Development and characterization of Hanwoo oviduct epithelial cell line for heat stress study

Objective: Heat-induced stress (HS) amid global warming compromise cattle reproductive performance causing economic loss including the vulnerable Korean native beef cattle, Hanwoo.Nevertheless, knowledge of response and effect in cattle oviduct epithelial cells (OEC) under HS remains lacking due to restricted commercial cell line and multi-omics data available.This research attempts to develop immortalized OEC derived from Hanwoo cattle as HS cell culture model and to investigate its HS-specific response by utilizing biomarkers comprising transcriptomic and proteomic analysis.Methods: Primary OEC was isolated from fresh Hanwoo oviduct and immortalized by using piggyBac transposon-mediated SV40T expression system.HS optimization was performed by detection of reactive oxygen species (ROS) and oxidative-endoplasmic reticulum (ER) stress biomarkers under varied HS and recovery time (4, 12, and 24 h).Optimized period was applied to generate differentially expressed genes (DEG) and protein (DEP) analyzed using gene ontology (GO) and pathway enrichment.Results: Immortalized OEC was successfully developed under high level of CDH1 positivity (98.2%) sorting.Increased intracellular ROS was detected at 4 h HS followed by rapid substantial upregulation of HSP70 and BiP protein and delayed upregulation of oxidative stress genes (SOD1, CAT, GPX1) peaked at optimized 24 h HS period.In total there were 510 DEG (fold change ≥|2|) and 258 DEP (fold change ≥|1.5|) were significantly altered under 24 h HS condition.Furthermore, GO and pathway enrichment analysis of the DEG-DEP list revealed highlighted upregulation process related to oxidative-thermal stress and protein folding.Other altered processes involve immune response, cellular senescence, response to starvation, and extracellular matrix structures as enriched terms suggesting possible hindering effects to OEC reproductive role.Conclusion: Developed immortalized OEC presents stable cell culture system to evaluate HS response in cattle oviductal tract.Transcriptomic and proteomic data generated in this study are valuable source for future research regarding targeted molecular approach attenuating detrimental HS effect in cattle reproduction.