Reproduction in Domestic Animals

Use of Microarray Technology for Reproduction in Cattle 363the inner cell mass and subsequent elongation. Analysisof expression patterns of developmentally importantgenes provides a useful tool to assess the normality ofin vitro produced embryos alongside their in vitroderivedcounterparts and may provide a blueprintagainst which to manipulate culture conditions in orderto improve embryo quality.Gene expression studies in cattle embryos have beenconducted employing qualitative, semi-quantitative andquantitative approaches. Most studies have focused on asmall set of ‘candidate’ genes putatively important forembryogenesis including growth factors, metabolicenzymes, transcription factors involved in the regulationof early developmental events and imprinted geneproducts (see Wrenzycki et al. 2005 for review). A muchmore limited, but increasing, number of studies haveused cDNA microarrays to examine global gene expressionchanges in cattle embryos (Pfister-Genskow et al.2005; Smith et al. 2005; Corcoran et al. 2006; Somerset al. 2006; Adjaye et al. 2007).Ushizawa et al. (2004) used a custom-designed uteroplacentalcDNA microarray to study gene expression inbovine embryos on days 7, 14 and 21, extra-embryonicmembranes on day 28 and foetuses on day 28 ofpregnancy. Comparison of day 7 and day 14 revealedmost genes increased during this period, and a smallnumber of genes exhibiting altered expression decreasedas gestation progressed (Ushizawa et al. 2004). Theexpression of trophoblast cell-specific molecules such asplacental lactogens, prolactin-related proteins, interferon-tauand adhesion molecules was enhanced duringthe pre-implantation period suggesting a pivotal role inthe preparation needed for implantation.We recently compared mRNA expression across awide range of biological processes in bovine blastocystsderived from either culture in vivo or in vitro usingbovine cDNA microarrays (BOTL). The vast majority(85%) of the differentially expressed transcripts betweenthe two embryo populations were downregulated inin vitro cultured blastocysts, suggesting that the primaryreason why in vitro embryos are of inferior developmentalcompetence compared to in vivo cultured may bebecause of a deficiency of the machinery associated withtranscription and translation (Corcoran et al. 2006).Based on previous data showing the feasibility andhigh reproducibility of cross species hybridizationapproach using a human cDNA array to study geneexpression in bovine tissues (Adjaye et al. 2004), Adjayeet al. (2007) used a human 15 529 chip (the ENSEMBLchip) to compare gene expression in bovine oocytes andblastocysts; 164 and 1324 genes were expressed exclusivelyat the oocyte or blastocyst stage, respectively,while 419 genes expressed in both stages. Few studieshave attempted to correlate the differences in mRNAabundance observed at the blastocyst stage, such asthose outlined earlier, with the ability of the embryo toestablish a pregnancy. One such recent study (El-Sayedet al. 2006) addressed the relationship between transcriptionalprofile of embryos and the pregnancy successbased on gene expression analysis of biopsies fromblastocysts taken prior to transfer to recipients. Microarraydata (BlueChip) analysis revealed a total of 52differentially regulated genes between embryos resultingin a calf delivery vs those not resulting in a pregnancy(El-Sayed et al. 2006). Biopsies from embryos that weretransferred to recipients and resulted in calf deliverywere enriched with genes necessary for implantation(COX2 and CDX2), carbohydrate metabolism(ALOX15), signal transduction (PLAU) and placentaldevelopment (PLAC8), while those failing to establish apregnancy were enriched with transcripts for an inflammatorycytokine (TNF), protein amino acid binding(EEF1A1), transcription factors (MSX1 and PTTG1),glucose metabolism (PGK1 and AKR1B1) and CD9which is an inhibitor of implantation (El-Sayed et al.2006).Bovine embryos produced by nuclear transfer (NT)are capable of developing to the blastocyst stage with anefficiency of 30–50% which is comparable to thatachieved with routine IVF (Yang et al. 2007). However,following somatic cell cloning and transfer to surrogaterecipients a very limited percentage (0.5–5%) of theembryos completes full-term development. This ismainly due to a high frequency of post-implantationdevelopmental arrest. Such losses are predominantlyduring the first trimester of pregnancy but can occurmuch later (Heyman et al. 2002) and are often associatedwith aberrant placental development (Hill et al.2000). The high incidence of pregnancy loss andneonatal death following somatic cell NT (SCNT) arehypothesized to result from incomplete nuclear reprogramming;several authors have convincingly demonstratedthat the donor somatic cell is reprogrammedsuch that expression pattern at the blastocyst stage issubstantially different from that of the somatic cell priorto NT (Smith et al. 2005; Beyhan et al. 2007). Therehave been several recent cDNA microarray studiesexamining differential gene expression in NT-derivedembryos (Pfister-Genskow et al. 2005; Smith et al. 2005;Somers et al. 2006) in an effort to explain the poorgestational success.In one of the first reports comparing individualembryos produced by NT and IVF using cDNAmicroarray technology for any species, Pfister-Genskowet al. (2005) found 18 genes differentially expressedbetween NT- and IVF-produced embryos includingthree intermediate-filament protein genes (cytokeratin8, cytokeratin 19 and vimentin), three metabolic genes(phosphoribosyl pyrophosphate synthetase 1, mitochondrialacetoacetyl-coenzyme A thiolase and alpha-glucosidase),two lysosomal-related genes (prosaposin andlysosomal-associated membrane protein 2), and a geneassociated with stress responses (heat shock protein 27)along with major histocompatibility complex class I,nidogen 2, a putative transport protein, heterogeneousnuclear ribonuclear protein K, mitochondrial 16S rRNA,and ES1 (a zebrafish orthologue of unknown function).Smith et al. (2005) used microarray technology toanalyze global differences in gene expression betweenblastocysts derived from (i) natural fertilization in vivo,(ii) in vitro fertilization or (iii) NT. Perhaps surprisingly,the NT embryos most closely resembled true in vivoembryos in terms of numbers of differentially expressedgenes. They reported a low variability in the pattern ofmRNA expression among individual in vivo-derivedblastocysts and among NT blastocysts when comparedÓ 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag