Embryo and Pluripotency : Epigenetic and Environment (EPEE)

Team leader : Alice Jouneau (alice.jouneau@inrae.fr)

The EPEE team consists of 21 people. It includes INRAE staff from the "Animal Physiology and Breeding Systems (PHASE)" department, the National Veterinary School of Alfort (ENVA) and the private company ALLICE, bringing together skills in Developmental Biology, Molecular and Cellular Biology, Genome Editing, Epigenetics, Imaging, Morphokinetics, Micromanipulations, Microfluidics, Embryo Biotechnology, Cell Culture, Transcriptomics and Metabolomics. Several members of the team are involved in platforms or shared facilities (MIMA2 platform -https://www6.jouy.inrae.fr/mima2_eng/MIMA2_platform - , BREED unit ruminant embryo production facility).

Composition au 1er juin 2020 : Véronique Duranthon (DR2, HDR, animatrice de l'équipe), Gilles Charpigny (CRHC), Alice Jouneau (CRHC, HDR), Amélie Bonnet Garnier (CRN, HDR), Sophie Calderari (CRN), Alline de Paula Reis (MC ENVA), Laurent Boulanger (IR1), Pierre Adenot (IR1- MIMA2), Bernadette Banrezes (IR2), Fabienne Nuttinck (IR ENVA), Adrien Acquistapace (AI), Catherine Archilla (AI), Thierry Sainte Beuve (AI), Martine Letheule (AI- MIMA2), Marine Foritano (CDD-AI), Marie-Christine Deloche (TR- ALLICE), Ludivine Laffont (TR), Olivier Dubois (TR), Jean-François Oudin (TR). Mélanie Pailles (doctorante), Romina Via-y- Rada-Fernandez (doctorante).

Main question

During fertilisation, the fusion of the highly differentiated gametes results in the formation of a totipotent embryo. The totipotent embryonic cells will then re-differentiate during preimplantation development to give the different cell lines of the embryo. This implies an architectural "remodelling" and epigenetic reprogramming of the embryonic genome, necessary for its transcriptional activation and then for the establishment at the blastocyst stage of the pluripotent cells of the inner cell mass (ICM) and the first differentiated cells of the trophectoderm. Within the ICM, the precursors of the pluripotent epiblast that will form the foetus and those of the hypoblast (or primitive endoderm) that will contribute to some extra-embryonic appendages can be distinguished. The trophectoderm will form most of the placenta. Understanding and controlling these early stages of embryonic development is crucial for the reproductive efficiency of animals. In addition, the conditions under which these early stages take place can affect the phenotype of unborn individuals. The research carried out by the team is therefore in line with the dual perspective of understanding and improving the fertility of animals, and characterising the early determinants of the development of the individual phenotype. 

Scientific questions

• Epigenetic reprogramming of the embryonic genome: Totipotency, pluripotency and first differentiations

After fertilisation, the remodelling of the parental genomes leads to the formation of a totipotent embryo whose transcriptional and epigenetic state will change during early embryonic development. Pluripotency, which takes place in the cells of the ICM, is an evolutionary process which ends at gastrulation, when the embryonic lineages differentiate. A distinction is made between the naïve state, which characterises the epiblast precursors in the ICM and the Embryonic Stem Cells (derived from the epiblast precursors), and the primed state corresponding to the pre-gastrulation epiblast cells and the Epiblast Stem Cells (derived from this primed epiblast).

Our objective is to understand the mechanisms, notably epigenetic, involved in remodelling parental genomes and leading to totipotency. We are studying how these mechanisms cooperate with signalling pathways to control the transition between the different states of pluripotency and then towards the differentiation of the first embryonic lineages.

To analyse these phenomena, we compare rabbit, cattle and mouse. We use in vitro derived pluripotent cells (ESC and EpiSC) in mice to study the evolution of pluripotency. 3D culture systems are being developed to make these in vitro models more representative of the embryo. 

Only naïve pluripotent cells derived in vitro present all the potentialities of differentiation in vitro as well as in vivo and make it possible to form germinal chimeras after aggregation with a host embryo. These cells are therefore of great interest for obtaining genetically modified animals and as a source for manufacturing organoids. They do not currently exist in domestic animals. In rabbit and cattle, we analyse how pluripotent cells appear and are maintained in the embryo and characterise the transcriptome of candidate pluripotent cells derived in vitro. Our dual objective is to understand how pluripotency is regulated in different species and to obtain naive pluripotent cells in vitro in non-rodent mammals.

• Effect of the micro-environment: Periconceptional programming.

Another aspect of our fundamental activity concerns the study of alterations induced by the microenvironment of the oocyte and embryo in vitro but also in vivo (e.g. the nutritional status of the mother) both in terms of epigenetic modifications and gene expression.

It has now been clearly demonstrated that the environment in which oogenesis and the very first stages of embryonic development take place influences the phenotype (and health) of the unborn individual, with manifestations that can be detected during development but may not be revealed until adulthood (concept of periconceptional programming). Changes in nutritional diet or maternal metabolic status may affect the composition of the in vivo microenvironment of the oocyte and embryo. Furthermore, the implementation of embryo biotechnology, both in agronomy and in human medicine, involves significant changes in the environment of the oocyte and embryo, both of which are manipulated in vitro.

Our work aims to characterise the metabolic, molecular and epigenetic alterations induced by modifications of the early microenvironment, which may affect the phenotype of the individual.

We are interested in different types of modifications of the in vitro environment and at different periods sensitive to these modifications: oocyte maturation (composition of the maturation medium), fertilisation (calcium signals, redox potential and metabolite composition of the fertilisation medium) and early stages of development (composition of the culture medium). In vivo, we are interested in the impacts of metabolic disorders (high-fat diet for example).

In the team we are more specifically studying the consequences of these disorders on the reprogramming of parental genomes and the establishment of first lineages. Their effects on foetal, placental and longer-term development are also taken into account, in collaboration with other teams of the UMR.

In cattle, pre-implantation development is characterized by a significant phase of trophoblast elongation. This phase is highly dependent on interactions with the maternal endometrium and cannot be mimicked in vitro. It is accompanied by numerous transcriptional events that participate in the "dialogue" between the maternal and the embryonic tissues. The team studies the maternal components produced by the endometrium that contribute to the differentiation of the embryo and its appendices. It is involved in the development of culture models associating endometrial organoids and embryos to understand how the interactions between these two organisms may affect the phenotype of the embryo.

• Improvement of embryo biotechnology

For several decades, knowledge about the embryo and its micro environment has made it possible to develop different reproductive biotechnologies such as In Vitro Maturation (IVM), In Vitro Fertilisation (IVF), Intra-Cytoplasmic Sperm Injection (ICSI), in vitro culture of the embryo up to the blastocyst stage. These biotechnologies are of great economic and public health interest because of their zootechnical (in vitro embryo production in cattle) and medical applications in humans (infertility treatment). However, the gestation rates obtained after transfer of in vitro produced embryos remain low and variable in these two species. The safety of the procedures remains also a matter of concern.

One of the applied objectives of our research is the improvement of these technologies with a view to better valorisation in the field. Two levers are currently favoured:

(i) improvement of the composition of the maturation, fertilisation or in vitro culture media in order to make them more reliable and to improve the aptitude for subsequent development of the embryos produced in vitro;

(ii) improvement of the selection of in vitro produced embryos according to their viability (ability to produce gestation and birth). Our work aims to identify different early and non-invasive markers (metabolic products and morphokinetic profile) and to relate them to different developmental potentials. The objective is to build and validate a predictive tool enabling users of these biotechnologies to better select the embryos to be transferred.

Animal models

Depending on the scientific question, the team chooses to use rabbit, mouse or bovine embryos.

Methodological approaches

• Production and micromanipulation of mammalian embryos:
  • Production of bovine embryos: in vitro oocyte maturation, fertilisation and development
  • Microinjection in the nucleus and cytoplasm, ICSI, biopsy, microdissection, chimera generation. Robotic controlled microfluidic culture.
• Imaging:
  • Immunofluorescence, DNA and RNA-FISH on 3D conserved embryos. Ex vivo imaging and morphokinetics using time-lapse microscopy (white and fluorescent microscopy).
  • Semi-automated image analyses, deep-learning.
• Cell and molecular biology:
  • Derivation and culture of stem cells from mouse (ESC, EpiSC, TSC), rabbit (TSC) embryos. Genome and epigenome editing.
  • RNA-seq, microarrays dedicated to rabbit and bovine, RT-qPCR, ddPCR, all adapted to small quantity of material present in the embryo.
  • ATAC-seq, with ongoing miniaturization
  • Metabolomic (1H RMN)

Networks

•  REVIVE-2 Labex : stem cells and regenerative medicine (directed by the Pasteur Institute)
• Researchers’ groups (GDR): ADN (nuclear architecture), Functional microscopy of live organisms, Repro (reproduction), Organoids
• PHC campus France (bilateral exchange program): Stephanik (2020-2021)
• European network COST action: Epiconcept, RGB-Net, CellFit
• SFEF (Société Française pour l'Etude de la Fertilité), SF DoHad (Société Francophone Origines Développementales de la Santé), FSSCR (French Society of Stem Cell Research), AETE -European Embryo Technology Association

Collaborations

• Region: 
  • Experimental units of INRAE,  Jouy-en Josas : SAAJ and IERP
  • UMR INRAE GABI Jouy
  • UMR INRAE MaIAGE
• National:
  • UMR INRAE DMEM
  • UMR INRAE PRC
  • INSERM U 846 Lyon Stem Cell and Brain Research
• Internationale :
  • Enrique Gomez Centro de Biotecnología Animal-SERIDA Gijon, Spain
  • Veronique Azuara, Imperial College, London, UK.
  • Hendrick Marks, Radboud University, Nijmegen, the Netherlands
  • Poul Hyttel, Department of Veterinary and Animal Sciences, University of Copenhagen Denmark
  • Jozef Laurencik Constantine the Philosopher University, Nitra, Slovak Republic
  • Leif Andersson, Uppsala University, Sweden
  • Frederick Lanner, Karolinska Institut, Stockolm, Sweden
  • P. Humblot, Uppsala University, Sweden

Private partner

• ALLICE : équipe d'accueil de personnel ALLICE : www.allice.fr

Publications (2016-2020)

2020

• Burton A, Brochard V, Galan C, Ruiz-Morales ER, Rovira Q, Rodriguez-Terrones D, Kruse K, Le Gras S, Udayakumar VS, Chin HG, Eid A, Liu X, Wang C, Gao S, Pradhan S, Vaquerizas JM, Beaujean N, Jenuwein T, Torres-Padilla ME. Heterochromatin establishment during early mammalian development is regulated by pericentromeric RNA and characterized by non-repressive H3K9me3.  Nat Cell Biol. 2020 Jul;22(7):767-778. doi: 10.1038/s41556-020-0536-6.
• Peyny M, Jarrier-Gaillard P, Boulanger L, Daniel N, Lavillatte S, Cadoret V, Papillier P, Monniaux D, Peynot N, Duranthon V, Jolivet G, Dalbies-Tran R. Investigating the role of BCAR4 in ovarian physiology and female fertility by genome editing in rabbit. Sci Rep. 2020;10(1):4992. doi: 10.1038/s41598-020-61689-6.
• Bell E, Curry EW, Megchelenbrink W, Jouneau L, Brochard V, Tomaz RA, Mau KHT, Atlasi Y, de Souza RA, Marks H, Stunnenberg HG, Jouneau A, Azuara V. Dynamic CpG methylation delineates subregions within super-enhancers selectively decommissioned at the exit from naive pluripotency. Nat Commun. 2020;11(1):1112. doi: 10.1038/s41467-020-14916-7.

2019

• Dirks RAM, van Mierlo G, Kerstens HHD, Bernardo AS, Kobolák J, Bock I, Maruotti J, Pedersen RA, Dinnyés A, Huynen MA, Jouneau A, Marks H. Allele-specific RNA-seq expression profiling of imprinted genes in mouse isogenic pluripotent states. Epigenetics Chromatin. 2019 ;12(1):14. doi: 10.1186/s13072-019-0259-8.
• Sanz G, Daniel N, Aubrière MC, Archilla C, Jouneau L, Jaszczyszyn Y, Duranthon V, Chavatte-Palmer P, Jouneau A. Differentiation of derived rabbit trophoblast stem cells under fluid shear stress to mimic the trophoblastic barrier. Biochim Biophys Acta Gen Subj. 2019 ; 1863(10):1608-1618. doi: 10.1016/j.bbagen.2019.07.003. Epub 2019 Jul 3.
• Rousseau-Ralliard D, Couturier-Tarrade A, Thieme R, Brat R, Rolland A, Boileau P, Aubrière MC, Daniel N, Dahirel M, Derisoud E, Fournier N, Schindler M, Duranthon V, Fischer B, Santos AN, Chavatte-Palmer P. A short periconceptional exposure to maternal type-1 diabetes is sufficient to disrupt the feto-placental phenotype in a rabbit model. Mol Cell Endocrinol. 2019;480:42-53. doi: 10.1016/j.mce.2018.10.010.
• May-Panloup P, Brochard V, Hamel JF, Desquiret-Dumas V, Chupin S, Reynier P, Duranthon V. Maternal ageing impairs mitochondrial DNA kinetics during early embryogenesis in mice. Hum Reprod. 2019;34(7):1313-1324. doi: 10.1093/humrep/dez054.

2018

• Bernardo AS*, Jouneau A*, Marks H, Kensche P, Kobolak J, Freude K, Hall V, Feher A, Polgar Z, Sartori C, Bock I, Louet C, Faial T, Kerstens HHD, Bouissou C, Parsonage G, Mashayekhiv K, Smith JC, Lazzari G, Hyttel P, Stunnenberg HG, Huynen M, Pedersen RA, Dinnyes A. Mammalian embryo comparison identifies novel pluripotency genes associated with the naïve or primed state. Biology Open 2018 doi: 10.1242/bio.033282.
• Bonnet-Garnier A, Kiêu K, Aguirre-Lavin T, Tar K, Flores P, Liu Z, Peynot N, Chebrout M, Dinnyés A, Duranthon V, Beaujean N. Three-dimensional analysis of nuclear heterochromatin distribution during early development in the rabbit. Chromosoma. 2018;127(3):387-403. doi: 10.1007/s00412-018-0671-z.
• Canon E, Jouneau L, Blachère T, Peynot N, Daniel N, Boulanger L, Maulny L, Archilla C, Voisin S, Jouneau A, Godet M, Duranthon V. Progressive methylation of POU5F1 regulatory regions during blastocyst development. Reproduction. 2018;156(2):145-161. doi: 10.1530/REP-17-0689.
• Fabrèges D, Daniel N, Duranthon V, Peyriéras N. Control of the proportion of inner cells by asymmetric divisions and the ensuing resilience of cloned rabbit embryos. Development.2018 145(8). doi: 10.1242/dev.152041
• Tosolini M, Brochard V, Adenot P, Chebrout M, Grillo G, Navia V, Beaujean N, Francastel C, Bonnet-Garnier A, Jouneau A. Contrasting epigenetic states of heterochromatin in the different types of mouse pluripotent stem cells. Sci Rep. 2018;8(1):5776. doi: 10.1038/s41598-018-23822-4.
• Duranthon V, Chavatte-Palmer P. Long term effects of ART: What do animals tell us? Mol Reprod Dev. 2018 (4):348-368. doi: 10.1002/mrd.22970. 23. Review.

2017

• Tapponnier Y, Afanassieff M, Aksoy I, Aubry M, Moulin A, Medjani L, Bouchereau W, Mayère C, Osteil P, Nurse-Francis J, Oikonomakos I, Joly T, Jouneau L, Archilla C, Schmaltz-Panneau B, Peynot N, Barasc H, Pinton A, Lecardonnel J, Gocza E, Beaujean N, Duranthon V, Savatier P. Reprogramming of rabbit induced pluripotent stem cells toward epiblast and chimeric competency using Krüppel-like factors. Stem Cell Res. 2017:106-117. doi: 10.1016/j.scr.2017.09.001.
• Nuttinck F, Jouneau A, Charpigny G, Hue I, Richard C, Adenot P, Ruffini S, Laffont L, Chebrout M, Duranthon V, Guienne BM. Prosurvival effect of cumulusprostaglandin G/H synthase 2/prostaglandin2 signaling on bovine blastocyst: impact on in vivo posthatching development. Biol Reprod. 2017;96(3):531-541.
• Carvalho AV, Canon E, Jouneau L, Archilla C, Laffont L, Moroldo M, Ruffini S, Corbin E, Mermillod P, Duranthon V. Different co-culture systems have the same impact on bovine embryo transcriptome. Reproduction. 2017;154(5):695-710. doi: 10.1530/REP-17-0449.
• Lelièvre JM, Peynot N, Ruffini S, Laffont L, Le Bourhis D, Girard PM, Duranthon V. Regulation of heat-inducible HSPA1A gene expression during maternal-to-embryo transition and in response to heat in in vitro-produced bovine embryos. Reprod Fertil Dev. 2017;29(9):1868-1881. doi: 10.1071/RD15504.

2016

• Sepulveda-Rincon LP, Dube D, Adenot P, Laffont L, Ruffini S, Gall L, Campbell BK, Duranthon V, Beaujean N, Maalouf WE. Random Allocation of Blastomere Descendants to the Trophectoderm and ICM of the Bovine Blastocyst. Biol Reprod.2016.
• Osteil P, Moulin A, Santamaria C, Joly T, Jouneau L, Aubry M, Tapponnier Y, Archilla C, Schmaltz-Panneau B, Lecardonnel J, Barasc H, Mouney-Bonnet N, Genthon C, Roulet A, Donnadieu C, Acloque H, Gocza E, Duranthon V, Afanassieff M, Savatier P. A Panel of Embryonic Stem Cell Lines Reveals the Variety and Dynamic of Pluripotent States in Rabbits. Stem Cell Reports. 2016;7(3):383-98.
• Kone MC, Fleurot R, Chebrout M, Debey P, Beaujean N, Bonnet-Garnier A. 3D Distribution of UBF and Nopp140 in Relationship to rDNA Transcription During Mouse Preimplantation Development. Biol Reprod.2016;94(4):95. doi: 10.1095/ biolreprod.115.136366.
• Salvaing J, Peynot N, Bedhane MN, Veniel S, Pellier E, Boulesteix C, Beaujean N, Daniel N, Duranthon V. Assessment of 'one-step' versus 'sequential' embryo culture conditions through embryonic genome methylation and hydroxymethylation changes. Hum Reprod. 2016.
• Chavatte-Palmer P, Tarrade A, Kiefer H, Duranthon V, Jammes H. Breeding animals for quality products: not only genetics. Reprod Fertil Dev. 2016;28(1-2):94-111.
• Chavatte-Palmer P, Vialard F, Tarrade A, Dupont C, Duranthon V, Lévy R. DOHaD and pre- or peri-conceptional programming. Med Sci (Paris). 2016;32(1):57-65.