Hif-1α/Hsf1/Hsp70 signaling pathway regulates redox homeostasis and apoptosis in large yellow croaker(Larimichthys crocea)under environmental hypoxia

Oxygen is an essential molecule for animal respiration,growth,and survival.Unlike in terrestrial environments,contamination and climate change have led to the frequent occurrence of hypoxia in aquatic environments,thus impacting aquatic animal survival.However,the adaptative mechanisms underlying fish responses to environmental hypoxia remain largely unknown.Here,we used large yellow croaker(Larimichthys crocea)and large yellow croaker fry(LYCF)cells to investigate the roles of the Hif-1α/Hsf1/Hsp70 signaling pathway in the regulation of cellular redox homeostasis,and apoptosis.We confirmed that hypoxia induced the expression of Hif-1α,Hsf1,and Hsp70 in vivo and in vitro.Genetic Hsp70 knockdown/overexpression indicated that Hsp70 was required for maintaining redox homeostasis and resisting oxidative stress in LYCF cells under hypoxic stress.Hsp70 inhibited caspase-dependent intrinsic apoptosis by maintaining normal mitochondrial membrane potential,enhancing Bcl-2 mRNA and protein expression,inhibiting Bax and caspase3 mRNA expression,and suppressing caspase-3 and caspase-9 activation.Hsp70 suppressed caspaseindependent intrinsic apoptosis by inhibiting nuclear translocation of apoptosis-inducing factor(AIF)and disturbed extrinsic apoptosis by inactivating caspase-8.Genetic knockdown/overexpression of Hif-1αand dual-luciferase reporter assay indicated that Hif-1αactivated the Hsf1 DNA promoter and enhanced Hsf1 mRNA transcription.Hsf1 enhanced Hsp70 mRNA transcription in a similar manner.In summary,the Hif-1α/Hsf1/Hsp70 signaling pathway plays an important role in regulating redox homeostasis and anti-apoptosis in L.crocea under hypoxic stress.

Scavenging reactive oxygen species is a potential strategy to protect Larimichthys crocea against environmental hypoxia by mitigating oxidative stress

The large yellow croaker(Larimichthys crocea),which is an economically important mariculture fish in China,is often exposed to environmental hypoxia.Reactive oxygen species(ROS)homeostasis is essential for the maintenance of normal physiological conditions in an organism.Direct evidence that environmental hypoxia leads to ROS overproduction is scarce in marine fish.Furthermore,the sources of ROS overproduction in marine fish under hypoxic stress are poorly known.In this study,we investigated the effects of hypoxia on redox homeostasis in L.crocea and the impact of impaired redox homeostasis on fish.We first confirmed that hypoxia drove ROS production mainly via the mitochondrial electron transport chain and NADPH oxidase complex pathways in L.crocea and its cell line(large yellow croaker fry(LYCF)cells).We subsequently detected a marked increase in the antioxidant systems of the fish.However,imbalance between the pro-oxidation and antioxidation systems ultimately led to excessive ROS and oxidative stress.Cell viability showed a remarkable decrease while oxidative indicators,such as malondialdehyde,proteincarbonylation,and8-hydroxy-2 deoxyguanosine,showed a significant increase after hypoxia,accompanied by tissue damage.Nacetylcysteine(NAC)reduced ROS levels,alleviated oxidative damage,and improved cell viability in vitro.Appropriate uptake of ROS scavengers(e.g.,NAC and elamipretide Szeto-Schiller-31)and inhibitors(e.g.,apocynin,diphenylene iodonium,and 5-hydroxydecanoate)may be effective at overcoming hypoxic toxicity.Our findings highlight previously unstudied strategies of hypoxic toxicity resistance in marine fish.

Evidence for the role of KIF17 in fish spermatid reshaping:expression pattern of KIF17 in Larimichthys polyactis spermiogenesis

The homodimeric kinesin-2 protein KIF17 functions in intracellular transport and spermiogenesis in mammals.However,its role in fish spermiogenesis has not been reported.Here,we aimed to clone full-length kif17 cDNA and determine the molecular characteristics and expression patterns of KIF17 in Larimichthys polyactis spermiogenesis.The full-length cDNA of L.polyactis kif17(Lp-kif17)was sequenced and found to contain a 332-bp 5′untranslated region,480-bp 3′untranslated region,and 2433-bp open reading frame encoding 810 amino acids.Bioinformatics analyses showed that L.polyactis KIF17(Lp-KIF17)shared high sequence similarity with homologs in other animals and possessed an N-terminal motor domain with microtubule-binding sites and adenosine triphosphate(ATP)hydrolysis sites,a stalk domain containing two coiled-coil regions,and a C-terminal tail domain.The Lp-kif17 mRNA was widely expressed in various tissues,with the highest level in the brain,followed by that in the testis.Fluorescence in situ hybridization(FISH)analysis revealed that Lp-kif17 was continuously expressed in spermiogenesis,showing that it had potential functions in this process.Using immunofluorescence(IF)analysis,we found that Lp-KIF17 colocalized with tubulin and was transferred from the perinuclear cytoplasm to the side of spermatid where the tail forms during spermiogenesis.These findings suggested that KIF17 is involved in L.polyactis spermiogenesis.In particular,it may participate in nuclear shaping and tail formation by interacting with perinuclear microtubules during spermatid reshaping.In addition to providing evidence for the role of KIF17 in fish spermatid reshaping,this study provides important data for studies of reproductive biology in L.polyactis.

Characterization of ghrelin mRNA expression in fasting Larimichthys crocea juveniles

Larimichthys crocea is a marine fish species cultured in China.Short-term starvation is often applied to improve the quality of cultured L.crocea,and the expression of ghrelin in tissues of stomach,muscle,brain,intestines,liver,and kidney,involved in starvation response,under starvation conditions were studied to understand the effect of starvation on the expression of ghrelin in L.crocea juveniles.The ghrelin expression was tissue-specific,and expression was significantly higher in the stomach compared to other tissues(P<0.01).Additionally,ghrelin expression in different tissues changed along with prolongation of fasting.In the stomach,ghrelin expression levels increased gradually at the beginning of the fast,and then declined after eight days of fasting.Gene expression in the brain and intestines increased at the beginning of the fast,and then decreased with longer fasting time.Interestingly,ghrelin expression declined at the beginning of the fast,then increased with longer fasting in the kidneys and muscles.These results suggest that ghrelin is involved in starvation response in L.crocea juveniles.This study provids insights into ghrelin function and an important reference for the development of reasonable feeding strategies for L.crocea juveniles.

Environmental hypoxia induces apoptosis in large yellow croaker Larimichthys crocea via both intrinsic and extrinsic pathways

Hypoxia has become an unfavorable factor affecting the sustainable development of the large yellow croaker Larimichthys crocea,an economically important mariculture fish in China.Apoptosis is a consequence of hypoxia on fish.However,the effects of hypoxia stress on apoptosis in L.crocea remain largely unknown.We investigated the effect of environmental hypoxia on apoptosis in L.crocea.Results show that hypoxia induced apoptosis in L.crocea both in vivo and in vitro.The mitochondrial membrane potential was significantly reduced in large yellow croaker fry(LYCF)cells.The expression levels of Bcell lymphoma/leukemia-2(Bcl-2)m RNA and protein were also significantly decreased in the liver and LYCF cells during 96 h and 48 h of hypoxia stress,respectively,whereas the expression level of Bcl-2 associated X(Bax)mRNA,Casp3 mRNA,and activity of caspase-3/7/9 were significantly increased,indicating that hypoxia induced caspase-dependent intrinsic apoptosis in L.crocea.The expression level of the apoptosis-inducing factor(AIF)protein was significantly increased in the liver and LYCF cells.The level of AIF protein was significantly decreased in the cytoplasm but increased in the nuclei of L.crocea,demonstrating that hypoxia induced the AIF-mediated caspase-independent intrinsic apoptosis.In addition,the activity of caspase-8 was significantly increased,indicating that hypoxia stress induced extrinsic apoptosis in L.crocea.Therefore,hypoxia induced apoptosis in L.crocea through both the intrinsic and extrinsic pathways.The present study accumulated basic biological information to help elucidate the mechanism of hypoxia response in marine fish.