Transcriptome responses to heat stress in hypothalamus of a meat-type chicken
© Sun et al.; licensee BioMed Central. 2015
Received: 4 May 2014
Accepted: 29 January 2015
Published: 17 February 2015
Heat stress has resulted in great losses in poultry production. To address this issue, we systematically analyzed chicken hypothalamus transcriptome responses to thermal stress using a 44 k chicken Agilent microarray,
Hypothalamus samples were collected from a control group reared at 25°C, a heat-stress group treated at 34°C for 24 h, and a temperature-recovery group reared at 25°C for 24 h following a heat-stress treatment. We compared the expression profiles between each pair of the three groups using microarray data.
A total of 1,967 probe sets were found to be differentially expressed in the three comparisons with P < 0.05 and a fold change (FC) higher than 1.5, and the genes were mainly involved in self-regulation and compensation required to maintain homeostasis. Consistent expression results were found for 11 selected genes by quantitative real-time PCR. Thirty-eight interesting differential expression genes were found from GO term annotation and those genes were related to meat quality, growth, and crucial enzymes. Using these genes for genetic network analysis, we obtained three genetic networks. Moreover, the transcripts of heat-shock protein, including Hsp 40 and Hsp 90, were significantly altered in response to thermal stress.
This study provides a broader understanding of molecular mechanisms underlying stress response in chickens and discovery of novel genes that are regulated in a specific thermal-stress manner.
With increasing importance of global warming issues, heat stress, also known as hyperthermia, could play a vital factor in affecting animal performance. Although chickens can use a variety of physiological mechanisms to regulate their body temperatures, heat stress could adversely affect chickens, especially broilers, in matters such as high mortality and consistent decrease in egg weight, shell thickness, rate of egg production, growth rate, meat quality, body weight [1,2], and immune capability as well as blood biochemical indicators [3,4]. St. Pierre et al. , reported that heat stress caused economic losses of between $1.69 billion and $2.36 billion per year in livestock industries in United States, with $51.8 million in the broiler sector alone.
During the past few decades, researchers have paid increased attention to physiological, biochemical, and immune capability changes of heat-stressed chickens. Deyhim and Teeter  found physiological responses to heat stress involve changes in respiration rate and blood pH, plasma concentration of ions, cardiovascular functions and hormonal effects. Heat stress also increased glucose and circulatory cortisol levels and decreased blood protein level. Khajavi et al. , reported that occurrence of CD4+ and CD8+ lymphocytes and antibody production decreased under heat stress.
Recently, transcriptome comparison has become a popular methodology for studying heat stress because it can elucidate the heat stress mechanism based on genetic level. Li et al. , using broiler breast tissue, investigated 110 differentially expressed genes during chronic cyclic heat stress, including 4 new genes that were related to heat stress. Wang et al. , reported 169 up-regulated and 140 down-regulated genes responded to acute heat stress; most of the differential expression genes were related to transport, signal, and metabolism.
Previous transcriptome research studies have chosen liver, breast, and testis as materials [8-10] to decipher heat stress, but no one has used the hypothalamus as a subject for studying the mechanism of heat stress and genetic networks. The hypothalamus, linking the nervous system to the endocrine system via the pituitary gland, plays the role of a central regulator in temperature regulation in poultry. It is well known the preoptic area (POA) and anterior hypothalamic area (AH) of the hypothalamus is the thermoregulatory center  and that hypothalamic areas such as the paraventricular hypothalamic nucleus (PVN), supraoptic nucleus (SO) [12-14], and lateral hypothalamic (LH) are activated under heat stress [15,16].
In addition, although RNA sequencing is currently a popular technology for studying transcriptome, it is very costly and results in less mature analysis than a microarray. The objective of this study is thus to use a microarray to detect the versatile gene expression profile in the hypothalamus, to determine the genes associated with heat stress, and to gain insight into the potential mechanisms underlying chickens’ response to heat stress. Characterizing the hypothalamus transcriptome of heat-stressed broilers will help clarify the effects of heat stress on broiler nerve centre and signal transduction. This information will also provide a platform for future investigation into genetic networks relevant to commercial broilers’ response to heat stress.
Materials and methods
Experimental animals and sampling
All experimental protocols were approved by the Committee for the Care and Use of Experimental Animal at Anhui Agricultural University. The purpose of this study was to find how broiler hypothalamus responds to heat stress in influencng growth rate and meat quality at the genetic level. Moreover, considering the particular economic value of hens, our priority was to use hen broilers as research subjects. Because broilers don’t have sweat glands, it is difficult for them to shed heat in hot weather (30-35°C). At a temperature of 34°C with 10-50% relative humidity, 50-80% of the heat loss was by evaporative heat shed through panting (http://www.heatstress.info/HeatStressExplained/BodyHeatregulationinPoultry.aspx). In this study, we used a full-sib and half-sib chicken population to reduce the impact of genetic background variation. Thus, twelve 56-day-old full-sib and half-sib commercial female broilers were randomly divided into three groups, including a control group, a heat-treated group, and a temperature-recovery group with four replicates in each group. All the birds were initially reared in an artificial climate chamber (relative humidity of 40-60%) with free access to food and water at 25°C. At the age of 56 days, both the heat-treated (HS) group and the temperature-recovery (TR) group were subjected to a temperature of 34°C for 24 h. After heat stress, hypothalamuses of four HS hens were dissected from their brains. The other chickens as a control group (CL) were further kept at a normal temperature of 25°C for 24 h. After exposure to heat, the hypothalamuses of CL and TR groups were collected. The hypothalamus collection procedure was as follows: (1) After decapitation, carefully remove the brain; then bisect it by making a section perpendicular to the cortical surface at the caudal end of the hemisphere. (2) Before identifying the hypothalamus, carefully remove the meninges and then, using tweezers, carefully pull the meninges layers away from the brain. (3) When the hypothalamus has been found, carefully remove the whole specimen. All the tissues were preserved overnight in RNAfixer (BioTeke Co. Ltd, Beijing, China) at 4°C and transferred to −80°C until RNA isolation. The hypothalamuses from the three groups were then subjected to gene expression analysis.
Total RNA isolation, labeling, and microarray hybridization
Total RNA was isolated from the hypothalamus using TRIZOL Reagent according to manufacturer’s instructions (Life Technologies, CA, US). RNA quantity and purity of each sample were detected using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc, Delaware, US). The RNA integrity of each sample was determined using an Agilent 2100 Bioanalyzer. The processed RNA had a 28S/18S > 1.5 and the RNA Integrity Numbers (RINs) for the samples were all higher than 7.6, indicating that the quality of the samples was suitable for microarray analysis. Four biological replicates were used in each group and cyanine 3-labeled nucleotide was conducted to prevent dye bias during sample labeling. A 2 μg isolated total RNA from each tissue sample was reverse-transcribed into cDNA, then into cRNA incorporating cyanine 3-labelled nucleotide. The labeled RNA was purified using QIAGEN RNeasy® Mini Kit (Qiagen, CA, US). A total of 875 ng Cy3 labeled cRNA was hybridized to 44 K chicken Agilent arrays (ID: 026441). Equal amounts of cRNA were hybridized using a Gene Expression Hybridization Kit (5188–5242, Agilent, USA). Arrays were performed at 60°C for 17 h in Agilent microarray hybridization chambers. The hybridized slides were washed using a Gene Expression Wash Buffer Kit (Agilent technologies, Santa Clara, CA, US) before stabilization and drying solutions were applied (Agilent Technologies, CA, US). Arrays were scanned using an Agilent Microarray Scanner (Agilent Technologies, Santa Clara, CA, US) with a scan resolution of 5 μmol/L, PMT 100%, 10%. The data were then compiled with Feature Extraction software 10.7 (Agilent Technologies, Santa Clara, CA, US).
Microarray data analysis
Array normalization and error detection analysis was carried out using a Quantile algorithm supplied with Gene Spring Software 11.0 (Agilent Technologies, CA, US). The “filter on flags feature” with software algorithms determined whether spots were “present”, “marginal”, or “absent” and values representing poor intensity and low dependability were removed from the raw data. The “absent” spots represented signals not positive, significant or above background level. A “present” spot was one for which the output was uniform, saturated, and significantly above background level. Spots were deemed “marginal” when the signals satisfied the main requirements but were outliers relative to the typical values for the other genes. We used only the items categorized as “present” and “marginal” for further analysis and compared the differential expression transcripts between each pair of the three groups. i.e., HS compared to CL (HS vs. CL), HS compared to TR (HS vs. TR), or TR compared to CL (TR vs. CL).
Principal-component analysis (PCA) and heat-map analysis were performed by SAS software from SHANGHAI BIOTECHNOLOGY CORPORATION eBioService (http://www.ebioservice.com). The differentially-expressed (DE) genes in each comparison were selected using SAS online software (http://www.ebioservice.com) and a local R package. With respect to the number of expression genes in the hypothalamus, we set the cutoff as P < 0.05, FDR < 0.15 and fold change (FC) > 1.5 to obtain more DE genes. Although this FDR cutoff is much higher than the usual FDR < 0.05, there are many good published papers using a variety of different FDR cutoff values according to reported experiments [17,18]. The significant DE gene clusters from heat-map results were used to do pathway analysis using Ingenuity Pathway Analysis (IPA) (Ingenuity® Systems, www.ingenuity.com). IPA is a very useful online tool for analyzing biological and molecular networks; it transforms gene network into relevant pathways using the genes’ functional annotation and known molecular interactions [19-21].
Quantitative real time PCR (qRT-PCR)
Primer sequences for qRT-PCRs
Product size, bp
Effects of heat stress on gene expression in different comparison groups
Heat map and Principal component analysis
Functional categories of differentially expressed genes
Validation of quantitative real time PCR results
Fold change of 11 selected genes in each comparison group by microarray and qRT-PCR
HS vs CL fold-change
HS vs TR fold-change
TR vs CL fold-change
Much literature has shown that meat quality and growth can be influenced by environmental heat stress [22-25], but few studies explain this phenomenon at the gene level for broilers, especially with a focus on the hypothalamus. In this study we found 24 DE genes related to meat quality, 8 DE genes in the growth category, and 6 crucial DE enzyme genes associated with acute heat stress and based on heat-map gene clusters and GO term annotation. In addition, these meat quality and growth related DE genes, together with the crucial enzyme genes and other DE genes were subjected to genetic network analysis through IPA. We found 3 networks: Cardiovascular Disease, Lipid Metabolism, Molecular Transport; Cell-to-Cell Signaling and Interaction, Molecular Transport, Small Molecule Biochemistry; and Lipid Metabolism, Molecular Transport, Small Molecule Biochemistry.
Gene network analysis
Cardiovascular Disease, Lipid Metabolism, Molecular Transport. The hub in the “Cardiovascular Disease, Lipid Metabolism, Molecular Transport” network is prostaglandin G/H synthase 2 precursor (PTGS2), also known as COX2, an enzyme that can catalyse the biosynthesis of prostaglandins (PGs) . PTGS2 directly interacts in this network with heme oxygenase (decycling) 1 (HMOX1 or HO1), platelet-derived growth factor beta (PDGFB), insulin (Ins), vascular endothelial growth factor (VEGF), signal transducer and activator of transcription 5B (STAT5B), and so on. HMOX1, PDGFB, Ins, VEGF, and STAT5B are key nodes in the “Cardiovascular Disease, Lipid Metabolism, Molecular Transport” network and also interact with other DE genes.
Arnaud et al. , reported that PTGS2 had capability to participate in heat stress-induced myocardial preconditioning in vivo. HMOX1 also has an important role in cellular homeostasis because of its pro- and antioxidative function [28,29]. Bloomer et al. , observed that HMOX1 was sensitive to temperature and its expression was increased immediately after heat stress. PTGS2 can regulate HMOX1 expression and this expression is up-regulated in cardiac fibroblasts when the broilers were under stress . In addition, HMOX1 can be regulated by c-fos induced growth factor (FIGF), a member of the PDGF/VEGF family,that can associate with mitogenic and morphogenic activity on fibroblast cells [32,33]. PDGFB can be affected by PTGS2, HMOX1, actin, alpha, cardiac muscle 1 (ACTC1), myosin, heavy chain 11, smooth muscle (MYH11), bone morphogenetic protein 3 (osteogenic) (BMP3). Signoroni et al. , demonstrated that PTGS2 can modulate constitutive interaction of a PDGF receptor. ACTC1 plays a crucial role in early muscle development and fetal development [35,36], and reduced ACTC1 expression had an essential role in congenital heart disease and atrial septa defect [37,38]. ACTC1 also is involved in “Cell-to-Cell Signaling and Interaction, Molecular Transport, Small Molecule Biochemistry” and “Lipid Metabolism, Molecular Transport, Small Molecule Biochemistry”. Many research studies [39,40] have indicated that gene ACTC1 contributes to muscle development and meat quality. MYH11 is a unique gene of smooth muscle cells. Landreh et al. , suggested that the cultured PDGF receptor α- positive peritubular cell (PTC) can increase MYH11 (PTC-specific gene) expression. PDGF receptorα- positive PTC is a steroidogenic member of the stem Leydig cell (SLCs). BMP3 has been identified as playing a crucial role in both skeletal development and bone homeostasis .
In this study, we found that PTGS2, FIGF, ACTC1, MYH11, and BMP3 were up-regulated under heat stress and their fold changed from 1.51 to 15.14 while the expression of HMOX1 and PDGFB decreased during heat stress and their fold changes were −1.64 and −1.80, respectively. All these DE genes are in HS vs. CL or HS vs. TR. However, except for HMOX1, those DE genes have not been reported as relating to heat stress. This might be relatively few researchers have studied meat quality and growth under heat stress at the gene level. Thus, based on this study, we can infer that heat stress significantly influences broiler body temperature, meat quality, sperm quality, growth, and skeletal quality through up-regulation or down-regulation of unique genes in the hypothalamus. Furthermore, the changes of expression of muscle-related genes indicate that muscle development is inhibited to a certain degree in the short term after heat treatment.
Heat stress also can change the hormone level. Tyrosine hydroxylase (TH), the precursor of catecholamines (the sympathetic nervous system) (SNS) neurotransmitters, is a rate-limiting enzyme for catecholamine biosynthesis and biomarker enzyme for adrenaline expression [16,43]. In this study, TH was a key node and involved in “Cardiovascular Disease, Lipid Metabolism, Molecular Transport” and “Lipid Metabolism, Molecular Transport, Small Molecule Biochemistry” networks. It can catalyse tyrosine into dopa that can be transformed into catecholamine by the dopamine enzyme. TH can also regulate the receptor of dopa (DRD2) amd dopa could also be formed into noradrenaline and adrenaline through a special enzyme. Garcia et al. , reported that TH expression was decreased under chronic heat stress. However, in our acute heat-stress study, TH and DRD2 were both significantly expressed with 14.91 and 1.63 FC, respectively. These genes are DE expressed in both HS vs. CL and HS vs. TR. Our current findings reveal that host-enhanced SNS activity leads to an up-regulated catecholamine metabolic pathway and increases noradrenaline and adrenaline to reinforce a body’s resistance to heat stress.
There are also still a number of interested DE genes in the three gene networks but for reasons of space not discussed here; they included SDC1, EMP1, EDN3, DBI, INSIG1, HDL, CETP, PDK4 in “Cardiovascular Disease, Lipid Metabolism, Molecular Transport”, GCH1, LMX1A, MOV10L1, GDPD5, JPH, AOAH in “Cell-to-Cell Signaling and Interaction, Molecular Transport, Small Molecule Biochemistry”, CUBN, MYH7, CROT, LPAP4, PLA1A in “Lipid Metabolism, Molecular Transport, Small Molecule Biochemistry”. All these DE genes were related to meat quality, growth, and enzyme; for example, MYH7 is related to drip loss and cooking loss [45,46]. More details are given in a supplemental file. Moreover, some of those DE genes have been identified as being associated with meat quality under heat stress by other researchers. For example, Yu et al. , reported that myogenic factor 6 (MYF6) and fatty acid binding protein 4, adipocyte (FABP4) were involved in meat quality under heat stress.
Heat shock protein
During heat stress, a class of heat shock protein family could also be significantly changed. When a host encounters heat stress, the expression of highly-conserved heat-shock protein (HSP) will be either increased or decreased in various tissues and cells to protect the body from excessive damage . HSP are ubiquitous in organisms ranging from from bacteria to mammals throughout the whole biosphere. They can help the body adapt to adverse environments by serving as molecular chaperone, antioxidant, synergistic immune, and anti-apoptotic . HSP70 can also play a significant role in heat stress, but we didn’t find that it was DE in the current study. This might be because of the distribution of HSP70 in terms of different expression in diverse tissues or organs, perhaps especially enriching heart, liver, kidney, and spleen but not gathering in hypothalamus. In addition, HSP70 usually changes within eighteen hours, while after eighteen hours its expression may be back to normal . In this study we found that the expression of HSPCB (HSP90) and DNAJC13 (HSP40) underwent significant changes. HSPCB families can play a key role in DNA replication and transcription, protein folding and mutation, protein transport, proteolysis, cell signaling, and immune response [51,52]. HSPCB is also necessary in hormone receptor and tyrosine protein kinase [53,54]. DNAJC13 can interact with HSP70 in renaturation of heat-denatured proteins  and DNAJC13 can also bind directly to HSP70 by recruiting specific substrates to stimulate both its intrinsically weak ATPase activity and its interaction with polypeptide substrates [56,57]. HSPCB and DNAJC13 are also DE in HS vs. CL and HS vs. TR. The expression of DNAJC13 was up-regulated while the expression of HSPCB was down-regulated in the heat-stress process, indicating that heat stress can exert great harm to the body and the body can actively produce various mechanisms to resist heat stress.
Transcriptome of the chicken hypothalamus could be altered extensively when chickens are exposed to heat stress. We performed global gene-expression analysis of hypothalamus from chickens subjected to heat stress, temperature recovery, and normal temperature. We used bird hypothalamus as material and used a microarray to detect the versatile transcript changes respond to broiler heat stress. In hypothalamus, the TR group and the CL group behaved similarly to the HS group. Various enzymes are changed following the level of hormone under heat stress. At the same time, a number of DE genes related to meat quality and growth were significantly DE. The expression of heat shock protein families also presented certain changes, especially in DNAJC13 and HSPCB. The results of this study provides novel insight into the effects of heat stress on meat quality, growth, and essential enzyme for hormone pathway by using hypothalamus transcriptome analysis; it offers a platform for future investigations into genetic networks for studying broiler heat-stress response.
This work was financially supported by Beijing innovation team attached to poultry industry technology system (CARS-PSTP).
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