Transcriptional dynamics of porcine granulosa cells during cellular acclimation to thermal challenge

Abstract

Investigations on heat stress induced transcriptomic changes is critical to characterization of candidate genes for thermal adaptability in livestock. Continues spells of high ambient temperature due to climate change has amplified reproductive dysfunctions, necessitating immediate attention. The present study aimed to explore the transcriptomic signature of heat stressed granulosa cells (GCs) and signalling pathways regulating their adaptability to thermal challenge. The GCs were collected from small follicles (3–6 mm) of pig ovary. The GCs primary culture was subjected to in vitro heat stress challenge at 42 ^OC for 6 h. RNA sequencing was conducted for heat stress (treated) and non-heat stress (control) groups using Illumina NextSeq2000 sequencing platform. The significant DEGs were selected using NOISeq R package with cut-offs, probability value ≥ 0.95 and log₂ fold change ≥1. Bioinformatics analysis was conducted for exploring gene ontology enrichment, functional pathways, hub genes in protein-protein interaction network and functional clusters regulating cellular homeostasis and survivability during heat stress challenge. The analysis pipeline yielded a total of 12156 protein coding transcripts, which were expressed during heat stress challenge in GCs, out of which 4904 were differentially (prob. ≥ 0.95) expressed; 2936 were upregulated and 1968 were downregulated. The large number of DEGs and gene ontologies in the study specifies the concerted mechanisms involving multiple signalling pathways like MAPK, HIPPO, WNT, PI3-AKT, NFKB, NOTCH and many more operating in the cell to maintain cellular homeostasis. Thermal stress induced differentially expressed hub genes HSP90, HSPA8, HSPA5, TGFB1 and PPARG are key elements in stress, regulating multiple pathways and expression of transcription factors. The TNF signalling pathway, phosphatidyl inositol signalling system and DERL3 gene network linked ubiquitin-dependent endoplasmic reticulum associated protein degradation pathway, which regulates cell viability, proliferation, apoptosis and estrogen synthesis, can be regarded as novel regulators involved in stress adaptation in pigs.