Mitochondrial responses to constant and cyclic hypoxia depend on the oxidized fuel in a hypoxia-tolerant marine bivalve Crassostrea gigas.

Sessile benthic organisms like oysters inhabit the intertidal zone, subject to alternating hypoxia and reoxygenation (H/R) episodes during tidal movements, impacting respiratory chain activities and metabolome compositions. We investigated the effects of constant severe hypoxia (90 min at ~ 0% O2 ) followed by 10 min reoxygenation, and cyclic hypoxia (5 cycles of 15 min at ~ 0% O2 and 10 min reoxygenation) on isolated mitochondria from the gill and the digestive gland of Crassostrea gigas respiring on pyruvate, palmitate, or succinate. Constant hypoxia suppressed oxidative phosphorylation (OXPHOS), particularly during Complex I-linked substrates oxidation. It had no effect on mitochondrial reactive oxygen species (ROS) efflux but increased fractional electron leak (FEL). In mitochondria oxidizing Complex I substrates, exposure to cyclic hypoxia prompted a significant drop after the first H/R cycle. In contrast, succinate-driven respiration only showed significant decline after the third to fifth H/R cycle. ROS efflux saw little change during cyclic hypoxia regardless of the oxidized substrate, but Complex I-driven FEL tended to increase with each subsequent H/R cycle. These observations suggest that succinate may serve as a beneficial stress fuel under H/R conditions, aiding in the post-hypoxic recovery of oysters by reducing oxidative stress and facilitating rapid ATP re-synthesis. The impacts of constant and cyclic hypoxia of similar duration on mitochondrial respiration and oxidative lesions in the proteins were comparable indicating that the mitochondrial damage is mostly determined by the lack of oxygen and mitochondrial depolarization. The ROS efflux in the mitochondria of oysters was minimally affected by oxygen fluctuations indicating that tight regulation of ROS production may contribute to robust mitochondrial phenotype of oysters and protect against H/R induced stress.

Effects of hypoxia and reoxygenation on mitochondrial functions and transcriptional profiles of isolated brain and muscle porcine cells.

Oxygen fluctuations might occur in mammalian tissues under physiological (e.g. at high altitudes) or pathological (e.g. ischemia-reperfusion) conditions. Mitochondria are the key target and potential amplifiers of hypoxia-reoxygenation (H-R) stress. Understanding the mitochondrial responses to H-R stress is important for identifying adaptive mechanisms and potential therapeutic solutions for pathologies associated with oxygen fluctuations. We explored metabolic response to H-R stress in two tissue types (muscle and brain) with different degrees of hypoxia tolerance in a domestic pig Sus scrofa focusing on the cellular responses independent of the systemic regulatory mechanisms. Isolated cells from the skeletal muscle (masseter) and brain (thalamus) were exposed to acute short-term (15 min) hypoxia followed by reoxygenation. The mitochondrial oxygen consumption, reactive oxygen species (ROS) production rates and transcriptional profiles of hypoxia-responsive mRNA and miRNA were determined. Mitochondria of the porcine brain cells showed a decrease in the resting respiration and ATP synthesis capacity whereas the mitochondria from the muscle cells showed robust respiration and less susceptibility to H-R stress. ROS production was not affected by the short-term H-R stress in the brain or muscle cells. Transcriptionally, prolyl hydroxylase domain protein EGLN3 was upregulated during hypoxia and suppressed during reoxygenation in porcine muscle cells. The decline in EGLN3 mRNA during reoxygenation was accompanied by an upregulation of hypoxia-inducible factor subunit α (HIF1A) transcripts in the muscle cells. However, in the brain cells, HIF1A mRNA levels were suppressed during reoxygenation. Other functionally important transcripts and miRNAs involved in antioxidant response, apoptosis, inflammation, and substrate oxidation were also differentially expressed between the muscle and brain cells. Suppression of miRNA levels during acute intermittent hypoxia was stronger in the brain cells affecting ~ 55% of all studied miRNA transcripts than in the muscle cells (~ 25% of miRNA) signifying transcriptional derepression of the respective mRNA targets. Our study provides insights into the potential molecular and physiological mechanisms contributing to different hypoxia sensitivity of the studied tissues and can serve as a starting point to better understand the biological processes associated with hypoxia stress, e.g. during ischemia and reperfusion.

Tissue- and substrate-dependent mitochondrial responses to acute hypoxia-reoxygenation stress in a marine bivalve (Crassostrea gigas).

Hypoxia is a major stressor for aquatic organisms, yet intertidal organisms such as the oyster Crassostrea gigas are adapted to frequent oxygen fluctuations by metabolically adjusting to shifts in oxygen and substrate availability during hypoxia-reoxygenation (H/R). We investigated the effects of acute H/R stress (15 min at ∼0% O2 and 10 min reoxygenation) on isolated mitochondria from the gill and the digestive gland of C. gigas respiring on different substrates (pyruvate, glutamate, succinate, palmitate and their mixtures). Gill mitochondria showed better capacity for amino acid and fatty acid oxidation compared with mitochondria from the digestive gland. Mitochondrial responses to H/R stress strongly depended on the substrate and the activity state of mitochondria. In mitochondria oxidizing NADH-linked substrates, exposure to H/R stress suppressed oxygen consumption and generation of reactive oxygen species (ROS) in the resting state, whereas in the ADP-stimulated state, ROS production increased despite little change in respiration. As a result, electron leak (measured as H2O2 to O2 ratio) increased after H/R stress in the ADP-stimulated mitochondria with NADH-linked substrates. In contrast, H/R exposure stimulated succinate-driven respiration without an increase in electron leak. Reverse electron transport (RET) did not significantly contribute to succinate-driven ROS production in oyster mitochondria except for a slight increase in the OXPHOS state during post-hypoxic recovery. A decrease in NADH-driven respiration and ROS production, enhanced capacity for succinate oxidation and resistance to RET might assist in post-hypoxic recovery of oysters mitigating oxidative stress and supporting rapid ATP re-synthesis during oxygen fluctuations, as is commonly observed in estuaries and intertidal zones.

Identification and Allelic Variants Associated With Cold Tolerance of PmPIAS in Pinctada fucata martensii.

The protein inhibitor of activated STAT (PIAS) functions in diverse aspects, including immune response, cell apoptosis, cell differentiation, and proliferation. In the present study, the PIAS in the pearl oyster Pinctada fucata martensii was characterized. The sequence features of PmPIAS were similar to that of other PIAS sequences with PIAS typical domains, including SAP, Pro-Ile-Asn-Ile-Thr (PINIT), RLD domain, AD, and S/T-rich region. Homologous analysis showed that PmPIAS protein sequence showed the conserved primary structure compared with other species. Ribbon representation of PIAS protein sequences also showed a conserved structure among species, and the PINIT domain and RLD domain showed the conserved structure compared with the sequence of Homo sapiens. The expression pattern of PmPIAS in different tissues showed significant high expression in the gonad. PmPIAS also exhibited a significantly higher expression in the 1 and 2 days after cold tolerance stress (17°C) and showed its potential in the cold tolerance. The SNP analysis of the exon region of PmPIAS obtained 18 SNPs, and among them, 11 SNPs showed significance among different genotypes and alleles between cold tolerance selection line and base stock, which showed their potential in the breeding for cold tolerance traits.

Characterization of cyclin dependent kinase-7 and its differential response to grafting challenge in the black shell colored selected line of pearl oyster Pinctada fucata martensii.

Cyclin dependent kinase-7 (Cdk-7) is a protein kinase associated with regulating the cell cycle, cell differentiation and proliferation, apoptosis and inflammatory response. This study characterized the full cDNA sequence of Cdk-7 in Pinctada fucata martensii (PmCdk-7). A full length sequence of 1473bp with an open reading frame (ORF) of 915bp and encodes a 304aa, 5'-UTR of 58bp and a 3'-UTR of 500bp was obtained. The construed amino acid sequence of PmCdk-7 comprised of a Serine/Threonine protein kinases, catalytic (S_TKc) domain with a protein kinases ATP-binding region signature (14-38aa) and the serine/Threonine protein kinases active-site signature (129-141aa) within the domain. Tissue distribution analysis revealed a high relative mRNA expression of PmCdk-7 within haemocytes. Following the insertion operation (grafting), the relative expression levels of PmCdk-7 in the haemocyte was expressed differentially among the studied groups; the black shell colored selected line (BS) and the control group (CG). High expression was recorded between 12 h and 5d with a peak at 3d suggesting a heightened level of DNA replication and inflammatory response during the pearl-sac formation and this expression was higher in BS than CS showcasing, the heightened immune capacity of BS to grafting operation. Immune stimulation experiment with bacterial endotoxin and a viral mimic revealed PmCdk-7 response to pathogenic stress. The results from our study showed that PmCdk-7 performs a vital function during the cell cycle by aiding DNA replication and also aid response to inflammations generated due to the incision from the grafting operation and long exposure to immune-stimulants (pathogens).

Characterization of thioredoxin-like PROTEIN-5 (TRXLP-5) and its differential response to grafting challenge in the black coloured selected line and control stocks of Pinctada fucata martensii.

Thioredoxin-like protein 5 (Trxlp-5) is a thioredoxin isoform associated with cellular redox homeostasis through the activity of thiol-disulfide reductase. In our study, Trxlp-5 was identified and characterized in Pinctada fucata martensii. The expression of PmTrxlp-5 was detected in response to polyinosinic: polycytidylic acid (poly I:C) and lipopolysaccharides (LPS) stimulation. The differences in PmTrxlp-5 expression were evaluated between the black coloured selected line and the control stock after grafting operation. The open reading frame (ORF) consisted of 1167bp encoding a 388 amino acid, 5'-UTR of 41bp and a 3'-UTR of 846bp. PmTrxlp-5 exhibited a conserved WCXXC functional motif similar to thioredoxins from other species. Tissue analysis showcased the highest relative mRNA expressions of PmTrxlp-5 in the haemocytes. Interestingly, after the grafting operation, mRNA expression of PmTrxlp-5 in the haemocytes was differentially expressed post grafting with a peak 6 h after grafting suggesting the high involvement of the gene in immune response in the early stage after grafting. The black coloured selected line group (BS) had significantly higher expression than the control group (CG) at 24 h, 6 d and 30 d after grafting operation. PmTrxlp-5 also showed a wave-like pattern in mRNA expression after bacterial endotoxin LPS and viral mimic poly I:C. These results suggested that PmTrxlp-5 plays a vital function in cellular redox homeostasis and immune response against grafting operation and pathogenic infections and can be used as a gene marker for selective breeding programs.

Survival, retention rate and immunity of the black shell colored stocks of pearl oyster Pinctada fucata martensii after grafting operation.

We have developed a black shell colored selected line observed to have higher survival ability. In this study, to understand its immune capacity, total carotenoid content (TCC) of the black shell colored line (BG) and the control group (CG) were compared. Survival and retention rates, immunity and antioxidant capacity of BG were compared relative to CG at different times after grafting operation. The results showed that BG had significantly larger TCC than CG (P < 0.05). BG had significantly higher survival and retention rates than CG on days 7, 30 and 360 after grafting (P < 0.05). On days 360, BG had significantly larger pearl thickness than CG (P < 0.05). BG exhibited increased ACP, AKP, SOD, CAT, TAOC and LZ activity than the CG on 0 h, 12 h, 1 d, 3 d, 5 d, 7 d and 30 d after grafting. BG had higher expression levels of Fascin, SOD, CDK-7, CDAP-1, IRAK-1, α2m, GST-1, TRAF-3 and Caspase-2 than CG. The results suggested that BG had higher immune competence and pearl production performances, which is promising to improve pearl quality and production.