Transcriptomics Reveals Granulosa Cell’s Coping through Redox, Inflammatory, Metabolic and Cytoskeleton Mechanisms under Acute Heat Stress

Abstract

Heat stress affects the granulosa cells (GCs) and 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 ℃ for 2 h) in-vitro and gave essential insights into the general interaction at cell–stress nexus. Heat-stressed GCs exhibited transient proliferation senescence, 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 heat stress group. In spite of the upregulation of pro-apoptotic and caspase executioner genes, antioxidants and anti-apoptotic genes were also upregulated in heat-stressed GCs. Progesterone and Estrogen hormones along with steroidogenic genes expression, declined significantly, in spite of the upregulation of genes involved in cholesterol synthesis. Out of 12385 differentially expressed genes (DEGs), 330 significant DEGs (75 upregulated, 225 downregulated) were subjected to KEGG functional pathway annotation, gene ontology enrichment, and STRING network analyses. Based on the manual query of DEGs, pathway and enrichment analyses, a vast interplay observed among all major signaling pathways strongly evidence the repression of cellular transcriptional and proliferation activity, averting the effects of heat stress through remodeling of cellular structural proteins and energetic-homeostasis. This study presents detailed responses of acute heat-stressed GCs at physical, transcriptional, and pathway levels and presents interesting insights into future studies regarding GCs adaptation and their interaction with oocyte and reproductive system at ovarian level.

Joint Transcriptome and Metabolome Analysis Prevails Biological Mechanisms Underlying the Prosurvival Fight in Heat Stressed Granulosa Cells

“…Granulosa cells present in the ovarian follicles are important for oocyte development due to their specialized steroidogenesis role and cross talk with oocytes [1,2]. Besides our studies [4,21], earlier studies also extensively reported the adverse effect of varying degree of heat stress on granulosa cells [3,19,25]. Where, heat stress promoted oxidative stress, inflammatory cytokine signaling, an evidence of intricate survival fight in the presence of pro-apoptotic and anti-apoptotic genes, and energetic support pathways [4,21].…”

“…While in our LC-MS based untargeted metabolomics study, important metabolites, metabolic pathways and their interplay in cellular mechanisms were reported in acute heat stressed bovine granulosa cells [22]. Our transcriptome study reported evidences of an intricate fight between pro-survival and pro-death pathways, while metabolome study suggested differential metabolites directed towards bioenergetics support mechanisms in acute heat stressed bovine granulosa cells [21,22]. Integrated transcriptome and metabolome analyses involving correlation and network analysis, is an efficient strategy to explore the mechanisms underlying a biological system.…”

“…Therefore, we performed another comprehensive study of acute heat stress (43 °C) to granulosa cells where a transient cellular senescence and later resumption of proliferation was observed in heat stressed granulosa cells. Our previous transcriptome level studies involving extensive range of temperatures gave useful biological insights into the cellular mechanisms, metabolic level changes and apoptotic and antioxidant pathways [4,21]. While in our LC-MS based untargeted metabolomics study, important metabolites, metabolic pathways and their interplay in cellular mechanisms were reported in acute heat stressed bovine granulosa cells [22].…”

“…Bovine granulosa were exposed to in vitro heat stress treatment (43 °C), while the control group remained at 38 °C [21,22]. Cells in the control group maintained steady proliferation activity (Figure 1A), while no change was observed until 24 h in treatment group (Figure 1A).…”

“…Integrated transcriptome and metabolome analyses involving correlation and network analysis, is an efficient strategy to explore the mechanisms underlying a biological system. Therefore, this study intends to evaluate cellular physical responses and integrate the transcriptome and metabolome data from acute heat stressed bovine granulosa cells [21,22], to narrow down and possibly explore new biological insights regarding important genes, metabolites, adaptive pathways involved in aftermath of acute heat stress to bovine granulosa cells.…”