71 research outputs found
Transcriptome analysis of scarce biological material: a pragmatic approach to increase differential screening performance
National audienc
DĂ©finition des Ă©tats de pluripotence in vivo dans lâembryon prĂ©implantatoirede lapin
National audienceChez le lapin, diffĂ©rentes lignĂ©es de cellules souches pluripotentes (PSC) sont accessibles Ă diffĂ©rents niveaux de pluripotence. De plus, les cellules embryonnaires pluripotentes de lapin sont facilement accessibles du fait de lâimplantation tardive chez le lapin et du grand nombre dâembryons produits par lapine. Enfin, le lapin est proche phylogĂ©nĂ©tiquement des rongeurs et des primates et son dĂ©veloppement embryonnaire prĂ©sente peu de particularitĂ©s par rapport aux autres mammifĂšres Ă©tudiĂ©s. Cependant, les diffĂ©rents processus rĂ©gulant la pluripotence au cours du dĂ©veloppement embryonnaire prĂ©-implantatoire du lapin sont relativement peu connus par rapport Ă ceux de la souris et des primates.Câest pourquoi nous avons rĂ©alisĂ© diffĂ©rentes analyses transcriptomiques sur lâembryon prĂ©-implantatoire de lapin, complĂ©tĂ©es par des analyses molĂ©culaires sur le mĂ©tabolisme et lâĂ©pigĂ©nome. GrĂące Ă ces donnĂ©es, nous avons pu dĂ©crire la sĂ©grĂ©gation des premiers lignages embryonnaires, ainsi que les processus rĂ©gulant le continuum de pluripotence dans lâembryon de lapin. Ces rĂ©sultats montrent que le continuum de pluripotence chez le lapin est trĂšs similaire Ă celui des autres espĂšces de mammifĂšres euthĂ©riens, lĂ oĂč celui de la souris possĂšde de nombreuses particularitĂ©s. Ces informations offrent Ă©galement de nouvelles pistes dans lâĂ©tude de la pluripotence in vitro. LâhĂ©tĂ©rochromatine et le cycle cellulaire semblent ĂȘtre des cibles prometteuses dans la manipulation de PSC de lapin. Des expĂ©riences prĂ©liminaires sur une lignĂ©e de PSC de lapin suggĂšrent qu'il est effectivement possible de manipuler lâĂ©tat de pluripotence des cellules traitĂ©es Ă lâaide dâinhibiteurs ciblant les enzymes favorisant la formation de lâhĂ©tĂ©rochromatine
Epigenetic programming of the rabbit preimplantation embryo in a diabetic context
International audienc
Early development of bovine embryos is influenced by the origin of feeders during in vitro culture
National audienceThe early development of embryo is clearly impacted by its environment and more specifically by the oviduct secretions in vivo. In cattle, an in vitro model of co-culture of bovine oviduct epithelial cells (BOEC) with the embryo has been developed to mimic the in vivo oviduct/embryo crosstalk. On the other hand, several other co-culture systems were previously developed including co-culture with monkey VERO fibroblasts to improve bovine embryo quality. Nevertheless, to the best of our knowledge, no comparison of co-culture systems using BOEC and other cells has been done yet to analyze if there is a specific impact of BOEC on bovine embryo. To answer to this question, we compared the influence of BOEC as well as VERO cells on bovine early development. Because co-culture with BOEC cells require culture at 20%O2, we included two control conditions: embryos cultured at 20%O2 and embryos cultured at 5% O2 in SOF medium + 5% SVF. No difference of cleavage rates were observed. No difference in the timing and rate of 16 cell stage development were observed despite this stage just follows EGA which is very sensitive to environmental conditions. Blastocyst rates are currently analyzed. To have new insight on the embryo quality at the 16-cell stage, a high throughput transcriptomic analysis was developed. Therefore, a new bovine microarray was designed including more than 26 700 transcripts and 250 retroviral EST. Considering an adjusted p value < 0.05, only one gene was differentially expressed between 5% O2 and 20%O2 conditions. Thus, 16-cell embryos seem to not respond to the induced oxidative stress. The direct comparison of the two co-culture conditions revealed only 19 differential expressed genes. Nevertheless, the presence of VERO or BOEC induced differential expression of 125 and 1162 genes respectively when compared to 5% O2 and 1209 and 2186 genes respectively when compared to 20% O2. Interestingly functional analysis using DAVID software pointed to different biological pathways according to the cell types used for co-culture. Further analyses at the blastocyst stage will be necessary to clarify the differential impact of cell types used in co-culture systems on embryo quality
Impact dâun environnement diabĂ©tique sur le dĂ©veloppement embryonnaire prĂ©coce
International audienceDiabĂšte et obĂ©sitĂ©, de prĂ©valence croissante, ont un abaissement de lâĂąge de survenue. Les 1ers signes affectent dĂ©sormais les femmes en Ăąge de procrĂ©er. De nombreuses grossesses sont alors affectĂ©es par une hyperglycĂ©mie associĂ©e Ă une hyperinsulinĂ©mie compensatrice en pĂ©riode pĂ©ri-conceptionnelle sans en connaĂźtre les consĂ©quences sur la descendance (1). Des perturbations de lâenvironnement in utero peuvent programmer un risque accru de dĂ©velopper ces mĂȘmes pathologies mĂ©taboliques Ă lâĂąge adulte (2-3). Nous Ă©tudions lâeffet dâune supplĂ©mentation en glucose (15mM) et/ou en insuline (1,7”M) sur le dĂ©veloppement in vitro dâembryon de lapin. Les embryons, collectĂ©s au stade 1 cellule, sont mis en culture jusquâau stade blastocyste, stade composĂ© de deux compartiments, la masse cellulaire interne (individu) et le trophectoderme (placenta). Les transcriptomes ont Ă©tĂ© rĂ©alisĂ©s par RNA-Seq. Un dĂ©veloppement en prĂ©sence de glucose induit un nombre de gĂšne diffĂ©rentiellement exprimĂ© (DEG) plus important dans le trophectoderme que dans la masse cellulaire interne (104vs35), gĂšnes impliquĂ©s dans la prolifĂ©ration, lâapoptose, la diffĂ©renciation et le remodelage de la chromatine. AprĂšs un dĂ©veloppement en prĂ©sence de glucose et dâinsuline, le nombre DEG est plus faible dans le trophectoderme que dans la masse cellulaire interne (15vs104). Les voies dĂ©rĂ©gulĂ©es sont impliquĂ©es dans la prolifĂ©ration, la diffĂ©renciation, la transcription, la phosphorylation oxydative et le stress oxydant. Dans les deux conditions, les embryons prĂ©sentent une augmentation dans la masse cellulaire interne de lâexpression de gĂšnes impliquĂ©s dans la diffĂ©renciation du trophectoderme. Nous montrons quâun dĂ©veloppement de lâembryon en condition (prĂ©)-diabĂ©tique induit une dĂ©rĂ©gulation de voies impliquĂ©es dans le mĂ©tabolisme Ă©nergĂ©tique, la prolifĂ©ration et la diffĂ©renciation avec une altĂ©ration de lâallocation des cellules au placenta, organe clĂ© dans la mĂ©diation de la programmation. La dĂ©rĂ©gulation des gĂšnes impliquĂ©s dans le remodelage de la chromatine suggĂšre un impact au niveau de la rĂ©gulation Ă©pigĂ©nĂ©tique de lâexpression des gĂšnes
Effects of BOEC and VERO co-culture systems on bovine early development
International audienceEmbryo development is known to be impacted by its environment and especially by oviductal secretions in vivo. In cattle, embryo co-culture with bovine oviduct epithelial cells (BOEC) has been developed to mimic the in vivo oviduct/embryo crosstalk. However, whether BOEC had a specific impact on embryo transcriptome hasnât been investigated yet. We thus compa-red bovine embryos co-cultured with BOEC to embryos co-cultured with VERO cells (a kid-ney epithelial cell line from monkey). Control embryos were obtained in SOF medium + 5% Fetal Calf Serum (FCS) at 5% O2. Because co-culture systems require 20% O2, embryos cul-tured at 20% O2 in SOF + 5% FCS were included as an additional control. No impact of co-culture systems was observed on timing and developmental rates. 16-cell stage embryos and day 8 blastocysts' transcriptomes were analyzed on a new customized bovine microarray (GPL21734). Comparing the two co-culture conditions revealed only 14 and 10 differentially expressed transcripts respectively at 16-cell and blastocyst stages suggesting almost no diffe-rence induced by the type of co-culture. Nevertheless, BOEC or VERO cells induced differential expression of 83 and 51 transcripts respectively when compared to 5% O2 and 218 and 309 transcripts respectively when compared to 20% O2 in 16-cell embryos and 192 and 229 transcripts respectively when compared to 5% O2 and 542 and 881 transcripts respectively when compared to 20% O2 in blastocysts. A large proportion of the transcripts affected by BOEC presence were also impacted by VERO cells. Several biofunctions relative to cell cycle regulation and lipid metabolism were impacted by both cell types when compared to culture in SOF without feeder cells. Collectively, co-culture systems, using BOEC or VERO cells, induce weak and closely related modifications of embryos transcriptome without improvement of cleavage and blastocyst rates when compared to standard 5% O2 culture conditions
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