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Effects Of Hypoxia And 4-Tert-Octylphenol On Gene Expression Profiles Of The Sheepshead Minnow (Cyprinodon Variegatus), Arthur Alan Karels
Effects Of Hypoxia And 4-Tert-Octylphenol On Gene Expression Profiles Of The Sheepshead Minnow (Cyprinodon Variegatus), Arthur Alan Karels
Dissertations
Hypoxia occurs in estuaries of northern Gulf of Mexico and world-wide, with increasing frequency/severity via eutrophication and anthropogenic influences. Hypoxia inducible factors (HIFs) form transcriptional complex and bind DNA at hypoxia responsive elements (HREs) in promoter regions of genes needed for systemic and cellular adaptation of fish to low dissolved oxygen (hypoxia, DO <2.0 mg/ml). Hypoxia-induced activation of HIF-αs can lead to a cascade of downstream activation, such as erythropoietin (EPO). Return to normal DO levels (normoxia), prolyl hydroxylases (PHDs) are activated to degrade HIF-αs back to baseline. Fish are affected by environmental estrogen mimics, like 4-tert-octylphenol (4tOP), binding estrogen receptor alpha (ERα) at estrogen responsive elements (EREs) and activating genes vitellogenin (VTG). Previous research showed overlap or crosstalk between these two mechanistic pathways. Hypoxia triggers unknown factors regulating ERE-mediated ERα signaling pathway, and stressor combinations could increase/decrease hypoxic or endocrine pathway. Research examined molecular/physiological effects of hypoxia (acute and chronic, moderate and severe) and 4tOP (~60μg/L)on adult male and/or female sheepshead minnow (Cyprinodon variegatus). Three genes identified, cloned, and sequenced (HIF-1α, HIF-2α, and PHD3), plus previously identified genes EPO and VTG, were examined in liver/testes exposed to hypoxia and/or 4tOP for cellular/physiological changes. Endpoints examined included mRNA expression from real-time PCR of HIF-1α, HIF-2α, PHD3, EPO, and VTG using cDNA from total RNA extracts, and microarray analyses of genes expressed during the transition from hypoxia back to normoxia. Phylogenetic analyses confirmed isolation of two HIF-α isofoms (HIF-1α and HIF-2α) and the PHD3 isoform. Significant up-regulation of PHD3 occurred within 10 hrs of chronic hypoxia, and persisted when severe (1.5 mg/L) and declined when moderate (~2.5mg/L). Significant up-regulation of HIF-1α and EPO occurred within 30 minutes to 2 hours of onset of acute severe and very severe (~1.08mg/L) hypoxia. Hypoxia acted similar to an estrogen mimic, with huge up-regulation of VTG gene expression in males, and increased VTG levels (additive effect) when hypoxia was combined with 4tOP. Microarray analyses showed 125 genes with significant transcriptional change, with up- or down-regulation from transitions of: (1) hypoxia (72 hrs) to normoxia (74 hrs) and (2) hypoxia+4tOP (72 hrs) to normoxia+4tOP (74 hrs).
Gene Expression Profiles As Molecular Indicators Of Dissolved Oxygen Stress In Grass Shrimp Palaemonetes Pugio Holthuis 1949, Tiandao Li
Dissertations
Occurrence and severity of hypoxia is increasing in coastal and estuarine environments, and recovery of impacted habitats and living resources is slow. Detection of early biological effects of hypoxia is needed for timely remedial action to be taken. The overall objectives of this research was to develop molecular indicators of dissolved oxygen stress to assess the biological impact of hypoxia in coastal estuaries and validate their use through a combination of laboratory and field studies. To achieve these goals, grass shrimp, Palaemonetes pugio, oxygen-sensitive and hypoxia-tolerant species abundant in estuarine systems, were exposed to hypoxia under controlled laboratory conditions, and …