Abstract
Caudal Type Homeobox 2 (CDX2) is a key regulator of trophectoderm formation and maintenance in preimplantation embryos. We previously demonstrated that supplementation of exogenous follistatin, during in vitro culture of bovine IVF embryos, upregulates CDX2 expression, possibly, via alteration of the methylation status of CDX2 gene. Here, we further investigated the effects of exogenous follistatin supplementation on developmental competence and CDX2 methylation in bovine somatic cell nuclear transfer (SCNT) embryos. SCNT embryos were cultured with or without follistatin for 72h, then transferred into follistatin free media until d7 when blastocysts were collected and subjected to CDX2 gene expression and DNA methylation analysis for CDX2 regulatory regions by bisulfite sequencing. Follistatin supplementation significantly increased both blastocyst development as well as blastocyst CDX2 mRNA expression on d7. Three different CpG rich fragments within the CDX2 regulatory elements; proximal promoter (fragment P1, -1644 to -1180; P2, -305 to +126) and intron 1 (fragment I, + 3030 to + 3710) were identified and selected for bisulfite sequencing analysis. This analysis showed that follistatin treatment induced differential methylation (DM) at specific CpG sites within the analyzed fragments. Follistatin treatment elicited hypomethylation at six CpG sites at positions -1374, -279, -163, -23, +122 and +3558 and hypermethylation at two CpG sites at positions -243 and +20 in promoter region and first intron of CDX2 gene. Motif analysis using MatInspector revealed that differentially methylated CpG sites are putative binding sites for key transcription factors (TFs) known to regulate Cdx2 expression in mouse embryos and embryonic stem cells including OCT1, AP2F, KLF and P53, or TFs that have indirect link to CDX2 regulation including HAND and NRSF. Collectively, results of the present study together with our previous findings in IVF embryos support the hypothesis that alteration of CDX2 methylation is one of the epigenetic mechanisms by which follistatin may regulates CDX2 expression in preimplantation bovine embryos.
Highlights
Somatic cell nuclear transfer (SCNT) is a powerful tool used to propagate valuable livestock species and to generate genetically modified animals that can be used in biomedical research and therapeutic cloning [1, 2]
We demonstrated that follistatin supplementation during the initial 72h of in vitro embryo culture (IVC1) upregulated Caudal Type Homeobox 2 (CDX2) expression in In Vitro Fertilization (IVF) blastocysts that was tightly associated with DNA methylation at specific CpG sites within the regulatory elements of CDX2 gene including the proximal promoter and intron 1
The objectives of the present study were to analyze the beneficial effects of follistatin on developmental competence and CDX2 expression in bovine SCNT embryos and to investigate whether these effects are linked to changes in DNA methylation as we previously reported in IVF embryos [16]
Summary
Somatic cell nuclear transfer (SCNT) is a powerful tool used to propagate valuable livestock species and to generate genetically modified animals that can be used in biomedical research and therapeutic cloning [1, 2]. We demonstrated that follistatin supplementation during the initial 72h of in vitro embryo culture (IVC1) upregulated CDX2 expression in IVF blastocysts that was tightly associated with DNA methylation at specific CpG sites within the regulatory elements of CDX2 gene including the proximal promoter and intron 1. These CpG sites contain putative binding sites for several key transcription factors implicated in regulating Cdx expression in mouse embryos and trophoblast stem cells [16]. Results show that follistatin treatment induces changes in DNA methylation at specific CpG sites within CDX2 regulatory regions in SCNT blastocysts, these changes are associated with upregulation of CDX2 expression
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