PRC, INRA, CNRS, IFCE, Université de Tours, Nouzilly, France.
Dept of Arctic and Marine Biology UiT-The Arctic University of Norway, Tromsø, Norway.
PLoS One. 2018 May 10;13(5):e0197123. doi: 10.1371/journal.pone.0197123. eCollection 2018.
This study investigated Vegfa expression in the pars tuberalis (PT) of the pituitary and medio-basal hypothalamus (MBH) of sheep, across seasons and reproductive states. It has recently been proposed that season impacts alternative splicing of Vegfa mRNA in the PT, which shifts the balance between angiogenic VEGFAxxx and anti-angiogenic VEGFAxxxb isoforms (with xxx the number of amino acids of the mature VEGFA proteins) to modulate seasonal breeding. Here, we used various RT-PCR methodologies and analysis of RNAseq datasets to investigate seasonal variation in expression and splicing of the ovine Vegfa gene. Collectively, we identify 5 different transcripts for Vegfa within the ewe PT/MBH, which correspond to splicing events previously described in mouse and human. All identified transcripts encode angiogenic VEGFAxxx isoforms, with no evidence for alternative splicing within exon 8. These findings led us to investigate in detail how "Vegfaxxxb-like" PCR products could be generated by RT-PCR and misidentified as endogenous transcripts, in sheep and human HEK293 cells. In conclusion, our findings do not support the existence of anti-angiogenic VEGFAxxxb isoforms in the ovine PT/MBH and shed new light on the interpretation of prior studies, which claimed to identify Vegfaxxxb isoforms by RT-PCR.
本研究调查了绵羊垂体柄（PT）和中基底下丘脑（MBH）中 Vegfa 的表达，横跨季节和生殖状态。最近有人提出，季节会影响 PT 中 Vegfa mRNA 的选择性剪接，从而改变血管生成 VEGFAxxx 和抗血管生成 VEGFAxxxb 同工型之间的平衡，从而调节季节性繁殖。在这里，我们使用了各种 RT-PCR 方法和 RNAseq 数据集的分析来研究绵羊 Vegfa 基因的表达和剪接的季节性变化。总的来说，我们在母羊 PT/MBH 中鉴定出了 5 种不同的 Vegfa 转录本，这些转录本对应于先前在小鼠和人类中描述的剪接事件。所有鉴定的转录本都编码血管生成的 VEGFAxxx 同工型，没有证据表明第 8 外显子内存在选择性剪接。这些发现促使我们详细研究 RT-PCR 如何产生“Vegfaxxxb 样”PCR 产物，并在绵羊和人类 HEK293 细胞中被错误识别为内源性转录本。总之，我们的研究结果不支持在绵羊 PT/MBH 中存在抗血管生成的 VEGFAxxxb 同工型，并为先前声称通过 RT-PCR 鉴定 Vegfaxxxb 同工型的研究提供了新的见解。
