National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics, Breeding and Reproduction, MARA, College of Animal Sciences and Technology, China Agricultural University, Beijing 100193, China.
Embryo Biotechnology and Reproduction Laboratory, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
Cells. 2022 Apr 25;11(9):1443. doi: 10.3390/cells11091443.
Heat stress affects granulosa cells (GCs) and the ovarian follicular microenvironment, causing poor oocyte developmental competence and fertility. This study aimed to investigate the physical responses and global transcriptomic changes in bovine GCs to acute heat stress (43 °C for 2 h) in vitro. Heat-stressed GCs exhibited transient proliferation senescence and resumed proliferation at 48 h post-stress, while post-stress immediate culture-media change had a relatively positive effect on proliferation resumption. Increased accumulation of reactive oxygen species and apoptosis was observed in the heat-stress group. In spite of the upregulation of inflammatory (CYCS, TLR2, TLR4, IL6, etc.), pro-apoptotic (BAD, BAX, TNFSF9, MAP3K7, TNFRSF6B, FADD, TRADD, RIPK3, etc.) and caspase executioner genes (CASP3, CASP8, CASP9), antioxidants and anti-apoptotic genes (HMOX1, NOS2, CAT, SOD, BCL2L1, GPX4, etc.) were also upregulated in heat-stressed GCs. Progesterone and estrogen hormones, along with steroidogenic gene expression, declined significantly, in spite of the upregulation of genes involved in cholesterol synthesis. Out of 12,385 differentially expressed genes (DEGs), 330 significant DEGs (75 upregulated, 225 downregulated) were subjected to KEGG functional pathway annotation, gene ontology enrichment, STRING network analyses and manual querying of DEGs for meaningful molecular mechanisms. High inflammatory response was found to be responsible for oxidative-stress-mediated apoptosis of GCs and nodes towards the involvement of the NF-κB pathway and repression of the Nrf2 pathway. Downregulation of MDM4, TP53, PIDD1, PARP3, MAPK14 and MYC, and upregulation of STK26, STK33, TGFB2, CDKN1A and CDKN2A, at the interface of the MAPK and p53 signaling pathway, can be attributed to transient cellular senescence and apoptosis in GCs. The background working of the AMPK pathway through upregulation of AKT1, AMPK, SIRT1, PYGM, SLC2A4 and SERBP1 genes, and downregulation of PPARGCIA, IGF2, PPARA, SLC27A3, SLC16A3, TSC1/2, KCNJ2, KCNJ16, etc., evidence the repression of cellular transcriptional activity and energetic homeostasis modifications in response to heat stress. This study presents detailed responses of acute-heat-stressed GCs at physical, transcriptional and pathway levels and presents interesting insights into future studies regarding GC adaptation and their interaction with oocytes and the reproductive system at the ovarian level.
热应激会影响颗粒细胞（GCs）和卵巢卵泡微环境，导致卵母细胞发育能力和生育能力下降。本研究旨在探讨体外急性热应激（43°C 2 小时）对牛 GCs 的物理反应和全转录组变化。热应激的 GCs 表现出短暂的增殖衰老，在应激后 48 小时恢复增殖，而应激后即时更换培养基对恢复增殖有相对积极的影响。在热应激组中观察到活性氧和细胞凋亡的增加。尽管炎症（CYCS、TLR2、TLR4、IL6 等）、促凋亡（BAD、BAX、TNFSF9、MAP3K7、TNFRSF6B、FADD、TRADD、RIPK3 等）和半胱天冬酶执行器基因（CASP3、CASP8、CASP9）上调，但抗氧化剂和抗凋亡基因（HMOX1、NOS2、CAT、SOD、BCL2L1、GPX4 等）也上调。尽管胆固醇合成相关基因上调，但孕激素和雌激素激素以及类固醇生成基因表达显著下降。在 12385 个差异表达基因（DEGs）中，有 330 个显著 DEGs（75 个上调，225 个下调）进行了 KEGG 功能途径注释、基因本体富集、STRING 网络分析和 DEGs 手动查询，以寻找有意义的分子机制。高炎症反应被认为是导致 GCs 氧化应激介导的细胞凋亡的原因，并且 NF-κB 途径和 Nrf2 途径的抑制涉及到该途径的节点。MDM4、TP53、PIDD1、PARP3、MAPK14 和 MYC 的下调，以及 STK26、STK33、TGFB2、CDKN1A 和 CDKN2A 的上调，都可以归因于 GCs 中的短暂细胞衰老和凋亡。AMPK 途径通过上调 AKT1、AMPK、SIRT1、PYGM、SLC2A4 和 SERBP1 基因，下调 PPARGCIA、IGF2、PPARA、SLC27A3、SLC16A3、TSC1/2、KCNJ2、KCNJ16 等基因，在 AMPK 途径的背景下工作，证明了细胞转录活性的抑制和对热应激的能量稳态修饰。本研究在物理、转录和途径水平上详细描述了急性热应激 GCs 的反应，并为未来关于 GC 适应及其与卵母细胞和卵巢水平生殖系统相互作用的研究提供了有趣的见解。
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