Biomedical subjects

S Rousseaux

Publications and source records attributed to S Rousseaux.

At least 19 recordsLinked to original sources

PubMed2006

[Epigenetics of the sperm cell].

In addition to genetic information, the spermatozoon carries another type of information, named epigenetic, which is not associated with variations of the DNA sequence. In somatic cells, it is now generally admitted that epigenetic information is not only regulated by DNA methylation but also involves modifications of the genome structure, or epigenome. During male germ cell maturation, the epigenome is globally re-organized, since most histones, which are associated to DNA in somatic cells, are removed and replaced by sperm specific nuclear proteins, the protamines, responsible for the tight compaction of the sperm DNA. However, a small proportion of histones, and probably other proteins, are retained within the sperm nucleus, and the structure of the sperm genome is actually heterogeneous. This heterogeneity of the sperm epigenome could support an epigenetic information, transmitted to the embryo, which could be crucial for its development. Although it is nowadays possible to appreciate the global structure of the sperm genome, the precise constitution of the sperm epigenome remains unknown. In particular, very recent data suggest that specific regions of the genome could be associated with particular proteins and define specific structures. This structural partitioning of the sperm genome could convey important epigenetic information, crucial for the embryo development.

DNA↗

PubMed2006

Histone acetylation-mediated chromatin compaction during mouse spermatogenesis.

One of the most dramatic chromatin remodelling events takes place during mammalian spermatogenesis involving massive incorporation of somatic and testis-specific histone variants, as well as generalized histone modifications before their replacement by new DNA packaging proteins. Our data suggest that the induced histone acetylation occurring after meiosis may direct the first steps of genome compaction. Indeed, a double bromodomain-containing protein expressed in postmeiotic cells, Brdt, shows the extraordinary capacity to specifically condense acetylated chromatin in vivo and in vitro. In elongating spermatids, Brdt widely co-localizes with acetylated histones before accumulating in condensed chromatin domains. These domains preferentially maintain their acetylation status until late spermatogenesis. Based on these data, we propose that Brdt mediates a general histone acetylation-induced chromatin compaction and also maintains differential acetylation of specific regions, and is therefore involved in organizing the spermatozoon's genome.

Acetylation↗

PubMed2004

[Organizing the sperm nucleus].

Thanks to the success of new assisted reproductive technology, including sperm microinjection (i.c.s.i.), men with severe spermatogenesis impairments can now become biological fathers. Whether the germinal cell used for i.c.s.i. is conveying appropriate genetic and epigenetic information is an important concern. However, to date, there is a huge lack of data on which information is epigenetically conveyed to the offspring and how. The basic support for epigenetic marks is the nucleus structure. During spermatogenesis, a major re-organization of the male germ cells nucleus structure occurs, which includes a global condensation associated with a removal of most core somatic histones and their replacement by sperm-specific nuclear proteins. The available data on the molecular mechanisms involved in this process and how it could relate to the setting of male-specific epigenetic information is reviewed and discussed in light of our current knowledge about nuclear structure and functions.

Cell Nucleus↗

PubMed2004

Use of PCR-restriction fragment length polymorphism of inlA for rapid screening of Listeria monocytogenes strains deficient in the ability to invade Caco-2 cells.

A PCR-restriction fragment length polymorphism (RFLP) method was developed in order to screen a large number of strains for impaired adhesion to epithelial cells due to expression of truncated InlA. inlA polymorphism was analyzed by PCR-RFLP in order to correlate inlA PCR-RFLP profiles and production of truncated InlA. Thirty-seven Listeria monocytogenes strains isolated from various sources, including five noninvasive and two invasive reference strains, were screened. Two endonucleases (AluI and Tsp509I) were used, and they generated five composite profiles. Thirteen L. monocytogenes isolates were characterized by two specific PCR-RFLP profiles similar to PCR-RFLP profiles of noninvasive reference strains previously described as strains that produce truncated InlA. Ten of the 13 isolates showed low abilities to invade human epithelial Caco-2 cells. However, 4 of the 13 isolates were able to invade Caco-2 cells like reference strains containing complete InlA. Sequencing of inlA and Western blot analysis confirmed that truncated InlA was expressed in the 10 L. monocytogenes strains which were isolated from food. This PCR-RFLP method allowed us to identify 10 new strains expressing a truncated internalin. Based on the results obtained in this study, the PCR-RFLP method seems to be an interesting method for rapidly screening L. monocytogenes strains deficient in the ability to invade Caco-2 cells when a sizeable number of strains are studied.

Bacterial Proteins↗

PubMed2003

Inoculation of an atrazine-degrading strain, Chelatobacter heintzii Cit1, in four different soils: effects of different inoculum densities.

The possibility to improve atrazine degradation in soils by bioaugmentation was studied. The atrazine-mineralizing strain, Chelatobacter heintzii Cit1, was inoculated in four sterile and four non-sterile soils, at varying inoculum densities. Two soils, which had shown enhanced atrazine mineralization, were used to determine which inoculum density was capable of restoring their original mineralizing capacity after sterilization. The two other soils, with intermediate and low capacity to mineralize atrazine, were used in order to demonstrate that atrazine mineralization in such soils could be improved by inoculation. Mineralization kinetics were fitted using the Gompertz model. In the case of soils adapted to atrazine mineralization, inoculation of C. heintzii did not accelerate the rate of atrazine mineralization, which was essentially performed by the indigenous microflora. However, with soils that did not mineralize atrazine, the introduction of 10(4) cfug(-1) resulted in a 3-fold increase of atrazine mineralization capacity.

Atrazine↗

PubMed2003

Misregulation of histone acetylation in Sertoli cell-only syndrome and testicular cancer.

In many species, including humans, chromatin remodelling during spermiogenesis is initiated with a marked increase in histone acetylation in elongating spermatids. We have investigated whether this process is disturbed when spermatogenesis is defective or in human testicular tumours. For this purpose, the presence of highly acetylated histone H4 was detected on testicular sections from men with a severe impairment of spermatogenesis of several origins, as well as in different types of testicular tumours. In most tubules devoid of germinal cells (including SCO, Sertoli cell only syndromes) or lacking spermatocytes and spermatids, the Sertoli cells' nuclei showed a global increase in histone H4 acetylation. A similar observation was made in the peritumoral seminiferous tubules of testicular tumour tissues, whenever they were lacking germinal cells, with carcinoma in situ (CIS) cells being hypoacetylated. The global hyperacetylation of elongating spermatids during spermatogenesis could be part of an intercellular signalling pathway involving Sertoli cells and germinal cells, which could be disturbed in cases of severe spermatogenesis impairment, as well as in tubes surrounding germ cells in testicular tumours.

Acetylation↗

PubMed2002

Polyploidy in large-headed sperm: FISH study of three cases.

BACKGROUND: Macrocephalic or large headed sperm with multiflagella is a rare abnormality often associated with infertility. Sperm chromosomal abnormalities could be associated with this specific morphological abnormality. METHODS: The cytogenetic content of large-headed sperm was assessed by dual and three-colour fluorescence in-situ hybridization in three patients carrying this specific morphological abnormality. RESULTS: In all patients nearly all sperm contained at least one copy of each sex chromosome, and in more than half of them at least two copies of either chromosome 1 or 18 were identified. In some sperm a tetraploidy was found. CONCLUSIONS: These observations suggested that both meiotic I and II divisions were affected by incomplete partition of homologous chromosomes during meiosis I and of sister chromatids during meiosis II associated with a failure of nuclear cleavage. Furthermore, they provide evidence for a clear relationship between a specific morphological abnormality of the sperm and their abnormal cytogenetic content. The treatment of infertility using ICSI would probably be unsuccessful and have a high genetic risk in these cases.

Adult↗

PubMed2001

Risk of trisomy 21 in offspring of patients with Klinefelter's syndrome.

Intracytoplasmic sperm injection (ICSI) has given some patients with Klinefelter's syndrome (ie, men with an XXY sex-chromosome profile) the chance to become fathers, but the genetic makeup of the spermatozoa used for the injection is a concern. We studied the segregation of the sex chromosomes and chromosomes 1 and 21 by multicolour fluorescence in-situ hybridisation in a patient with non-mosaic Klinefelter's syndrome who was a candidate for ICSI. As other workers have found, we saw a higher rate of 24,XX and 24,XY spermatozoa in the patient than in controls. However, we also found a much higher frequency of disomy 21 in the spermatozoa of this patient than in controls (6.2 vs 0.4%). Any child conceived by ICSI using this man's sperm will thus have a proportionally higher risk of trisomy 21.

Chromosome Aberrations↗

PubMed2001

Isolation and characterisation of new Gram-negative and Gram-positive atrazine degrading bacteria from different French soils.

The capacity of 12 soils to degrade atrazine was studied in laboratory incubations using radiolabelled atrazine. Eight soils showed enhanced degradation of this compound. Twenty-five bacterial strains able to degrade atrazine were isolated by an enrichment method from 10 of these soils. These soils were chosen for their wide range of physico-chemical characteristics. Their history of treatment with atrazine was also variable. The genetic diversity of atrazine degraders was determined by amplified ribosomal restriction analysis (ARDRA) of the 16S rDNA gene with three restriction endonucleases. The 25 bacterial strains were grouped into five ARDRA types. By sequencing and aligning the 16S rDNA genes, the isolates were shown to belong to the Gram-negative species Chelatobacter heintzii, Aminobacter aminovorans, Stenotrophomonas maltophilia and to the Gram-positive genus Arthrobacter crystallopoietes. These species were not described previously as being capable of atrazine degradation. Most Gram-negative bacteria could mineralise (14)C ring labelled atrazine and carried the atzA, atzB, atzC and trzD genes. Gram-positive strains could convert atrazine to cyanuric acid and carried only the atzB and atzC genes. In this study, we describe the atrazine degradation capacities and corresponding genes in bacterial species that were not known as atrazine degraders. We report for the first time the occurrence of the trzD gene in these atrazine-mineralising bacteria and we demonstrate the potential use of colony hybridisation to isolate bacteria involved in atrazine degradation.

Journal Article↗

PubMed2001

Identification of components of the murine histone deacetylase 6 complex: link between acetylation and ubiquitination signaling pathways.

The immunopurification of the endogenous cytoplasmic murine histone deacetylase 6 (mHDAC6), a member of the class II HDACs, from mouse testis cytosolic extracts allowed the identification of two associated proteins. Both were mammalian homologues of yeast proteins known to interact with each other and involved in the ubiquitin signaling pathway: p97/VCP/Cdc48p, a homologue of yeast Cdc48p, and phospholipase A2-activating protein, a homologue of yeast UFD3 (ubiquitin fusion degradation protein 3). Moreover, in the C-terminal region of mHDAC6, a conserved zinc finger-containing domain named ZnF-UBP, also present in several ubiquitin-specific proteases, was discovered and was shown to mediate the specific binding of ubiquitin by mHDAC6. By using a ubiquitin pull-down approach, nine major ubiquitin-binding proteins were identified in mouse testis cytosolic extracts, and mHDAC6 was found to be one of them. All of these findings strongly suggest that mHDAC6 could be involved in the control of protein ubiquitination. The investigation of biochemical properties of the mHDAC6 complex in vitro further supported this hypothesis and clearly established a link between protein acetylation and protein ubiquitination.

Acetylation↗

PubMed2000

Active maintenance of mHDA2/mHDAC6 histone-deacetylase in the cytoplasm.

The intracellular localization, and thereby the function, of a number of key regulator proteins tagged with a short leucine-rich motif (the nuclear export signal or NES) is controlled by CRM1/exportin1, which is involved in the export of these proteins from the nucleus [1]. A common characteristic of these regulators is their transient action in the nucleus during either a specific phase of the cell cycle or in response to specific signals [1]. Here, we show that a particular member of the class II histone-deacetylases mHDA2/mHDAC6 [2] belongs to this family of cellular regulators that are present predominantly in the cytoplasm, but are also capable of shuttling between the nucleus and the cytoplasm. A very potent NES present at the amino terminus of mHDAC6 was found to play an essential role in this shuttling process. The sub-cellular localization of mHDAC6 appeared to be controlled by specific signals, since the arrest of cell proliferation was found to be associated with the translocation of a fraction of the protein into the nucleus. Data presented here suggest that mHDAC6 might be the first member of a functionally distinct class of deacetylases, responsible for activities not shared by other known histone deacetylases.

Amino Acid Sequence↗

PubMed2000

Genome organization in the human sperm nucleus studied by FISH and confocal microscopy.

The sperm nucleus has a unique chromatin structure where the DNA is highly condensed and associated with specific proteins, the protamines. It is a nondividing cell which is also transcriptionally inactive. After fusion with an oocyte, the sperm nucleus undergoes decondensation and, in the same time, starts replication and transcription. It has been suggested that somatic chromosomes during interphase are organized in territories which display a cell type and cell cycle specific distribution. The purpose of this work was to investigate whether chromosomes would also have a specific distribution in the sperm nucleus, which could be related to its inactive state, and have implications on the early stages of fertilization. In the present study, centromeric and telomeric sequences were detected by fluorescent techniques performed on human decondensed spermatozoa. Chromosome painting probes were used to detect the chromosome X and chromosome 13 on interphase sperm nuclei. The fluorescent signals were captured in 3D with a confocal microscope. For each of these chromatin structures, the volume, position, and distribution of the signals were analyzed in samples of 30 nuclei with the help of image analysis software. The centromeres appeared grouped in several foci that were randomly distributed within the sperm nucleus. The telomeres gave an approximately haploid number of small signals, evenly distributed throughout the nucleus. The chromosomes X and 13 occupied 4.7% and 3. 7% of the total nuclear volume, respectively. Interestingly, the X chromosome territory showed a preferential position in the anterior half of the volume of the nucleus, whereas chromosome 13 had a random position. This work shows a particular distribution of chromosome territories in the human sperm nucleus that could be related to mechanisms implicated in its specific functions. The analysis of more chromosomes and chromosomal structures, including the Y chromosome, would help to understand the structure of the human sperm chromatin, and its fundamental and clinical implications.

Cell Nucleus↗

PubMed2000

Regulated hyperacetylation of core histones during mouse spermatogenesis: involvement of histone deacetylases.

Here we report a detailed analysis of waves of histone acetylation that occurs throughout spermatogenesis in mouse. Our data showed that spermatogonia and preleptotene spermatocytes contained acetylated core histones H2A, H2B and H4, whereas no acetylated histones were observed throughout meiosis in leptotene or pachytene spermatocytes. Histones remained unacetylated in most round spermatids. Acetylated forms of H2A and H2B, H3 and H4 reappeared in step 9 to 11 elongating spermatids, and disappeared later in condensing spermatids. The spatial distribution pattern of acetylated H4 within the spermatids nuclei, analyzed in 3D by immunofluorescence combined with confocal microscopy, showed a spatial sequence of events tightly associated with chromatin condensation. In order to gain an insight into mechanisms controlling histone hyperacetylation during spermiogenesis, we treated spermatogenic cells with a histone deacetylase inhibitor, trichostatin A (TSA), which showed a spectacular increase of histone acetylation in round spermatids. This observation suggests that deacetylases are responsible for maintaining a deacetylated state of histones in these cells. TSA treatment could not induce histone acetylation in condensing spermatids, suggesting that acetylated core histones are replaced by transition proteins without being previously deacetylated. Moreover, our data showed a dramatic decrease in histone deacetylases in condensing spermatids. Therefore, the regulation of histone deacetylase activity/concentration appears to play a major role in controling histone hyperacetylation and probably histone replacement during spermiogenesis.

Acetylation↗

PubMed1998

Disomy rates for chromosomes 14 and 21 studied by fluorescent in-situ hybridization in spermatozoa from three men over 60 years of age.

In order to further investigate the paternal-age effect on meiotic non-disjunction rates for the chromosomes 14 and 21, we examined spermatozoa from three men aged > 60, using multicolour fluorescent in-situ hybridization (FISH). More than 10,000 sperm cells were analysed for each of the three subjects (A, B and C), by simultaneously hybridizing two YAC probes specific for chromosomes 14 and 21 respectively using two-colour FISH. The results show that the disomy 21 rates observed in the spermatozoa of two out of the three men aged > 60 years were higher (1.02 and 1.17% respectively) than the rates observed in eight control adults aged < 30 years (mean frequency 0.48%) analysed under similar conditions. These results suggest that there may be a small effect of age on male non-disjunction rates for chromosome 21. However, before any firm conclusions could be drawn, a much bigger sample of older men would have to be compared with a paired control population using the same FISH experimental approach.

Adult↗

PubMed1998

Application of fluorescence in situ hybridization to the identification of different marker chromosomes.

Chromosome studies performed on lymphocyte culture of a baby with specific dysmorphism and congenital anomalies suggestive of trisomy 21 revealed a mosaicism: 46,XY,rea(21q21q) [25]/47,XY,rea(21q21q),+mar1[25]. The karyotype of the mother is normal, but the father's karyotype presents an supernumerary chromosome greater and different from the marker of his son: 47,XY,+mar2 (100%). The identification of the two marker chromosomes by standard cytogenetic techniques followed by molecular techniques is essential for the identification of the origin of these two chromosomes. The unusual presence of two different markers one in the father and one in the son, as well as the clinical features of the child, are presented. The possible role of the paternal marker, in the de novo chromosomal rearrangement in his child will be discussed.

Abnormalities, Multiple↗

PubMed1997

Dynamic molecular combing: stretching the whole human genome for high-resolution studies.

DNA in amounts representative of hundreds of eukaryotic genomes was extended on silanized surfaces by dynamic molecular combing. The precise measurement of hybridized DNA probes was achieved directly without requiring normalization. This approach was validated with the high-resolution mapping of cosmid contigs on a yeast artificial chromosome (YAC) within yeast genomic DNA. It was extended to human genomic DNA for precise measurements ranging from 7 to 150 kilobases, of gaps within a contig, and of microdeletions in the tuberous sclerosis 2 gene on patients' DNA. The simplicity, reproducibility, and precision of this approach makes it a powerful tool for a variety of genomic studies.

Calpain↗

PubMed1997

A 1.7-megabase sequence-ready cosmid contig covering the TSC1 candidate region in 9q34.

The disease gene TSC1 has been genetically mapped to human chromosome region 9q34, in a 4-cM interval between the markers D9S149 and D9S114. Within this interval there is conflicting genetic evidence as to the finer localization of the gene. We have used finger-printing methods and hybridization to produce a 1.7-Mb overlapping clone map covering the TSC1 candidate region, with a single gap of 20 kb. We have localized 12 previously cloned genes and 17 genetic markers on this map and have confirmed the order of the genetic map. This deep set of overlapping clones is now ready to be used for candidate gene isolation, for transcription studies, or for sequencing.

Chromosome Mapping↗

PubMed1997

Meiotic behaviour of sex chromosomes investigated by three-colour FISH on 35,142 sperm nuclei from two 47,XYY males.

Meiotic segregation of sex chromosomes from two fertile 47,XYY men was analysed by a three-colour fluorescence in situ hybridisation procedure. This method allows the identification of hyperhaploidies (spermatozoa with 24 chromosomes) and diploidies (spermatozoa with 46 chromosomes), and their meiotic origin (meiosis I or II). Alpha-satellite probes specific for chromosomes X, Y and 1 were observed simultaneously in 35,142 sperm nuclei. For both 47,XYY men (24,315 sperm nuclei analysed from one male and 10,827 from the other one) the sex ratio differs from the expected 1:1 ratio (P < 0.001). The rates of disomic Y, diploid YY and diploid XY spermatozoa were increased for both 47,XYY men compared with control sperm (142,050 sperm nuclei analysed from five control men), whereas the rates of hyperhaploidy XY, disomy X and disomy 1 were not significantly different from those of control sperm. These results support the hypothesis that the extra Y chromosome is lost before meiosis with a proliferative advantage of the resulting 46,XY germ cells. Our observations also suggest that a few primary spermatocytes with two Y chromosomes are able to progress through meiosis and to produce Y-bearing sperm cells. A theoretical pairing of the three gonosomes in primary spermatocytes with an extra sex chromosome, compatible with active spermatogenesis, is proposed.

Adult↗