Meng Li, Wu Zhenfang, Zhao Kun, Tao Jian, Chit Tam, Zhang Shouquan, Wang Chi Chiu, Teerds Katja
National Engineering Research Center of Breeding Swine Industry, College of Animal Science, South China Agricultural University, Guangzhou 510642, China.
Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding, Key Lab of Chicken Genetics, Breeding and Reproduction, Ministry of Agriculture, South China Agricultural University, Guangzhou 510642, China.
Antioxidants (Basel). 2020 Dec 28;10(1):22. doi: 10.3390/antiox10010022.
One of the main causes of female infertility is a deregulated antral follicular atresia, a process of which the underlying molecular mechanisms are largely unknown. Our objective was therefore to characterize the complex transcriptome changes in porcine granulosa cells of healthy antral (HA) and advanced antral atretic (AA) follicles, using ELISA and RNA-Seq followed by qRT-PCR and immunohistochemistry. Granulosa cell RNA-Seq data revealed 2160 differentially expressed genes, 1483 with higher and 677 with lower mRNA concentrations in AA follicles. Bioinformatic analysis showed that the upregulated genes in AA follicles were highly enriched in inflammation and apoptosis processes, while the downregulated transcripts were mainly highlighted in the steroid biosynthesis pathway and response to oxidative stress processes including antioxidant genes (e.g., , , , , ) involved in the glutathione metabolism pathway and other redox-related genes (e.g., , ). These observations were confirmed by RT-qPCR and immunohistochemistry. Additionally, the granulosa cells of AA follicles express significantly stronger 8-OHdG immunostaining, a marker of oxidative DNA damage, implicating that oxidative stress may participate in follicular atresia. We hypothesize that the decrease in anti-apoptotic factors and steroid hormones coincides with increased oxidative stress markers and the expression of pro-apoptotic factors, all contributing to antral follicular atresia.
女性不孕的主要原因之一是窦状卵泡闭锁失调，而该过程的潜在分子机制在很大程度上尚不清楚。因此，我们的目标是利用酶联免疫吸附测定（ELISA）和RNA测序，随后进行定量逆转录聚合酶链反应（qRT-PCR）和免疫组织化学，来表征健康窦状（HA）卵泡和晚期窦状闭锁（AA）卵泡的猪颗粒细胞中复杂的转录组变化。颗粒细胞RNA测序数据显示有2160个差异表达基因，其中1483个在AA卵泡中的mRNA浓度较高，677个在AA卵泡中的mRNA浓度较低。生物信息学分析表明，AA卵泡中上调的基因在炎症和凋亡过程中高度富集，而下调的转录本主要集中在类固醇生物合成途径以及对氧化应激过程的反应中，包括参与谷胱甘肽代谢途径的抗氧化基因（如 、 、 、 、 ）和其他氧化还原相关基因（如 、 ）。这些观察结果通过逆转录定量聚合酶链反应（RT-qPCR）和免疫组织化学得到了证实。此外，AA卵泡的颗粒细胞表达明显更强的8-羟基脱氧鸟苷（8-OHdG）免疫染色，这是氧化DNA损伤的标志物，表明氧化应激可能参与卵泡闭锁。我们推测，抗凋亡因子和类固醇激素的减少与氧化应激标志物的增加以及促凋亡因子的表达同时出现，所有这些都导致了窦状卵泡闭锁。
