[Environmental endocrine disruptors and fertility]. / Perturbateurs endocriniens environnementaux et fertilité.

Endocrine disruptor chemicals (EDCs) are ubiquitous contaminants in the environment, wildlife, and humans. During the last 20 years, several epidemiological, clinical and experimental studies have demonstrated the role of EDCs on the reduction of male and female fertility. The concept of foetal origins of adult disease is particularly topical in the field of reproduction. Moreover, exposure to EDCs during pregnancy has been shown to influence epigenetic programming of endocrine signalling and other important physiological pathways, and provided the basis for multi- and transgenerational transmission of adult diseases. However, the large panel of EDCs simultaneously present in the air, sol and water makes the quantification of human exposition still a challenge. Gas chromatography coupled with mass spectrometry, the measurement of total plasmatic hormonal bioactivity on stably transfected cell lines as well as the EDC analysis in hair samples are useful methods of evaluation. More recently, microRNAs analysis offers a new perspective in the comprehension of the mechanisms behind the modulation of cellular response to foetal or post-natal exposure to EDCs. They will help researchers and clinicians in identifying EDCs exposition markers and new therapeutic approaches in the future.

Esterases in marine dinoflagellates and resistance to the organophosphate insecticide parathion.

Esterases are involved in the susceptibility or resistance of organisms to organophosphate pesticides. We have examined the action of parathion on the marine dinoflagellates Crypthecodinium cohnii and Prorocentrum micans by looking at their esterases. One-dimensional gel electrophoresis, immunoblotting and cytochemistry plus image analysis were used to characterize the nature and distribution of the enzymes. Esterases were found in both species, but there appeared to be no particular intracellular localization. The esterase activity of the heterotrophic species Crypthecodinium cohnii was 30-fold greater than that of the autotrophic Prorocentrum micans and had an antigenic site in common with mosquito esterase. The resistance of Crypthecodinium cohnii to parathion was specific and reversible. Less parathion entered the parathion-resistant Crypthecodinium cohnii cells than the untreated control cells. Parathion-resistant cell extracts of Crypthecodinium cohnii analyzed after immunoblotting also contained an additional band of esterase activity. These results confirm the importance of esterases in toxicological studies of organophosphate insecticides, especially those of marine dinoflagellates.

Morphology and behaviour of dinoflagellate chromosomes during the cell cycle and mitosis.

The morphology and behaviour of the chromosomes of dinoflagellates during the cell cycle appear to be unique among eukaryotes. We used synchronized and aphidicolin-blocked cultures of the dinoflagellate Crypthecodinium cohnii to describe the successive morphological changes that chromosomes undergo during the cell cycle. The chromosomes in early G(1) phase appeared to be loosely condensed with numerous structures protruding toward the nucleoplasm. They condensed in late G(1), before unwinding in S phase. The chromosomes in cells in G(2) phase were tightly condensed and had a double number of arches, as visualised by electron microscopy. During prophase, chromosomes elongated and split longitudinally, into characteristic V or Y shapes. We also used confocal microscopy to show a metaphase-like alignment of the chromosomes, which has never been described in dinoflagellates. The metaphase-like nucleus appeared flattened and enlarged, and continued to do so into anaphase. Chromosome segregation occurred via binding to the nuclear envelope surrounding the cytoplasmic channels and microtubule bundles. Our findings are summarized in a model of chromosome behaviour during the cell cycle.

Characterization of p80, a novel nuclear and cytoplasmic protein in dinoflagellates.

The presence of myosin in dinoflagellates was tested using an anti-Acanthamoeba castellanii myosin II polyclonal antibody on the heterotrophic dinoflagellate Crypthecodinium cohnii Seligo. Western blots revealed the presence of a unique band of 80 kDa in total protein extracts and after immunoprecipitation. Expression of this 80 kDa protein appeared constant during the different phases of the cell cycle. In protein extracts from various other dinoflagellates, this 80 kDa protein was detected only in the autotrophic species Prorocentrum micans Ehr. Screening of a C. cohnii cDNA expression library with this antibody revealed a cDNA coding for an amino acid sequence without homology in the databases. However, particular regions were detected: - a polyglutamine repeat domain in the N-terminal part of the protein, - four peptide sequences associated with GTP-binding sites, - a sequence with slight homology to the rod tail of Caenorhabditis elegans myosin II, -a sequence with homology to a human kinesin motor domain. Immunocytolocalization performed on C. cohnii thin sections with a polyclonal antibody raised against the recombinant protein showed p80 to be present both within the nucleus and in the cytoplasm. Labelling was widespread in the nucleoplasm and more concentrated at the periphery of the permanently condensed chromosomes. In the cytoplasm, labelling appeared in a punctate region close to the nucleus and in the flagellum. Potential functions of this novel protein are discussed.

Cyclic expression of a nuclear protein in a dinoflagellate.

Nuclei of the dinoflagellate Crypthecodinium cohnii strain Whd were isolated and nuclear proteins were extracted in three fractions, corresponding to the increasing affinity of these proteins to genomic DNA. One fraction contained two major bands (48- and 46-kDa) and antibodies specific to this fraction revealed two major bands by Western blot on nuclear extracts, corresponding to the 46- and 48-kDa bands. The 48-kDa protein was detected in G1 phase but not in M phase cells. An expression cDNA library of C. cohnii was screened with these antibodies, and two different open reading frames were isolated. Dinoflagellate nuclear associated protein (Dinap1), one of these coding sequences, was produced in E. coli and appeared to correspond to the 48-kDa nuclear protein. No homologue of this sequence was found in the data bases, but two regions were identified, one including two putative zinc finger repeats, and one coding for two potential W/W domains. The second coding sequence showed a low similarity to non-specific sterol carrier proteins. Immunocytolocalization with specific polyclonal antibodies to recombinant Dinap1 showed that the nucleus was immunoreactive only during the G1 phase: the nucleoplasm was immunostained, while chromosome cores and nuclear envelopes were negative.

Dinoflagellate chromosome behaviour during stages of replication.

In most dinoflagellate species, chromosomes are characterized by an almost continuous condensation of the nucleofilaments throughout the cell cycle and the absence of longitudinal differentiation as Q, G, or C banding. Their supercoiled architecture is maintained by divalent cations and structural RNAs. Their chromatin is devoid of histones and nucleosomes and their DNA composition is distinctive: in several species, more than 60% of thymines are replaced by a rare base, hydroxymethyluracil. We report here an immunofluorescence (conventional and confocal laser scanning microscopy, CLSM) and immunogold transmission electron microscopy (TEM) analysis of some stages of the early replication process in Prorocentrum micans dinoflagellate cells, after long pulse incorporation (3, 6 or 9 days) with 50 micrograms/ml bromodeoxyuridine (BrdU) in the presence of 5-fluoro-2'-deoxyuridine (FUdR) and BrdU antibody technique (BAT) detection. The large DNA content (45 pg per nucleus) of P. micans cells is compacted on 100 chromosomes, 10 microns in length. In early S-phase, DNA replication sites are revealed as fluorescent domains organized in clusters, which appear in the periphery of the nucleus unlike other eukaryotes. In late S-phase, the number of labelled clusters increased; helically distributed, they did not appear synchronously in the whole chromosome. Under TEM, spherical domains of equivalent diameter appeared located all along the chromosomes after 6 days BrdU pulse. Replication occurs, but in our experimental conditions, segregation of daughter chromosomes was never observed. The blockade of the cell cycle after BrdU incorporation intervening just before the segregation of daughter chromosomes is discussed.

Colocalization of the cyclin B homologue p56 and beta-tubulin during the cell cycle in a unicellular eucaryote dinoflagellate.

We provide evidence for an unusual behavior of the cyclin B homologue, p56, in the dinoflagellate Crypthecodinium cohnii. p56, of which we previously demonstrated the presence in this original eukaryotic protist, is present all along the cell cycle progression, and is exclusively cytoplasmic as revealed after immunofluorescence labeling with anti-p56 Ab and counterstaining with Dapi. It was never found in the nucleus as is the case in higher eukaryotic cells. During mitosis, p56 was essentially associated with the mitotic apparatus: centrosomes and mitotic spindle, as shown after double immunofluorescence labeling with anti p56 and anti beta-tubulin Ab. Using high pressure freeze fixation, we clearly detected in transmission electron microscopy (TEM) the localization of p56 cyclin B homologue and beta-tubulin: single immunogold labeling demonstrated that p56 is localized along the whole cell cortex, along the cleavage furrow of anaphase to cytokinesis cells and into cytoplasmic channels passing throughout the mitotic nucleus where is located the mitotic spindle. Double immunogold labeling realized with anti-p56 and anti-beta-tubulin antibodies confirm that p56 antigens colocalize with beta-tubulin in many sites. The significance of the exclusively cytoplasmic localization of the cyclin B homologue is discussed.

Cloning, characterization and chromosomal localization of a repeated sequence in Crypthecodinium cohnii, a marine dinoflagellate.

Genomic DNA of Crypthecodinium cohnii has been extracted in the presence of cetylmethylammonium bromide and hydrolysed by 13 restriction enzymes. No typical ladder-like pattern or isolated band of satellite sequences were found with any of these enzymes. A "mini" genomic DNA library had been made and screened by reverse hybridization to isolate highly repeated sequences. Seven such DNA fragments were sequenced. The copy number of one of them (Cc18), 226 bp long, was estimated at around 25,000, representing 0.06% of the total genome. Cc18 was found to be included in a higher fragment of 3.0 kb by Southern blot analysis after cleavage by PstI. This higher molecular weight fragment could be composed either of tandemly repeated Cc18 sequences, or by only one or a very low copy number of Cc18. In this latter case, these fragments, also repeated 25,000 times would represent 1 to 2% of the total genome. Genomic localization of Cc18 by in situ hybridization on squashed C. cohnii cells showed that it was widely distributed on the different chromosomes. All the chromosomes observed displayed Cc18 labeling, which appeared homogeneously distributed. The ability of Cc18 to be a specific molecular marker to distinguish sibling C. cohnii species is discussed.

Cyclin B (p56cdc13) localization in the yeast Schizosaccharomyces pombe: an ultrastructural and immunocytochemical study.

The eucaryote cell cycle is driven by a set of cyclin dependent kinases (CDKs) associated to cyclins, which confer not only the activity but also the substrate specificity and the proper localization of the kinase activity. In the fission yeast Schizosaccharomyces pombe, only one cyclin, the product of the cdc13 gene (p56cdc13), is required to be associated with p34cdc2, to control the complete cell cycle. Earlier studies have localized this complex mainly in the nucleus and its periphery. Using new improved electron microscopy (EM) technologies, based on high pressure freezing fixation, we refined previous studies, evidencing cytoplasmic localization of p56cdc13, in addition to the nuclear localization previously observed. Further immunofluorescence studies, performed on aldehydically fixed cells, confirmed our EM results, emphasizing the major cytoplasmic localization of p56cdc13 in interphase cells and the relocalization towards the nucleus in mitotic cells, suggesting that the S pombe cyclin B localization is cell cycle-regulated.

Nuclear and cytoplasmic actin in dinoflagellates.

Experiments using monoclonal and polyclonal anti-actin antibodies allowed us to demonstrate the presence of F- or G-actin in original protists, dinoflagellates, either by biochemistry, immunofluorescence and in TEM. SDS-PAGE electrophoresis and immunoblottings made either from total or nuclear protein extracts revealed the presence of a 44-kDa band reacting with monoclonal anti-actin antibody in two species, Prorocentrum micans and Crypthecodinium cohnii, and thus demonstrated the presence of actin in nuclear and cytoplasmic fractions. After squash preparation of P micans cells, actin was identified within the nucleus and in some regions of the cytoplasm by immunofluorescence microscopy. Labelling of both the nucleolus and the centrosome region was evident together with amorphous nucleoplasmic material surrounding the chromosomes. The use of cryosections of intact P micans and C cohnii cells for immunofluorescence along with staining with DAPI to delineate the chromosomes themselves, yielded finer resolution of the intranuclear network labelling pattern and allowed us to complete our observations, in particular on the cytoplasmic labelling. In P micans, in addition to the centrosome region, the cytoplasmic channels passing through the nucleus in dividing cells are labelled. In C cohnii, the cortex, the centrosome region, the cytoplasmic channels, the region surrounding the nucleus, the filaments linking it to the cortex and the cleavage furrow are also labelled. In the nucleus of the two species, there is a prominent "weft' of fine actin filaments in the nucleoplasm forming a matrix of varying density around the persistent chromosomes. This actin matrix, of unknown function, is most conspicuous at the end of the S-phase of the cell cycle. Fluorescent derivatives of phalloidin, used as diagnostic cytochemical probes for polymeric actin (F-actin), gave similar results. Positive TEM immunolabelling of intranuclear actin confirms its presence in the nucleoplasm, in the nucleolus where the preribosomal region is labelled while C cohnii chromosomes are unlabelled and the P micans chromosomes very slightly. In the cytoplasm, lips of the cleavage furrow and kinetosome regions are labelled as well as the centrosome region. The possible functions of this protein located in several compartments of dinoflagellate cells are discussed.

Osmium ammine: review of current applications to visualize DNA in electron microscopy.

This review has been collectively written. The contribution of the authors is mentioned for each part. References have been grouped at the end of the review. The objective of this review is to outline the principle of the method for electron microscopy, to emphasize the major applications and recent developments of this technique for DNA detection and finally to compare this technique with some other methods of DNA detection.

Identification of an HSP70-related protein associated with the centrosome from dinoflagellates to human cells.

The monoclonal antibody CTR210 raised against isolated human centrosomes strongly decorates the centrosome and more weakly a domain congruent with the Golgi apparatus in several animal cells (HeLa, 3T3, CHO, PtK2). Both decorations resist Triton extraction in conditions which totally extract the Golgi apparatus, as judged by galactosyltransferase decoration. A 67 kDa centrosomal antigen can be demonstrated in human cells with this antibody. CTR210 also decorates the centrosome or associated structures in several systems, including unicellular eukaryotes such as dinoflagellates or ciliates. A 72 kDa antigen has been identified and purified from the dinoflagellate C. cohnii and its NH2-terminal sequence partially established. It shows a close homology with HSP70 proteins. The possibility that the 72 kDa antigen belongs to this chaperone family was further supported using a mAb reacting, in most species, with HSP70. A polyclonal antibody raised against the 72 kDa antigen from C. cohnii decorates the centrosome in human cells and reacts with the CTR210 centrosomal 67 kDa antigen. These results suggest that specific chaperone proteins are associated with the centrosome in eukaryotic cells. The centrosomal chaperones could participate in the microtubule nucleation reaction or in the process of centrosome assembly.

Changes in ATP concentration, mitochondrial structures, and rhodamine 123 binding in two marine dinoflagellates cultured in the presence of parathion.

Parathion, an organophosphorous insecticide, is highly toxic to the two free-living marine dinoflagellates Prorocentrum micans Ehrenberg (autotrophic) and Crypthecodinium cohnii Biechler (heterotrophic). To study its non-antiacetylcholinesterase action we assessed its effect on the mitochondrial system, as shown by changes in intracellular ATP concentration and in rhodamine 123 fluorescence evaluated by image analysis. The technique of image analysis permits direct assessment of changes in the overall activity of mitochondria in living cells. Mitochondrial structures were also examined in the electron microscope. The three methods of investigation yielded complementary results. In P. micans, parathion noticeably altered mitochondria but did not significantly alter ATP concentrations. In C. cohnii, however, mitochondrial disturbance was slight, whereas ATP increased greatly. We think, therefore, that parathion has different effects on mitochondria in the two organisms, and in particular that it increases mitochondrial activity in C. cohnii.

Fluorescence induction used to measure parathion toxicity in the marine dinoflagellate Prorocentrum micans: a possible model for biodetection.

The marine dinoflagellate Prorocentrum micans Ehrenberg was used as a test organism to determine the conditions of use of fluorescence induction kinetic measurements in the study of parathion phytotoxicity. Measurements were taken of the kinetics of slow and fast fluorescence induction in whole cells and isolated chloroplast fragments at various concentrations of parathion. In both types of induction, the addition of parathion greatly decreased the fluorescence yield, indicating either inhibition of electron transport or physical changes. The action of parathion was a function of its concentration and was similar to the action of DCMU. Fluorescence induction was clearly less in isolated chloroplast fragments than in whole cells.

Microtubule organization during the cell cycle of the primitive eukaryote dinoflagellate Crypthecodinium cohnii.

The complete microtubular system of the dinoflagellate Crypthecodinium cohnii Biecheler is described, as seen by confocal laser scanning fluorescence microscopy and labelling with anti-beta-tubulin antibody. This technique allowed us to observe the organization of the subcortical and internal cytoskeletons and the mitotic microtubular system, and their changes during the cell cycle. These observations are compared with those made in cryosections by light microscopy and in fast-freeze-fixed, cryosubstituted cells by electron microscopy. We show the organization of the cortical microtubules, and in particular of the thick microtubular bundles arranged as a three-pronged fork from which they seem to emanate. This fork emerges from a peculiar cytoplasmic zone at the pole of the cell and is in contact with the region of the kinetosomes, at the cingulum. During the G1 phase, only a single, radial microtubular bundle (a "desmose") is observable in the inner part of the cytoplasm. One of its ends is near the flagellar bases and the other end is close to the nucleus in the centrosome region. During the S phase, the flagella drop off, the cell encysts and the kinetosomes duplicate. In mitosis, the cortical microtubules and the intracytoplasmic microtubular bundles do not depolymerize. The microtubular fork, desmose and centrosome double and migrate, while the divided kinetosomes stay in the same place. Later, the centrosomes organize the extranuclear spindle, which is connected to the kinetosome region by the microtubular desmose. The convergent end of the three-pronged fork seems to be in contact with the centrosome region. In early and mid-prophase, thick microtubular bundles pass through the nucleus in cytoplasmic channels and converge towards the two poles. Asters were never seen at the spindle poles. The channels and microtubular bundles in the spindle double in number during late prophase and lengthen in early anaphase. The spindle bundles diverge in late anaphase, extend to very near the plasma membrane and depolymerize during telophase. The cleavage furrow in which tubulin and actin are characterized appears in anaphase, formed by invagination of plasma membrane in the kinetosome region. The structure and rearrangements of the Crypthecodinium cohnii microtubular system are compared with those of other dinoflagellates and protists and of higher eukaryotes.

Molecular cloning and immunolocalization of two variants of the major basic nuclear protein (HCc) from the histone-less eukaryote Crypthecodinium cohnii (Pyrrhophyta).

Two clones that encode variants (HCc1 and HCc2) of the major basic nuclear protein of the dinoflagellate Crypthecodinium cohnii, were identified by immunoscreening of a cDNA expression library. The first clone carries a full-length cDNA with an open reading frame (HCc1) encoding 113 amino acids. The cDNA from the second clone lacks some of the 5' end, and the coding sequence is only 102 residues. The two proteins display 77% sequence similarity and their NH2-ends are homologous to the NH2-peptide of the HCc protein determined by P. Rizzo. The amino acid composition, which confirms the basic nature of lysine-rich HCc proteins, differs markedly from other known DNA-binding proteins such as histones, HMGs or prokaryotic histone-like proteins. No convincing homology was found with other proteins. HCc antigens were localized on C. cohnii by immunofluorescence, and by electron microscopy (EM) with immunogold labelling. HCc proteins are mainly detected at the periphery of the permanently condensed chromosomes, where active chromatin is located, as well as in the nucleolar organizing region (NOR). This suggests that these basic, non-histone proteins, with a moderate affinity for DNA, are involved at some level in the regulation of gene expression.

Microtubular spindle and centrosome structures during the cell cycle in a dinoflagellate Crypthecodinium cohnii B.: an immunocytochemical study.

In order to determine if a mitotic spindle organizing center is present in dinoflagellate cells, we used a library of 12 monoclonal antibodies obtained by immunizing mice with isolated human centrosomes. When tested by immunofluorescence on cryosections of the dinoflaggelate Crypthecodinium cohnii B., a positive labeling was obtained with three of these antibodies. In interphase cells, the anti-centrosome antibodies labeled structures located either in the cell periphery, corresponding probably to both basal bodies (i.e. kinetosomes) and in the perinuclear area. In the latter case, two punctate structures were observed near the nuclear envelope. They have never been described, either in light, or in electron microscopic studies of dinoflagellates. We have designated them as centrosome-like structures. A microtubular desmose reacting positively with anti-tubulin Ab was also visible, linking kinetosomes and centrosome-like structures. During mitosis, the double punctate structures were observed at the poles of the nucleus. Double immunolabeling with tubulin and anti-centrosome Ab was also carried out and strongly suggested that in mitotic cells, centrosome-like structures, located at the poles of the mitotic spindle, were associated with microtubular bundles and probably organize and polarize them. These data indicate the existence of centrosome-like structures in C. cohnii cells and the strong conservation of some centrosomal epitopes from dinoflagellates to human. One of the antibodies (CTR 210) recognized by immunoblotting, a single protein band at 72 kDa from a total protein extract. The direct demonstration that this protein is located at the centrosome-like structures and at the kinetosomes deserves further study.

Nucleolar localization of rRNA coding sequences in Prorocentrum micans Ehr. (dinomastigote, kingdom Protoctist) by in situ hybridization.

To define the molecular mechanisms of ribosome biogenesis and to find out in which nucleolar compartment transcription of rDNA occurs, we have performed in situ hybridization (ISH) of RNase-treated cryosections using biotinylated rRNA coding sequences as a probe and the eukaryotic dinoflagellate nucleolar system as a model. Recent data from ISH of eukaryotic ribosomal genes by electron microscopy (EM) has so far failed to establish a consensus which clearly defines the function of the three compartments of the nucleolus. Dinomastigote protoctists are the only known eukaryotes whose chromatin is totally devoid of nucleosomes. Their chromosomes remain permanently condensed during the entire cell cycle and active nucleoli arise from an unwound part of some of the otherwise compact chromosomes. In this work, DNA-DNA hybrids were detected either by fluorescent avidin or by indirect immunogold staining procedures in EM; this is the first use of cryosections to detect hybrids in EM not only in the nucleolus sensu lato but also in a dinomastigote cell. Coding sequences of ribosomal genes were detected both in the periphery of the nucleolar organizer region (NOR), which corresponds to the unwound part of the nucleolar chromosome, and in the proximal part of the fibrillo-granular (FG) region. These results suggest that the rRNA gene transcription predominantly occurs at the periphery of the NOR where the coding sequences are located. A predictive model summarizes and allows discussions and comparisons with other eukaryotes in which nucleolar mechanisms were previously studied. This leads to the conclusion that dinoflagellate cells constitute an excellent model for the study of the functional structure of the eukaryotic nucleolus.(ABSTRACT TRUNCATED AT 250 WORDS)