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Ontogeny of the Heat Shock Protein, hsp70 and hsp60, Response and Developmental Effects of Heat-Shock in the Teleost, Medaka (Oryzias latipes)
Abstract
Cellular biomarkers such as stress proteins (heat-shock proteins, hsp) have been extensively investigated for
their use as sublethal indicators of environmental stress. As more refined methods such as developmental
assays are employed in toxicity testing, an understanding of the expression of molecular biomarkers at various
stages of organism development can yield valuable information about their role in normal and altered
development. In addition, the ability of an organism to synthesize stress proteins at specific stages may
influence its tolerance and/or susceptibility to environmental stressors. This study characterizes the hsp70 and
hsp60 response in eight developmental stages of Japanese medaka (Oryzias latipes). Neither hsp70 nor hsp60
was inducible in early embryos (stage 11 - early gastrula), and these embryos proved considerably less tolerant
to heat stress than did embryos at stage 19 and older. Although individuals from all post-hatch stages were
able to raise a stress protein response, they were more susceptible to heat-shock than were embryonic stages
(with the exception of stage 11). One hsp70 isoform was detected in embryonic stages. Additional hsp70
isoform(s) were detected in medaka larvae and adults. Two isoforms of hsp60 were detected in embryonal
stages, but not in larval or adult medaka. Results indicated that hsp60 was less inducible by heat-shock than
hsp70, particularly in later stages. Constitutive levels of soluble hsp70 and hsp60 proteins were relatively high
just before (stage 35) and after hatching (24–36 h old larvae), and in adult fish. The inability of early embryos
to induce hsp70 and hsp60 stress proteins could render them more susceptible to a variety of environmental
stressors including chemical pollutants.
... Previous studies on the effects of elevated temperature during embryogenesis have observed faster development with smaller fry at hatch and increased abnormalities and mortality when the temperature exceeds suboptimal levels (Lahnsteiner et al., 2012;Ojanguren and Brana, 2003;Prokešová et al., 2015). The embryonic HSR has been studied in some species of fish including medaka (Oryzias latipes) (Werner et al., 2001), Atlantic cod (Gadus morhua) (Takle et al., 2005), Atlantic salmon (Salmo salar) (Skjaerven et al., 2011), green sturgeon (Acipenser medirostris) (Werner et al., 2006) and lake whitefish (Coregonus clupeaformis) (Sessions, 2015;Stefanovic et al., 2016). All species were able to elicit a HSR during development, but the stage at which this was observed, temperature of induction and the nature of the response was species-specific. ...
... A 2-fold increase in hsp70 mRNA levels was observed in embryos sampled immediately following a 1 h, +8°C TS at the gastrula and 15th somite stages, but at other time points the response was either delayed or smaller in magnitude, with the exception of the 1st and 20th somite stage where exposure to TS did not induce hsp70 (Takle et al., 2005). Medaka did not induce the expression of hsps until gastrulation was completed (Werner et al., 2001). Lake whitefish have been shown to be able to initiate a HSR during embryogenesis, which could be attenuated when subjected to repeated TS events, although lake whitefish studies to date have only focused on the latter part of development (Sessions, 2015;Stefanovic et al., 2016). ...
... Collectively the data presented herein show that lake whitefish embryos can induce multiple hsps in response to TS during development and their HSR may be either attenuated or supressed by exposure to multiple TS events; thus indicating plasticity. There is limited information available on the HSR in early embryogenesis and the developmental time point at which a HSR can be induced is species-specific Skjaerven et al., 2011;Werner et al., 2001). The data from these two experiments provides novel information on the response of early stage lake whitefish embryos to TS and how repeated TS can alter the HSR. ...
Lake whitefish (Coregonus clupeaformis) embryos were exposed to thermal stress (TS) at different developmental stages to determine when the heat shock response (HSR) can be initiated and if it is altered by exposure to repeated TS. First, embryos were subject to one of three different TS temperatures (6, 9, or 12 °C above control) at 4 points in development (21, 38, 60 and 70 days post-fertilisation (dpf)) for 2 h followed by a 2 h recovery to understand the ontogeny of the HSR. A second experiment explored the effects of repeated TS on the HSR in embryos from 15 to 75 dpf. Embryos were subjected to one of two TS regimes; +6 °C TS for 1 h every 6 days or +9 °C TS for 1 h every 6 days. Following a 2 h recovery, a subset of embryos was sampled. Our results show that embryos could initiate a HSR via upregulation of heat shock protein 70 (hsp70) mRNA at all developmental ages studied, but that this response varied with age and was only observed with a TS of +9 or +12 °C. In comparison, when embryos received multiple TS treatments, hsp70 was not induced in response to the 1 h TS and 2 h recovery, and a downregulation was observed at 39 dpf. Downregulation of hsp47 and hsp90α mRNA was also observed in early age embryos. Collectively, these data suggest that embryos are capable of initiating a HSR at early age and throughout embryogenesis, but that repeated TS can alter the HSR, and may result in either reduced responsiveness or a downregulation of inducible hsps. Our findings warrant further investigation into both the short- and long-term effects of repeated TS on lake whitefish development.
... were extracted from frozen tissues using T-PER tissue protein extraction reagent (Pierce, Rockford, Illinois) with added protease inhibitor mix (Halt Protease Inhibitor Cocktail; Thermo Scientific, Rockford, Illinois) in disposable 1.5-mL tubes using disposable pellet pestles (Fisher Scientific, Hampton, New Hampshire). Total protein concentration of the supernatant was determined using a Qubit 2.0 fluorometer (Life Technologies, Grand Island, New York), and 25 μg of total protein were used in a western blot following a modified protocol described by Werner et al. (2001). Protein quantification was carried out in accordance with the technique of Cassinelli and Moffitt (2010). ...
... This is likely due to the different perfusion, clearance, and induction rates of different tissue types (Dyer et al. 1991;Dietz and Somero 1993;Williams et al. 1995Williams et al. , 1996, with liver being more perfuse and having faster clearance than muscle. Other stressors have been shown to increase hsp70 abundance in fishes (Sanders 1993;Forsyth et al. 1997;Wang et al. 2013), but thermal stress has been of primary interest (Werner et al. 2001;Cassinelli and Moffitt 2010;Kammerer and Heppell 2013b). Increased temperatures were shown to increase hsp70 expression in Atlantic Cod (Aursnes et al. 2011) but without a measurable increase in hsp70 abundance (Zakhartsev et al. 2005). ...
... In Atlantic salmon the HSR was found to be dependent on the stage of development as some stages (1st and 25th somite) showed no increase in hsp70 mRNA expression in response to HS (Takle et al., 2005). Correspondingly, Werner et al. (1986) reported developmental stage-dependent responses to HS in the warm water Medaka (Oryzias latipes) where a 30 min, 15°C HS resulted in an increase in Hsp70 and Hsp60 proteins in late, but not early embryos (stage 11 or early gastrula). Likewise, lake whitefish embryos may show stage-dependent responses to HS. ...
... Takle et al. (2005) observed elevated hsp70 mRNA in early stage (gastrula to 45th somite) Atlantic salmon embryos for 24 h following a 1 h HS from an 8°C control temperature. The embryonic period (especially early embryogenesis) is very susceptible to the teratogenic effects of thermal stress (Werner et al., 1986;Krone et al., 2003;Hattori et al., 2004) and the role of Hsps in protecting against thermally induced teratogenesis has been well-documented (Edwards et al., 1997;Mirkes, 1997;Luft and Dix, 1999). Thus, given the reduction in metabolic rate associated with lower temperatures (Johnston and Dunn, 1987;Podrabsky and Somero, 2004), our results are consistent with those of other vertebrates. ...
We investigated the effects of thermal stress on embryonic (fin flutter, vitelline circulation stage) and young of the year (YOY) juvenile lake whitefish by characterizing the kinetics of the heat shock response (HSR). Lake whitefish were subjected to one of three different heat shock (HS) temperatures (3, 6, or 9°C above control) for six different lengths of time (0.25, 0.50, 1, 2, 3, or 4 h) followed by a 2 h recovery period at the control temperature of 2°C or 14°C for embryos and YOY juveniles, respectively. The duration of the HSR was examined by allowing the fish to recover for 1, 2, 4, 8, 12, 16, 24, 36, or 48 h following a 2 h HS. In embryos, at the fin flutter stage, only hsp70 mRNA levels were upregulated in response to the various HS treatments. By comparison, all three typically inducible hsps, hsp90α, hsp70 and hsp47, were upregulated in the YOY juveniles. In both instances the HSR was long lasting, but much more so in embryos where hsp70 mRNA levels continued to increase for 48 h after a 2 h HS and remained significantly higher than untreated controls. Collectively our data indicate that both embryo and YOY juvenile lake whitefish have a robust HSR which permits them to survive a 4 h, 9°C HS. Moreover, both life history stages are capable of triggering a HSR following a moderate 3°C HS which is likely an important protective mechanism against environmental stressors during embryogenesis and early life history stages of lake whitefish.
... Thus, the Hsp70 levels observed herein in fertilized eggs may indicate a role in S. dumerili early ontogenesis. Likewise, Japanese medaka (Oryzias latipes) exhibited non-inducible Hsp70 and Hsp60 in early embryos, while levels increased later in posthatching stages (Werner et al. 2001). ...
Cell growth and differentiation signals of insulin-like growth factor-1 (IGF-1), a key regulator in embryonic and postnatal development, are mediated through the IGF-1 receptor (IGF-1R), which activates several downstream pathways. The present study aims to address crucial organogenesis and development pathways including Akt, MAPKs, heat shock response, apoptotic and autophagic machinery, and energy metabolism in relation to IGF-1R activation during five developmental stages of reared Seriola dumerili: 1 day prior to hatching fertilized eggs (D-1), hatching day (D0), 3 days post-hatching larvae (D3), 33 (D33) and 46 (D46) days post-hatching juveniles. During both the fertilized eggs stage and larval-to-juvenile transition, IGF-1R/Akt pathway activation may mediate the hypertrophic signaling, while p44/42 MAPK phosphorylation was apparent at S. dumerili post-hatching processes and juvenile organs completion. On the contrary, apoptosis was induced during embryogenesis and autophagy at hatching day indicating a potential involvement in morphogenetic rearrangements and yolk-sac reserves depletion. Larvae morphogenesis was accompanied by a metabolic turnover with increased substantial energy consumption. The findings of the present study demonstrate the developmental stages-specific shift in critical signaling pathways during the ontogeny of reared S. dumerili.
... Collectively these studies demonstrate that thermal history, Hsp expression and HSR plasticity are linked, and that the threshold of induction for the HSR can shift in response to thermal history; however, the majority of these studies have focused on adult fishes and chronic temperature shifts. Although data are more limited, embryos from a variety of fish species have been shown to mount a HSR following exposure to TS, including zebrafish (Danio rerio; Krone et al., 1997); Atlantic salmon (Salmo salar; Takle et al., 2005) and Japanese medaka (Oryzias latipes; Werner et al., 2001). Likewise, embryonic lake whitefish can induce a HSR following exposure to a TS of 3-9 o C above control temperature (Stefanovic et al., 2016;Whitehouse et al., 2017). ...
We examined the impact of repeated thermal stress on the heat shock response (HSR) of thermally sensitive lake whitefish (Coregonus clupeaformis) embryos. Our treatments were designed to mimic temperature fluctuations in the vicinity of industrial thermal effluents. Embryos were either maintained at control temperatures (3 oC) or exposed to a repeated thermal stress (TS) of 3 or 6 oC above control temperature every 3 or 6 days throughout embryonic development. At 82 days post-fertilisation, repeated TS treatments were stopped and embryos received either a high level TS of 12, 15, or 18 oC above ambient temperature for 1 or 4 h, or no additional TS. These treatments were carried out after a 6 h recovery from the last repeated TS. Embryos in the no repeated TS group responded, as expected, with increases in hsp70 mRNA in response to 12, 15 and 18 oC high-level TS. However, exposure to repeated TS of 3 or 6 ⁰C every 6 days also resulted in a significant upregulation of hsp70 mRNA relative to the controls. Importantly, these repeated TS events and the associated elevations in hsp70 attenuated the upregulation of hsp70 in response to a 1 h, high-level TS of 12 oC above ambient, but not to either longer (4 h) or higher (15 or 18 oC) TS events. Conversely, hsp90α mRNA levels were not consistently elevated in the no repeated TS groups exposed to high-level TS. In some instances, hsp90α levels appeared to decrease in embryos exposed to repeated TS followed by a high-level TS. The observed attenuation of the HSR in lake whitefish embryos demonstrates that embryos of this species have plasticity in their HSR and repeated TS may protect against high-level TS, but the response differs based on repeated TS treatment, high-level TS temperature and duration, and the gene of interest.
... Typically, they are proteins constitutively expressed in the cytoplasm that co-localize to the nucleus under stress induced by physical and chemical insult. Among them, HSP90 and HSP27 are associated with poor prognosis and likely play an important role in drug-resistant breast cancer (223), as well as serve as a biomarker due to toxicant exposure (224). A previous study showed that the gene expression of HSP27 and HSP90 was upregulated by malathion or parathion, alone or combined with estrogen (20). ...
Cancer development is a multistep process that may be induced by a variety of compounds. Environmental substances, such as pesticides, have been associated with different human diseases. Organophosphorus pesticides (OPs) are among the most commonly used insecticides. Despite the fact that organophosphorus has been associated with an increased risk of cancer, particularly hormone‑mediated cancer, few prospective studies have examined the use of individual insecticides. Reported results have demonstrated that OPs and estrogen induce a cascade of events indicative of the transformation of human breast epithelial cells. In vitro studies analyzing an immortalized non‑tumorigenic human breast epithelial cell line may provide us with an approach to analyzing cell transformation under the effects of OPs in the presence of estrogen. The results suggested hormone‑mediated effects of these insecticides on the risk of cancer among women. It can be concluded that, through experimental models, the initiation of cancer can be studied by analyzing the steps that transform normal breast cells to malignant ones through certain substances, such as pesticides and estrogen. Such substances cause genomic instability, and therefore tumor formation in the animal, and signs of carcinogenesis in vitro. Cancer initiation has been associated with an increase in genomic instability, indicated by the inactivation of tumor‑suppressor genes and activation of oncogenes in the presence of malathion, parathion, and estrogen. In the present study, a comprehensive summary of the impact of OPs in human and rat breast cancer, specifically their effects on the cell cycle, signaling pathways linked to epidermal growth factor, drug metabolism, and genomic instability in an MCF‑10F estrogen receptor‑negative breast cell line is provided.
... Few studies have evaluated hsp60 modulation in response to changes in temperature in early stages in teleost fish. In medaka eggs, an increase in HSP60 protein levels has been previously observed in response to high temperature (Werner et al., 2001), showing a time-associated temperature response depending on the developmental stage. Concordantly, we observed in our study an increased gene expression of hsp60, but in G. chilensis a faster hsp60 response (6 h) was observed compared to medaka (12 h), ...
Red cusk‐eel (Genypterus chilensis) is a native species with potential for Chilean aquaculture diversification. However, no information exists on the effects of temperature on oxidative stress and eggs quality markers in post‐ovulatory eggs and ovary of this species. We determine that high and low temperature generate oxidative damage on post‐ovulatory eggs, with no effect on ovary. Temperature induces thermal stress markers expression on post‐ovulatory eggs, and modulates antioxidant and eggs quality markers on post‐ovulatory eggs and ovary, information to consider for quality evaluation in the red cusk‐eel management.
... Many of the chemical toxicants are known to induce the typical heat shock response as an evolutionarily conserved cellular mechanism to cope with the adverse effects of a variety of external stressors (Hightower 1991;Nover 1991;Sanders 1993). It acts through a specific set of proteins, called heat shock proteins (HSPs) or molecular chaperones, exhibiting wide-ranging cellular functions, including protection from stress-induced injury (Feige et al. 1996;Ryan and Hightower 1996;Werner et al. 2001). Among the HSPs, HSP70 is widely suggested to protect cells from the injury inflicted upon by a variety of stressors by preventing misfolding or aggregation of misfolded protein, proteolytic degradation of unstable proteins, or catalyzing proper folding of nascent polypeptides to their native functional state (Feder and Hofmann 1999;Iwama et al. 1999;Krebs and Feder 1997;Morimoto et al. 1994;Nadeau et al. 2001;Nover 1991;Sejerkilde et al. 2003). ...
The effect of two most commonly used and highly toxic organic pesticides, endosulphan (organochlorine) and monocrotophos (organophosphate), was studied in a blowfly, Lucilia cuprina, to test whether these pesticides induce the stress response and, if so, whether the intensity of the response, in terms of induction of heat shock proteins (HSPs), HSP60 and HSP70 in particular, is pesticide concentration dependent. The in vitro exposure of larval and adult tissues to varying concentrations of these pesticides (endosulphan: 1.0-4.0 ppm for larva and 0.05-0.50 ppm for adult; monocrotophos: 0.0005-0.0050 ppm for larva and 0.0001-0.0010 ppm for adult) revealed that both compounds were able to induce the expression of HSP60 and HSP70 proteins. Western blot analysis of these HSPs indicated that the induction of expression was tissue-specific. The trypan blue staining of pesticide-exposed tissues demonstrated monocrotophos to exert more severe effect than endosulphan, as the former compound induced both HSP60 and HSP70 significantly at a much lower concentration than that of the later. The pattern of expression of these HSPs, in general, appeared in direct correlation with the pesticide concentration. Gut tissues were found relatively more sensitive to pesticide toxicity than other tissues, as revealed by trypan blue staining, and hence, they might serve as primary targets for early detection of pesticide toxicity. The results indicated that either of these HSPs or both could serve as a potential biomarker toward assessment and monitoring of toxicity induced by these pesticides.
As poikilotherms, fish health is compromised by exposure to elevated temperatures (e.g. climate change-related warming, anthropogenic thermal pollution, and/or hatchery processes). While fish thermotolerance has been demonstrated to be plastic, the downstream impacts of early life-stage high temperature exposure are not known. In the present study, we investigated the thermotolerance of rainbow trout (Oncorhynchus mykiss) fry 2 months after being exposed to elevated temperature (22°C) for 96 h. Exposed fry demonstrated a reduced critical thermal maxima (CTmax) in comparison to control fish. Using the RNase H-dependent quantitative PCR method, expression of rainbow trout hsp70 isoforms was determined immediately after the acute thermal stress and immediately following the thermotolerance trials. The lowered CTmax was associated with a reduced ability to upregulate the hsp70b gene during the thermotolerance trials, whereas no changes in hsp70a were observed. Overall, these results indicate that exposure to thermal stress in early life-stages of rainbow trout can have negative effects on future physiological function.
Water temperature is one of the most important factors in fish physiology; thus, it is important to identify genes that respond to changes in water temperature. In this study, we identified a warm- temperature acclimation-associated 65-kDa protein (Wap65) in the Kumgang fat minnow Rhynchocypris kumgangensis, a small, cold-freshwater fish species endemic to Korea. Kumgang fat minnow Wap65-1 (kmWap65-1) was cloned using polymerase chain reaction (PCR)-based strategies, and was found to be highly homologous with teleost Wap65-1 and mammalian hemopexin, a heme-binding protein that transfers plasma heme into hepatocytes. kmWap65-1 mRNA was expressed mainly in the liver and its expression levels were significantly increased by both short- and long-term exposure to high temperature, which was evaluated by real-time quantitative PCR. Furthermore, the expression levels of kmWap65-1 were highly elevated by exposure to bacterial lipopolysaccharide. These results indicate that kmWap65-1 expression is associated with environmental stresses such as increases in water temperature and bacterial infection. J. Exp. Zool. 9999A:XX-XX, 2015. © 2015 Wiley Periodicals, Inc.
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