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

Abstract

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 with high 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 24 h HS period. In total, 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 upregulation processes 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 The immortalized OEC provides a stable system to evaluate HS response in cattle oviducts. Transcriptomic and proteomic data from this study offer valuable resources for targeted molecular approach to attenuate detrimental HS effect in cattle reproduction.