Applying biotechnologies to understand fertility in cattle

Transcription

1 ABIC 2008 (Agricultural Biotechnology International Conference) Agricultural Biotechnology for a competitive and sustainable future Cork, Ireland 24 to 27 Aug 2008 Applying biotechnologies to understand fertility in cattle Alex Evans Niamh Forde, Paul McGettigan, Pat Lonergan Reproductive Biology Research Cluster School of Agriculture Food Science and Veterinary Medicine University College Dublin, Ireland

2 Reproductive Biology 45% 35% Fertility (pregnancy rates to a single insemination) 25% 40% 85% 90%

3 Dairy cow reproduction Aim to have a calf per cow per year with peak lactation to correspond with peak grass growth

4 Milk Production and Fertility in Dairy Cows CR (%) CR Milk Production Milk Production (kg/yr) Data from Herds in NY, USA WR Butler (unpublished)

5 Reproductive wastage in dairy cows Fertilization Failure 10 to 20 % Embryo mortality 25 to 45% Reproductive wastage Insemination to Recognition of Pregnancy: 35 to 65% Diskin et al 2006

6 Establishment of pregnancy 1. Ovarian Follicle Development 3. Embryo Development ools/echnologies Physiology 2. Oocyte Maturation 4. Uterine Function Cell Biology Endocrinology Biotechnologies

7 Comprehensive and systematized analysis of important systems and molecules governing reproductive fitness in Dairy cattle Proteomics ranscriptomics Glycomics Metabolomics

8 Analysis of bovine endometrial tissue by off-line MudPI: Proof of principle experiment 1)ryptic digest of tissue 2) Strong cationic exchange HPLC; Collect fractions, desalt, concentrate 4) Identification (Sequest) ~14000 peptides/run ~3500 unique peptides ~500 proteins 3) Reverse Phase HPLC MS/MS of each fraction

9 Glycobiology - Cervical Mucus Mucus = hydrated polymeric mucins Secreted and membrane-bound forms Heavily O-glycosylated Glycosylation influences charge, cohesion & hydration.

10 2 Bovine microarrays in use Bovine cdna microarray BOL library + additional genes ~1,400 genes / ESs Bovine Affymetrix Gene Chip ~19,000 genes/ess

11 Global gene expression during ovarian follicle development Ovarian Follicle Development

12 Dominant versus subordinate follicles Microarray (BOL4) comparison of Significantly greater expression newly selected Dominant and Dominant Subordinate follicles 15 - Granulosa 59 - heca 38 - Granulosa 58 - heca 106 otal 261 Greater expression in Dominant follicles 10 ranslation regulator activity, 5% Greater expression in Subordinate follicles Calcium ion binding, 3% st ra di ol Calcium ion binding, 3% 5 Oe Follicle diameter (mm) Subordinate Number Other, 8% Structural molecule activity, 1% ransporter activity, 2% ranslation regulator activity, 1% Other, 4% Calcium ion binding, 5% None, 26% ranscription regulator activity, 3% Protein binding, 32% ranscription regulator activity, 8% Nucleotide binding, 9% None, 58% Nucleotide binding, 24% Nucleic acid binding, 14% Days Nucleic acid binding, 11% Catalytic activity, 18% Catalytic activity, 39% Signal transducer activity, 42% Signal transducer activity, 12% Red = fewer, Blue = more than expected Protein binding, 15% Park et al. unpublished

13 Apoptosis genes validated Q-R-PCR 53 apoptosis genes on the array 21 genes identified from the array 15 confirmed by Q-R-PCR *P<0.05, **P<0.02 Evans et al., 2004

14 Dom v Sub follicles (P<0.05, Q-R-PCR) Signal transduction FIBP G CAM kinase 1 RanGPase G FRAP Cyclophilin G BCAR1 G FIBP FGF-I Intracellular Binding Protein Greater expression in Sub than Dom follicles sirna granulosa cells in vitro (a) GF-betaIIR alpha Gi3 alpha protein WN2B Integrin alpha 2 DGCR2 EphA4 Prion protein * (b) * * * 25 0 (c) 8 E2 Conc e ntr ation pe r v ia ble c e lls FIBP mrna e x pres s ion re la tiv e to b -Ac ti n Syntaxin Control FSH RC β-glycan FIBP sirna sirna 45 (d) Control FSH RC FIBP sirna G Granulosa heca Forde et al. 2007, 2008

15 Dom v Sub follicles (P<0.05, Q-R-PCR) BOL XH08R BOL XF04R BOL _G06 BOL _G04 BOL _G02 BOL XH04R G BOL XC03R G BOL XA05R G BOL XG12R G G Granulosa heca BC1D1 = Bos aurus BC1 (tre-2/usp6, BUB2, cdc16) domain family, member 1 Differentiation & regulation cell growth Signal transduction Expression relative to β-actin Unknown Emergence Selection Dominance Stage of the follicle wave Zielak et al. 2007

16 Global gene expression during embryo development Embryo Development

17 Embryo development (good v bad) In vitro culture - BAD 2-cell 4-cell 8-cell 16-cell Early morula Compact morula Blastocyst Comparison of mrna IVM IVF Zygote 2-cell 4-cell 8-cell 16-cell Early morula Compact morula Blastocyst In vivo culture (ewe oviduct) - GOOD Day 0 Day 1 Day 7

18 Indicators of embryo quality after in vivo vs. in vitro culture IVM & IVF produced and cultured In vitro In vivo Similar Similar Ultrastructure Poor Good Cryotolerance Poor Good Candidate genes Poor Good Low (~35%) Higher (~55%) Blastocyst yield Pregnancy rate Lonergan et al.

19 Global gene expression: in vivo vs. in vitro cultures embryos cdna microarray: 384 significant genes (n = 384) Greater gene expression in embryos cultured in vivo 0% 20% 40% in vitro 60% 80% 100% Corcoran et al., 2006

20 mrna expression in in vivo relative to in vitro cultured blastocysts 0 ranscription or ranslation Cellular Metabolism 6XE03R 13_A08 HEBP1 bikk beta GALE GCGR 9_G 06 In vivo 3XC10R REA EEF1G GNBL2 PABPN1 6 CCR4-NO FOXO 3A DO1L HMG 2 Q-R-PCR validation 7 In vitro Unknown Corcoran et al., 2006

21 Global gene expression in the uterine endometrium Uterine Function

22 Pregnancy Rate Effect of pregnancy and progesterone on gene expression in the uterine endometrium Embryo survival Day 5 progesterone concentration (ng/ml) Day 7 progesterone concentration (ng/ml) Starbuck et al. (1999); Diskin et al., (2003) Mann et al (1999)

23 Effect of pregnancy and progesterone on gene expression in the uterine endometrium Groups (2x2): High & Normal P4; Pregnant & Cycling Collect endometrium on Day 5, Day 7, Day 13, Day 16 Afymetrix bovine Gene Chip 2.5 PRID in P r oge s te r one (ng/m l) 3 2 Pregnant High Pregnant Normal 1.5 Control High Control Normal Day of cycle/pregnancy Carter et al 2008

24 Progesterone Regulated Genes (endometrium) reatment (i) H v N D5 (ii) H v N D7 Differentially expressed genes (iii) H v N D13 (iv) H v N D16 otal Up and Down Regulated Genes H = High Progesterone N = Normal Progesterone D = Day P<0.01 Forde et al

25 Pregnancy Induced Genes (endometruim) reatment (i) P v C D5 (ii) P v C D7 Differentially expressed genes (iii) P v C D13 (iv) P v C D16 otal Up and Down Regulated Genes P = Pregnant C = Cycling D = Day P < 0.01 Forde et al

26 Digital Gene Expression (Illumina Genome Analyzer) Bovine Endometrium tissue Day 13 Pregnant (n=4) vs. Cycling (n=3) Differentially expressed (FDR <= 0.001) Affymetrix DGE 0 genes 1025 genes

27 Progesterone, Pregnancy, Uterine Function Working Hypothesis Progesterone Endometrial gene expression Endometrial secretions (histotroph) into uterine lumen Stimulates embryo growth Embryo IFNτsecretion and Maternal Recognition of Pregnancy

28 Reproduction in cattle