Genome-wide expression analysis of the heat stress response in dermal fibroblasts of Tharparkar (zebu) and Karan-Fries (zebu × taurine) cattle

Abstract

The present study sought to evaluate mRNA expression profiles in the cultured dermal fibroblasts of Tharparkar (zebu) and Karan-Fries (zebu, Tharparkar × taurine, Holstein Friesian) cattle in response to heat stress. Bioinformatics' analysis identified temperature-regulated biological processes and pathways. Biological processes overrepresented among the earliest genes induced by temperature stress include regulation of stress responses, protein repair, metabolism, protein transport, cell division, and apoptosis. The present microarray platform contains 51,338 synthesized oligonucleotide probes corresponding to at least 36,713 unigenes. A total of 11,183 and 8126 transcripts were differentially expressed with a fold change of ≥ 2 in Tharparkar and Karan-Fries cattle, respectively. Randomly selected real-time validation showed 83.33% correlation with microarray data. Functional annotation and pathway study of the differentially expressed transcripts or genes (DEGs) reveal that upregulated genes significantly (P < 0.05) affect protein processing and NOD-like receptor pathways (NLRs), while downregulated genes were significantly (P < 0.05) found to be associated with cell cycle, metabolism, and protein transport. Gene expression changes include activation of heat shock factors (HSFs), increased expression of heat shock proteins (HSPs), and apoptosis, while decreasing protein synthesis and another metabolism. These findings provide insights into the underlying mechanism of the physiology of heat stress in Tharparkar and Karan-Fries cattle. Understanding the biology and mechanisms of heat stress is critical to developing approaches to ameliorate current production issues for improving animal performance and agriculture economics in tropical climatic conditions. In conclusion, the present study indicates that heat stress differentially affects the expression of the significant number of genes associated with stress response, metabolism, apoptosis, and protein transport in dermal fibroblasts of Tharparkar and Karan-Fries cattle.

Keywords: Apoptosis; Dermal fibroblasts; Heat stress; Metabolism; Stress response; cDNA microarray.

Fig. 1

Dermal fibroblast cultures: a skin biopsies in culture media (× 10); b growth of primary layer (× 10); c growth of dermal fibroblasts from primary layer (× 10); d growth of dermal fibroblast (× 10); e 70–80% cellular confluence (× 10; used for subculture of dermal fibroblasts); f dermal fibroblasts with fusiform morphology (× 40); g dermal fibroblasts with cytoplasmic prolongations (× 40); h dermal fibroblasts in suspension with spherical morphology (× 40)

Fig. 2

Effects of heat shock at 44 °C for 3 h on a reactive oxygen species (μM), b cytotoxicity (%), and c cell viability (%) compared with control at 37 °C in dermal fibroblasts of Tharparkar and Karan-Fries cattle. Asterisk indicates statistically significant change: *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Representative hierarchical clustering of microarray data of gene expression in dermal fibroblasts of control (TP-37 Tharparkar control; KF-37 Karan-Fries control) vs heat shock at 44 °C for 3 h (TP-44 Tharparkar heat shock; KF-44 Karan-Fries heat shock). Clustering was done by linking similarly expressed genes together and having fold change ≥ 2

Fig. 4

Differentially expressed genes in dermal fibroblasts of control (TP-37 Tharparkar control; KF-37 Karan-Fries control) and heat shock at 44 °C for 3 h (TP-44 Tharparkar heat shock; KF-44 Karan-Fries heat shock) are hierarchically clustered based on their intensity values

Fig. 5

Effects of heat shock at 44 °C for 3 h on apoptosis in dermal fibroblasts of Tharparkar and Karan-Fries cattle. Apoptosis was assessed using the TUNEL assay. Microscopic pictures of FITC-positive apoptotic dermal fibroblasts (green) and counterstaining with propidium iodide (red)

Fig. 6

Molecular network analysis of upregulated genes due to heat shock at 44 °C for 3 h in dermal fibroblast of Tharparkar cattle

Fig. 7

Molecular network analysis of upregulated genes due to heat shock at 44 °C for 3 h in dermal fibroblast of Karan-Fries cattle

Fig. 8

qRT-PCR validation of the genes randomly selected from microarray data. Genes were validated by relative expression; GAPDH was used as housekeeping gene. Two-way ANOVA was used for statistical analysis. The difference between the means was compared using least significance difference (LSD) at P ≤ 0.05. Experimental data were expressed as the mean ± SEM. Asterisk indicates statistically significant change: *P < 0.05, **P < 0.01, ***P < 0.001