Reproduction in Domestic Animals

364 ACO Evans, N Forde, GM O’Gorman, AE Zielak, P Lonergan and T Fairwith in vitro produced blastocysts suggesting a relativelyuniform reprogramming mechanism in the NT embryosby the blastocyst stage. Despite this apparent similarityin gene expression pattern between NT- and AI-derivedblastocysts, the low pregnancy rate and developmentalanomalies associated with NT suggest that reprogrammingof the donor genome is incomplete. To investigatethe possible causes of these losses, transcript profiling oftrophoblast and embryonic disc tissue collected fromday 25 conceptuses derived by NT or AI was conductedusing a microarray consisting of 13 257 oligonucleotides-derivedfrom cattle gene sequences. Approximately9000 genes were differentially expressed between trophoblastand disc demonstrating major gene expressiondifferences in embryonic and extra-embryonic tissues.Comparing NT with AI, 188 genes were differentiallyexpressed in trophoblast and 10 in the embryonic disc,providing evidence that nuclear reprogramming isdefective in a large proportion of NT-derived clonesand that aberrant gene expression in the trophoblastcontributes to pregnancy failure at day 25 and beyond(Everts et al. 2005).Using Affymetrix GeneChip bovine arrays, Beyhanet al. (2007) compared global gene expression of twodifferent somatic cell lines whose capacity to generatehealth NT-derived calves is significantly different, thecloned blastocysts derived from the cell lines and IVFblastocysts in order to elucidate some of the key genesresponsible for successful reprogramming. The majorityof genes in SCNT blastocysts had comparable expressionpatterns to IVF blastocysts, with the exception of asmall number of genes whose relative expression valueswere similar to their corresponding donor cells, indicatinga failure of reprogramming in SCNT blastocysts(Beyhan et al. 2007).Global Gene Expression in the UterineEndometriumThe study of endometrial function is necessary tounderstand the factors associated with the establishmentof pregnancy and to get an insight into the causes ofearly embryo loss. The endometrium is responsible forthe secretion of the numerous cytokines, growth factorsand proteins that together make are collectively termedthe histotroph. The histotroph is secreted from theglandular epithelium in the endometrium into the lumenof the uterus and it is in this environment that theembryo develops. Studies involving endometrial geneexpression in cattle have mainly focused on the changesthat occur at different stages of the oestrous cycle. Somestudies have examined the mRNA expression profiles ofbovine epithelial cells of the ipsilateral vs the contralateraloviduct (Bauersachs et al. 2003) and cells from theipsilateral oviduct (Bauersachs et al. 2004) or endometrium(Bauersachs et al. 2005) at oestrus (low progesterone)and dioestrus (high progesterone), to identifychanges in gene expression at different stages of theoestrous cycle in cattle.Microarray studies that give an insight into thechanges that occur in gene expression in the endometrium,which have a functional consequence for embryosurvival, have been performed in humans (Horcajadaset al. 2006), mice (Kashiwagi et al. 2007), sheep (Chenet al. 2007) and pigs (Ross et al. 2007). Although there isscant literature on the use of microarrays to studyendometrial gene expression in cattle, two papers(Bauersachs et al. 2006; Klein et al. 2006) have takentwo different animal model approaches to address thedifferences in endometrial gene expression betweenpregnant and cycling animals on day 18. In one study,monozygotic twin recipients were used along within vitro-derived embryos and suppression subtractivehybridization to enrich the cDNA for transcripts thatwere upregulated in the endometrium of pregnantanimals before microarray hybridization (Klein et al.2006). They identified 87 different genes ⁄ mRNAs, ofwhich 80 were known bovine genes or were inferredfrom human orthologues. Ontological classification ofthese genes showed that the largest class was type Iinterferon-stimulated genes. Of these differentially expressedgenes, several have already been described asbeing involved in changes in endometrial gene expressionin other species. For example, interferon-stimulatedgene-15 has shown to be expressed in the endometriumof cows (Austin et al. 2004), sheep (Johnson et al. 2000),pigs (Joyce et al. 2003), mice (Austin et al. 2003) andhumans and baboons (Bebington et al. 1999). However,the power of the microarray technology allowed for theidentification of genes that are involved in cell adhesion(connective tissue growth factor – CTGF, glycosylphosphatidylinositolspecific phospholipase D1 – GPLD1,milk fat globule-EGF factor 8 protein – MFGE8) aswell as genes involved in endometrial remodelling(matrix metalloproteinase 19 – MMP19, tissue inhibitorof metalloproteinase 2 – TIMP2) that have not previouslybeen described as being differentially regulated inthe endometrium (Klein et al. 2006). The second modelutilized SSH technology also, but used in vivo-derivedembryos (Bauersachs et al. 2006). This study identified109 transcripts that were differentially regulated in day18 pregnant animals when compared with day 18 cyclinganimals. Of these 109 transcripts, 34, 28 and 3 geneswere enriched in the GO terms for immune responsegenes, response to stimulus and antigen presentation,respectively.The earlier studies looked at differences in geneexpression in the endometrium associated with pregnancyon a day when maternal recognition had alreadyoccurred. However, in cattle, the majority of embryonicloss occurs prior to maternal recognition of pregnancy(Dunne et al. 2000; Humblot 2001). Retrospectivestudies have shown a correlation between concentrationsof progesterone and pregnancy rates both in beefand dairy cattle (Diskin et al. 2006). Other studies incattle have shown that progesterone supplementationincreases embryo size on day 14 of pregnancy (Garrettet al. 1988). Recent studies in our laboratory (Carteret al. 2008), Forde et al. (2008b) have tried to addressthe issue of early embryo mortality by examining thedifferential effects of elevated progesterone in theimmediate post-conception period and pregnancy onendometrial gene expression in cycling and pregnantanimals at different physiological stages of the earlydevelopmental axis (i.e. days 5, 7, 13 and 16 postconception,corresponding to the 16-cell ⁄ early morulaÓ 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag