Expression of Prolyl Hydroxylase Domains (PHDs), the upstream regulator of HIF, in the Brain of the Anoxia-Tolerant Crucian Carp during Anoxia-Reoxygenation.

The hypoxia-inducible factor (HIF) is considered key in the transcriptional response to low oxygen. Yet, HIF's role in absence of oxygen (anoxia) and in preparation for reoxygenation remains unclear. Recent studies suggest that mounting a HIF response may be counterproductive for anoxia survival. We here studied the champion of anoxia survival, the crucian carp ( Carassius carassius ), and hypothesized that expression of prolyl hydroxylase domains, PHDs (the upstream regulators of HIF), are upregulated to circumvent an energy-costly activation of HIF in anoxia and to prepare for reoxygenation. We measured whole-brain mRNA and protein levels of the three isoforms PHD1,2 and 3, coded for by multiple paralogs of the genes egln2 , 1 and 3 , using qPCR and Western blotting in brain of crucian carps exposed to 5 days normoxia or anoxia, and 5 days anoxia followed by 3- or 24-hours reoxygenation. The mRNA level of most egln paralogs were increased in anoxia and upon reoxygenation, with egln3 showing the largest increase in mRNA level (up to 20-fold) and highest relative mRNA abundance (up to 75% of expressed eglns ). The protein level of all PHDs was maintained in anoxia and increased upon reoxygenation. We then explored PHDs distribution in different brain regions and found PHDs-immunoreactivity to be associated with axonal branches and showing region-specific changes during anoxia-reoxygenation. Our results support an overall upregulation of eglns under prolonged anoxia and PHDs upon reoxygenation in crucian carp, likely aimed at suppressing HIF responses, although regional differences are apparent in such a complex organ as the brain.