Oxygen deprivation is an environmental condition organisms may encounter in their natural habitat, thus mechanisms evolved to respond to and survive oxygen deprivation. Hypoxia and anoxia are both terms used to describe oxygen deprivation. Hypoxia has been defined in several ways including: 1. When O2 deprivation limits electron transport, 2. A state of reduced O2 availability or decreased oxygen partial pressures (pO2), 3. When a decrease in O2 results in an abolishment or reduction of functions in organs, tissues or cells. Anoxia is sometimes referred to as a state of "severe hypoxia" yet the term anoxia typically describes the absence of detectable O2 in either the tissue or the environment that an organism is exposed to [1–3].
In regards to human health, oxygen deprivation is central to the pathology of several diseases including myocardial infarction, pulmonary disease, and solid tumor progression. Oxygen deprivation can also cause severe cellular damage as a result of trauma due to blood loss, suffocation or drowning. Thus, it is of interest to identify the molecular responses to oxygen deprivation. Several model systems are used to understand the physiological response organisms have to oxygen deprivation [4, 5]. For example, anoxia tolerant organisms are capable of decreasing energy usage by stopping non-essential cellular functions, maintain stable and low permeability of membranes, and produce ATP by glycolysis . However, the sub-cellular response to oxygen deprivation, in developing embryos, is less understood.
Oxygen deprivation influences the growth, development, and behavior of the soil nematode Caenorhabditis elegans. For example, C. elegans exposed to anoxia (<.001 kPa O2) in laboratory culture conditions displays the remarkable characteristic of suspended animation in which embryonic development and cell cycle progression arrests and post-embryonic nematodes arrest development, feeding, movement, and in the case of adults, do not lay eggs [7, 8]. These arrested biological processes in the nematode resume upon re-exposure to normoxia. Several organisms are capable of arresting embryonic development and cell cycle progression in response to oxygen deprivation [9–11]. Blastomeres of C. elegans and D. melanogaster embryos exposed to anoxia arrest during interphase, some stages of mitosis, predominately prophase and metaphase, but not anaphase [7, 10]. D. melanogaster embryos exposed to hypoxia arrest in interphase and the metaphase stage of mitosis [12–14]. In comparison, blastomeres of zebrafish embryos exposed to anoxia arrest during interphase . Analysis of interphase blastomeres of C. elegans, zebrafish and Drosophila embryos exposed to anoxia indicates that the chromatin appears condensed and is not uniformly distributed throughout the nucleus [7, 10, 11]. Thus, not only is the phenomena of anoxia-induced suspended animation conserved but some of the cellular responses and mechanisms involved with suspended animation are evolutionarily conserved.
The use of genetic model systems has increased our understanding of the mechanisms regulating oxygen deprivation sensing and survival [15–20]. For example, in C. elegans, an RNA interference (RNAi) genomic screen provided evidence that the spindle checkpoint proteins, SAN-1 and MDF-2, homologous to the yeast MAD3 and MAD2 spindle checkpoint proteins respectively, are active in the early embryo and are required for anoxia-induced suspended animation [21–23]. Additionally, the Drosophila spindle checkpoint protein Mps1, is required for hypoxia-induced arrest . Thus, the oxygen-deprivation induced cell cycle arrest by the spindle checkpoint proteins is likely a conserved process. The spindle checkpoint components monitor kinetochore-microtubule attachment and tension and delay the metaphase to anaphase transition until chromatids are adequately attached to microtubules [25–27]. The requirement of spindle checkpoint proteins for oxygen deprivation arrest supports the idea that an oxygen-sensing pathway regulates the spindle checkpoint components, however the mechanism for such a pathway is not understood.
Analysis of the cellular structures in blastomeres of embryos deprived of oxygen will provide insight into how oxygen-deprivation tolerant organisms, such as C. elegans, respond to and survive oxygen deprivation. The C. elegans embryo exposed to 24-hours of anoxia displays specific distinguishing features such as dephosphorylation of cell cycle regulated proteins including histone H3 and proteins recognized by the MPM-2 antibody, kinetochore rearrangements, and astral microtubule depolymerization [7, 28]. In this report, we expand the analysis of the sub-cellular changes in C. elegans embryos exposed to anoxia for various periods of time. The cellular structures examined include: nuclear localization of chromatin, phosphorylation of histone H3, spindle microtubule structure, and SAN-1 localization pattern. Also, we demonstrate that the cellular-structures differ depending upon anoxia exposure time. We suggest that microtubule and SAN-1 alterations are involved with the arrest of metaphase blastomeres, because we find that in embryos exposed to brief periods of anoxia the spindle microtubules depolymerize, SAN-1 localization to the kinetochore is altered and SAN-1 is required for brief periods of anoxia. The work presented here raises the idea that analysis of cellular structures can be used as hallmarks of oxygen deprivation exposure time. Moreover, identifying the temporal order of cellular changes in response to anoxia yields a framework to differentiate the specific mechanisms required for the establishment or maintenance of anoxia-induced suspended animation.