Marie-Odile Soyer-Gobillard

Prevalence of hypospadias in grandsons of women exposed to diethylstilbestrol during pregnancy: a multigenerational national cohort study

Prenatal diethylstilbestrol (DES)-exposed mice have raised the suspicion of a transgenerational effect in the occurrence of genital malformation in males. This nationwide cohort study in collaboration with a French association of DES-exposed women studied 529 families and showed that a significant proportion of boys born to DES daughters exhibited hypospadias with no other molecular defects identified.

A Detailed Study of the Complex Cell Cycle of the Dinoflagellate Crypthecodinium cohnii Biecheler and Evidence for Variation in Histone H1 Kinase Activity

ABSTRACT By adding the protein synthesis inhibitor, emetine (10‐4 M) to a highly synchronized population of Crypthecodinium cohnii Biecheler 1938 at different phases of its cycle, we were able to determine: 1. The existence and the lengthening of the G2‐Phase (30 min) in the first cycle (cycle with swimming G1 phase). 2. The time of the second cell cycle phases (cycle in the cyst): G1, 30 min; S, 1.5 h; G2, 2 h and M, 2 h. These results, together with the estimation of the cell volume of the two and four swimming daughter cells emerging from the cysts, allowed us to state the existence of two transition points: G1/S and G2/M, which are necessary for completion of mitosis. We completed this refined approach of the cell cycle in studying the activities of the histone H1 kinase either in dividing or in non‐dividing Crypthecodinium cohnii cells with either total soluble proteins or the isolated mitotic kinase complex. The H1 kinase activity of this purified complex is noticeably higher (twice as high) in the dividing cells than in the non‐dividing ones. These data are discussed in the light of the basic characteristics of the dinokaryon, and also compared with recent biochemical observations on the same organism and studies on other higher eukaryotic protists and metazoa.

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.

Immunocytochemical localization of the DNA-binding protein HCc during the cell cycle of the histone-less dinoflagellate protoctista Crypthecodinium cohnii B

Summary— The major basic nuclear protein HCc (previously named Histone‐like) of the dinoflagellate Crypthecodinium cohnii B was immunolocalized in light and electron microscopy using an affinity‐purified polyclonal antibody. Complementary conventional and cryo‐techniques were used to study the distribution of the DNA‐binding protein in interphase cells and to follow its behaviour throughout the mitotic cycle. In non‐dividing cells, the HCc protein was found to be located on extra‐chromosomal loops and chromosomal nucleofilaments dispersed in the nucleoplasm. In mitotic cells, from prophase to early telophase, it was homogeneously distributed in the (whole) dividing chromosomes. HCc protein was also detected in two compartments of all the permanently observable nucleoli: the nucleolar organizing region and the fibrillo‐granular region. In this paper we discuss the hypothetical roles, structural and/or functional, of this DNA‐binding protein, which is specific to dinoflagellates, the only eukaryotes whose chromatin is devoid of histones and nucleosomes.

The toxic dinoflagellate Prorocentrum lima and its associated bacteria: I. An ultrastructural study

Summary Prorocentrum lima Ehrenberg (Dodge) and its associated bacteria were examined by scanning and transmission electron microscopy. The absence of trichocysts in this species is confirmed. The peculiarities of this P. lima strain (PL2V) were an abundance of intracellular reserves and extensive development of fibrous bodies, probably constituting a part of the pusule. Associated bacterial microflora was abundant. Various morphological types of free-living bacteria were found free-floating in the liquid culture medium. Numerous bacteria were directly attached to the dinoflagellate cell or agglomerated, forming large aggregates in the P. lima mucus. Bacteria-like inclusions were visible in a few percent of the P. lima cells observed. We suggest that they were intracellular bacteria, and that bacterial infection within this toxic dinoflagellate is possible, but occurs only rarely. This SEM and TEM study provides the first evidence for an association between this toxic dinoflagellate and surrounding bacteria.

Cyclic Expression of A Nuclear Protein In A Dinoflagellate

ABSTRACT. 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 (Dinapl), 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 Dinapl showed that the nucleus was immunoreactive only during the G1 phase: the nucleoplasm was immunostained. while chromosome cores and nuclear envelopes were negative.

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)

The toxic dinoflagellate Prorocentrum lima and its associated bacteria: II. Immunolocalization of okadaic acid in axenic and non-axenic cultures

Summary An immunofluorescence method, using a monoclonal antibody against okadaic acid (OA) toxin and DNA staining, was used to visualize the toxin and extranuclear DNA which could possibly be of intracellular bacterial origin, in the dinoflagellate species Prorocentrum lima Ehrenberg (Dodge). Okadaic acid (OA) was detected within the cytoplasm of the dinoflagellate cell, frequently near the cytoplasmic membrane in the cell periphery and occasionally close to the nuclear membrane in the center of the cytoplasm. These results suggest that OA production could be related to the peripheral chloroplast and that OA accumulation may be associated with membrane lipids. OA is not co-localized with extra-nuclear DNA, implying that intracellular bacteria do not contain or accumulate the toxin. Comparison between axenic and non-axenic dinoflagellate cultures showed no differences in immunolabelling, suggesting that extracellular associated bacteria are not essential to okadaic acid production by the P. lima culture. These results tend to show that the dinoflagellate cell is able to produce OA autonomously.

Argyrophilic proteins in Dinoflagellates: An ultrastructural, cytochemical and immunocytochemical study

Summary— Dinoflagellate protists constitute an original eukaryotic phylum and have an ancestor in common with ciliates. They are important tools in studies of structure and function of the nucleus because they present a mixing of prokaryotic characteristics such as chromatin devoid of histones and nucleosomes, eukaryotic characteristics such as the presence of a nuclear membrane, nucleoli and AgNOR‐like proteins and original characteristics of their own. Among them are the permanent compaction of the chromosomes, the presence of a nuclear envelope during the whole cell cycle, rare bases in their DNA, as well as an original mitosis. We have studied the distribution of the nuclear argyrophilic proteins (AgP) in three genera of Dinoflagellates (Prorocentrum, Crypthecodinium and Amphidinium) by means of light microscopy (LM) and electron microscopy (EM), using cytochemical silver staining and immunocytochemical reactions following various preparation procedures. By means of the silver staining reaction, we determined by LM the distribution of nucleoli in the three non‐synchronized cell populations and localized by EM the presence of AgP. These are always found in the nucleolar fibrillo‐granular compartment (FG) and partly in the chromosomes and in the nucleolar UCh (unwound region of the nucleolar chromosome corresponding to the NOR); the chromosomes and the UCh are always stained in P micans, under special conditions in C cohnii but never in A carterae. To determine whether these nucleolar and chromosomal proteins are similar or different, we modified the conditions of the silver staining reaction by acidic, alkaline or enzymatic pretreatments and changes in the reactions temperature. Our results suggested that these proteins belong to different groups. We have characterized one of these proteins using a mammalian anti‐B23 Ab in P micans cells. Positive labeling was mostly detected in chromosomes and UCh and in a smaller amount in the nucleolar FG and G compartments, co‐locating with end‐products of the silver staining reaction. This suggests that: i) one among the dinoflagellate chromosomal AgP is analogous to the B23 mammalian protein; and ii) this B23‐like protein is probably a DNA partner.

Association between fetal DES-exposure and psychiatric disorders in adolescence/adulthood: evidence from a French cohort of 1002 prenatally exposed children

Abstract In utero diethylstilbestrol (DES) exposure has been demonstrated to be associated with somatic abnormalities in adult men and women. Conversely, the data are contradictory regarding the association with psychological or psychiatric disorders during adolescence and adulthood. This work was designed to determine whether prenatal exposure to DES affects brain development and whether it is associated with psychiatric disorders in male and female adolescents and young adults. HHORAGES Association, a national patient support group, has assembled a cohort of 1280 women who took DES during pregnancy. We obtained questionnaire responses from 529 families, corresponding to 1182 children divided into three groups: Group 1 (nu2009=u2009180): firstborn children without DES treatment, Group 2 (nu2009=u2009740): exposed children, and Group 3 (nu2009=u2009262): children born after a previous pregnancy treated by DES. No psychiatric disorders were reported in Group 1. In Group 2, the incidence of disorders was drastically elevated (83.8%), and in Group 3, the incidence was still elevated (6.1%) compared with the general population. This work demonstrates that prenatal exposure to DES is associated with a high risk of psychiatric disorders in adolescence and adulthood.