MeDIP-sequencing for profiling global DNA methylation in buffalo embryos produced by in vitro fertilization

2021 ◽  
pp. 1-17
Author(s):  
Shivani Malpotra ◽  
Manoj Kumar Singh ◽  
Prabhat Palta
2020 ◽  
Vol 114 (3) ◽  
pp. e153-e154
Author(s):  
Saúl Lira-Albarrán ◽  
Xiaowei Liu ◽  
Paolo Rinaudo

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Congru Li ◽  
Yang Yu

Abstract Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in women of childbearing age and is the main cause of anovulatory infertility. To increase the number of oocytes obtained, controlled ovarian stimulation (COS) has become a routine choice for in vitro fertilization-embryo transfer (IVF-ET), which is one of the common assisted reproductive technologies for PCOS patients. However, for these patients, there is a high risk of ovarian hyperstimulation syndrome (OHSS). Obtaining in vitro maturation (IVM) of immature oocytes, and then in vitro fertilization and embryo transfer of mature oocytes provides a possible way for people to solve the above problems. Since the IVM technology will expose oocytes to in vitro conditions for a longer period of time, theoretically increasing the risk of the oocytes being affected by the culture environment, further research and explorations are needed for study in gene programming, epigenetics, etc. Therefore, to explore the impact of IVM operation on embryonic development is of great significance for further clarifying assisted reproductive safety and improving IVM operation conditions. Here we focused on DNA methylation reprogramming process which was essential for embryonic development. We tested the DNA methylation of sperm, IVM oocytes and IVM generated early stage embryos including pronucleus, 4cell, 8cell, morula, inner cell mass, trophoectoderm (TE) as well as six-week embryos by Nimble Gen Human DNA Methylation 3x729K CpG Island Plus RefSeq Promoter Array and compared the data with our published genome-wide DNA methylomes of human gametes and early embryos generated from in vivo maturation oocytes. We showed that IVM embryos show abnormal DNA methylation reprogramming pattern. By analyzing the abnormally reprogrammed promoters, we further found that IVM may affect the functions of demethylation related genes. Oocytes from IVM manipulation were tested with higher DNA methylation levels, and their abnormal methylated promoters mainly enriched in immune and metabolism pathways. Furthermore, we investigated the DNA methylation of TE, which was directly related with implantation process and revealed the abnormal methylated promoters were related with metabolism pathway too. Our data support that IVM may influence the DNA methylome of oocytes, which in turn affects the methylome of their embryos. However, due to the limited number of samples and the inability of the chip to cover all CpG sites, the results of this study require further research and validation.


Author(s):  
Saúl Lira-Albarrán ◽  
Xiaowei Liu ◽  
Seok Hee Lee ◽  
Paolo Rinaudo

Abstract Offspring generated by in vitro fertilization (IVF) are believed to be healthy but display a possible predisposition to chronic diseases, like hypertension and glucose intolerance. Since epigenetic changes are believed to underlie such phenotype, this study aimed at describing global DNA methylation changes in the liver of adult mice generated by natural mating (FB group) or by IVF. Embryos were generated by IVF or natural mating. At 30 weeks of age, mice were sacrificed. The liver was removed, and global DNA methylation was assessed using whole-genome bisulfite sequencing (WGBS). Genomic Regions for Enrichment Analysis Tool (GREAT) and G:Profilerβ were used to identify differentially methylated regions (DMRs) and for functional enrichment analysis. Overrepresented gene ontology terms were summarized with REVIGO, while canonical pathways (CPs) were identified with Ingenuity® Pathway Analysis. Overall, 2692 DMRs (4.91%) were different between the groups. The majority of DMRs (84.92%) were hypomethylated in the IVF group. Surprisingly, only 0.16% of CpG islands were differentially methylated and only a few DMRs were located on known gene promoters (n = 283) or enhancers (n = 190). Notably, the long-interspersed element (LINE), short-interspersed element (SINE), and long terminal repeat (LTR1) transposable elements showed reduced methylation (P < 0.05) in IVF livers. Cellular metabolic process, hepatic fibrosis, and insulin receptor signaling were some of the principal biological processes and CPs modified by IVF. In summary, IVF modifies the DNA methylation signature in the adult liver, resulting in hypomethylation of genes involved in metabolism and gene transcription regulation. These findings may shed light on the mechanisms underlying the developmental origin of health and disease.


2007 ◽  
Vol 19 (1) ◽  
pp. 143
Author(s):  
M. Kawasumi ◽  
Y. Unno ◽  
M. Nishiwaki ◽  
K. Matsumoto ◽  
M. Anzai ◽  
...  

Cloning by adult somatic cell nuclear transfer (SCNT) has proven to be successful for the production of clones from many species (Keith 2004 Cytogenet. Genome Res. 105, 285). However, somatic cloning is currently highly inefficient. One of the reasons for this is that SCNT is believed to be associated with epigenetic errors including abnormal DNA methylation of the reconstructed embryo. The Oct-3/4 gene, a member of the POU transcription factor family, is expressed throughout the pre-implantation embryo. Abnormal expression of the Oct-3/4 gene in the nuclear-transferred embryo is either directly or indirectly caused by nuclear transfer and is suggested to be indicative of a general failure to reset the genetic program (Boiani et al. 2002 Genes Dev. 16, 1209). In this study, we investigated the DNA methylation profiles of the Oct-3/4 gene in the genome of SCNT embryos, using bisulfite sequencing analysis. Then, we observed the detailed subcellular localization of Oct-3/4 proteins in SCNT embryos using immunocytochemical (ICC) analysis. Nuclear transfer of cumulus cell nuclei was carried out as previously described (Wakayama et al. 1998 Nature 394, 369). After nuclear transfer, embryos were subsequently cultured in KSOM media to the morula and blastocyst stages. We compared the methylation profiles of 3 transcriptional control elements (distal enhancer, DE; proximal enhancer, PE; and promoter) of the upstream region of the Oct-3/4 gene with the genome of in vitro fertilization (IVF) and SCNT embryos. The methylation rate of CpG sites in the DE and promoter regions of both IVF and SCNT embryos was low at both the morula and the blastocyst stages. What&apos;s interesting is that there was a significant difference in the methylation level on CpG sites in the PE element between IVF and SCNT embryos. At the morula stage, the methylation level on CpG sites in the PE element was very low in the IVF embryo and moderately high in the SCNT embryo (0.9&percnt; and 26.3&percnt;). Conversely, at the blastocyst stage, CpG sites in the PE element showed high methylation in the IVF embryo and low methylation in the SCNT embryo (55.2&percnt; and 10.5&percnt;). CpG sites in the PE element were lightly methylated (3.0&percnt;) in the inner cell mass (ICM) of the IVF embryo. This means that the main portion of methylation in the IVF blastocyst embryo occurred at the trophectoderm (TE). On the other hand, in ICM of the SCNT embryo, the methylation level of each embryonic cell was almost the same in the whole blastocyst embryo (9.8&percnt; and 10.5&percnt;). As a result, it is highly possible that the CpG sites in the PE element of ICM were methylated as in the TE. ICC analysis revealed that some SCNT embryos showed aberrant Oct-3/4 expression in the TE. These results indicate that the methylation of CpG sites in the Oct-3/4 PE element may be related to expression of Oct-3/4 in the mouse IVF and SCNT embryos. These differences in methylation level between IVF and SCNT embryos were reflected as abnormal expressions of Oct-3/4 on SCNT embryos. This study was supported by the 21st COE Program of MEST. M.K. is a JSPS Research Fellow and supported by Grant-in Aid for Scientific Research (No. 1751132) of JSPS.


2012 ◽  
Vol 97 (1) ◽  
pp. 147-153.e7 ◽  
Author(s):  
Verity F. Oliver ◽  
Harriet L. Miles ◽  
Wayne S. Cutfield ◽  
Paul L. Hofman ◽  
Jackie L. Ludgate ◽  
...  

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