Identification of various testicular cell populations in pubertal and adult cockerels

Precise identification of the male germinal stem cell population is important for their practical use in programs dedicated to the integration of exogenous genetic material in testicular tissues. In the present study, our aim was to identify germinal cell populations in the testes of pubertal and adult cockerels based on the detection of the nuclear DNA content by fluorescence-activated cell sorting (FACS) and on the expression of the Dazl and Stra8 genes in singlecell suspensions of testicular tissues. Cells with a tetraploid DNA content (4c) represent a small and equal fraction of the total germinal cell population in both pubertal and adult males. In contrast, the diploid (2c) and haploid (c) subpopulations differ significantly between ages as a consequence of different degrees of sexual maturation. A specific subpopulation of testicular cells, the side-scatter subpopulation of cells, or side population (SP), was identified at the junction between the haploid and diploid cell populations. The percentage of this cell subpopulation differs significantly in pubertal and adult cockerels, accounting for 4.1% and 1.3% of the total cell population, respectively. These four testicular cell populationswere also tested for the expression of Dazl and Stra8 genes known to be expressed in premeiotic cells including stem spermatogonia. Both genes were expressed in SP, whereas the expression of either Dazl or Stra8 genes was detected only in the 4c and in the 2c testicular cell subpopulations, respectively. The correlation between the cell ploidy and Dazl/Stra8 expression was the same at both male ages. We conclude that SP cells might represent a subpopulation of germinal cells enriched in stem spermatogonia, which can be of great importance for transgenesis in chicken

Etude de la solubilisation des membranes lipidiques (mécanismes impliqués dans la transition magnétoliposomes-micelles)

Les liposomes sont des objets artificiels de taille variable possédant une bicouche lipidique fermée sur un milieu aqueux. Des molécules hydrophobes peuvent être insérées dans cette bicouche lipidique et des molécules hydrophiles peuvent être encapsulées dans le milieu aqueux, ce qui fait de ces objets de précieux vecteurs de médicaments et autorisent de nombreuses études tant dans les domaines pharmaceutiques que physico-chimiques et chimiques. Les vésicules géantes, objets phares de ce travail sont utilisées comme modèles cellulaires simples. Cependant, leur utilisation pour l étude des phénomènes de solubilisation, qui est le processus de transformation de vésicules en micelles mixtes sous l effet d un détergent, est encore méconnue. Au cours de notre étude, des vésicules géantes (GUV) observables au microscope optique et enfermant dans leur milieu aqueux une dispersion colloïdale magnétique (ferrofluide) vont servir de modèle pour l étude du processus de solubilisation. Ce dernier comporte trois domaines : le domaine vésiculaire, le domaine de coexistence vésicules/micelles mixtes et le domaine micellaire. Cette étude s est concentrée à l exploration du domaine vésiculaire. L interaction de la membrane avec un détergent a été suivie dans un premier temps par mesure de turbidité sur des vésicules de taille moyenne (LUV) non magnetiques et dans un second temps par microscopie optique sur un système mixte comportant les GUV magnétiques et les LUV non magnétiques. Les modifications morphologiques, conséquences de cette interaction (augmentation de surface apparente, changement de courbure, ouverture/fermeture de pores, vésiculation) ont ainsi été mises en évidence. Les nanoparticules confèrent aux GUV des propriétés magnétiques et les rendent ainsi déformables sous l action d une tension magnétique. On va ainsi avoir accès à des paramètres physiques (élasticité, tension de membrane ) de la membrane mixte détergent phospholipide.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Overexpression of TET dioxygenases in seminomas associates with low levels of DNA methylation and hydroxymethylation.

Germ cell tumors and particularly seminomas reflect the epigenomic features of their parental primordial germ cells, including genomic DNA hypomethylation and expression of pluripotent cell markers. Because the DNA hypomethylation might be a result of TET dioxygenase activity, we examined expression of TET1-3 enzymes and the level of their product, 5-hydroxymethylcytosine, in a panel of histologically characterized seminomas and non-seminomatous germ cell tumors. Expression of TET dioxygenase mRNAs was quantified by real-time PCR. TET1 expression and the level of 5-hydroxymethylcytosine were examined immunohistochemically. Quantitative assessment of 5-methylcytosine and 5-hydroxymethylcytosine levels was done by the liquid chromatography-mass spectroscopy technique. We found highly increased expression of TET1 dioxygenase in most seminomas and strong TET1 staining in seminoma cells. Isocitrate dehydrogenase 1 and 2 mutations were not detected, suggesting the enzymatic activity of TET1. The levels of 5-methylcytosine and 5-hydroxymethylcytosine in seminomas were found decreased in comparison to non-seminomatous germ cell tumors and healthy testicular tissue. We propose that TET1 expression should be studied as a potential marker of seminomas and mixed germ cell tumors and we suggest that elevated expression of TET dioxygenase enzymes is associated with the maintenance of low DNA methylation levels in seminomas. This "anti-methylator" phenotype of seminomas is in contrast to the CpG island methylator phenotype observed in a fraction of tumors of various types

The role of DNA methylation in expression and transmission of porcine endogenous retrovirus

Porcine endogenous retroviruses (PERV) represent a major safety concern in pig-to-human xenotransplantation. To date, no PERV infection of a xenograft recipient has been recorded; however, PERVs are transmissible to human cells in vitro. Some recombinants of the A and C PERV subgroups are particularly efficient in infection and replication in human cells. Transcription of PERVs has been described in most pig cells, but their sequence and insertion polymorphism in the pig genome impede identification of transcriptionally active or silenced proviral copies. Furthermore, little is known about the epigenetic regulation of PERV transcription. Here, we report on the transcriptional suppression of PERV by DNA methylation in vitro and describe heavy methylation in the majority of PERV 5′ long terminal repeats (LTR) in porcine tissues. In contrast, we have detected sparsely methylated or nonmethylated proviruses in the porcine PK15 cells, which express human cell-tropic PERVs. We also demonstrate the resistance of PERV DNA methylation to inhibitors of methylation and deacetylation. Finally, we show that the high permissiveness of various human cell lines to PERV infection coincides with the inability to efficiently silence the PERV proviruses by 5′LTR methylation. In conclusion, we suggest that DNA methylation is involved in PERV regulation, and that only a minor fraction of proviruses are responsible for the PERV RNA expression and porcine cell infectivity