Digital Commons @ George Fox University Faculty Publications - Department of Biology and Department of Biology and Chemistry Chemistry 2010 Mitochondrial Physiology of Diapausing and Developing Embryos of the Annual Killifsh Austrofundulus Limnaeus: Implications for Extreme Anoxia Tolerance Jeff Duerr George Fox University, [email protected] Jason E. Podrabsky Portland State University Follow this and additional works at: http://digitalcommons.georgefox.edu/bio_fac Part of the Biology Commons, Cell and Developmental Biology Commons, and the Physiology Commons Recommended Citation Previously published in Journal of Comparative Physiology B, 2010, 180(7), pp. 991-1003 http://link.springer.com/article/10.1007/ s00360-010-0478-6 This Article is brought to you for free and open access by the Department of Biology and Chemistry at Digital Commons @ George Fox University. It has been accepted for inclusion in Faculty Publications - Department of Biology and Chemistry by an authorized administrator of Digital Commons @ George Fox University. Mitochondrial physiology of diapausing and developing embryos of the annual killiWsh Austrofundulus limnaeus: implications for extreme anoxia tolerance JeVrey M. Duerr · Jason E. Podrabsky Abstract Diapausing embryos of the annual killiWsh with the ATP synthase exhibiting the greatest inhibition Austrofundulus limnaeus have the highest reported anoxia during diapause II. Mitochondria isolated from diapause II tolerance of any vertebrate and previous studies indicate embryos are not poised to produce ATP, but rather to shut- modiWed mitochondrial physiology likely supports anoxic tle carbon and electrons through the Kreb’s cycle while metabolism. Functional mitochondria isolated from diapa- minimizing the generation of a proton motive force. This using and developing embryos of the annual killiWsh exhib- particular mitochondrial physiology is likely a mechanism ited VO2, respiratory control ratios (RCR), and P:O ratios to avoid production of reactive oxygen species during consistent with those obtained from other ectothermic ver- large-scale changes in Xux through oxidative phosphoryla- tebrate species. Reduced oxygen consumption associated tion pathways associated with metabolic transitions into with dormancy in whole animal respiration rates are corre- and out of dormancy and anoxia. lated with maximal respiration rates of mitochondria isolated from diapausing versus developing embryos. P:O Keywords ATP synthase · Metabolic depression · ratios for developing embryos were similar to those Diapause · Anoxia obtained from adult liver, but were diminished in mito- chondria from diapausing embryos suggesting decreased oxidative eYciency. Proton leak in adult liver corresponded Introduction with that of developing embryos but was elevated in mito- chondria isolated from diapausing embryos. In metaboli- The embryo of the annual killiWsh Austrofundulus limnaeus cally suppressed diapause II embryos, over 95% of the represents the most anoxia-tolerant vertebrate and can sur- mitochondrial oxygen consumption is accounted for by vive for months in the complete absence of oxygen at 25°C proton leak across the inner mitochondrial membrane. (Podrabsky et al. 2007). This level of anoxia tolerance is Decreased activity of mitochondrial respiratory chain com- two orders of magnitude higher than other anoxia-tolerant plexes correlates with diminished oxidative capacity of iso- vertebrates and suggests new mechanisms for supporting lated mitochondria, especially during diapause. Respiratory anoxia tolerance in this species. Survival of anoxia is a nat- complexes exhibited suppressed activity in mitochondria ural part of the life of A. limnaeus embryos and is associ- ated with entry of the embryos into diapause, a pre-emptive form of metabolic dormancy that is a natural part of their developmental program. Diapause represents a form of intrinsic metabolic depression since its onset is not trig- J. M. Duerr (&) Department of Biology, George Fox University, gered by environment cues. Entry into diapause is associ- Newberg, OR 97132, USA ated with a severe metabolic depression as estimated by e-mail: [email protected] both oxygen consumption and heat dissipation (Podrabsky and Hand 1999). In addition, calorimetric:respirometric J. E. Podrabsky Department of Biology, Portland State University, ratios are elevated during diapause suggesting a signiWcant P.O. Box 751, Portland, OR 97207, USA contribution of anaerobic metabolism to overall heat dissipation even under aerobic conditions. Thus, it appears diapause II embryos of A. limnaeus, which maintain high that the metabolic changes associated with dormancy [ATP]/[ADP] ratios and adenylate energy charge in the during diapause may also serve to prepare embryos for face of over a 90% reduction in oxygen consumption subsequent exposures to anoxia. The mechanisms that sup- (Podrabsky and Hand 1999). The extent to which control of press oxidative metabolism during diapause are currently mitochondrial metabolism may be implicated in supporting unknown and led us to investigate the physiology of mito- metabolic depression during diapause has yet to be chondria isolated from developing and diapausing embryos explored in A. limnaeus. of A. limnaeus. During the past decade, both overwintering frogs (Rana) There are a maximum of three distinct diapause phases and estivating snails (Helix) have been employed to investi- that may occur in annual killiWsh, only two of which occur gate mitochondrial respiration during metabolic depression. in embryos of A. limnaeus, termed diapause II and III In frogs, isolated skeletal muscle mitochondria exhibited (Wourms 1972a, b). Diapause II is developmentally pre- decreases in rates of oxygen consumption and proton leak dictable in A. limnaeus and occurs in embryos possessing during overwintering (Boutilier and St-Pierre 2002). In the 38–40 pairs of somites, a beating heart and the foundations snail, respiration rates of whole hepatopancreas cells were of a central nervous system; 80% or more of the embryos measured (Bishop and Brand 2000). In control cells, non- will enter diapause II after 24–26 days of development at mitochondrial oxygen consumption accounted for 45% and 25°C. Diapause III is obligate for most embryos and occurs mitochondrial 55% of total oxygen consumption; in cells just prior to hatching when the embryo is fully developed. from estivating snails (overall metabolism 33% of normal), This stage of diapause is generally compared with aestiva- non-mitochondrial and mitochondrial respiration each tion in frogs. Diapause II embryos are tolerant of long-term accounted for 50% of the total respiration rate. In both anoxia, while diapause III embryos are not (Podrabsky cases, the strong depression of metabolism reported was the et al. 2007). Importantly, the extreme anoxia tolerance result of a concomitant decrease in rates of both oxygen observed in diapause II embryos is retained for at least consumption and proton leak. Suppression of mitochondrial 4 days of post-diapause II development, but is lost after respiration may be achieved in three ways: (1) a decrease in 8 days of post-diapause II development (Podrabsky et al. the area of the imm, (2) a decrease in substrate oxidation 2007). Thus, in this species extreme anoxia tolerance is and a subsequent decline in the pmf, and (3) a decrease in gained as embryos develop toward and enter diapause II, the proton conductance of the imm. Data collected in anu- and then lost by the time embryological development is ran amphibians suggest that option 2 is the case: a decrease complete. in the reactions that catalyze substrate oxidation was mea- Two main strategies for metabolic suppression in sured in Bufo (Trzcionka et al. 2008) and Rana (St-Pierre anoxia-tolerant animals have been proposed: (1) a reduc- et al. 2000a, b). However, it is worth noting that several tion in ATP turnover and (2) improved ATP yield of anaer- investigations have found very little diVerence between obic metabolism (Hochachka and Somero 2002; Storey and proton leak rates in mitochondria isolated from dormant Hochachka 1974). ATP-consuming reactions in the cell can versus active animals. For example, mitochondria isolated be divided into two groups. The Wrst comprises 70% of from Artemia embryos during diapause exhibit similar total respiration and includes powering the Na+/K+-ATPase respiratory and proton leak rates despite a 97% reduction in on the plasma membrane, protein synthesis, mRNA synthe- metabolism (Reynolds and Hand 2004). sis and Ca2+ cycling. A large proportion of the remaining Our investigation focuses on the bioenergetic status of 30% is spent on proton leak at the inner mitochondrial mitochondria isolated from diapausing embryos of the membrane (imm) (Rolfe and Brand 1996). Proton leak annual killiWsh A. limnaeus. We isolated intact mitochon- occurs as a result of limited permeability of the imm to pro- dria from six developmental stages corresponding to peri- tons and the proton motive force (pmf) that exists across ods of signiWcant change in whole animal metabolism and that membrane in energized mitochondria. Pathways that morphology: 10 days post-fertilization (dpf), diapause II, require oxygen (respiration) can also be divided into two post-diapause II, diapause III, larvae, and adults (liver). We groups: mitochondrial (ATP-producing) and non-mito- measured oxygen consumption rates, phosphate:oxygen chondrial (which includes oxygen-dependent, non-phos- ratios, proton leak rates,