Although, some reports are available in bovine and other species on the DNA methylation in cloned compared to IVF embryos at different developmental stages, these reports provide a limited insight on the aberrations in DNA methylation in cloned embryos since low throughput techniques like immunostaining and RT-PCR were used for examining the methylation status in these studies 52, 53, 54. To our information, there is no report in any species on the complete comparative global DNA methylation profile of cloned and IVF embryos at different developmental stages using a high throughput technique like MeDIP-Seq.
Over the years, many methods have been developed for analyzing genome-wide DNA methylome. These include whole genome bisulfite sequencing (WGBS), reduced representation bisulfite sequencing (RRBS), TET‑assisted bisulfite sequencing (TAB‑seq), comprehensive high‑throughput arrays for relative methylation (CHARM), methylation array and MeDIP-SEq. However, each method has its own merits and demerits when it comes to sequence whole genome. WGBS is considered to be the best method for examination of DNA methylation profile of mammalian genome due to its high resolution. But it is costly, time consuming and generates complicated data that further needs comprehensive bioinformatics analysis which limits the popularity of this technique. MeDIP-Seq is a widely used affinity-based method for examining DNA methylation. Global DNA methylation analysis is feasible with MeDIP-Seq even with very low amount of starting (as low as 1 ng) DNA sample 55, 56. Many recent studies have used MeDIP-Seq to generate relative genome-wide DNA methylation profile 51, 57, 58. Therefore, we selected this method to study genome-wide DNA methylation in cloned embryos in comparison with IVF embryos in buffalo.
In the present study, we reported for the first time, genome-wide DNA methylome in cloned and IVF embryos. We generated 130 GB data from two types of embryos (cloned and IVF) each at 2-cell, 8-cell and blastocyst stages. The total number of CpG islands (CpGis) in the genome was found to be 2,17,246 out of which 1,04,188 islands belonged to cloned embryos and 1,13,058 to IVF embryos CpG islands related to cloned embryos showed much higher methylation when compared with IVF counterparts. Distribution of hypermethylated CpGs sites was found to be more prevalent in the 2-cell stage embryos (46.06 %), which reduced at the 8-cell stage (36.94%) and was highest at the blastocyst stage (53.07%) in cloned relative to IVF embryos. Distribution of CpG islands in the repetitive elements of the genome (i.e. LINE, SINE, repetitive DNA, LTR, simple repeats & low complexity DNA) was found to different for different developmental stage of cloned and IVF embryos. At 2-cell stage embryos, maximum distribution of CpGis was observed in LTRs (23.8%) in cloned group compared to 30.4% for the IVF counterparts. Cloned embryos at the 8-cell stage showed maximum distribution of CpGis in simple repeats (21.3%) whereas, the IVF counterparts showed it in LTRs (19.3%). Blastocyst-stage embryos had major distribution of CpGis in LINE (25.4%) in cloned and low complexity DNA (18.8%) in IVF embryos.
A total of 8,07,160 DMRs were identified at the 2-cell stage in cloned embryos out of which, maximum number of DMRs were found to be hypermethylated with respect to IVF counterparts. However, the difference in the magnitude of hypermethylation in the two groups of embryos decreased significantly at the 8-cell stage followed by a significant increase at the blastocyst stage. It was observed that a major portion of hypermethylated DMRs belonged to intergenic region at upstream and downstream 1 KB and 2KB region of genome at each developmental stage. Finally, we validated our MeDIP-Seq data by carrying out direct bisulfite sequencing PCR with randomly selected genes irrespective of their methylation status.
An unusual pattern of de-methylation and re-methylation in cloned embryos has been reported in several 5-methylcytosine immunofluorescence-based studies in past years 46, 52, 53, 54. Our study was not immunofluorescence-based but our MeDIP-Seq data is in agreement with the results of these low-throughput studies as cloned embryos showed hypermethylation of DNA at blastocyst stage in comparison to IVF counterparts.
Dean et al. (2001) 54 reported that cloned bovine embryos lack passive demethylation mechanism and have high expression of DNMT3A and DNMT3B which is responsible for de novo methylation at 4-cell and 8-cell stages. Likewise, in the present study, 23,621 genes were identified at the 8-cell stage in cloned embryos out of which 14,594 showed hypermethylation. This scenario reflects re-methylation of genes due to re-establishment of methylation marks that coincides with time of embryonic genome activation (EGA). Memili et al. (2000) 59 suggested the same possible reason for this pattern of methylation at 8-cell stage as this stage involves major change in transcriptional activation of the embryonic genome which might activate Dnmt3a and Dnmt3b enzymes.
Dean et al. (2001)54 reported increased methylation level in the bovine blastocyst stage embryos specifically in the trophectoderm cells. Contrary to this, we found that the less number of genes were showing hypermethylation (9111) in cloned embryos relative to IVF embryos at the blastocyst stage.
Using single-cell RRBS to generate DNA methylomes for SCNT embryos in mice, Liu et al. (2016) 60 reported abnormally high levels of DNA methylation in 2-cell and 4-cell stage SCNT embryos. It was found that, SCNT embryos at 4-cell stage were relatively hypermethylated compared with 2-cell stage embryos which opposed the demethylation pattern of normal embryos. Also, the aberrant DNA methylation in arrested SCNT embryos was attributed to differences in expression of Dnmt1 and Tet1 enzymes. However, our study showed a slightly different trend as we found that 27,517 genes were identified in 2-cell stage cloned embryo, with 12,392 genes showing hypermethylation at 2-cell stage cloned embryos in comparison to IVF counterparts. This suggests that the majority of genes undergo demethylation at early developmental stages during nuclear reprogramming. In our data, DNMT1 and TET1 showed hypermethylation which could be the reason for lower methylation level of genes in 2-cell stage cloned embryos relative to their IVF counterparts. It is suggested that cytoplasmic factors of oocytes might help in triggering demethylation with remodelling of chromatin.
To our information, there is only one study in which genome-wide DNA methylation has been studied in SCNT preimplantation embryos but it is focused on specific parts of genome. Zhang et al. (2016)46 compared DNA methylation reprogramming in bovine SCNT and IVF preimplantation embryos and analyzed the influence of vitamin C (VC) on the reprogramming of DNA methylation by using bisulfite sequencing. The results showed that global DNA methylation followed a typical pattern of demethylation and remethylation in IVF preimplantation embryos however, the global genome remained hypermethylated in SCNT preimplantation embryos. Compared with the IVF group, pluripotency genes POU5F1 and NANOG showed insufficient demethylation and hypermethylation in the SCNT group. Similarly, in our study, we found that pluripotency genes (POU5F1, TCF3, LIN28b, and DUSP1) showed high number of regions with hypermethylation which suggests lower expression of these genes.
Gene ontology analysis (GO) of differentially methylated genes in the present study showed 483 biological process, 85 molecular function and 121 cellular components to be affected by hypermethylation of genes in cloned 2-cell stage embryos relative to their IVF counterparts. Similarly, in the 8-cell stage cloned embryos, 526 biological process, 97 molecular function and 115 cellular components were found to be affected by hypermethylation of genes with respect to IVF embryos. GO analysis for cloned blastocyst-stage embryos relative to IVF embryos revealed 594 biological process, 131 molecular function and 176 cellular component to be affected by hypermethylation.
In the present study, 15 pathways were found to be affected at the 2-cell stage, 15 at the 8-cell stage and 27 at the blastocyst stage in cloned embryos relative to IVF counterparts. For all the developmental stages covered under present study, the major pathways affecting embryonic development in cloned embryos relative to IVF counterparts were found to be Wnt signaling pathway, inflammation mediated by chemokine & cytokine signalling pathways, gonadotropin-releasing hormone receptor pathway and Integrin signalling pathway. These pathways were affected by hypermethylation of DNA.
It was found in the present study that imprinting genes such as IGF2R, PEG10, GRB10, MEST, and MAOA showed presence of hypermethylated regions that suggest lower gene expression at the blastocyst stage in cloned relative to IVF embryos. Some important apoptosis-related genes such as CASP9 and DFFA, FAS, APAF1, and PTEN were found to have hypermethylation in gene region in blastocyst stage cloned embryos compared to their IVF counterparts.
Similarly, cell cycle-related genes such as MDM2, and NPAT showed presence of hypermethylation in gene regions in blastocyst stage cloned embryos relative to IVF counterparts.
We found that the methylation status of methylation-specific genes viz. DNMT1, DNMT3A, DNMT3B, TET1, EZH2, and MBD3 showed no methylation in gene regions with whereas TET2 and TET3 gene found to have hypermethylation in gene regions in blastocyst stage cloned embryos relative to their IVF counterparts. This pattern of methylation reflects high expression of abovementioned genes in the cloned blastocyst-stage embryos which might be responsible for the abnormal hypermethylation observed in cloned blastocysts in comparison with IVF counterparts.
There are some functionally important genes which contribute to embryonic development in bovine embryos. We found that YY1, SOD1, IL6, G6PD, PCGF2, MCL1, and IFN-τ represented no methylation in the gene regions whereas TIMP2, MTPN, COL4A1, TCF7, AQP9, BMP7, FGF7, WNT2, DKK3, CRABP2, PPARA, DYSF, LEF1 and HDAC8 showed high number of regions with hypermethylation in blastocyst stage cloned embryos compared to their IVF counterparts. Shyam et al. (2020) 61 also observed a high expression of WNT signalling pathway genes i.e. TCF7 and LEF1 in cloned buffalo blastocyst-stage embryos. They also reported lower gene expression level of IFN-τ (primary maternal signal for recognition of pregnancy in ruminants) in cloned blastocyst was lower than in IVF counterparts.
Similarly, Sood et al. (2019) 62 also reported down- regulated expression of IFN-τ in cloned embryos in comparison with IVF counterparts. In contrast, present study indicates hypermethylation of TCF7 and LEF1 at gene level which suggests lower expression of these genes in cloned embryos. Also, we found the gene region for IFN-τ to be unmethylated which points to a high expression of this gene in cloned compared to IVF embryos. Thus, our results regarding methylation status of TCF7, LEF1 and IFN-τ genes were found to be not in agreement with above mentioned studies.
Kim et al. (2009) 63 reported abnormally up-regulated expression of tissue inhibitor of metalloproteinase-2 (TIMP-2) and superoxide dismutase (SOD) in the placentae of SCNT cloned Korean native cattle that died immediately after birth and in normal placentae obtained by artificial insemination. Abnormal expression of these two genes reported to be partly responsible to for abnormal placental function and low survivability of cloned claves. Results from our study regarding these two gene showed that showed hypermethylation in gene region of TIMP-2 gene and no methylation in gene regions of SOD1 gene in cloned blastocysts relative to IVF counterparts. For TIMP2 gene, our results are not in agreement with those of above study but our results for SOD1 gene, are in agreement with above study as its unmethylated status can suggesting high level of gene expression.
Gene such as AQP9 encode a water channels protein which is involved in coordinating urea transport. Gao et al. (2019) 64 reported abnormally lower expression level of AQP9 gene in placenta of SCNT cattle by both RNA-seq and q-PCR. Similarly, in present study, methylation level of AQP9 gene was found to be very high which suggests lower expression level of this gene in cloned embryos relative to IVF counterparts. Sood et al. (2019)62 also reported down-regulated expression of DKK3 gene in cloned embryos in comparison with IVF embryos. Likewise, our study showed high number of hypermethylated regions of DKK3 gene in cloned blastocysts relative to IVF counterparts that might indicate towards low expression level of this gene. This study can be sum up with the following conclusions: there is a profound difference in the global DNA methylation profile between cloned and IVF embryos. These differences are manifested throughout the course of embryonic development. Cloned embryos differ from their IVF counterparts in the enriched GO terms of Biological Process, Molecular Function and Cellular Component categories in terms of the differentially methylated regions in cloned and IVF embryos. A large number of pathways are affected by hypermethylation in cloned relative to IVF embryos. Among these, the major pathways related to embryonic development are Integrin signalling pathway, Wnt signaling pathway, apoptosis signaling pathway, inflammation mediated by chemokine & cytokine signaling pathway, gonadotropin-releasing hormone receptor pathway, CCKR signaling map, angiogenesis. There is a need of further more targeted studies stimulated by this study to evaluate exact genomic locations, genes and other factors that are responsible for low efficiency of blastocyst rate and live birth rate of cloned offsprings.