claire ponsart unceia - | icar · fertilization oocyte capture gamete maturation source cordova...
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Phenotyping the reproduction function in ttl i t f f ti lcattle: inputs from functional
genomicsgenomics
Claire Ponsart UNCEIAClaire Ponsart, UNCEIA
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Phenotyping the reproduction function : a l d l lmultidimensional scale
2.FUNCTIONAL2.FUNCTIONAL2.FUNCTIONAL 2.FUNCTIONAL CONTINUUMCONTINUUM
3.INPUTS IN 3.INPUTS IN GENETICGENETICGENETIC GENETIC SELECTIONSELECTION
1.TIME1.TIME‐‐SPACE CONTINUUMSPACE CONTINUUM
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Reproduction function : a space‐time continuump p
GametesGametes survivalsurvival
FertilizationFertilizationand and meiosismeiosis
OviductalOviductaltransporttransport
andand cellcell divisionsdivisionsand and cellcell divisionsdivisions
GametesGametes maturationmaturation
BlastocystBlastocyst stagestage
FERTILITYFERTILITY
éclosionéclosionEmbryoEmbryo elongatingelongatingorganogenesisorganogenesisimplantationimplantation
FERTILITYFERTILITY
PlacentaPlacenta
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Phenotyping early embryo mortality : bl k b i i t b d !a black box remaining to be opened !
BreedingEarly : 75%Milk Progesterone
Late: 25%Proteins
Gamete quality I l i
Time Implantation
Source HUMBLOT
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How may functional genomics help to h h ?characterise this time‐space continuum ?
• New phenotyes refined– More precise : successive events can be identified / p /isolated
– New phenotypes : « omics »New phenotypes : « omics »
• Very powerful techniques : lots of information !
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ID /Position”OMICS”
ID /Position GENOMICS
G E i+
Gene Expression TRANSCRIPTOMICS
Proteins+
Proteins PROTEOMICS
Small Molecules+ FUNCTION
Small MoleculesMETABOLOMICS Source HUMBLOT
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Phenotyping early embryo mortality : focus on l d l b d loocyte quality and early embryo development
GametesGametes survivalsurvival
FertilizationFertilizationand and meiosismeiosis
OviductalOviductaltransporttransport
andand cellcell divisionsdivisionsand and cellcell divisionsdivisions
GametesGametes maturationmaturationEMMAEMMA
BlastocystBlastocyst stagestage
OVOGENAEOVOGENAE
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Oocyte quality impacts embryo developmentEmbryo developmentFolliculogenesis Ovulation
Fertilization
Oocyte quality impacts embryo development
8 16t 2 4 l bl t
Fertilization
8c 16czygote 2c 4c morula blasto
Croissance Maturation
Start of embryonic genome
• Nuclear maturation
ption
• Nuclear maturation
•Chromatine condensation
•MeiosisVG
transcrip
•Cytoplasmic maturation‐Regulation of RNA andproteins synthesis
MII
No transcription
Source ANGULO Leslie
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OVOGENAE : genes related to oocye quality(Rozenn Trans‐Dalbies, INRA)
Gene expression profiles related toTranscriptomics
p pembryo development competence Profiles compared in oocyte populations
presenting contrasted developmentcompetence
Candidate genes
Functional approach of candidate genes Functional approach of one gene specificallyexpressed in oocytes
Source ANGULO LeslieSource ANGULO Leslie
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CombiningCombining «« omicsomics » » approachesapproaches and and embryoembryorelatedrelated biotechnologies : abiotechnologies : a powerfulpowerful modelmodelrelatedrelated biotechnologies : a biotechnologies : a powerfulpowerful modelmodel
Multiple Ovulation AI Multiple Ovulation AI 5 embryos / cow1 flushing every 2 months1 flushing every 2 months
RepeatedRepeated In vitro production In vitro production 2 embryos / cow2 embryos / cow1 session / week (immature oocytes)
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Transcriptomic approach
0 % ‐ 17 %36% ‐ 55%
DonorDonor cowscows withwith contrastedcontrastedembryoembryo production (UNCEIAproduction (UNCEIA)
40264026 52545254 7019701970597059 96669666 94349434
RNA extractionRNA extraction
A lifi iA lifi i
2 replicates ; 15 oocytes/stage/donor
AmplificationAmplification
Cy 3Cy 5 RT + P33
ARNa ARNa
MMi 22k
ADNc ‐ P33Source ANGULO Leslie MacroarrayMicroarray 22k
+ANGULO Leslie
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Transcriptomic approachMature oocytesMature oocytes 22122212 spots / spots / 20182018 genesgenes withwith differentdifferent expression profilesexpression profiles
Transcriptomic approach
974 974 genesgenes upup‐‐regulatedregulated in oocytes in oocytes fromfrom donordonor cowscows withwith highhighdevelopmentdevelopment competencecompetence ( B<M) :( B<M) :
12381238 genesgenes pp reg latedreg lated in ooc tesin ooc tes fromfrom donordonor co sco s ithith lolo1238 1238 genesgenes upup‐‐regulatedregulated in oocytes in oocytes fromfrom donordonor cowscows withwith lowlowdevelopmentdevelopment competencecompetence (B>M) :(B>M) :
ll h l lh l l hh ??
GENE
Implication in Implication in physiologicalphysiological pathwayspathways ??Links Links withwith fertilityfertility QTLsQTLs ? ?
Oocyte stage Genes in fertility QTLsFUNCTION
y g y QB>M B<M
Immature (108) (123)M (180) (149)
Source ANGULO Leslie
Mature (180) (149)
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Functional approach : one gene specificallydexpressed in oocytes
• Gene specifically expressed in oocytes• Gene specifically expressed in oocytes
• Transcripts and proteins abundance investigatedamong early steps of development
Antibody (Eurogentec)
0 4
0.6
0.8
1.01.2
ative level
RNA RNA
OI OM Z 2C 4C 8C Mo Bex Bec
14 kDa
Antibody (Eurogentec)Protein
0.00.2
0.4
rela
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Phenotyping early embryo mortality : focus on l d l b d loocyte quality and early embryo development
GametesGametes survivalsurvival
FertilizationFertilizationand and meiosismeiosis
OviductalOviductaltransporttransport
andand cellcell divisionsdivisionsand and cellcell divisionsdivisions
GametesGametes maturationmaturationEMMAEMMA
BlastocystBlastocyst stagestage
OVOGENAEOVOGENAE
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Transport in oviduct : genes related tob l i iembryo‐maternal communication (P Mermillod, INRA PRC)
• Functional approach• Functional approach– Specific functions of the oviduct– Signals related to embryo-maternal communication
• Transcriptomics– Oviduct + embryo viability
• Proteomics– Investigation of proteins related to embryo-maternalg f p y
communication
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Oviduct : different successive events / functions in different oviduct parts
Embryo transport
Early embryo development
different oviduct parts
OviductOviduct
Early embryo development
UterusUterusOvary UterusUterusOvary
Fertilization
Oocyte capture
Gamete maturation
Source Cordova Amanda
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Transport in oviduct : genes related toembryo maternal communicationembryo‐maternal communication (P Mermillod, INRA PRC)
Use of an original in vitro embryo culture system
(%)In vitro survival following embryo thawing
with BOEC (bovine oviductepithelial cells)compared to classical culture p
system (SOF)
SOFSOF
5 h following thawing
Hours following thawing
SOF + Boec
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PROTEOMICS : different proteins are synthetised in specific parts of the oviduct
bb
(n:462)(n:541)Blastocyst
rateIS AM
a(n:461)rate
(%)
1 2 3
Cells issued from different oviduct parts
Proteins synthetised using an in vitro BOEC culture system with cells issued
from different oviduct partsSource Cordova Amanda
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Clinics
Reproductive
Physiology
OmicsGenetics
How « Omics » can help to improvep pgenetic selection for fertility ???
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Genifer : Phenotyping pregnancy failure occurring within 90 days following first postpartum insemination in Holsteindays following first postpartum insemination in Holstein
• 3508 Holstein (62%• 3508 Holstein (62% primiparous), 984 herds, 17 insemination centers
• 12 sires pooled in three FI groups, low: [‐0.7; ‐0.5]; a erage [ 0 1 +0 3] highaverage: [‐0.1;+0.3]; high: [0.5;+1.0]
• Phenotypes of pregnancy• Phenotypes of pregnancy failure between first AI (D0) and D90 (failure):
• Environment and milk production data recorded
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1 bl d l
Genifer : Phenotyping pregnancy failure occurring within 90 days following first
t t i i ti i H l t i
2 ilk l2 Pregnancy assessments
1 blood sample(genotyping)
postpartum insemination in Holstein
2 milk samples
1rst AI RETURN0 21-23 35-45 d 80-100 d
D 0 (IA)
D 21-23
Return Pregnancy D35
Pregnancy D80
AI d i l t l h 6 3 %AI during luteal phase +
Early emb. death - - Regular
6,3 %
35,4 %
16 7 %Late emb. death - + delayed Not pregnant
Foetal death - + delayed Pregnant Not pregnant
16,7 %
3,6 %
July 2008 WBC 2008
Pregnancy - + No return Pregnant Pregnant37 %
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Estimated incidence of pregnancy failure between p g yD0 and D90, a vs b: p≤0.05; a vs c: p≤0.01; avs d: p≤0.001
Source Ledoux 2011
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Impacts on genomic selectionImpacts on genomic selection
i ( f f il ) l d i• 7 traits (type of pregnany failure) analysed in 2669 females with 306 SNPs (from 13 h )chromosomes).
• Fertility QTL on chromosome 3 more preciselylocated
• Effects of QTLs confirmed / isolated– 6 on calving rate– 3 on EEM, 3 on LEM, 8 on global EM, – 2 on FM– 3 on aborts
Source : Lefevre, 3R 2011
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ConclusionConclusion
• Use of « omics » : lots of positive interactions with genomic selectiong– Refining new phenotypes : bood markers, genes, proteins…proteins…
– Better evaluation of performances reliability, genetic improvementgenetic improvement
• More direct applications : prevention, treatment (for fertility ?)
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Thanks !Thanks !
• INRA PRC, Nouzilly– Rozenn Dalbiès‐Tran, Xavier Druart, Joelle Dupont, Pascal Mermillod
• INRA BDR, Jouy– ,Véronique Duranthon, Isabelle Hue, Hélène Jammes, Svetlana , q , , ,
Uzbekova, Fabienne Nuttinck, Gilles Charpigny, Olivier Sandra –
• INRA GABI, Jouy, y– Didier Boichard, Mekki Boussaha, Rachel Lefevre
• ENVABénédicte Grimard Dorothée Ledoux– Bénédicte Grimard, Dorothée Ledoux
• UNCEIA– Sébastien Fritz, Aurélien Capitan,
h l l l– Catherine Joly, Brigitte Leguienne, Pascal Salvetti– Patrice Humblot (SLU)