It has been reported that in vitro culture conditions alter gene expression and may lead to developmental aberrations in IVF derived cattle, commonly referred to as the large offspring syndrome [38
]. In the case of embryos produced by SCNT, besides the alterations due to in vitro culture conditions, gene expression defects may be caused by improper silencing and activation of specific genes, altered chromatin remodelling, and epigenetic alterations [41
]. But identifying key genes responsible for the general developmental failure in cloned embryos is not an easy task, since the alterations may be caused by a variety of factors including donor cell type, cell cycle stage, nuclear transfer protocol, source of the oocytes, embryo culture system, embryo transfer procedure, recipient cows management, and operators' skills [42
]. Consequently, there is a big variety of alterations that are not always shared by all cloned embryos. Still, the common thread uniting many of the SCNT failures can be traced to epigenetic alterations, specifically failures in chromatin remodelling and DNA and histone methylation [13
]. The fetal fibroblast cells used in this study are not fully representative of adult somatic cells. However, these cells were chosen because of their practicality and higher efficiency in SCNT studies.
Microarray analysis has been used to explore the transcriptome profile of cloned embryos relative to that of the donor cells and IVF embryos as a control. However, the appropriate microarray platform is crucial in order to detect changes in particular genes. Smith and colleagues reported similar transcriptome profiles for cloned blastocysts and blastocysts produced by artificial insemination [15
]. However, the cDNA microarray used by Smith and colleagues consisted of placenta and spleen cDNA libraries, lacking embryonic genes, which therefore were not analysed. The results from the present study show an extensive reprogramming in cloned embryos by the blastocyst stage. However, the data point to a group of differentially expressed transcripts between IVF and cloned blastocysts.
Serial cloning is often performed for the production of transgenic animals. Although apparently healthy animals can be obtained after serial cloning, the efficiency of cloning decreases from generation to generation despite comparable blastocyst and early pregnancy rates. This increase in pregnancy losses and perinatal deaths could be caused by gene expression defects accumulated throughout the serial cloning procedures, which could be detected in blastocysts, although no phenotypic alterations are observed at this stage. Furthermore, it has been proposed that the extended culture, associated with transfection and selection procedures, may induce changes in the donor cells [36
]. The present studies show that serial cloning does not significantly affect transcriptional reprogramming of cloned blastocysts. The global transcriptome profile of blastocysts from four consecutive rounds of cloning did not significantly differ from the one obtained from blastocysts after only one round of cloning. However, for a set of genes, misregulation was significantly greater in the blastocysts obtained from four rounds of cloning (see Tables and ). However the observed differences between blastocysts from the first and fourth rounds of cloning could be due to the fact that these are different donor cells and not of the same clonal origin.
To our knowledge this is the first study to focus on the influence of serial chromatin transfer on global transcriptome profile of embryos and donor cells. Only a small proportion of the data set generated by the present study corresponded to fully annotated bovine genes (Table ). The rest of the probe sets were excluded from further analyses due to lack of annotations. Progress in the annotation of the bovine genome will greatly facilitate global gene expression studies in the bovine species.
In the present study, multiple comparisons revealed five distinctive patterns of differential gene expression among all embryos and donor cells. The first pattern
corresponded to 1,183 transcripts (30.74% of the data set) that had similar abundance in all five groups. Housekeeping genes like GAPD and Actin showed this pattern of expression. The second pattern
corresponded to genes that had similar expression in IVF and CT embryos, but had a very different pattern of expression in both donor cell lines. We hypothesised that these are genes that switched from the "donor cell gene expression mode" to the "embryo gene expression mode". The majority of the genes in the data set (76.49%) showed this pattern, including some imprinted and embryonic specific genes such as the Oct-4 protein coding gene (POU5F1), which has been reported as differentially expressed for cloned embryos in previous studies [21
]. Placenta specific 8 (PLAC8) also shows this pattern of expression (Figure ). It is possible that some genes, due to their methylation pattern in the somatic cells or to their location in the chromosome, are more likely to be reprogrammed by the oocyte factors.
The third pattern corresponded to genes with a similar pattern of expression for CT embryos and donor cells, and a very different expression pattern in IVF embryos. These were 147 (3.81%) genes with apparently incomplete reprogramming, probably with a somatic cell pattern of expression. The heat shock 70 kD protein 1 (HSPA1A), involved in cell protection from stress and apoptosis was significantly higher in IVF embryos when compared to CT embryos and donor cells (Figure ). Important embryonic genes showed this pattern of expression. Desmocollin 3 (DSC3) a transmembrane glycoprotein, involved in cell adhesion that belongs to the cadherin family, was present in IVF embryos but was absent in CT embryos and donor cells. The signal transducer and activator of transcription 3 (STAT3), was significantly upregulated in IVF embryos when compared to both groups of cloned embryos and donor cells. A similar pattern was observed for high mobility group nucleosomal binding domain 3 (HMGN3) a gene involved in chromatin remodelling, a vital process during embryonic genome activation (Figure ). The importance of both genes during morulae and blastocyst formation could make them good candidates in understanding the lower developmental rates of cloned embryos.
group of genes corresponded to only 85 probe sets (2.21%) with a marked differential expression in all cloned embryos compared to the one observed in both IVF embryos and donor cells. The misregulation of these genes could point to a compensation mechanism after chromatin transfer. Genes with this kind of expression pattern included prostaglandin-endoperoxide synthase (PTGS2) and the transcription factor GATA-2. Both genes had a greater microarray signal in all CT embryos, but low expression in IVF and donor cells. The imprinted gene glycine amidinotransferase (GATM), showed significantly greater values in the cloned embryos compared to IVF embryos and donor cells. Two interesting genes in this group were DNMT3A and DNMT3B transcripts, which are responsible for de novo methylation. Both genes were significantly greater in CT-1 and CT-4 embryos compared to IVF blastocysts (Figure and ), which is consistent with the hypermethylation often reported in cloned blastocysts. These results do not agree with previous findings, in which DNMT3A was downregulated in NT embryos compared to IVF embryos [21
]. Zhou et al., reported similar levels of DNMT3B for embryos produced in vivo
, in vitro
, and by different nuclear transfer methods, including chromatin transfer [9
]. These contrasting results confirm that alterations greatly vary and are not shared by all cloned embryos. One limitation of our study is that we have not used in vivo blastocysts which might have provided more biological means and as the physiological standard against in vitro culture conditions.
A fifth pattern corresponded to genes that had an increasing or a decreasing pattern of expression from IVF embryos through donor cells showing an intermediate pattern of expression in CT embryos. In total, 245 probe sets showed this pattern of expression with 119 (3.08%) increasing, and 126 (3.28%) decreasing from IVF through DC. It could be assumed that these genes have been partially reprogrammed, since their transcript abundance is in between IVF and donor cells. The imprinted gene insulin-like growth factor 2 receptor (IGF2R), one of the most studied genes in the large offspring syndrome, showed similar expression values in IVF and CT1 embryos, but significantly higher signals in CT4 embryos, and very high signals in both donor cells (Figure ). These higher mRNA levels in the fourth generation of cloning could indicate a cumulative misregulation of this gene. The Bcl-2 inhibitor of transcription (BIT1) showed the greatest values in IVF embryos, intermediate values in CT embryos and the lowest values in donor cells (Figure ). The nuclear transcription factor Y, alpha (NFYA), showed a similar expression pattern in both IVF and CT1 embryos; although it was significantly lower in CT4 embryos and donor cells. Neuroguidin (NGDN), an eukaryotic translation initiation factor with important functions in embryonic development was another gene with a decreasing pattern of expression (Figure ). Genes with and increasing pattern of expression included F-box protein 9 (FBXO9), and guanine nucleotide binding protein alpha inhibiting activity polypeptide 2 (GNAI2) represented in Figure and Figure , respectively. Transcripts for the progesterone receptor (PGR) were significantly higher in IVF embryos compared to CT embryos and donor cells (Figure ). Among this group of transcripts could be genes that are cumulatively affected be serial cloning.
Based on the difference in gene expression for RARB, CRAB1, THBS, SERPINB5, and HLA-A, Beyhan et al. suggest a possible role for the retinoic acid signalling pathway in the failures observed in cloned bovine embryos [22
]. However the bovine GeneChip does not contain a Retinoic Acid Receptor Beta (RARB) probe set. It only contains a probe set that corresponds to a bovine EST with similarity to the rat RARB (Bt.21044.2.A1_at). In the present data, CRAB1 and THBS2 were slightly higher in IVF embryos, although without statistical significance. They also found differential gene expression among several genes in both donor cells (CDKN1C, COPG2, DCN, GATM, MEST, NDN, NNAT, PON3, and SGCE). In the current study GATM was significantly downregulated in donor cells from the fifth successive generation of chromatin transfer (Figure ).
At the blastocyst stage there is an extensive reprogramming of cloned embryos leading to very similar transcriptomes in IVF and CT blastocysts. However, there were around 200 differentially expressed genes in both CT embryos compared to IVF. For some genes, the differences were significantly greater in CT4 when compared to CT1, suggesting a possible cumulative missregulation caused by serial cloning. Genes involved in transcription, cellular proliferation, embryonic development, cellular death, and response to stress are over represented in IVF embryos; many of these genes are present in the nucleus, which was the cell component overrepresented in IVF embryos. Genes involved in cell morphology, cell development, and metabolism were over expressed in donor cells and in cloned embryos when compared to IVF, suggesting that they were not properly silenced in the donor nucleus. The up regulation of genes involved in metabolism should be further explored as it could be linked to the large size of cloned animals.