Phenotyping the reproduction function in ttl i t f f ti lcattle: inputs from functional

genomicsgenomics

Claire Ponsart UNCEIAClaire Ponsart, UNCEIA

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

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

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

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 !

ID /Position”OMICS”

ID /Position  GENOMICS

G E i+

Gene Expression TRANSCRIPTOMICS

Proteins+

Proteins PROTEOMICS

Small Molecules+ FUNCTION

Small MoleculesMETABOLOMICS Source HUMBLOT

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

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

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

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)

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

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)

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

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

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

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

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

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

Clinics

Reproductive

Physiology

OmicsGenetics

How « Omics » can help to improvep pgenetic selection for  fertility ???

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

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 %

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

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

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 ?)

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)