Impact of Heat Stress on Cellular and Transcriptional Adaptation of Mammary Epithelial Cells in Riverine Buffalo (Bubalus Bubalis)

Abstract

The present study aims to identify the heat responsive genes and biological pathways in heat stressed buffalo mammary epithelial cells (MECs). The primary mammary epithelial cells of riverine buffalo were exposed to thermal stress at 42°C for one hour. The cells were subsequently allowed to recover at 37°C and harvested at different time intervals (30 min to 48 h) along with control samples (un-stressed). In order to assess the impact of heat stress in buffalo MECs, several in-vitro cellular parameters (lactate dehydrogenase activity, cell proliferation assay, cellular viability, cell death and apoptosis) and transcriptional studies were conducted. The heat stress resulted in overall decrease in cell viability and cell proliferation of MECs while induction of cellular apoptosis and necrosis. The transcriptomic profile of heat stressed MECs was generated using Agilent 44 K bovine oligonucleotide array and at cutoff criteria of ≥3-or ≤3 fold change, a total of 153 genes were observed to be upregulated while 8 genes were down regulated across all time points post heat stress. The genes that were specifically up-regulated or down-regulated were identified as heat responsive genes. The upregulated genes in heat stressed MECs belonged to heat shock family viz., HSPA6, HSPB8, DNAJB2, HSPA1A. Along with HSPs, genes like BOLA, MRPL55, PFKFB3, PSMC2, ENDODD1, ARID5A, and SENP3 were also upregulated. Microarray data revealed that the heat responsive genes belonged to different functional classes viz., chaperons; immune responsive; cell proliferation and metabolism related. Gene ontology analysis revealed enrichment of several biological processes like; cellular process, metabolic process, response to stimulus, biological regulation, immune system processes and signaling. The transcriptome analysis data was further validated by RT-qPCR studies. Several HSP (HSP40, HSP60, HSP70, HSP90, and HSPB1), apoptotic (Bax and Bcl2), immune (IL6, TNFα and NF-kβ) and oxidative stress (GPX1 and DUSP1) related genes showed differential expression profile at different time points post heat stress. The transcriptional data strongly indicated the induction of survival/apoptotic mechanism in heat stressed buffalo MECs. The overrepresented pathways across all time points were; electron transport chain, cytochrome P450, apoptosis, MAPK, FAS and stress induction of HSP regulation, delta Notch signaling, apoptosis modulation by HSP70, EGFR1 signaling, cytokines and inflammatory response, oxidative stress, TNF-alpha and NF- kB signaling pathway. The study thus identified several genes from different functional classes and biological pathways that could be termed as heat responsive in buffalo MEC. The responsiveness of buffalo MECs to heat stress in the present study clearly suggested its suitability as a model to understand the modulation of buffalo mammary gland expression signature in response to environmental heat load.

Effect of Heat Stress on the Expression of HSP70, UCP3 and CYP450 Genes in Liver; Longissimus Dorsi and Semitendinosus Muscle of Growing Pigs

“…Acute exposure of mouse to 40-42°C increased the expression of CYP3A in liver and promoted hepatocyte proliferation, but the exposure to 44-46°C inhibited proliferation and promoted apoptosis of hepatocytes (Li et al, 2012). Altered electron transport by cytochrome P450 in mammary epithelial cell culture exposed to 42°C during 1 h was also reported (Kapila et al, 2016). In the present experiment, the expression of CYP3A was detected only in liver, which is in agreement with other authors (Shang et al, 2013;Nielsen et al, 2017).…”

The Bos taurus–Bos indicus balance in fertility and milk related genes

“…One enriched GO term from this list is GO:0071822 (Protein complex subunit organization) with a FDR p-value = 0.00505. Worth mentioning is DNAJC18 (DnaJ heat shock protein family member C18) due to its recently reported association with heat stress in contrasting Bos taurus and Bos indicus cattle [ 45 , 46 ]. Also noteworthy is SLC25A33 estimated to have a contribution to Bos indicus of 100% and encoded at 44.8 Mb of BTA16 in a hard-sweep region recently reported to be shared among four Bos taurus breeds [ 23 ] and possibly related to the initial cattle domestication events.…”

Physiological implications, cellular responses and lactational performance of Saanen goats under heat stress

“…According to the same authors, the apoptosis regulation of mammary cells is a multifactorial event and depends on various pathways (GREEN and STREULI, 2004). However, in buffalo the expression of Bax gene increased and Bcl-2 gene decreased immediately after heat stress (KAPILA et al, 2016). Similar to our results, Bomfim et al (2018) reported that cortisol increased Bax expression without Bcl-2 change of mammary epithelial cells (MEC) in vitro.…”

“…In vitro, HSP70 and other HSPs (HSPA6, HSPB8, DNAJB2) were expressed in buffalo mammary epithelial cell exposed to thermal stress at 42°C (KAPILA et al, 2016). Although, HSP70 plays important role to recovery cells damage and inhibit different apoptosis pathways (OHNISHI et al, 1999;.…”