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.

中文翻译：

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卵泡抑素治疗可改变牛植入前胚胎中 CDX2 基因的 DNA 甲基化。


CDX2 在植入前胚胎滋养外胚层上皮的形成和维持中起着至关重要的作用。在体外培养的前 72 小时内补充卵泡抑素可显着增加囊胚率、 CDX2表达和滋养外胚层细胞数量。然而，卵泡抑素上调CDX2表达的潜在表观遗传机制尚不清楚。在这里，我们研究了卵泡抑素的刺激作用是否与CDX2基因关键调控区域内 DNA 甲基化的改变有关。体外受精 (IVF) 受精卵在有或没有 10 ng/ml 重组人卵泡抑素的情况下培养 72 小时，然后在没有卵泡抑素的情况下培养直至第 7 天。亚硫酸氢盐测序分析显示，在特定 CpG 位点上存在差异甲基化 (DM)。卵泡抑素治疗后的CDX2启动子和内含子 1。这些DM CpG位点包括启动子区域中位置-1384、-1283、-297、-163和-23的五个低甲基化位点，以及位于-1501、-250、-243和+20位的四个高甲基化位点。内含子 1 中 +3060、+3105、+3219、+3270 和 +3545 位点有 5 个低甲基化位点。使用 MatInspector 结合文献检索对转录因子结合位点进行分析，揭示了差异甲基化 CpG 位点与假定的 CpG 位点之间的潜在关联。参与调节CDX2表达的关键转录因子的结合位点。低甲基化位点是 FXR、STAF、OCT1、KLF、AP2 家族和 P53 蛋白的假定结合位点，而高甲基化位点是 NRSF 的假定结合位点。 总的来说，我们的结果表明，卵泡抑素可能至少部分通过调节关键调控区域的 DNA 甲基化来增加早期牛胚胎中CDX2 的表达。