasing transcription. Upon major activation, newly synthesized RNA/proteins are essential for further embryonic development. EGA is dependent on the availability and activity of the basal transcriptional machinery components but also on the structural modifications of the nuclei after fertilization. Indeed, during the first embryonic cycles, the maternal and paternal genome undergo intense chromatin remodeling that could be a key regulator of embryonic transcription.

Spontaneous calcium oscillations and nuclear PLC-beta1 in human GV oocytes.

Our aim was to investigate if human oocytes, like mouse oocytes, exhibit spontaneous Ca(2+) oscillations and nuclear translocation of PLC-beta1 prior to germinal vesicle breakdown (GVBD), and to correlate these events with the evolution of chromatin configuration as a landmark for the meiosis resumption kinetics. Human germinal vesicle (GV) oocytes were either loaded with Fluo-3 probe to record Ca(2+) signals or fixed for subsequent fluorescent labeling of both chromatin and PLC-beta1, and immunogold labeling of PLC-beta1. Here for the first time, we show that human oocytes at the GV-stage exhibit spontaneous Ca(2+) oscillations. Interestingly, only oocytes with a large diameter and characterized by a compact chromatin surrounding the nucleolus of the GV could reveal these kind of oscillations. We also observed a translocation of PLC-beta1 from the cytoplasm towards the nucleus during in vitro maturation of human oocytes. Spontaneous calcium oscillations and nuclear translocation of PLC-beta1 may reflect some degree of oocyte maturity. The impact of our results may be very helpful to understand and resolve many enigmatic problems usually encountered during the in vitro meiotic maturation of human GV oocytes.

Multiparameter assessment of mouse oogenesis during follicular growth in vitro.

Comparison of oocyte development within the follicle in vitro and in vivo has a major impact on research into ovarian physiology and clinical practice. Despite major differences in ovarian physiology between rodents and humans, mice provide a useful model for studies of the endocrine and paracrine mechanisms controlling follicular development. In this study, early preantral follicles were isolated from 12-day-old mice and cultured individually in microdrops under oil during 6, 9 or 12 days. Taking into account previous observations, several oocyte criteria (diameter, chromatin configuration, transcriptional activity, intracytoplasmic calcium signalling and ability to undergo meiosis) were assessed to check that the development pattern of oocytes during follicle growth in vitro was similar to that already observed for oocytes developing in vivo, and that they reached the fertilizable oocyte stage. Results indicate that, during the 12-day-culture period, the oocytes grew until 74.3 +/- 4.2 microm, they became transcriptionally quiescent with a surrounded nucleolus (SN) chromatin organization, 50% of them exhibited regular calcium signals and 73.4% of them resumed meiosis. These data demonstrate that the protocol used generates oocytes with characteristics similar to oocytes allowed to mature fully in vivo and that it could be useful to set up the experimental culture of human ovarian follicles.

CD4+ T cell-mediated immunity against prion proteins.

The prion protein (PrP(C)) is essential for susceptibility to transmissible spongiform encephalopathies. A specific conformer of this protein (PrP(Sc)) is, according to the 'protein only' hypothesis, the principal or only component of the infectious agent, designated prion. Transmission of prions between species is often inefficient, resulting in low attack rates and/or prolonged incubation times and is ascribed to a 'species barrier' caused by differences in the amino acid sequence of PrP between recipient and donor. In this report, we demonstrate that these differences in amino acid sequence result in presentation of distinct peptides on major histocompatibility complex class II molecules. These peptides result in activation of specific CD4+ T cells which leads to the induction of an effective immune response against foreign PrP as demonstrated by antibody production. Therefore, CD4+ T cells represent a crucial component of the immune system to distinguish between foreign and self PrP.

Mouse oocytes and preimplantation embryos bear the two sub-units of interferon-gamma receptor.

Cytokines and growth factors play important roles in implantation and maintenance of pregnancy, but also during early development. Among them interferon-gamma (IFNgamma) is highly expressed by mammalian trophoblast cells during implantation and seems to be involved in some cases of pregnancy loss. In the present study we investigated the possible presence of IFNgamma receptors (IFNGR) on mouse oocytes and preimplantation embryos. The two receptor chains IFNgammaRalpha (IFNGR-1) and IFNgammaRbeta (IFNGR-2) have been detected by indirect immunofluorescence at the surface of mouse oocytes (in germinal vesicle and metaphase II stages), as well as at all stages of in vitro embryo development from the one-cell to blastocyst stage. IFNGR appeared to colocalize partly with ganglioside GM1 at the cell surface of oocytes and embryos, indicating a possible preferential localization of this receptor in "rafts" microdomains. This was analyzed in more detail using software developed in the laboratory. IFNgamma was found to bind to its receptor at all stages analyzed. RT-PCR and Southern blot experiments confirmed the presence of the transcriptionally regulated IFNGR-2 chain mRNA, in mouse oocytes and preimplantation embryos. These results show, for the first time, that mouse oocytes and preimplantation embryos bear a complete and theoretically functional IFNGR, suggesting that this cytokine could play a role during early development.

Sheep prion protein synthetic peptide spanning helix 1 and beta-strand 2 (residues 142-166) shows beta-hairpin structure in solution.

According to the "protein only" hypothesis, a conformational conversion of the non-pathogenic "cellular" prion isoform into a pathogenic "scrapie" isoform is the fundamental event in the onset of prion diseases. During this pathogenic conversion, helix H1 and two adjacent surface loops L2 and L3 of the normal prion protein are thought to undergo a conformational transition into an extended beta-like structure, which is prompted by interactions with the pre-existing beta-sheet. To get more insight into the interaction between the helix and one of the beta-strands in the partially unfolded prion protein, the solution structure of a synthetic linear peptide spanning helix H1 and beta-strand S2 (residues 142-166 in human numbering) was studied by circular dichroism and nuclear magnetic resonance spectroscopies. We found that, in contrast to many prion fragments studied earlier, this peptide (i) is highly soluble and does not aggregate up to a millimolar concentration range in aqueous medium and (ii) exhibits an intrinsic propensity to a beta-hairpin like conformation at neutral pH. This beta-propensity can be one of the internal driving forces of the molecular rearrangement responsible for the pathogenic conversion of the prion protein.

Zinc binding to Alzheimer's Abeta(1-16) peptide results in stable soluble complex.

Aggregation of the human amyloid beta-peptide (Abeta) into insoluble plaques is a key event in Alzheimer's disease. Zinc sharply accelerates the Abeta aggregation in vitro, and the Abeta region 6-28 was suggested to be the obligatory zinc binding site. However, time-dependent aggregation of the zinc-bound Abeta species investigated so far prevented their structural analysis. By using CD spectroscopy, we have shown here for the first time that (i) the protected synthetic peptide spanning the fragment 1-16 of Abeta binds specifically zinc with 1:1 and 1:2 stoichiometry under physiologically relevant conditions; (ii) the peptide-zinc complex is soluble and stable for several months; (iii) zinc binding causes a conformational change of the peptide towards a more structured state. These findings suggest the region 1-16 to be the minimal autonomous zinc binding domain of Abeta.

Electric field-mediated BrUTP uptake by mouse oocytes, eggs, and embryos.

Electropermeabilization was used to introduce 5-bromouridine 5'-triphosphate (BrUTP) into mouse oocytes, zygotes, 2-cell embryos, and parthenogenetic eggs containing nuclei transferred from 3T3 cells. BrUTP incorporated into nascent RNA was detected by indirect immunofluorescence. Two electric pulses of 100 micros duration and of 20 V strength applied at 10 mM concentration of BrUTP loaded most efficiently all cell types tested. Zygotes loaded with BrUTP developed for the next 20 hr in vitro and cleaved to 2-cell stage. The parameters of electric field which promoted BrUTP uptake were also efficient in inducing fusion of blastomeres of 2-cell embryos.

Different initiation of pre-TCR and gammadeltaTCR signalling.

Lineage choice is of great interest in developmental biology. In the immune system, the alphabeta and gammadelta lineages of T lymphocytes diverge during the course of the beta-, gamma- and delta-chain rearrangement of T-cell receptor (TCR) genes that takes place within the same precursor cell and which results in the formation of the gammadeltaTCR or pre-TCR proteins. The pre-TCR consists of the TCRbeta chain covalently linked to the pre-TCRalpha protein, which is present in immature but not in mature T cells which instead express the TCRalpha chain. Animals deficient in pre-TCRalpha have few alphabeta lineage cells but an increased number of gammadelta T cells. These gammadelta T cells exhibit more extensive TCRbeta rearrangement than gammadelta T cells from wild-type mice. These observations are consistent with the idea that different signals emanating from the gammadeltaTCR and pre-TCR instruct lineage commitment. Here we show, by using confocal microscopy and biochemistry to analyse the initiation of signalling, that the pre-TCR but not the gammadeltaTCR colocalizes with the p56lck Src kinase into glycolipid-enriched membrane domains (rafts) apparently without any need for ligation. This results in the phosphorylation of CD3epsilon and Zap-70 signal transducing molecules. The results indicate clear differences between pre-TCR and gammadeltaTCR signalling.

Functional and molecular reorganization of the nucleolar apparatus in maturing mouse oocytes.

In mammalian preovulatory oocytes, rRNA synthesis is down-regulated until egg fertilization and zygotic genome reactivation, but the underlying regulatory mechanisms of this phenomenon are poorly characterized. We examined the molecular organization of the rRNA synthesis and processing machineries in fully grown mouse oocytes in relation to ongoing rDNA transcription and oocyte progression throughout meiosis. We show that, at the germinal vesicle stage, the two RNA polymerase I (RNA pol I) subunits, RPA116 and PAF53/RPA53, and the nucleolar upstream binding factor (UBF) remain present irrespective of ongoing rDNA transcription and colocalize in stoichiometric amounts within discrete foci at the periphery of the nucleolus-like bodies. These foci are spatially associated with the early pre-rRNA processing protein fibrillarin and in part with the pre-ribosome assembly factor B23/nucleophosmin. After germinal vesicle breakdown, the RNA pol I complex disassembles in a step-wise manner from chromosomes, while UBF remains associated with chromosomes until late prometaphase I. Dislodging of UBF, but not of RNA pol I, is impaired by the phosphatase inhibitor okadaic acid, thus strengthening the idea of a relationship between UBF dynamics and protein phosphorylation. Since neither RNA pol I, UBF, fibrillarin, nor B23 is detected at metaphase II, i.e., the normal stage of fertilization, we conclude that these nucleolar proteins are not transported to fertilized eggs by maternal chromosomes. Together, these data demonstrate an essential difference in the dynamics of the major nucleolar proteins during mitosis and meiosis.

Induction of early transcription in one-cell mouse embryos by microinjection of the nonhistone chromosomal protein HMG-I.

In the mouse embryo, the onset of zygotic transcription occurs at the end of the first cell cycle, upon completion of DNA replication. We show that the nonhistone chromosomal protein HMG-I, whose translocation into the pronuclei of one-cell embryos is linked to this first round of DNA synthesis, plays a critical role in the activation of zygotic transcription. Indeed, microinjection of purified HMG-I results in a higher nuclear accumulation of the protein and triggers an earlier activation of zygotic transcription, an effect which is abolished by the preincubation of the protein with a specific antibody directed against its AT-hook DNA-binding motifs. Significantly, microinjection of this antibody also prevents the normal onset of transcription in the embryo, suggesting that endogenous HMG-I is similarly involved in this process. Finally, microinjection of the exogenous protein modifies chromatin structure as measured by in situ accessibility to DNase I. We propose that general chromosomal architectural factors such as HMG-I can modulate the accessibility of chromatin to specialized regulatory factors, thereby promoting a transcriptionally competent state.

High yield purification and physico-chemical properties of full-length recombinant allelic variants of sheep prion protein linked to scrapie susceptibility.

Sheep susceptibility to scrapie is governed by polymorphisms at two major sites, codons 136 and 171, of the prp gene. To get more insight into the prion protein (PrP) sequence-linked basis of differential scrapie susceptibility, a high yield one-step method for the purification (over 99% final purity) of the full-length recombinant sheep PrP was developed, based on the affinity of the conserved octapeptide repeats for transition-metal cations. Thermal and chemical denaturation experiments and limited proteolysis studies were performed on the natural variants (A136R171, V136Q171 and A136Q171) and a recombinant PrP mutated at position 136 (V136R171). Results revealed the influence of mutations in positions 136 and 171 on the folding thermodynamic parameters and on the conformation of the C-terminal domain. Together, our results show that the VQ cellular protein linked to higher scrapie susceptibility is intrinsically more compact and/or stable than the resistance-linked AR counterpart. This might lead to a lower in vivo clearance rate of VQ and a consequently higher probability of occurrence of pathological events.

Structural changes induced by binding of the high-mobility group I protein to a mouse satellite DNA sequence.

Using spectroscopic methods, we have studied the structural changes induced in both protein and DNA upon binding of the High-Mobility Group I (HMG-I) protein to a 21-bp sequence derived from mouse satellite DNA. We show that these structural changes depend on the stoichiometry of the protein/DNA complexes formed, as determined by Job plots derived from experiments using pyrene-labeled duplexes. Circular dichroism and melting temperature experiments extended in the far ultraviolet range show that while native HMG-I is mainly random coiled in solution, it adopts a beta-turn conformation upon forming a 1:1 complex in which the protein first binds to one of two dA.dT stretches present in the duplex. HMG-I structure in the 1:1 complex is dependent on the sequence of its DNA target. A 3:1 HMG-I/DNA complex can also form and is characterized by a small increase in the DNA natural bend and/or compaction coupled to a change in the protein conformation, as determined from fluorescence resonance energy transfer (FRET) experiments. In addition, a peptide corresponding to an extended DNA-binding domain of HMG-I induces an ordered condensation of DNA duplexes. Based on the constraints derived from pyrene excimer measurements, we present a model of these nucleated structures. Our results illustrate an extreme case of protein structure induced by DNA conformation that may bear on the evolutionary conservation of the DNA-binding motifs of HMG-I. We discuss the functional relevance of the structural flexibility of HMG-I associated with the nature of its DNA targets and the implications of the binding stoichiometry for several aspects of chromatin structure and gene regulation.

Intracellular pH in one-cell mouse embryo differs between subcellular compartments and between interphase and mitosis.

The pH-sensitive dual-emission fluorophore SNARF-1 coupled with a laser confocal microspectrofluorimeter was used to measure the internal pH (pHi) in different subcellular and subnuclear compartments of early mouse embryos. By this method we analysed the first cell cycle of naturally fertilised embryos in order to detect possible pHi changes correlated to cellular events, particularly the onset of replication or transcription and the first mitosis. Throughout interphase, significant differences of pHi were observed between cytoplasm and pronuclei, and, even more striking, between these compartments and nucleolus precursor bodies, whose pHi was systematically lower. We could detect significant pHi change neither during the replication phase nor at the onset of zygotic transcription, but pHi increased at the end of the one-cell stage in both cytoplasm and chromatin regions, a process that seemed specifically correlated with mitosis.

An unexpected effect of an ouabain-sensitive ATPase activity on the amount of antigen-antibody complexes formed in situ.

A classical method of indirect immunofluorescence was applied on various kinds of lightly fixed and permeabilized cells to analyze the formation of the complexes between a nuclear antigen and its antibody (AAC). The amount of AAC decreased dramatically when the incubation with the first antibody was realized in the presence of ATP in a sodium-rich medium with 0.5 mM KCl. Addition of sodium vanadate, a general inhibitor of ATPases, ouabain or tetrabutylammonium ion, specific inhibitors of the Na+,K+-ATPase, prevented this effect. The established role of this enzyme is to increase free-K+ concentration and decrease free Na+ concentration in the cell. It is not surprising to find an ATPase still active since light fixation and permeabilization do not destroy phosphatases. But it is rather surprising to find something looking like Na+,K+-ATPase activity in permeabilized cells. The importance of potassium in this puzzling result is suggested by the fact that appearance of ACC was equally suppressed if the incubation was made in the absence of ATP in a potassium-rich medium without sodium. Results are discussed, taking into account the properties of cell-associated water and recently found interrelation between Na+,K+-ATPase and tubulin. In any case, the results seem interesting in the field of immunocytochemistry.

Pressure-sensitivity of endoplasmic reticulum membrane and nucleolus as revealed by electron microscopy.

Murine erythroleukemia cells submitted to high hydrostatic pressure (up to 110 MPa, 17 min. at room temperature) remain viable (Mentré et al., 1997) but present, at the ultrastructure level, perturbations which are documented in this work. In cells submitted to 50 MPa, endoplasmic reticulum membranes displayed a characteristic rigid aspect, whereas plasma membrane remained unaffected up to 110 MPa. This result is in agreement with: i) the liquid-crystalline --> gel state transition undergone by phospholipid bilayers under pressure, which involves the structuration of water at the polar-apolar interface; ii) the low concentration in cholesterol of endoplasmic reticulum membranes compared to plasma membrane, and iii) the ability of cholesterol to protect membranes against the effects of high hydrostatic pressure. Nucleoli displayed a remarkable compact aspect above 80 MPa, involving the disappearance of vacuoles and the diminution of fibrillar component, but the retention of granular component. Pressure inhibition of translation is advanced as a possible cause of this perturbation.

Differential transcriptional activity associated with chromatin configuration in fully grown mouse germinal vesicle oocytes.

It was previously shown that fully grown ovarian germinal vesicle (GV) oocytes of adult mice exhibit several nuclear configurations that differ essentially by the presence or absence of a ring of condensed chromatin around the nucleolus. These configurations have been termed, respectively, SN (surrounded nucleolus) and NSN (nonsurrounded nucleolus). Work from our and other laboratories has revealed ultrastructural and functional differences between these two configurations. The aims of the present study were 1) to analyze the equilibrium between the SN and the NSN population as a function of the age of the mice and the time after hCG-induced ovulation and 2) to study the polymerase I (pol I)- and polymerase II (pol II)-dependent transcription in both types of oocytes through the detection of bromouridine incorporated into nascent RNA. We show 1) that ovarian GV oocytes exhibiting the SN-type configuration can be found as soon as 17 days after birth in the C57/CBA mouse strain and 2) that the SN:NSN ratio of ovarian GV oocytes is very low just after hCG-induced ovulation and then increases progressively with the time after ovulation. Furthermore, we demonstrate that the SN configuration correlates strictly with the arrest of both pol I- and pol II-dependent transcription in mice at any age. Finally, we show that ribosomal genes are located at the outer periphery of the nucleolus in the NSN configuration and that pol I-dependent perinucleolar transcription sites correspond to specific ultrastructural features of the nucleolus. Altogether, these results provide clear-cut criteria delineating transcriptionally active GV oocytes from those that are inactive, and confirm that the SN-type configuration is mostly present in preovulatory oocytes.

DNA content by in situ fluorescence imaging and S-phase detection, with chromatin structure preserved.

OBJECTIVE: To test the feasibility of in situ DNA quantitation of adherent cells' nuclei by fluorescence imaging, preserving chromatin structure and to follow-up S phase, in relation to DNA content, in order to assess the precision of DNA measurements. STUDY DESIGN: Double labeling experiments involved total DNA staining with Hoechst 33342 and BrdU immunostaining (after either Br photolysis and DNA strand break labeling by terminal transferase or acid denaturation) to detect replicating DNA. An epifluorescence microscope was used, images captured with a CCD camera and quantitative total DNA measurements done in 12 bits with IPLab software. BrdU results were related to DNA content on an individual cell basis. Cell cycle analyses were run with Imastat software (developed in the laboratory) on Hoechst-stained cells and on double labeled cells. RESULTS: In cells progressing through the cycle, as assessed by BrdU, a corresponding increase in DNA content was measured. Early S differed from G1 (P < .05). Imastat analyses gave a CV for GI peak of 6-7%. CONCLUSION: Quantitative fluorescence imaging allows a sensitive determination of DNA content for adherent-cell nuclei in situ. Topologic analyses of nuclear components will be possible in relation to DNA content.

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.

Nuclear envelope removal/maintenance determines the structural and functional remodelling of embryonic red blood cell nuclei in activated mouse oocytes.

Nuclei of embryonic red blood cells (e-RBC) from 12-day mouse fetuses are arrested in G0 phase of the cell cycle and have low transcriptional activity. These nuclei were transferred with help of polyethylene glycol (PEG)-mediated fusion to parthenogenetically activated mouse oocytes and heterokaryons were analysed for nuclear structure and transcriptional activity. If fusion proceeded 25-45 min after oocyte activation, e-RBC nuclei were induced to nuclear envelope breakdown and partial chromatin condensation, followed by formation of nuclei structurally identical with pronuclei. These 'pronuclei', similar to egg (female) pronuclei, remained transcriptionally silent over several hours of in vitro culture. If fusion was performed 1 h or later (up to 7 h) after activation, the nuclear envelope of e-RBC nuclei remained intact and nuclear remodelling was less spectacular (slight chromatin decondensation, formation of nucleolus precursor bodies). These nuclei, however, reinforced polymerase-II-dependent transcription within a few hours of in vitro culture. Our present experiments, together with our previous work, demonstrate that nuclear envelope breakdown/maintenance are critical events for nuclear remodelling in activated mouse oocytes and that somatic dormant nuclei can be stimulated to renew transcription at a time when the female pronucleus remains transcriptionally silent.