The Dnmt3b Splice Variant is Specifically Expressed in In Vitro-manipulated Blastocysts and Their Derivative ES Cells

Abstract

Abstract. Manipulation of preimplantation embryos in vitro, such as in vitro fertilization (IVF), in vitro culture (IVC), intracytoplasmic sperm injection (ICSI), somatic cell nuclear transfer (SCNT) and other assisted reproduction technologies (ART), has contributed to the development of infertility treatment and new animal reproduction methods. However, such embryos often exhibit abnormal DNA methylation patterns in imprinted genes and centromeric satellite repeats. These DNA methylation patterns are established and maintained by three DNA methyltransferases: Dnmt1, Dnmt3a and Dnmt3b. Dnmt3b is responsible for the creation of methylation patterns during the early stage of embryogenesis and consists of many alternative splice variants that affect methylation activity; nevertheless, the roles of these variants have not yet been identified. In this study, we found an alternatively spliced variant of Dnmt3b lacking exon 6 (Dnmt3bΔ6) that is specific to mouse IVC embryos. Dnmt3bΔ6 also showed prominent expression in embryonic stem (ES) cells derived from in vitro manipulated embryos. Interestingly, IVC blastocysts were hypomethylated in centromeric satellite repeat regions that could be susceptible to methylation by Dnmt3b. In vitro methylation activity assays showed that Dnmt3bΔ6 had lower activity than normal Dnmt3b. Our findings suggest that Dnmt3bΔ6 could induce a hypomethylation status especially in in vitro manipulated embryos. Key words: DNA methylation, Dnmt3b, In vitro culture (J. Reprod. Dev. 57: 579-585, 2011) he 5th position cytosine residues in CpG sequences are often methylated in vertebrate genomic DNA [1]. DNA methylation plays an essential role in the normal development of mammalian embryos by regulating gene expression through genomic imprinting, X chromosome inactivation and genomic stability [2][3][4][5][6]. In vertebrates, two types of DNA methyltransferase activity have been reported, the de novo and maintenance types. In mice, de novo-type DNA methylation activity creates gene-specific methylation patterns during the implantation stage of embryogenesis, while maintenance-type activity ensures clonal transmission of lineage-specific methylation patterns during replication. Dnmt1 is responsible for the latter activity. On the other hand, two DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for creation of methylation patterns during the early stages of embryogenesis [7,8] and have been shown to possess de novo-type DNA methylation activity in vitro [9][10][11][12]. Recent studies have shown that Dnmts function in cooperation with each other to facilitate DNA methylation in both humans and mice [13][14][15].In mice, Dnmt3b is the major de novo DNA methyltransferase in E (embryonic stage) 4.5-7.5 embryos, and its expression is downregulated after midgestation [8,16]. Disruption of Dnmt3b results in embryonic lethality at E13.5 and hypomethylation of centromeric minor satellite repeats [8]. In humans, DNMT3B mutations have been shown to cause ICF (immunodeficiency, chromosomal instabi...

Origins of Lifetime Health Around the Time of Conception: Causes and Consequences

“…Importantly, even a very limited culture period is sufficient to induce epigenetic changes 81 . Embryo culture exposure also modifies expression and methylation of nonimprinted genes in mice and alters expression of DNA methyltransferases 84 . For example, in mouse models ART affects the endothelial nitric oxide synthase (eNOS) gene implicated in vascular dysfunction and modification of culture media composition may prevent this effect 85 .…”

Regulation of CpG methylation by Dnmt and Tet in pluripotent stem cells

“…For example, blastocyst-stage embryos derived from in vitro fertilized zygotes or parthenogenetically activated oocytes showed a significant loss of CpG methylation in major and minor satellite repeats or in differentially methylated regions of several imprinted genes [42, 43]. This hypomethylation may be caused by artificial treatment, such as superovulation [44] and in vitro culture [45].…”

“…In particular, Dnmt3b has nearly 40 different isoforms generated by alternative splicing and/or alternative promoter usage, and the ratio of its isoforms is different among cell types [55,56,57]. We recently found that mouse Dnmt3b lacking exon 6 (Dnmt3bΔ6) is highly expressed in in vitro manipulated embryos and their derivative ESCs, which exhibit a loss of CpG methylation [43]. In vitro methylation activity assays revealed that Dnmt3bΔ6 had lower methylation activity than the normal Dnmt3b.…”

Embryonic Stem Cells Derived from In Vivo or In Vitro-Generated Murine Blastocysts Display Similar Transcriptome and Differentiation Potential

“…More specifically the authors found modifications of the methyltransferase Dnmt3b in in vitro cultured blastocysts when compared to in vivo blastocysts. These differences persisted in mESC derived from in vitro generated blastocysts [43]. If present, epigenetic changes in our system do not appear to result in alterations of significant gene expression.…”

“…Given the results showing transcriptome similarity, we did not examine the methylation status of genes in this study. Prior work has examined DNA methylation differences in mESC generated in vivo or in vitro at selected loci [42,43]. These authors found epigenetic differences between mESC originated from in vivo or in vitro blastocysts only after the initial passages, while the differences disappeared at later passages, suggesting an effect of extended culture on mESC epigenome [42].…”