X-Y chromosome univalency in the testes of hyperthermic mice: II. Light and electron microscopic analysis of synaptonemal complexes

Hyperthermia-induced X-Y dissociation has been observed in diakinesis-metaphase I sper-matocytes but not in pachytene spermatocytes, which have been implicated as highly susceptible to heat stress. To determine X-Y dissociation in pachytene spermatocytes and to compare levels of dissociation between pachytene and diakinesis-metaphase I spermatocytes male ICR mice were exposed to 35°C ± 0.07°C and 65% ± 0.14% relative humidity for 2 or 4 days. Control mice were housed at 24°C ± 0.04°C and 43% ± 0.58% relative humidity. Mice were killed 0, 3, 5, 8, or 10 days after stress and the testes processed for meiotic chromosome display at diakinesis-metaphase I and synaptonemal complex display at pachynema. Twenty-five to thirty cells per mouse at both stages of meiosis were observed with light microscopy, and pachytene spreads with transmission electron microscopy to determine heat-stress effects on synaptonemal complex structure. Statistical analyses revealed significant linear increases in X-Y dissociation with prolonged exposure to heat at pachynema and diakinesis-metaphase I. Levels of pachytene dissociation were one-half the levels of dissociation at diakinesis-metaphase I. The resolvable structure of the lateral elements of the synaptonemal complex was not affected by heat stress.     

Effects of dexamethasone and chlorpromazine treatment on X-Y dissociation and multinucleated giant cell formation in hyperthermic mice

The effects of anti-stress drugs on X-Y dissociation and multinucleated giant cell formation in the testes of hyperthermic mice were determined for the possible use of such drugs in animal production. Mice were injected with dexamethasone 1 mg/kg or 2 mg/kg, chlorpromazine 1 mg/kg or 4 mg/kg, or 0.85% saline, and heat stressed for 4 d at 35 +/- 1 C and 65 +/- 1 % relative humidity. Mice were killed 5 d after stress, and the testes processed for observation of meiotic chromosomes and testicular histology. Heat stress caused a significant increase in X-Y bivalent dissociation in diakinesis-metaphase I spermatocytes and a significant increase in formation of multinucleated giant cells. Drug treatment increased X-Y dissociation but had no significant effect on giant cell formation.     

X-Y chromosome dissociation in mouse strains difering in efficiency of spermatogenesis: Elevated frequency of univalents in pubertal males

Dissociation of the X-Y chromosome bivalent in diakinesis-metaphase I spermatocytes of adult mice was significantly more frequent in the CBA strain (29%) than in C57, KP, or KE strains (7–11%). Autosome dissociatio (1–5%) involved only the smallest chromosome pairs. Eleyatedfrequency of X-Y dissociation in the CBA strain correlates with significantly lower testes weight and lower yield of spermatogenesis, which suggests that sex bivalent dissociation man be responsible for some loss of spermatogenic cells. However, sperm quality is not affected, the percentage of normal spermatozoa and their fertlizing capacity being higher in CBA thatn in the remaining strains. Two congenic strains, KE and KE. CBA (the latter with the Y chromosome introduced from CBA), had the same level of X-Y dissociatios, suggesting that the Y chromosome plays no rle in the determination of this character. In comparison with adult males pubertal (27–29 day-old) males had twice as hig a frequency of X-Y dissociation in KE an KP strains, and combined frequeicies of dissociated sex and autosome bivalents were significantly higher in pubertal males of all tested strains. Although te level of chromosome dissociation is not sufficient to explain increased mortality of germ cells observed in pubertal males, it could be one of the contributing factors.     

Frequency of X-Y chromosome dissociation in mouse spermatocytes from interstrain crosses,recombinant inbred strains,and chimeras: Possible involvement of paternal genome imprinting

The frequency of dissociation of the X-Y chromosome bivalent in diakinesis-metaphase I spermatocytes differs significantly between two inbred mouse strains, CBA (29%) and KE (7%), that were used to obtain reciprocal F1 hybrids, and to develop recombinant inbred (R1) strains. The level of X-Y dissociation was significantly higher in (KExCBA)F1 hybrids sired by the CBA males (24%) than in reciprocal F1 hybrids (12%), revealing the inheritance after the father. Among 14 RI strains, nine were concordant with KE, one with CBA, and four had intermediate phenotype, significantly different from both progenitor strains. This shows that at least two genes are involved, and their possible linkage with agouti and Trf loci is suggested. The linkage with agouti was confirmed by testing additional 10 CBXE incipient RI strains. There was no significant difference in the level of X-Y dissociation between EXCB RI strains derived from the original cross sired by the CBA males and CBXE RI strains derived from the reciprocal cross. The involvement of the Y chromosome-linked factors was unlikely because it was found earlier (Krzanowska, 1989: Gamete Res 23:357–365) that two congenic strains, KE and KE.CBA, differing with respect to the source of the Y chromosome, had the same level of X-Y dissociation. Thus, the difference obtained between reciprocal F1 hybrids is interpreted in terms of paternal genome imprinting imposed by CBA males and propagated only in the presence of some alleles derived from this strain. Analysis of six KE ? CBA-T6 chimeras, among them three germ line chimeras, points to the conclusion that the tendency to low or high level of X-Y chromosome dissociation is expressed rather autonomously by KE or CBA-T6 spermatocytes (as recognized by a marker chromosome pair), respectively, and was not modified by the presence of somatic cells of the opposite strain. © 1994 Wiley-Liss, Inc.     

Heat stress effects on sink activity of developing maize kernels grown in vitro

The objective of this study was to determine the effect of short-term (4 days) and long-term (8 days) heat stress (35°C) on sink activity of maize (Zea mays L.) kernels. Beginning at 3 days after pollination (DAP) kernels were grown in vitro at 25°C and 24 h later were transferred to 35°C for either 4 or 8 days. Each treatment had a control that was maintained continously at 25°C. Two experiments were designed to examine the uptake and distribution of ¹⁴C among hexoses, sucrose and starch in the pedicel placento-chalazal (pedicel/p-c). endosperm, and pericarp tissues of kernels exposed to heat stress for 4 or 8 days. Kernels cultured in vitro were placed in ¹⁴C-sucrose medium either during the period of heat stress (experiment 1; 5 to 13 DAP) or immediately following heat-stress treatments (experiment 2; 10 to 22 DAP). In both experiments no significant effect of heat stress was observed on the total radioactivity accumulated in the kernels until about 17 DAP, after which heat-stressed kernels accumulated less ¹⁴C than the control. During the linear fill period, the endosperm of kernels exposed to heat stress accumulated more radioactivity associated with hexoses and sucrose and less radioactivity incorporated into starch, as compared to the control. Kernels heat stressed for 4 days showed a partial recovery in starch synthesis by 21 DAP, but to levels of only 65% of that of the control. Kernels heat stressed for 8 days did not recover. When ¹⁴C-sucrose was supplied during the heat stress period (5–13 DAP). kernels from all treatments accumulated more hexoses that sucrose in the pedicel/p-c. However, during the period following heat stress (10–22 DAP), pedicel/p-c accumulated sucrose, but only in kernels exposed to long-term heat stress. Soluble invertase activity was inhibited by both short-term and long-term heat stress, whereas the activity of insoluble invertase was affected only by long-term heat stress. These results support the hypothesis that the disruption of kernel growth and more particularly endosperm starch biosynthesis, in response to heat stress, is mainly associated with changes in carbon utilization and partitioning between the different nonstructural carbohydrates within the endosperm rather than with a limitation in carbon supply to the kernel. Therefore, the effect on sink activity does not seem to be attributable to a thermal disruption of kernel uptake of sugars, but rather it is a consequence of heat perturbation of other physiological processes such as endosperm sugar metabolism and starch biosynthesis.     

Lack of heat shock response triggers programmed cell death in a rat histiocytic cell line.

Stress response is a universal phenomenon. However, a rat histiocytic cell line, BC-8, showed no heat shock response and failed to synthesize heat shock protein 70 (hsp70) upon heat shock at 42 degrees C for 30 min. BC-8 is a clone of AK-5, a rat macrophage tumor line that is adapted to grow in culture and has the same chromosome number and tumorigenic potential as AK-5. An increase in either the incubation temperature or time or both to BC-8 cells leads to loss of cell viability. In addition, heat shock conditions activated apoptotic cell death in these cells as observed by cell fragmentation, formation of nuclear comets, apoptotic bodies, DNA fragmentation and activation of ICE-like cysteine proteases. Results presented here demonstrate that BC-8 cells cannot mount a typical heat shock response unlike all other eukaryotic cells and that in the absence of induction of hsps upon stress, these cells undergo apoptosis at 42 degrees C.     

Heat stress results in loss of chloroplast Cu/Zn superoxide dismutase and increased damage to Photosystem II in combined drought‐heat stressed Lotus japonicus

Drought and heat stress have been studied extensively in plants, but most reports involve analysis of response to only one of these stresses. Studies in which both stresses were studied in combination have less commonly been reported. We report the combined effect of drought and heat stress on Photosystem II (PSII) of Lotus japonicus cv. Gifu plants. Photochemistry of PSII was not affected by drought or heat stress alone, but the two stresses together decreased PSII activity as determined by fluorescence emission. Heat stress alone resulted in degradation of D1 and CP47 proteins, and D2 protein was also degraded by combined drought–heat stress. None of these proteins were degraded by drought stress alone. Drought alone induced accumulation of hydrogen peroxide but the drought–heat combination led to an increase in superoxide levels and a decrease in hydrogen peroxide levels. Furthermore, combined drought–heat stress was correlated with an increase in oxidative damage as determined by increased levels of thiobarbituric acid reactive substances. Heat also induced degradation of chloroplast Cu/Zn superoxide dismutase (SOD: EC 1.15.1.1) as shown by reduced protein levels and isozyme‐specific SOD activity. Loss of Cu/Zn SOD and induction of catalase (CAT: EC 1.11.1.6) activity would explain the altered balance between hydrogen peroxide and superoxide in response to drought vs combined drought–heat stress. Degradation of PSII could thus be caused by the loss of components of chloroplast antioxidant defence systems and subsequent decreased function of PSII. A possible explanation for energy dissipation by L. japonicus under stress conditions is discussed.     

Photosynthetic response of transgenic soybean plants, containing an Arabidopsis P5CR gene, during heat and drought stress

The biochemical basis of heat/drought tolerance was investigated by comparing the response of antisense and sense transgenic soybean plants (containing the L-delta1-pyrroline-5-carboxylate reductase gene) with non-transgenic wild-type plants. The plants were subjected to a simultaneous drought and heat stress of 2 days, whereafter they were rewatered at 25 degrees C. During this time the sense plants only showed mild symptoms of stress compared to the antisense plants which were severely stressed. Upon stress, nicotinamide adenine dinucleotide phosphate (NADP+) levels decreased in antisense while it increased in sense plants. Recovery with respect to NADP+ levels was best in sense plants. Sense plants had the highest ability to accumulate proline during stress and to metabolise proline after rewatering. Analyses of the fast phase chlorophyll-a fluorescence transients showed dissociation of the oxygen-evolving complex (OEC) upon stress in all plants tested. In the sense plants, which best resisted the stress, OEC dissociation was bypassed by proline feeding electrons into photosystem 2 (PSII), maintaining an acceptable nicotinamide adenine dinucleotide hydrogen phosphate (NADPH) level, preventing further damage. Upon recovery, NADPH is consumed during oxidation of accumulated proline providing high Levels of NADP+ to act as electron acceptor to PSII, which indirectly may ameliorate the inhibition and/or the effect of uncoupling of the OEC.     

Mouse heat-shock factor 1 (HSF1) is involved in testicular response to genotoxic stress induced by doxorubicin

Heat-shock factor 1 (HSF1) protects cells and organisms against various types of stress, either by triggering a complex response that promotes cell survival or by triggering cell death when stress-induced alterations cannot be rescued. Although this dual role of HSF1 was observed in spermatogenesis exposed to heat shock or proteotoxic stress, HSF1 was also reported to contribute to cell resistance against genotoxic stress, such as that caused by doxorubicin, an anticancer drug in common clinical use. To better understand the stress/cell-dependent functions of HSF1, we used wild-type and Hsf1(tm1Ijb)/Hsf1(tm1Ijb) males to determine the role of HSF1 in the genotoxic stress response elicited in spermatogenic cells. Within 2 days after a single intraperitoneal injection of doxorubicin (DOXO; 5 mg/kg), proliferation of Hsf1+/+ but not Hsf1-/- spermatogenic cells was significantly reduced, whereas cell death was increased in mitotic germ cells and metaphase I spermatocytes. By 21 days, meiotic cells were depleted in all treated Hsf1+/+ testes but not in Hsf1-/- ones. Nevertheless, after 3 mo, spermatogenesis showed better signs of recovery in Hsf1+/+ than in Hsf1-/- males. Taken together, these data indicate that acute response to genotoxic stress in the testis involves HSF1-dependent mechanisms that induce apoptotic cell death in a TRP53-independent manner, but also intervene on a longer term to restore seminiferous tubules.     

Stress protection by a fluorescent Hsp27 chimera that is independent of nuclear translocation or multimeric dissociation

A chimeric protein consisting of enhanced green fluorescent protein (EGFP) fused to the N-terminus of human Hsp27 conferred stress protection in human A549 lung carcinoma and murine L929 cells that were stably transfected to express the chimera constitutively. The resultant protection was comparable with that in the same cell lines when they were transfected to express corresponding levels of Hsp27. Unlike L929 cells, A549 cells exhibit endogenous Hsp27 expression, whose expression was inhibited in proportion to the amount of fluorescent chimera expressed, suggesting that the A549 cells recognized the latter as Hsp27. Upregulation of Hsp27 or chimeric Hsp27 in all transfected cell lines (stable or transient transfection) caused no measurable change in cellular glutathione levels, indicating that glutathione played no role in the stress protection associated with either protein. Chimeric Hsp27 had a monomeric molecular weight of 55 kDa (that of Hsp27 plus EGFP) in both cell types and formed a 16-mer complex twice as massive as that formed by Hsp27. Heat shock or sodium arsenite induced phosphorylation of both chimeric Hsp27 and Hsp27, which resulted in the disaggregation of Hsp27 multimers in both cell types and disaggregation of 20% of the chimeric multimers in L929 cells. But chimeric Hsp27 multimers did not disaggregate after stress in A549 cells. Epifluorescence and confocal microscopy demonstrated that chimeric Hsp27 was restricted to the cytoplasm under normal growth conditions and after heat shock in all cells. This study supports the conclusions that Hsp27 stress protection requires neither its translocation into the nucleus nor the dissociation of its multimeric complex. Furthermore, it demonstrates that fluorescent chimeras of heat shock proteins can be functional and used to observe the protein's distribution within living cells.     

The TX; Y test for the detection of nondisjunction and chromosome breakage in Drosophila melanogaster. I. Analysis of spontaneous events and results of male exposure

The translocation X; Y test is a selective system in Drosophila melanogaster designed to detect and distinguish among sex chromosome nondisjunction, chromosome breakage, and X-Y interchange. In the test, only exceptional progeny survive. This enables the investigator to score thousands of progeny with relative ease. The distribution of spontaneous events occurring in individual TX; Y males are analyzed in this paper. Evidence is obtained suggesting that the clusters of two products arising from a single nondisjunction can significantly affect the distribution of recovered chromosome gain or chromosome loss events. Non-parametric statistical methods are therefore recommended for the analysis of TX; Y data. In addition, use of the TX; Y test following exposures of pre-adult males to X-rays, heat shock, cold shock, colchicine, dimethyl sulfoxide (DMSO), and trifluralin are presented. Significant increases in nondisjunction (both gain and loss) were obtained following exposures to heat shock, cold shock, DMSO and trifluralin. Significant increases in chromosome breakage and X-Y interchange were obtained after exposures to X-rays and heat shock. These results indicate that the TX; Y test is an efficient method for detecting aneuploidy. Further work is needed, however, to fully validate this system for the routine screening of aneuploidy-inducing agents.     

Ultrasound stimulates proteoglycan synthesis in bovine primary chondrocytes

Mechanical forces can stimulate the production of extracellular matrix molecules. We tested the efficacy of ultrasound to increase proteoglycan synthesis in bovine primary chondrocytes. The ultrasound-induced temperature rise was measured and its contribution to the synthesis was investigated using bare heat stimulus. Chondrocytes from five cellular isolations were exposed in triplicate to ultrasound (1 MHz, duty cycle 20%, pulse repetition frequency 1 kHz) at average intensity of 580 mW/cm2 for 10 minutes daily for 1-5 days. Temperature evolution was recorded during the sonication and corresponding temperature history was created using a controllable water bath. This exposure profile was used in 10-minute-long heat treatments of chondrocytes. Heat shock protein 70 (Hsp70) levels after one-time treatment to ultrasound and heat was analyzed by Western blotting, and proteoglycan synthesis was evaluated by 35S-sulfate incorporation. Ultrasound treatment did not induce Hsp70, while heat treatment caused a slight heat stress response. Proteoglycan synthesis was increased approximately 2-fold after 3-4 daily ultrasound stimulations, and remained at that level until day 5 in responsive cell isolates. However, chondrocytes from one donor cell isolation out of five remained non-responsive. Heat treatment alone did not increase proteoglycan synthesis. In conclusion, our study confirms that pulsed ultrasound stimulation can induce proteoglycan synthesis in chondrocytes.     

Role of Heat Shock Proteins in the Effect of NMDA and KCl on Cerebellar Granule Cells Survival

Cerebellar granule cells (CGC) die apoptotically after five days in culture (DIV) at physiological concentrations of potassium (5 mM; K5). When CGC are depolarized (K25) or treated with NMDA (150 M) cell survival is increased. CGC changed from K25 to K5 die after 24–48 h. It is known that heat shock protein (HSP) may protect from cell death. Here, we found that cells in K5 showed an increase in HSP-70 levels after 3 DIV. Similarly, in cells changed from K25 to K5, HSP-70 levels were increased after 6 h. Neither NMDA nor K25 treatment affected HSP-70 levels from 2–7 DIV. Ethanol or thermal stress induced HSP-70, but cell survival was not affected in K5 medium. These results suggest that HSP, particularly HSP-70, are not involved in the mechanisms by which NMDA and KCl promote cell survival.     

A role of the Drosophila homeless gene in repression of Stellate in male meiosis

The homeless gene of Drosophila melanogaster encodes a member of the DE-H family of ATPase and RNA helicase proteins. Loss-of-function homeless mutations were previously found to cause female sterility with numerous defects in oogenesis, including improper formation of both the anterior-posterior and dorsal-ventral axes and failure to transport and localize key RNAs required for axis formation. One homeless mutation was also found to affect male meiosis, causing elevated X-Y nondisjunction. Here we further analyze the role of homeless in male meiosis. We show that homeless mutations cause a variety of defects in male meiosis including nondisjunction of the X-Y and 2-2 pair, Y chromosome marker loss, meiotic drive, chromosome fragmentation, chromatin bridges at anaphase, and tripolar meiosis. In addition, homeless mutations interact with an X chromosomal factor to cause complete male sterility. These phenotypes are similar to those caused by deletion of the Suppressor of Stellate [Su(Ste)] locus. Like Su(Ste) deficiencies, homeless mutants also exhibit crystals in primary spermatocytes and derepression of the X-linked Stellate locus. To determine whether the regulatory role of hls is specific for Stellate or includes other repeated sequences as well, we compared testis RNA levels for nine transposable elements and found that all but one, copia, were expressed at the same levels in hls mutants and wild type. Copia, however, was strongly derepressed in hls mutant males. We conclude that hls functions along with Su(Ste) and other recently described genes to repress the Stellate locus in spermatocytes, and that it may also play a role in repressing certain other repeated sequences.     

Aggregation of human red blood cells after moderate heat treatment

The aggregation behaviour of normal and heat treated (48.4 degrees C, 48.8 degrees C, 49.5 degrees C) red blood cells (RBCs) suspended in dextran-saline solutions (Dx 70, Dx 173) was investigated by a laser light reflectometric method over a wide range of bridging energies. The characteristic times of rouleau formation were found to be increased after RBC heat treatment. The disaggregation shear stress is not significantly different between normal RBCs and heat treated RBCs. The loss of cell deformability is nevertheless shown to improve slightly the dissociation efficiency of the flowing liquid in a shear flow resulting in a small reduction of the disaggregation shear rate after heat treatment. Heat treatment is also shown to alter the structure of RBC network at equilibrium. These results indicate that heat induced alterations of erythrocytes only affects the mechanical properties of the cell membrane without significant changes in the macromolecular bridging energy.     

Characterization of tpp1(+) as encoding a main trehalose-6P phosphatase in the fission yeast Schizosaccharomyces pombe

We have characterized an open reading frame of 2,454 bp on chromosome I of Schizosaccharomyces pombe as the gene encoding trehalose-6P phosphatase (tpp1(+)). Disruption of tpp1(+) caused in vivo accumulation of trehalose-6P upon heat shock and prevented cell growth at 37 to 40 degrees C. Accumulation of trehalose-6P in cells bearing a chromosomal disruption of the tpp1(+) gene and containing a plasmid with tpp1(+) under the control of the thiamine-repressible promotor correlated with tpp1(+) repression. The level of tpp1(+) mRNA rose upon heat shock, osmostress, or oxidative stress and was negatively controlled by cyclic AMP-dependent protein kinase activity. Expression of tpp1(+) during oxidative or osmotic stress, but not during heat shock, was under positive control by the wis1-sty1 (equivalent to phh1 and spc1) mitogen-activated protein kinase pathway. Analysis of Tpp1 protein levels suggests that the synthesis of trehalose-6P phosphatase may also be subjected to translational or posttranslational control.     

Heat stress interferes with formation of double-strand breaks and homolog synapsis

Meiotic recombination (MR) drives novel combinations of alleles and contributes to genomic diversity in eukaryotes. In this study, we showed that heat stress (36°C–38°C) over the fertile threshold fully abolished crossover formation in Arabidopsis (Arabidopsis thaliana). Cytological and genetic studies in wild-type plants and syn1 and rad51 mutants suggested that heat stress reduces generation of SPO11-dependent double-strand breaks (DSBs). In support, the abundance of recombinase DMC1, which is required for MR-specific DSB repair, was significantly reduced under heat stress. In addition, high temperatures induced disassembly and/or instability of the ASY4- but not the SYN1-mediated chromosome axis. At the same time, the ASY1-associated lateral element of the synaptonemal complex (SC) was partially affected, while the ZYP1-dependent central element of SC was disrupted, indicating that heat stress impairs SC formation. Moreover, expression of genes involved in DSB formation; e.g. SPO11-1, PRD1, 2, and 3 was not impacted; however, recombinase RAD51 and chromosome axis factors ASY3 and ASY4 were significantly downregulated under heat stress. Taken together, these findings revealed that heat stress inhibits MR via compromised DSB formation and homolog synapsis, which are possible downstream effects of the impacted chromosome axis. Our study thus provides evidence shedding light on how increasing environmental temperature influences MR in Arabidopsis.

Heat stress inhibits CO formation by affecting SPO11-dependent DSB formation and synapsis of homologous chromosomes, probably through its impact on chromosome axis.     

热胁迫下沙门菌细胞结构和特性变化研究进展

沙门菌(Salmonella)是一种非常重要的食源性致病菌。由于食品基质的保护作用,有些沙门菌可以抵抗热胁迫而存活下来。存活细胞往往因为热胁迫或应激而导致细胞结构、生理特性、基因及蛋白表达发生变化,并会进一步对食品原料和加工环境造成持续污染。本文主要综述沙门菌在热胁迫前后细胞形态、菌体组分、细胞壁和细胞膜结构等方面的变化,结合基因和蛋白表达改变,探讨沙门菌在热胁迫下引起的热休克反应、抗逆性和致病性分子机制。     

Effect of single and repeated heat stress on chemical signals of heat shock response cascade in the rat’s heart

Exposure to sublethal heat stress activates a complex cascade of signaling events, such as activators (NO), signal molecules (PKCε), and mediators (HSP70 and COX-2), leading to implementation of heat preconditioning, an adaptive mechanism which makes the organism more tolerant to additional stress. We investigated the time frame in which these chemical signals are triggered after heat stress (41?±?0.5°С/45 min), single or repeated (24 or 72 h after the first one) in heart tissue of male Wistar rats. The animals were allowed to recover 24, 48 or 72 h at room temperature. Single heat stress caused a significant increase of the concentration of HSP70, NO, and PKC level and decrease of COX-2 level 24 h after the heat stress, which in the next course of recovery gradually normalized. The second heat stress, 24 h after the first one, caused a significant reduction of the HSP70 levels, concentration of NO and PKC?, and significant increase of COX-2 concentration. The second exposure, 72 h after the first heat stress, caused more expressive changes of HSP70 and NO in the 24 h-recovery groups. The level of PKC? was not significantly changed, but there was significantly increased COX-2 concentration during recovery. Serum activity of AST, ALT, and CK was reduced after single exposure and increased after repeated exposure to heat stress, in both time intervals. In conclusion, a longer period of recovery (72 h) between two consecutive sessions of heat stress is necessary to achieve more expressive changes in mediators (HSP70) and triggers (NO) of heat preconditioning.