Personne :
Robert, Claude

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Structures organisationnelles
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Université Laval. Département des sciences animales
Identifiant Canadiana

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Voici les éléments 1 - 8 sur 8
  • Publication
    Accès libre
    Genetic influence on the reduction in bovine embryo lipid content by L-carnitine
    (Commonwealth Scientific and Industrial Research Organization, 2015-01-13) Baldoceda Baldeon, Luis Manuel; Robert, Claude; Gagné, Dominic; Ramires Ferreira, Christina
    The decreased rate of pregnancy obtained in cattle using frozen in vitro embryos compared with in vivo embryos has been associated with over-accumulation of intracellular lipid, which causes cell damage during cryopreservation. It is believed that the higher lipid content of blastomeres of bovine embryos produced in vitro results in darker-coloured cytoplasm, which could be a consequence of impaired mitochondrial function. In this study, l-carnitine was used as a treatment to reduce embryonic lipid content by increasing metabolism in cultured bovine embryos. We have observed previously that in vivo embryos of different dairy breeds collected from cows housed and fed under the same conditions differed in lipid content and metabolism. As such, breed effects between Holstein and Jersey were also examined in terms of general appearance, lipid composition, mitochondrial activity and gene expression. Adding l-carnitine to the embryo culture medium reduced the lipid content in both breeds due to increased mitochondrial activity. The response to l-carnitine was weaker in Jersey than in Holstein embryos. Our results thus show that genetics influence the response of bovine embryos to stimulation of mitochondrial metabolism.
  • Publication
    Accès libre
    Breed specific factors influence embryonic lipid composition : comparison between Jersey and Holstein
    (Commonwealth Scientific and Industrial Research Organization, 2015-01-15) Baldoceda Baldeon, Luis Manuel; Vigneault, Christian; Gilbert, Isabelle; Gagné, Dominic; Robert, Claude; Blondin, Patrick.; Ramires Ferreira, Christina
    Some embryos exhibit better survival potential to cryopreservation than others. The cause of such a phenotype is still unclear and may be due to cell damage during cryopreservation, resulting from overaccumulation and composition of lipids. In cattle embryos, in vitro culture conditions have been shown to impact the number of lipid droplets within blastomeres. Thus far, the impact of breed on embryonic lipid content has not been studied. In the present study were compared the colour, lipid droplet abundance, lipid composition, mitochondrial activity and gene expression of in vivo-collected Jersey breed embryos, which are known to display poor performance post-freezing, with those of in vivo Holstein embryos, which have good cryotolerance. Even when housed and fed under the same conditions, Jersey embryos were found to be darker and contain more lipid droplets than Holstein embryos, and this was correlated with lower mitochondrial activity. Differential expression of genes associated with lipid metabolism and differences in lipid composition were found. These results show genetic background can impact embryonic lipid metabolism and storage.
  • Publication
    Accès libre
    Exploring the function of long non-coding RNA in the development of bovine early embryos
    (CSIRO publishing, 2014-12-04) Gilbert, Isabelle; Caballero, Julieta; Fournier, Éric; Scantland, Sara; Gagné, Dominique; Robert, Claude; Macaulay, Angus
    Now recognised as part of the cellular transcriptome, the function of long non-coding (lnc) RNA remains unclear. Previously, we found that some lncRNA molecules in bovine embryos are highly responsive to culture conditions. In view of a recent demonstration that lncRNA may play a role in regulating important functions, such as maintenance of pluripotency, modification of epigenetic marks and activation of transcription, we sought evidence of its involvement in embryogenesis. Among the numerous catalogued lncRNA molecules found in oocytes and early embryos of cattle, three candidates chosen for further characterisation were found unexpectedly in the cytoplasmic compartment rather than in the nucleus. Transcriptomic survey of subcellular fractions found these candidates also associated with polyribosomes and one of them spanning transzonal projections between cumulus cells and the oocyte. Knocking down this transcript in matured oocytes increased developmental rates, leading to larger blastocysts. Transcriptome and methylome analyses of these blastocysts showed concordant data for a subset of four genes, including at least one known to be important for blastocyst survival. Functional characterisation of the roles played by lncRNA in supporting early development remains elusive. Our results suggest that some lncRNAs play a role in translation control of target mRNA. This would be important for managing the maternal reserves within which is embedded the embryonic program, especially before embryonic genome activation.
  • Publication
    Cumulus cell transcripts transit to the bovine oocyte in preparation for maturation
    (Society for the Study of Reproduction., 2015-11-13) Gilbert, Isabelle; Ettaoumi, Sara; Bastien, Alexandre; Gagné, Dominic; Ashkar, Fazl; Khandjian, Edward William; Macaulay, Angus; Shojaei Saadi, Habib Allah; Richard, François J.; Robert, Claude; Fournier, Éric; Sirard, Marc-André; Hyttel, P.
    So far, the characteristics of a good quality egg have been elusive, similar to the nature of the physiological, cellular, and molecular cues leading to its production both in vivo and in vitro. Current understanding highlights a strong and complex interdependence between the follicular cells and the gamete. Secreted factors induce cellular responses in the follicular cells, and direct exchange of small molecules from the cumulus cells to the oocyte through gap junctions controls meiotic arrest. Studying the interconnection between the cumulus cells and the oocyte, we previously demonstrated that the somatic cells also contribute transcripts to the gamete. Here, we show that these transcripts can be visualized moving down the transzonal projections (TZPs) to the oocyte, and that a time course analysis revealed progressive RNA accumulation in the TZPs, indicating that RNA transfer occurs before the initiation of meiosis resumption under a timetable fitting with the acquisition of developmental competence. A comparison of the identity of the nascent transcripts trafficking in the TZPs, with those in the oocyte increasing in abundance during maturation, and that are present on the oocyte's polyribosomes, revealed transcripts common to all three fractions, suggesting the use of transferred transcripts for translation. Furthermore, the removal of potential RNA trafficking by stripping the cumulus cells caused a significant reduction in maturation rates, indicating the need for the cumulus cell RNA transfer to the oocyte. These results offer a new perspective to the determinants of oocyte quality and female fertility, as well as provide insight that may eventually be used to improve in vitro maturation conditions.
  • Publication
    The gametic synapse : RNA transfer to the bovine oocyte
    (Oxford University Press, 2014-10-01) Gilbert, Isabelle; Caballero, Julieta; Tossou, Prudencio; Khandjian, Edward William; Macaulay, Angus; Richard, François J.; Barreto, Rodrigo; Clarke, Hugh James; Robert, Claude; Fournier, Éric; Sirard, Marc-André; Hyttel, P.
    Even after several decades of quiescent storage in the ovary, the female germ cell is capable of reinitiating transcription to build the reserves that are essential to support early embryonic development. In the current model of mammalian oogenesis, there exists bilateral communication between the gamete and the surrounding cells that is limited to paracrine signaling and direct transfer of small molecules via gap junctions existing at the end of the somatic cells' projections that are in contact with the oolemma. The purpose of this work was to explore the role of cumulus cell projections as a means of conductance of large molecules, including RNA, to the mammalian oocyte. By studying nascent RNA with confocal and transmission electron microscopy in combination with transcript detection, we show that the somatic cells surrounding the fully grown bovine oocyte contribute to the maternal reserves by actively transferring large cargo, including mRNA and long noncoding RNA. This occurrence was further demonstrated by the reconstruction of cumulus-oocyte complexes with transfected cumulus cells transferring a synthetic transcript. We propose selective transfer of transcripts occurs, the delivery of which is supported by a remarkable synapselike vesicular trafficking connection between the cumulus cells and the gamete. This unexpected exogenous contribution to the maternal stores offers a new perspective on the determinants of female fertility.
  • Publication
    Regulation of gap-junctional communication between cumulus cells during in vitro maturation in swine, a gap-FRAP study
    (Society for the Study of Reproduction, 2012-05-30) Santiquet, Nicolas; Richard, François J.; Develle, Yann; Robert, Claude; Laroche, Anthony
    Intercellular gap-junctional communication (GJC) plays an important role in ovarian cell physiology. Closure of GJC has been proposed to be involved in oocyte maturation, particularly in the resumption of meiosis, both in vivo and in vitro, by controlling the flow of meiosis inhibitors, such as cAMP and cGMP. Understanding how GJC dynamics are regulated during in vitro maturation (IVM) could provide a powerful tool for controlling meiotic resumption and oocyte maturation in vitro. Since little is known about the GJC dynamic regulation between cumulus cells, we have developed an assay based on recovery of calcein fluorescence in photobleached cumulus cells, a gap-FRAP assay. The GJC profile has been characterized during the first hours of porcine IVM. We showed that equine chorionic gonadotropin (eCG) and epidermal growth factor (EGF) down-regulated GJC effectiveness between cumulus cells. However, human chorionic gonadotropin was not down-regulating GJC effectiveness. We also showed that the GJC network expanded during this period and that this effect was not regulated by gonadotropins. Porcine follicular fluid present in the maturation medium also had an impact on GJC regulation, increasing GJC network establishment and the effectiveness of calcein transfer rate between cumulus cells. These results show that both eCG and EGF are regulating the decrease in GJC effectiveness after 4.5 h of IVM, while the network extension is gonadotropin independent. Regulation of GJC between cumulus cells would then be specifically regulated during in vitro IVM.
  • Publication
    Accès libre
    The dynamics of connexin expression, degradation and localisation are regulated by gonadotropins during the early stages of in vitro maturation of swine oocytes
    (Public Library of Science, 2013-07-04) Santiquet, Nicolas; Richard, François J.; Robert, Claude
    Gap junctional communication (GJC) plays a primordial role in oocyte maturation and meiotic resumption in mammals by directing the transfer of numerous molecules between cumulus cells and the oocyte. Gap junctions are made of connexins (Cx), proteins that regulate GJC in numerous ways. Understanding the dynamic regulation of connexin arrangements during in vitro maturation (IVM) could provide a powerful tool for controlling meiotic resumption and consequently in vitro development of fully competent oocytes. However, physiological events happening during the early hours of IVM may still be elucidated. The present study reports the dynamic regulation of connexin expression, degradation and localization during this stage. Cx43, Cx45 and Cx60 were identified as the main connexins expressed in swine COC. Cx43 and Cx45 transcripts were judged too static to be a regulator of GJC, while Cx43 protein expression was highly responsive to gonadotropins, suggesting that it might be the principal regulator of GJC. In addition, the degradation of Cx43 expressed after 4.5 h of IVM in response to equine chorionic gonadotropin appeared to involve the proteasomal complex. Cx43 localisation appeared to be associated with GJC. Taken together, these results show for the first time that gonadotropins regulate Cx43 protein expression, degradation and localisation in porcine COC during the first several hours of IVM. Regulation of Cx43 may in turn, via GJC, participate in the development of fully competent oocytes.
  • Publication
    Transplanted bone marrow cells do not provide new oocytes but rescue fertility in female mice following treatment with chemotherapeutic agents
    (Mary Ann Liebert, Inc., 2012-04-03) Santiquet, Nicolas; Richard, François J.; Robert, Claude; Vallières, Luc; Sirard, Marc-André; Pothier, François
    It is generally accepted that mammalian females are born with a finite pool of oocytes and that this is the sole source of ovules throughout the reproductive life of the adult. This dogma was shaken in 2003 when researchers showed that the oocyte stock might be renewable in adult mammals. It has been proposed that hematopoietic stem cells might be a source of new oocytes. These discoveries have puzzled many researchers and remain controversial. In our study, we attempted to determine if transplanted bone marrow cells could provide new oocytes in PU.1 mice and in severe combined immunideficiency (SCID) mice after treatment with chemotherapeutic agents. We also examined the possibility that grafted bovine embryonic ovarian cortex might provide an environment favoring such a response. We found no evidence that transplanted bone marrow cells provide new fertilizable oocytes in PU.1 mice, in SCID mice treated with chemotherapeutic agents, or with bovine embryonic ovarian tissue grafts. However, transplanted bone marrow cells have improved the fertility of SCID mice previously treated with chemotherapeutic agents. These data suggest that bone marrow cells cannot provide new oocytes but can positively influence ovarian physiology to improve the fertility of mice previously treated with chemotherapeutic agents.