Yang Cai-Xia, Song Zhi-Qiang, Pei Surui, Yu Xiao-Xia, Miao Jia-Kun, Liang Hao, Miao Yi-Liang, Du Zhi-Qiang
College of Animal Science, Yangtze University, Jingzhou, 434025, Hubei, China; College of Animal Science and Technology, Northeast Agricultural University, Harbin, 150030, Heilongjiang, China.
College of Animal Science and Technology, Northeast Agricultural University, Harbin, 150030, Heilongjiang, China.
Theriogenology. 2020 Nov;157:449-457. doi: 10.1016/j.theriogenology.2020.08.020. Epub 2020 Aug 21.
Oocytes of better quality and developmental competence are highly demanded, which is affected by many intrinsic and external factors, including environmental pollutants. We have previously demonstrated that 7, 12-dimethylbenz [a]anthracene (DMBA) reduces the developmental competence of porcine oocytes, by desynchronizing nuclear and ooplasmic maturation. However, the underlying molecular mechanism remains obscure. Here we performed single cell RNA-seq to study the transcriptome changes in DMBA-treated porcine MII oocytes, and identified 19 protein-coding genes and 156 novel long non-coding RNAs (lncRNAs) with abundance to be significantly different (P < 0.05), which enriched in signaling pathways such as glycosphingolipid biosynthesis, nicotine addiction, basal transcription factors and nucleotide excision repair. RT-qPCR on oocyte pools confirmed ornithine aminotransferase (Oat) and serine/arginine-rich splicing factor 4 (Srsf4) to be significantly up- and down-regulated, respectively (P < 0.05). Treating porcine COCs with MAPK and PLC pathway inhibitors suppressed DMBA's effects on increasing PB1 extrusion rate. In addition, DMBA co-incubation with 250 μM vitamin C derivative (l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, AA2P) and 100 μM co-enzyme Q10 (CoQ10) could significantly reduce the DMBA-induced high ROS level, and partially alleviate the DMBA-induced high PB1 rate, whereas the cleavage and blastocyst rates of parthenotes derived from treated mature oocytes remained to be low. Collectively, our findings indicate that single cell RNA-seq can help reveal the dynamics of molecular signaling pathways for porcine oocytes treated by DMBA, and supplement of anti-oxidative reagents could not sufficiently rescue DMBA-induced defects of porcine oocytes.
人们对质量更好、发育能力更强的卵母细胞有很高的需求，而卵母细胞的质量和发育能力受许多内在和外在因素影响，包括环境污染物。我们之前已证明，7,12-二甲基苯并[a]蒽（DMBA）会使猪卵母细胞核成熟和细胞质成熟不同步，从而降低其发育能力。然而，其潜在分子机制仍不清楚。在此，我们进行了单细胞RNA测序，以研究经DMBA处理的猪MII期卵母细胞的转录组变化，鉴定出19个蛋白质编码基因和156个新的长链非编码RNA（lncRNA），其丰度有显著差异（P<0.05），这些基因富集于糖鞘脂生物合成、尼古丁成瘾、基础转录因子和核苷酸切除修复等信号通路中。对卵母细胞池进行的RT-qPCR证实，鸟氨酸转氨酶（Oat）和富含丝氨酸/精氨酸的剪接因子4（Srsf4）分别显著上调和下调（P<0.05）。用MAPK和PLC通路抑制剂处理猪卵丘-卵母细胞复合体可抑制DMBA对提高第一极体（PB1）排出率的影响。此外，DMBA与250μM维生素C衍生物（l-抗坏血酸2-磷酸倍半镁盐水合物，AA2P）和100μM辅酶Q10（CoQ10）共同孵育可显著降低DMBA诱导的高活性氧水平，并部分缓解DMBA诱导的高PB1率，而来自经处理的成熟卵母细胞的孤雌胚胎的卵裂率和囊胚率仍然较低。总的来说，我们的研究结果表明，单细胞RNA测序有助于揭示DMBA处理的猪卵母细胞分子信号通路的动态变化，补充抗氧化剂不能充分挽救DMBA诱导的猪卵母细胞缺陷。
