Phenotyping the reproduction function in
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1 Phenotyping the reproduction function in cattle: inputs from functional genomics Claire Ponsart, UNCEIA
2 Phenotyping the reproduction function : a multidimensional lscale 2.FUNCTIONAL CONTINUUM 3.INPUTS IN GENETIC SELECTION 1.TIME SPACE CONTINUUM
3 Reproduction function : a space time continuum Gametes survival Fertilization and meiosis Oviductal transport and cell divisions Gametes maturation Blastocyst stage FERTILITY implantation organogenesis Embryo elongating éclosion Placenta
4 Phenotyping early embryo mortality : a black box remaining i to be opened! Breeding Early : 75% Milk Progesterone Late: 25% Proteins Gamete quality Time Implantation Source HUMBLOT
5 How may functional genomics help to characterise this time space continuum? New phenotyes refined More precise : successive events can be identified / isolated New phenotypes : «omics» Very powerful techniques : lots of information!
6 OMICS ID /Position GENOMICS Proteins + Gene Expression TRANSCRIPTOMICS + PROTEOMICS + FUNCTION Small Molecules METABOLOMICS Source HUMBLOT
7 Phenotyping early embryo mortality : focus on oocyte quality and early embryo development Gametes survival Gametes maturation Fertilization and meiosis Oviductal transport EMMA and cell divisions OVOGENAE Blastocyst stage
8 Oocytequality impacts embryo development Folliculogenesis Ovulation Fertilization Embryo development zygote 2c 4c 8c 16c morula blasto Croissance Maturation Start of embryonic genome Nuclear maturation Chromatine condensation transcrip ption VG MII Meiosis Cytoplasmic maturation Regulation of RNA and proteins synthesis No transcription Source ANGULO Leslie
9 OVOGENAE : genes related to oocye quality (Rozenn Trans Dalbies, INRA) Gene expression profiles related to embryo development competence Transcriptomics Profiles compared in oocyte populations presenting contrasted development competence Functional approach of candidate genes Candidate genes Functional approach of one gene specifically expressed in oocytes Source ANGULO Leslie
10 Combining «omics» approaches and embryo related biotechnologies : a powerful model Multiple Ovulation AI 5 embryos / cow 1 flushing every 2 months Repeated In vitro production 2 embryos / cow 1 session / week (immature oocytes)
11 Transcriptomic approach 36% 55% 0 % 17 % Donor cows with contrasted embryo production (UNCEIA) replicates ; 15 oocytes/stage/donor RNA extraction Amplification i Cy 3 Cy 5 RT + P 33 Source ANGULO Leslie ARNa ARNa Microarray 22k + Macroarray ADNc P 33
12 Transcriptomic approach Mature oocytes 2212 spots / 2018 genes with different expression profiles 974 genes up regulated in oocytes from donor cows with high development competence ( B<M) : 1238 genes up regulated inooc oocytes from donor cows with low development competence (B>M) : GENE Implication in physiological lpathways? Links with fertility QTLs? Oocyte stage Genes in fertility QTLs B>M B<M Immature (108) (123) Mature (180) (149) FUNCTION Source ANGULO Leslie
13 Functional approach : one gene specifically expressed in oocytes Gene specifically expressed in oocytes Transcripts and proteins abundance investigated among early steps of development rela ative level RNA 14 kda Antibody (Eurogentec) OI OM Z 2C 4C 8C Mo Bex Bec Protein
14 Phenotyping early embryo mortality : focus on oocyte quality and early embryo development Gametes survival Gametes maturation Fertilization and meiosis Oviductal transport EMMA and cell divisions OVOGENAE Blastocyst stage
15 Transport in oviduct : genes related to embryo maternal communication i (P Mermillod, INRA PRC) Functional approach Specific functions of the oviduct Signals related to embryo-maternal communication Transcriptomics Oviduct + embryo viability Proteomics Investigation of proteins related to embryo-maternal communication
16 Oviduct : different successive events / functions in differentoviduct parts Oviduct Embryo transport Early embryo development Ovary Uterus Fertilization Oocyte capture Gamete maturation Source Cordova Amanda
17 Transport in oviduct : genes related to embryo maternal maternal communication (P Mermillod, INRA PRC) Use of an original in vitro embryo culture system with BOEC (bovine oviduct epithelial cells) compared to classical culture system (SOF) (%) In vitro survival following embryo thawing SOF 5 h following thawing SOF + Boec Hours following thawing
18 PROTEOMICS : different proteins are synthetised in specific parts of the oviduct Blastocyst rate (%) (n:461) a (n:462) b (n:541) b IS AM Cells issued from different oviduct parts Source Cordova Amanda Proteins synthetised using an in vitro BOEC culture system with cells issued from different oviduct parts
19 Clinics Reproductive Physiology Genetics Omics How «Omics» can help to improve genetic selection for fertility???
20 Genifer : Phenotyping pregnancy failure occurring within 90 days following first postpartum insemination in Holstein 3508 Holstein (62% primiparous), 984 herds, 17 insemination centers 12 sires pooled in three FI groups, low: [ 0.7; 0.5]; aerage[ average: [ 0.1;+0.3]; 01+03] high: [0.5;+1.0] Phenotypes of pregnancy failure between first AI (D0) and D90 (failure): Environment and milk production data recorded
21 1bl blood sample (genotyping) Genifer : Phenotyping pregnancy failure occurring within 90 days following first postpartum t insemination i in Holstein 2 milk samples 2 Pregnancy assessments D 0 (IA) 63 6,3 % AI during luteal l phase + 35,4 % d d 1rst AI RETURN D Return Early emb. death - - Regular 16,7 % Pregnancy D35 Late emb. death - + delayed Not pregnant 3,6 % Pregnancy D80 Foetal death - + delayed Pregnant Not pregnant July % WBC 2008 Pregnancy - + No return Pregnant Pregnant
22 Estimated incidence of pregnancy failure between D0 and D90, a vs b: p 0.05; a vs c: p 0.01; avs d: p Source Ledoux 2011
23 Impacts on genomic selection 7 traits (type of pregnany fil failure) analysed din 2669 females with 306 SNPs (from 13 chromosomes). Fertility QTL on chromosome 3 more precisely located 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
24 Conclusion Use of «omics»: lots of positive interactions withgenomic selection Refining new phenotypes : bood markers, genes, proteins Better evaluation of performances reliability, genetic improvement More direct applications : prevention, treatment (for fertility?)
25 Thanks! INRA PRC, Nouzilly Rozenn Dalbiès Tran, Xavier Druart, Joelle Dupont, Pascal Mermillod INRA BDR, Jouy,Véronique Duranthon, Isabelle Hue, Hélène Jammes, Svetlana Uzbekova, Fabienne Nuttinck, Gilles Charpigny, Olivier Sandra INRA GABI, Jouy Didier Boichard, Mekki Boussaha, Rachel Lefevre ENVA Bénédicte Grimard, Dorothée Ledoux UNCEIA Sébastien Fritz, Aurélien Capitan, Catherine Joly, Brigitte Leguienne, Pascal Salvetti Patrice Humblot (SLU)
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