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Follistatin treatment modifies DNA methylation of the CDX2 gene in bovine preimplantation embryos.

ABSTRACT: CDX2 plays a crucial role in the formation and maintenance of the trophectoderm epithelium in preimplantation embryos. Follistatin supplementation during the first 72?hr of in vitro culture triggers a significant increase in blastocyst rates, CDX2 expression, and trophectoderm cell numbers. However, the underlying epigenetic mechanisms by which follistatin upregulates CDX2 expression are not known. Here, we investigated whether stimulatory effects of follistatin are linked to alterations in DNA methylation within key regulatory regions of the CDX2 gene. In vitro-fertilized (IVF) zygotes were cultured with or without 10?ng/ml of recombinant human follistatin for 72?hr, then cultured without follistatin until Day 7. The bisulfite-sequencing analysis revealed differential methylation (DM) at specific CpG sites within the CDX2 promoter and intron 1 following follistatin treatment. These DM CpG sites include five hypomethylated sites at positions -1384, -1283, -297, -163, and -23, and four hypermethylated sites at positions -1501, -250, -243, and +20 in the promoter region. There were five hypomethylated sites at positions +3060, +3105, +3219, +3270, and +3545 in intron 1. Analysis of transcription factor binding sites using MatInspector combined with a literature search revealed a potential association between differentially methylated CpG sites and putative binding sites for key transcription factors involved in regulating CDX2 expression. The hypomethylated sites are putative binding sites for FXR, STAF, OCT1, KLF, AP2 family, and P53 protein, whereas the hypermethylated sites are putative binding sites for NRSF. Collectively, our results suggest that follistatin may increase CDX2 expression in early bovine embryos, at least in part, by modulating DNA methylation at key regulatory regions.

SUBMITTER: Ashry M

PROVIDER: S-EPMC7670970 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Follistatin treatment modifies DNA methylation of the CDX2 gene in bovine preimplantation embryos.

Ashry Mohamed M Rajput Sandeep K SK Folger Joseph K JK Yang Chunyan C Knott Jason G JG Smith George W GW

Molecular reproduction and development 20200810 9

CDX2 plays a crucial role in the formation and maintenance of the trophectoderm epithelium in preimplantation embryos. Follistatin supplementation during the first 72 hr of in vitro culture triggers a significant increase in blastocyst rates, CDX2 expression, and trophectoderm cell numbers. However, the underlying epigenetic mechanisms by which follistatin upregulates CDX2 expression are not known. Here, we investigated whether stimulatory effects of follistatin are linked to alterations in DNA ...[more]

PMID: 32776625

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Project description: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.

Project description:BackgroundInfertility in dairy cattle is a concern where reduced fertilization rates and high embryonic loss are contributing factors. Studies of the paternal contribution to reproductive performance are limited. However, recent discoveries have shown that, in addition to DNA, sperm delivers transcription factors and epigenetic components that are required for fertilization and proper embryonic development. Hence, characterization of the paternal contribution at the time of fertilization is warranted. We hypothesized that sire fertility is associated with differences in DNA methylation patterns in sperm and that the embryonic transcriptomic profiles are influenced by the fertility status of the bull. Embryos were generated in vitro by fertilization with either a high or low fertility Holstein bull. Blastocysts derived from each high and low fertility bulls were evaluated for morphology, development, and transcriptomic analysis using RNA-Sequencing. Additionally, DNA methylation signatures of sperm from high and low fertility sires were characterized by performing whole-genome DNA methylation binding domain sequencing.ResultsEmbryo morphology and developmental capacity did not differ between embryos generated from either a high or low fertility bull. However, RNA-Sequencing revealed 98 genes to be differentially expressed at a false discovery rate < 1%. A total of 65 genes were upregulated in high fertility bull derived embryos, and 33 genes were upregulated in low fertility derived embryos. Expression of the genes CYCS, EEA1, SLC16A7, MEPCE, and TFB2M was validated in three new pairs of biological replicates of embryos. The role of the differentially expressed gene TFB2M in embryonic development was further assessed through expression knockdown at the zygotic stage, which resulted in decreased development to the blastocyst stage. Assessment of the epigenetic signature of spermatozoa between high and low fertility bulls revealed 76 differentially methylated regions.ConclusionsDespite similar morphology and development to the blastocyst stage, preimplantation embryos derived from high and low fertility bulls displayed significant transcriptomic differences. The relationship between the paternal contribution and the embryonic transcriptome is unclear, although differences in methylated regions were identified which could influence the reprogramming of the early embryo. Further characterization of paternal factors delivered to the oocyte could lead to the identification of biomarkers for better selection of sires to improve reproductive efficiency.

Project description:The methylation of cytosine at CG sites in the mammalian genome is dynamically reprogrammed during gametogenesis and preimplantation development. It was previously shown that oocyte-derived DNMT1 (a maintenance methyltransferase) is essential for maintaining and propagating CG methylation at imprinting control regions in preimplantation embryos. In mammalian somatic cells, hemimethylated-CG-binding protein UHRF1 plays a critical role in maintaining CG methylation by recruiting DNMT1 to hemimethylated CG sites. However, the role of UHRF1 in oogenesis and preimplantation development is unknown. In the present study, we show that UHRF1 is mainly, but not exclusively, localized in the cytoplasm of oocytes and preimplantation embryos. However, smaller amounts of UHRF1 existed in the nucleus, consistent with the expected role in DNA methylation. We then generated oocyte-specific Uhrf1 knockout (KO) mice and found that, although oogenesis was itself unaffected, a large proportion of the embryos derived from the KO oocytes died before reaching the blastocyst stage (a maternal effect). Whole genome bisulfite sequencing revealed that blastocysts derived from KO oocytes have a greatly reduced level of CG methylation, suggesting that maternal UHRF1 is essential for maintaining CG methylation, particularly at the imprinting control regions, in preimplantation embryos. Surprisingly, UHRF1 was also found to contribute to de novo CG and non-CG methylation during oocyte growth: in Uhrf1 KO oocytes, transcriptionally-inactive regions gained less methylation, while actively transcribed regions, including the imprinting control regions, were unaffected or only slightly affected. We also found that de novo methylation was defective during the late stage of oocyte growth. To the best of our knowledge, this is the first study to demonstrate the role of UHRF1 in de novo DNA methylation in vivo. Our study reveals multiple functions of UHRF1 during the global epigenetic reprogramming of oocytes and early embryos.

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Follistatin treatment modifies DNA methylation of the CDX2 gene in bovine preimplantation embryos.

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Follistatin treatment modifies DNA methylation of the CDX2 gene in bovine preimplantation embryos.

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