The bovine IGF2 locus is a genomic region with alternative transcripts controlled by five promoters (P0, P1, P2, P3 and P4). As transcriptional regulation can affect messenger RNA (mRNA) stability and translation, and thus, subsequent biological effects, this study evaluated the bovine IGF2 promoter-specific expression patterns in oocytes and pre-implantation embryos produced in vitro by our standard IVP procedures. Immature and matured oocytes, and pre-implantation embryos at the 1-, 2-, 4-, 8- and 16-cell, and at early morula, compact morula, blastocyst and expanded blastocyst stages were collected in three pools of five structures per stage, in four replicates. Total RNA was extracted and subjected to RT-qPCR, using four sets of IGF2 promoter-specific primers covering transcripts driven by promoters P0/P1, P2, P3 and P4, with fragments sequenced for confirmation. Expression of P2- and P4-derived transcripts showed an initial peak between immature (P4) or matured (P2/P4) oocytes and 2-cell embryos, gradually falling until embryo genome activation (EGA), rising again at compaction and cavitation. P0/P1-derived transcripts were identified after EGA, during compaction, whereas P3 activity was not detected at any stage. Our findings suggest that P0/P1 and P2 likely have secondary roles during early stages, whereas P3 may be more relevant later in development. P4 seems to be the main pathway for bovine IGF2 expression during oocyte maturation and embryo development and, therefore, the main target to influence IVP in modulation of embryo growth and in studies in developmental biology.

中文翻译：

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印迹 IGF2 基因在牛卵母细胞和植入前胚胎中的启动子特异性表达

牛IGF2 基因座是一个基因组区域，具有由五个启动子（P0、P1、P2、P3 和 P4）控制的替代转录本。由于转录调控会影响信使 RNA (mRNA) 的稳定性和翻译，从而影响随后的生物学效应，本研究评估了通过我们的标准 IVP 程序在体外产生的卵母细胞和植入前胚胎中的牛IGF2启动子特异性表达模式。未成熟和成熟的卵母细胞，以及 1、2、4、8 和 16 细胞以及早期桑椹胚、紧密桑椹胚、囊胚和扩张囊胚阶段的植入前胚胎被收集在三个池中，每个池中有五个结构。阶段，四次重复。提取总 RNA 并进行 RT-qPCR，使用四组IGF2启动子特异性引物覆盖由启动子 P0/P1、P2、P3 和 P4 驱动的转录本，片段测序用于确认。P2 和 P4 衍生转录本的表达在未成熟 (P4) 或成熟 (P2/P4) 卵母细胞和 2 细胞胚胎之间显示出初始峰值，逐渐下降，直到胚胎基因组激活 (EGA)，在压实和空化时再次上升。在 EGA 之后，在压实过程中鉴定了 P0/P1 衍生的转录物，而在任何阶段都未检测到 P3 活性。我们的研究结果表明，P0/P1 和 P2 可能在早期阶段具有次要作用，而 P3 可能在发育后期更相关。P4 似乎是牛IGF2的主要途径 在卵母细胞成熟和胚胎发育过程中表达，因此是影响 IVP 调节胚胎生长和发育生物学研究的主要目标。