| High temperature has a ubiquitous effect on the rate of physiological reactions, stability of macromolecular structures and therefore plays a vital role in restraining the geographic distribution ranges. Heat stress (HS), an array of disorders caused by hyperthermia includes heat cramps,heat injury and heat stroke. Investigation of the basic mechanism of thermoregulation is the very important concern to differentiate the patho-physiology of heat stress associatedt illnesses. HS is produced by different factors associated with inflammatory and homeostatic responses. HS results in responses of elevated temperature, heart rate and sweating. It might play a very important role in affecting animal performance. In the present studies impacts of thermally induced stress on some physiological parameters and the protein expression in mouse pituitary glandwere evaluated and studies were carried out as follows.1- Effects of thermally induced stress on animal behavior and some physiological parametersImpact of the thermally induces stress on the physiological parameter included behavior,body weight, water and feed intake and rectal temperature were evaluated. Adult male mice weighing about 50 grams were used as the experimental model. Mice were divided into 3 groups comprising of 12 mice in each group. Group A and B were exposed to 30℃ and 40℃ for 2 hours each day till 15 day, whereas group C was used as a control kept at room temperature(25℃). Rectal temperature was continuously monitored during all the duration of experimental period. Body weight was measured by keeping the differences in body weights before and after each heat treatment. Feed intake and water intake was also recorded each day during the whole study period. Significant behavioral changes that are found to be induced by heat stress were checked during and after the treatments. Rough, oily and greasy fur was observed as a physiological change and obnoxious activities like biting the cage wires in order to escape were also observed during the heat treatment. Rectal temperature was increased (36.57±0.57 to 38.01±0.45℃) after the exposure of heat at 40℃ and no significant difference was recorded at 30℃. Water intake was significantly increased in both stressed groups as compared to control.Significant body weight loss (46.86 to 42.15±3.55 g) was recorded in 40℃ group, whereas, at 30℃ average weight loss was recorded as 46.06 to 44.68±5.01g. There was no significant difference were observed in feed intake among the groups. Our finding indicated that heat stress at high temperature induced the physiological changes including escaping behavior, increased rectal temperature, and water intake and decreased body weight.2- Effects of heat stress on hormone profile and testicular histology of miceThe experiment was conducted to evaluate the effect of the heat stress on the endocrine function. Mice were divided into 3 groups comprising of 12 mice in each group. Group A and B were exposed to 30℃ and 40℃ for 2 hours each day till 15 day, whereas group C was used as a control kept at room temperature (25℃). On day 7 and 15 post thermal treatment serum samples were collected and adrenocorticotropic hormone (ACTH), testosterone and growth hormone was checked by radioimmunoassay kitby using the RIA counter. Our results revealed that, serum testosterone level was significantly decreased in both heat treatment groups at 15th day. At 7th day the low level of growth hormone was recorded, whereas, no significant difference was recorded at 15th day. ACTH level were significantly decreased in mice exposed to 30°C and 40℃at day 7. Whereas, ACTH hormone level in the high temperature group at 15th day was slightly increased. Impact of the thermally induced stress on testicular histology was also examined.After thermal treatment testes were collected, fixed in formalin. Formalin fixed testes were processed for dehydration and paraffin infiltration.Testicular tissues were embedded in paraffin and 5μm thick sections were cut, after removal of wax by xylene, the sections were rehydrated through a graded series of ethanol solutions. Hydrated sections were stained with hematoxylin and eosin (HE) and examined by light microscopy. Our results indicated that, high temperature(40℃) severely damages the testicular histology. The seminiferous tubules of stressed mice were shrunken and contained less spermatozoa, testicular degeneration, irregular appearance of the seminiferous tubules lining, spermatogenic arrest and atrophy of leydig cells were observed at 40 ℃. These histological and hormonal findings suggested that, thermally induced stress adversely affect the male reproductive performances with hormonal imbalances and decline the process of spermatogenesis which leads to the sterility.3- Proteomic response of mouse pituitary gland against heat stressIn this experiment, adult male mice about 50g weights were used as experimental animal. A total of 24 mice were used and divided into 2 groups comprising 12 animals per group. The animals were kept in a temperature controlled room at room temperature (20-25℃) before start hyper-thermal treatment. The temperature was increased 10℃/ hour up to 40℃ for group A.Group B was kept at RT as a control. All the animals were kept on this temperature for 2 hours per day for 15 days and after completion of the hyper-thermal treatment animals were scarified and pituitary gland were collected after decapitation and stored at -80℃ for further analysis.Proteomic response of mice pituitary gland against heat stress was evaluated by iTRAQ followed by LC-MS/MS. To determine the biological and functional properties of all the identified proteins, the identified protein sequences were mapped with Gene Ontology Terms. The GO term matching was performed with blast2go. To identify candidate biomarkers, we employed hypergeometric test to perform GO enrichment and Pathway enrichment. To find out the functional relationship among stress relevant proteins, protein sequences were imported by selecting Musmusculusas a model organism to STRING 9.1 http://string-db.org/ (a database of known and predicted protein interactions). The transcription levels of the proteins with differential expression during HS were investigated using real-time PCR. To that end, six proteins were selected for analysis of expression patterns at the mRNA level and four differentially expressed proteins of pituitary gland identified in the iTRAQ data were validated by Western blotting.Our study revealed that thermal induced stress actively regulates the stress related proteins in mice pituitary gland. A total of 564 proteins of pituitary gland were quantified, among them 374 proteins were identified as differentially expressed under the heat stress at 40℃ compared to control (RT). During the heat stress 89 proteins were upregulated and 285 were quantified as downregulated. 130 proteins related to stress response were identified out of 564 quantified proteins, among them 26 and 46 proteins were expressed up and down regulated respectively. Seven proteins related to heat shock protein family were identified differentially expressed, among them two proteins belongs to HSP70 family and one proteins belong to HSP90 family were identified upregulated. Whereas expression of Heat shock protein 90, alpha(Cytosolic), class A member 1, 60 kDa heat shock protein and heat shock protein 84b were found downregulated. In our study HSP70 (Hspalb) and a nicotinamide adenine dinucleotide biosynthetic enzymeNicotinamide mononucleotide adenylyltransferase 3 (Nmnat3) was found significantly overexpressed with fold ratio 6.9 and 6.64 respectively. Three proteins related to intermediate filaments protein family (IP) including, lamins (Prelamin-A/C and lamin B), Vimentin, Keratins were also found upregulated. Here we report, association of the intermediate filament proteins and HSPs as a biological markers for heat shock stress at proteomic level. The expression level of Apo A-IV was upregulated during heat stress in mice pituitary gland and might be one of the reasons of low feed intake during heat stress. Differentially expressed proteins were functionally characterized according to their gene ontology,37 upregulated proteins were assigned to response to stimulus, among them 26 were related to the response to stress. As concerned to downregulated proteins, 99 proteins were assigned to response to stimulus and 46 were annotated as response to stress.Transcriptional level of the differentially expressed proteins during heat stress were investigated by real-time PCR, for that purpose 6 gene encoding heat shock proteins were used. The results of study revealed that, gene expression level of HSP70 (HSP1B)showed significantly up regulation changes that correlated with the proteomic results. We also found that changes in the expression of HSPa5, HSP84b and HSP 90B did not corresponded with mRNA level.Whereas mRNA level of Heat shock protein 90, alpha (Cytosolic), class A member 1, 60 kDa heat shock protein HSP12a were correlated with the results of protein expression. In current study a nicotinamide adenine dinucleotide biosynthetic enzymeNicotinamide mononucleotide adenylyltransferase 3(NMNAT3) was found significantly up-regulated that indicated that, up-regulation of NMNAT3 under heat stress could induce neuroprotection against heat shock injury. Here we identified up-regulation of 3 intermediate filament (IF) proteins included prelamin A/C (Lmna, Lmn1), Lamin-B1 (Lmnb1), vimentins (Vim) and keratins (K). Here we report first time at proteomic level association between IFPs and HSPs are very important biomarkers for the heat stress in pituitary glands. Preproenkephalinprotein is an important modulator of the stress response. In the present study, we found that heat stress increased the expression of Proenkephalin-A (Penk) protein.Four differentially expressed proteins of pituitary gland identified in the iTRAQ data were validated by Western blotting. NMAT3 and HSP1ab were up-regulated, while HSP60 and HSP90a were down-regulated in 40°C group as compared to control (RT). Actin was used as internal control. Our findings were in accordance with the proteomics data and these results confirmed the reliability of the iTRAQ-based approach.According to our knowledge, this is the first research to provide insights into the differential expression of proteins in pituitary gland of mice against heat stress and provide the baseline for the understanding of the role of the stress related proteins and associations of the HSPs with intermediate filament proteins. Here we report important biological markers for heat stress. Our findings suggest that proteins differentially expressed under heat stress play important roles in combating to heat stress. The functional correlations of these differentially expressed proteins under heat stress particularly between HSPs and intermediate filament proteins need to be further researched. |
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