Establishment and characterization of Hanwoo cumulus cell line for heat stress studies

Heat stress (HS) negatively affects cattle reproduction, decreasing livestock productivity. HS response in cattle reproductive cumulus cells remain understudied due to limited cell lines and comprehensive omics data. This study aims to establish immortalized cumulus cells from Hanwoo cattle as a HS model and explores their HS response using molecular markers including transcriptomics and proteomics approaches.

Immortalized cumulus cells were established from primary cumulus isolated from fresh Hanwoo cattle follicles by using piggyBac transposon-mediated SV40T expression system. HS condition was optimized using reactive oxygen species (ROS) detection, quantitative reverse-transcription polymerase chain reaction, and Western blotting on oxidative and endoplasmic reticulum stress markers under different HS and recovery periods (4 h, 12 h, and 24 h). Transcriptomic and proteomic data were generated to identify HS-related differentially expressed genes (DEG) and proteins (DEP) utilized for gene ontology (GO) and pathway enrichment analysis.

We established the first immortalized cumulus cell line with high specificity (99% CD44[+]) for in vitro HS study. ROS accumulation was observed at 4 h HS, leading to immediate upregulation of HSP70 and BiP proteins, with oxidative stress gene expression (SOD1, CAT, GPX1) peaked at 12 h HS. We identified 754 DEGs (fold change≥|2|) and 357 DEPs (fold change≥|1.5|) related to HS in the cumulus cell line. Enrichment analysis exhibited upregulation in GO and pathways related to protein unfolding and cellular response to HS, respectively, from both the DEG and DEP list. Downregulated DEG and DEP showed significant enrichment in GO related to the extracellular matrix, potentially affecting cumulus-oocyte complex communication for reproductive function.

The cumulus cell line serves as a robust in vitro model for studying HS response in cattle reproductive system. The multi-omics data could help clarify the comprehensive HS response and identify possible molecular targets to mitigate negative HS effects in the future.