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 caspase-independent intrinsic apoptosis by inhibiting nuclear translocation of apoptosis-inducing factor (AIF) and disturbed extrinsic apoptosis by inactivating caspase-8. Genetic knockdown/overexpression of Hif-1α and dual-luciferase reporter assay indicated that Hif-1α activated 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, protein carbonylation, and 8-hydroxy-2 deoxyguanosine, showed a significant increase after hypoxia, accompanied by tissue damage. N-acetylcysteine (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.

Developmental and mRNA transcript relative abundance pattern of vitellogenin receptors, LR8-/Lrp13, during ovarian development in the large yellow croaker (Larimichthys crocea).

For vitellogenin (Vtg) absorption to occur, there needs to binding of the glycolipophosphoproteins to the oocyte membrane in oviparous species, including teleosts. The cDNAs encoding homologous Vtg receptors (VgRs) LR8- and Lrp13 were cloned from ovaries of the large yellow croaker (Larimichthys crocea), an economically important species in Chinese aquaculture. The full-length Lc-lr8-/lrp13 cDNAs contained 4266/3760 base pairs (bp) and the deduced Lc-LR8-/Lrp13 proteins had 844/1218 amino acids, respectively. The VgRs comprised a ligand-binding domain, an epidermal growth factor precursor homology domain, YWXD motifs forming a ß-propeller structure, and transmembrane and cytoplasmic domains. There was a marked relative abundance of Lc-lr8-/lrp13 transcripts in the tissues that were evaluated, with the largest abundance in the ovaries at Stage II of development. Furthermore, there was a lesser relative abundance of Lc-lr8-/lrp13 mRNA transcript during ovarian development (Stages II to IV). In situ hybridization technology was used to analyze decreasing relative abundance pattern of Lc-lr8-/lrp13 mRNA transcript during oogenesis in Stage II to IV of ovarian development. By combining mRNA relative abundance with morphological results, a model was developed to explain the reduction in Lc-lr8-/lrp13 mRNA transcript relative abundance during ovarian development. During the early developmental stages, transcription, translation, and differential accumulation of VgRs in previtellogenic and vitellogenic oocytes may occur and result in Vtg absorption in teleost oocytes. Overall, there is preliminary evidence indicating that at least two VgRs (Lc-LR8-/Lrp13) are present in the large yellow croaker and may be important for Vtg transport from the blood into the oocyte during ovarian development.