Mitochondrial physiology of diapausing and developing embryos of the annual killifish Austrofundulus limnaeus: implications for extreme anoxia tolerance

Abstract

Diapausing embryos of the annual killifish Austrofundulus limnaeus have the highest reported anoxia tolerance of any vertebrate and previous studies indicate modified mitochondrial physiology likely supports anoxic metabolism. Functional mitochondria isolated from diapausing and developing embryos of the annual killifish exhibited VO(2), respiratory control ratios (RCR), and P:O ratios consistent with those obtained from other ectothermic vertebrate species. Reduced oxygen consumption associated with dormancy in whole animal respiration rates are correlated with maximal respiration rates of mitochondria isolated from diapausing versus developing embryos. P:O ratios for developing embryos were similar to those obtained from adult liver, but were diminished in mitochondria from diapausing embryos suggesting decreased oxidative efficiency. Proton leak in adult liver corresponded with that of developing embryos but was elevated in mitochondria isolated from diapausing embryos. In metabolically suppressed diapause II embryos, over 95% of the mitochondrial oxygen consumption is accounted for by proton leak across the inner mitochondrial membrane. Decreased activity of mitochondrial respiratory chain complexes correlates with diminished oxidative capacity of isolated mitochondria, especially during diapause. Respiratory complexes exhibited suppressed activity in mitochondria with the ATP synthase exhibiting the greatest inhibition during diapause II. Mitochondria isolated from diapause II embryos are not poised to produce ATP, but rather to shuttle carbon and electrons through the Kreb's cycle while minimizing the generation of a proton motive force. This particular mitochondrial physiology is likely a mechanism to avoid production of reactive oxygen species during large-scale changes in flux through oxidative phosphorylation pathways associated with metabolic transitions into and out of dormancy and anoxia.

Anoxia Tolerance During Vertebrate Development - Insights from Studies on the Annual Killifish Austrofundulus limnaeus

“…In fact, citrate synthase activity is low throughout most of embryonic development in this species, especially in those developmental stages that exhibit the greatest tolerance of anoxia. This low level of citrate synthase activity is consistent with what is known about mitochondrial physiology in this species (see below, Duerr and Podrabsky, 2010). Calorimetric:respirometric (C:R) ratios can be used as an indicator of the contribution of anaerobic metabolism to total heat production.…”

“…The dependence of anoxia tolerant embryos on anaerobic metabolism even under aerobic conditions is supported by an extremely low activity of mitochondrial electron transport chain complexes and the almost complete absence of ATP synthase activity in mitochondria isolated from diapause II embryos ( Fig. 8; Duerr and Podrabsky, 2010). In fact, the extremely low rates of oxygen consumption exhibited by mitochondria isolated from diapausing embryos can be attributed almost exclusively to proton leak (Duerr and Podrabsky, 2010).…”

“…8; Duerr and Podrabsky, 2010). In fact, the extremely low rates of oxygen consumption exhibited by mitochondria isolated from diapausing embryos can be attributed almost exclusively to proton leak (Duerr and Podrabsky, 2010). This apparent waste of metabolic fuel to maintain proton leak and therefore some small level of oxidative metabolism appears paradoxical.…”

“…Diapausing embryos have reduced activity of enzyme complexes involved in mitochondrial electron transport, including extremely low levels of ATP synthase (complex V) activity. DII = diapause II, DIII = diapause III, post-DII embryos are WS 39, larvae are 1-2 days post-hatch, adult data are for isolated liver mitochondria (Duerr and Podrabsky, 2010).…”

Cell cycle regulation during development and dormancy in embryos of the annual killifishAustrofundulus limnaeus

“…2). 23 The metabolic capacity of diapause II embryos is highly biased toward anaerobic metabolic pathways as indicated by a 50-fold greater capacity for lactate dehydrogenase activity compared with citrate synthase activity. 24 Despite low mitochondrial activity and a low metabolic rate, cellular ATP levels remain high, and ATP/ADP ratios and adenylate energy charge indicate a very positive cellular energetic status with respect to phosphorylated adenylates.…”