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

Embryos of the annual killifish Austrofundulus limnaeus can enter into a state of metabolic dormancy, termed diapause, as a normal part of their development. In addition, these embryos can also survive for prolonged sojourns in the complete absence of oxygen. Dormant embryos support their metabolism using anaerobic metabolic pathways, regardless of oxygen availability. Dormancy in diapause is associated with high ATP and a positive cellular energy status, while anoxia causes a severe reduction in ATP content and large reductions in adenylate energy charge and ATP/ADP ratios. Most cells are arrested in the G₁/G₀ phase of the cell cycle during diapause and in response to oxygen deprivation. In this paper, we review what is known about the physiological and biochemical mechanisms that support metabolic dormancy in this species. We also highlight the great potential that this model holds for identifying novel therapies for human diseases such as heart attack, stroke and cancer.     

Depression of protein synthesis during diapause in embryos of the annual killifish Austrofundulus limnaeus

Rates of protein synthesis are substantially depressed in diapause II embryos of Austrofundulus limnaeus. Inhibition of oxygen consumption and heat dissipation with cycloheximide indicates that 36% of the adenosine triphosphate (ATP) turnover in prediapausing embryos (8 d postfertilization [dpf]) is caused by protein synthesis; the contribution of protein synthesis to ATP turnover in diapause II embryos is negligible. In agreement with the metabolic data, incorporation of amino acids (radiolabeled via (14)CO(2)) into perchloric acid-precipitable protein decreases by over 93% in diapause II embryos compared with embryos at 8 dpf. This result represents a 36% reduction in energy demand because of depression of protein synthesis during diapause. Adjusting for changes in the specific radioactivity of the free amino acid pool at the whole-embryo level yields rates of protein synthesis that are artifactually high and not supportable by the observed rates of oxygen consumption and heat dissipation during diapause. This result indicates a regionalized distribution of labeled amino acids likely dictated by a pattern of anterior to posterior cell cycle arrest. AMP/ATP ratios are strongly correlated with the decrease in rates of protein synthesis, which suggests a role for adenosine monophosphate (AMP) in the control of anabolic processes. The major depression of protein synthesis during diapause II affords a considerable reduction in energy demand and extends the duration of dormancy attainable in these embryos.     

Cell cycle arrest associated with anoxia-induced quiescence, anoxic preconditioning, and embryonic diapause in embryos of the annual killifish Austrofundulus limnaeus

Embryos of the annual killifish Austrofundulus limnaeus can enter into dormancy associated with diapause and anoxia-induced quiescence. Dormant embryos are composed primarily of cells arrested in the G(1)/G(0) phase of the cell cycle based on flow cytometry analysis of DNA content. In fact, most cells in developing embryos contain only a diploid complement of DNA, with very few cells found in the S, G(2), or M phases of the cell cycle. Diapause II embryos appear to be in a G(0)-like state with low levels of cyclin D1 and p53. However, the active form of pAKT is high during diapause II. Exposure to anoxia causes an increase in cyclin D1 and p53 expression in diapause II embryos, suggesting a possible re-entry into the cell cycle. Post-diapause II embryos exposed to anoxia or anoxic preconditioning have stable levels of cyclin D1 and stable or reduced levels of p53. The amount of pAKT is severely reduced in 12?dpd embryos exposed to anoxia or anoxic preconditioning. This study is the first to evaluate cell cycle control in embryos of A. limnaeus during embryonic diapause and in response to anoxia and builds a foundation for future research on the role of cell cycle arrest in supporting vertebrate dormancy.     

Patterns of ubiquitylation and SUMOylation associated with exposure to anoxia in embryos of the annual killifish Austrofundulus limnaeus

Embryos of the annual killifish Austrofundulus limnaeus acquire extreme tolerance to anoxia during embryonic development. These embryos can survive environmental and cellular conditions that would likely result in death in the majority of vertebrate cells, despite experiencing a massive loss of ATP. It is highly likely that the initial response to anoxia must quickly alter cellular physiology to reprogram cell signaling and metabolic pathways to support anaerobiosis. Covalent protein modifications are a mechanism that can quickly act to effect large-scale changes in protein structure and function and have been suggested by others to play a key role in mammalian ischemia tolerance. Using Western blot analysis, we explored patterns of protein ubiquitylation and SUMOylation in embryos of A. limnaeus exposed to anoxia and anoxic preconditioning. Surprisingly, we report stage-specific protein ubiquitylation patterns that suggest different mechanisms for altering protein turnover in dormant and actively developing embryos that both survive long-term anoxia. Anoxic preconditioning does not appear to alter levels of ubiquitin conjugates in a unique manner. Global SUMOylation of proteins does not change in response to anoxia, but there are stage-specific changes in SUMOylation of specific protein bands. Contrary to other systems, global changes in protein SUMOylation may not be required to support long-term tolerance to anoxia in embryos of A. limnaeus. These data lead us to conclude that embryos of A. limnaeus respond to anoxia in a unique manner compared to other vertebrate models of anoxia tolerance and may provide novel mechanisms for engineering vertebrate tissues to survive long-term anoxia.     

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

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 VO₂, 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.     

An inducible 70 kDa-class heat shock protein is constitutively expressed during early development and diapause in the annual killifish Austrofundulus limnaeus

The annual killifish Austrofundulus limnaeus inhabits ephemeral ponds in regions of northern South America, where they survive the periodic drying of their habitat as diapausing embryos. These diapausing embryos are highly resistant to a number of environmental insults such as high temperature, dehydration, anoxia, and increased salinity. Molecular chaperones are known to play a role in stabilizing protein structure and function during events of cellular stress. Relative levels of heat shock protein (Hsp)70 were measured in developing and diapausing embryos of A. limnaeus using quantitative Western blots. An inducible or embryo-specific form of Hsp70 is expressed during embryonic development in A. limnaeus and is elevated during diapause II in this species. Constitutive expression of Hsp70 during development may afford these embryos protection from environmental stresses during development more quickly than relying on the induction of a classic heat shock response.     

The effects of hypoxia and temperature on metabolic aspects of embryonic development in the annual killifish Austrofundulus limnaeus

Embryos of Austrofundulus limnaeus are exceptional in their ability to tolerate prolonged bouts of complete anoxia. Hypoxia and anoxia are a normal part of their developmental environment. Here, we exposed embryos to a range of PO₂ levels at two different temperatures (25 and 30 °C) to study the combined effects of reduced oxygen and increased temperature on developmental rate, heart rate, and metabolic enzyme capacity. Hypoxia decreased overall developmental rate and caused a stage-specific decline in heart rate. However, the rate of early development prior to the onset of organogenesis is insensitive to PO₂. Increased incubation temperature caused an increase in the developmental rate at high PO₂s, but hindered developmental progression under severe hypoxia. Embryonic DNA content in pre-hatching embryos was positively correlated with PO₂. Citrate synthase, lactate dehydrogenase, and phosphoenolpyruvate carboxykinase capacity were all reduced in embryos developing under hypoxic conditions. Embryos of A. limnaeus are able to develop normally across a wide range of PO₂s and contrary to most other vertebrates severe hypoxia is not a teratogen. Embryos of A. limnaeus do not respond to hypoxia through an increase in the capacity for enzymatic activity of the metabolic enzymes lactate dehydrogenase, citrate synthase, or phosphoenolpyruvate carboxykinase. Instead they appear to adjust whole-embryo metabolic capacity to match oxygen availability. However, decreased DNA content in hypoxia-reared embryos suggests that cellular enzymatic capacity may remain unchanged in response to hypoxia, and the reduced capacity may rather indicate reduced cell number in hypoxic embryos.     

Temperature preference and reproductive fitness of the annual killifish Austrofundulus limnaeus exposed to constant and fluctuating temperatures

Austrofundulus limnaeus thrive in ephemeral ponds that may experience temperatures spanning a range of over 20°C on a daily basis. We hypothesized that A. limnaeus may have mechanisms, either behavioral or physiological, that allow them to support successful reproduction in this environment. To evaluate this hypothesis, we exposed male and female adult A. limnaeus to constant 26°C and cycling 21–37°C acclimation regimes in the laboratory and then determined their temperature preference and reproductive fitness. Temperature preference was determined using a thermal gradient. We demonstrated that A. limnaeus is capable of accurate behavioral thermoregulation, has a final thermal preferendum near 26°C, and exhibits a daily cycle of temperature preference. Exposure to a cycling temperature regime has an acute effect on thermal preference that differs between the sexes. Reproductive capability was negatively affected by the cyclic temperature exposure. These findings suggest that thermal partitioning between males and females may be a natural part of the ecology of A. limnaeus. In addition, it appears that behavioral thermoregulation, or partitioning of reproductive events to the cool parts of the thermoperiod, are likely to be critical to support successful reproduction in natural populations of A. limnaeus.     

Differential effects of anoxia on heart rate in developmental stages of the annual killifish Austrofundulus limnaeus that differ in their tolerance of anoxia

Embryos of the annual killifish Austrofundulus limnaeus can experience oxygen deprivation as part of their normal developmental environment. We exposed embryos to anoxia and monitored heart activity for 48 hr, and subsequent aerobic recovery from anoxia for 40 hr. Embryos were tested at four different developmental stages that differ in their tolerance of anoxia. Our results indicate that high tolerance of anoxia is associated with an arrest of heart contractility during the first 24 hr of anoxia. These embryos recover to normoxic levels of heart rate within 16 hr of aerobic recovery. In contrast, embryos from later developmental stages that have a highly reduced ability to survive long-term anoxia experience a severe bradycardia but not an arrest of heart rate. These data illustrate a new and potentially powerful model for investigating the effects of anoxia on the developing cardiovascular system in vertebrates.     

Husbandry of the Annual Killifish Austrofundulus limnaeus with Special Emphasis on the Collection and Rearing of Embryos

Annual killifish development is unique compared to other teleosts and is characterized by the dispersion and subsequent reaggregation of pre-embryonic blastomeres and the occurrence of embryonic diapause. Austrofundulus limnaeus is an excellent species to use for studies of development and embryonic diapause in annual killifish. A. limnaeus has a high fecundity, reproduces readily in a laboratory environment, and has a relatively long laboratory life span compared to many other species of annual killifish. Methods are presented for rearing A. limnaeus in the laboratory with an emphasis on collecting and incubating large numbers of embryos for biochemical and physiological studies. Females produce an average of 29 eggs during a two to four hour spawning. Egg quality (% fertilization and survival) and egg production (eggs female^-1) are affected by the number of days between spawning events. Percent fertilization of eggs and survival of embryos decreases as the interval between spawning increases from two to eight days. The number of fertile embryos produced per female remains relatively constant as a function of spawning interval. Fertilization rates may be maintained at high levels by replacing aged males (1.5 years old) with younger males. An embryo medium was formulated to mimic the natural waters inhabited by A. limnaeus. The developmental rate and survival of embryos in the embryo medium was essentially equivalent when compared to Yamamoto's fish saline solution.     

The fate of mosaic aneuploid embryos during mouse development

More than any other species, humans have difficulty reproducing. As recent studies show that human infertility is ever increasing, much efforts are needed towards the understanding of our low fecundity. While aneuploidy is the leading cause of spontaneous pregnancy loss in humans, we still know surprisingly little about the developmental consequences of chromosomal abnormalities. We have here used a mouse model that spontaneously incites chromosomal primary aneuploidy in female haploid oocytes and find that after fertilization, these primary aneuploid cells become cytological unstable, generating diverse karyotypic mosaic embryos. The mosaic aneuploid embryos can develop and implant into the female uterine tissue and initiate the gastrulation process (E6.5) but quickly degrade and succumb by E8.0. We find that loss of embryo viability due to chromosomal mosaicism is caused by the activation of a spatially and temporally controlled p53-independent apoptotic mechanism and does not result from a failure to progress through mitosis. We conclude that an initial state of primary aneuploidy within an embryo results in a rapid evolution of mosaicism and early embryonic death. This gestational loss due to aneuploid mosaicism could account for the large proportion of human pregnancy loss prior to clinical recognition.     

Control of the annual testicular cycle of the marbled-newt by p53, p21, and Rb gene products

p53, p21, and Rb are proteins with an important role in cell-cycle control. The expression and distribution of these gene products and the apoptotic rate were studied in the marbled-newt testis along the annual cycle to know the role of these factors in the control of spermatogenesis and glandular tissue formation. The study was carried out using Western blot analysis and immunohistochemistry. The results differed from those, previously reported in mammals showing constant spermatogenesis. Greater expression of p53 and p21 was found in the quiescence period and was detected in PCGs (primordial germ cells), spermatogonia, follicular, interstitial cells, and glandular tissue. Greater expression of Rb and phosph-Rb was present in the proliferation period, in PCGs, and spermatogonia. Apoptosis was only detected in secondary spermatogonia (quiescence and spermiogenesis periods) and primary spermatocytes (proliferation and spermiogenesis periods). In the quiescence period, the increase in p53 expression activates p21 expression, which inhibits Rb phosphorylation and arrests the cell cycle in G1. In the proliferation period and, in a lesser degree, in the spermiogenesis period, the expressions of p53 and p21 decrease and phosph-Rb increases, enhancing cell proliferation. These gene products do not seem to be related to apoptosis.     

The regulation of APAF1 expression during development and tumourigenesis

Apoptosis Protease-Activating Factor 1, APAF1, was originally isolated four years ago and shown to be the mammalian homologue of the C. elegans pro-apoptotic ced4 gene. Since then, the expression of APAF1 has been demonstrated to be involved in several cell death pathways, including the induction of apoptosis by the p53 tumour suppressor protein and neuronal apoptosis. In this review we will focus on the regulation of APAF1 expression, in particular with regard to recent developments in our understanding of the role of APAF1 in both tumourigenesis and mammalian development.     

葡萄休眠的自然诱导因子及其对休眠诱导期冬芽呼吸代谢的调控

以高需冷量葡萄品种‘夏黑’为试材,研究短日照、长日照和自然光照3个条件下,葡萄冬芽休眠的自然诱导因子及其对休眠诱导期冬芽呼吸代谢的调控机制.结果表明: 自然低温、短日照2个环境因素单独或共同作用均能诱导葡萄冬芽进行自然休眠.短日照在诱导葡萄冬芽进入自然休眠的过程中起主导作用,自然低温起辅助作用;温度相同条件下,日照时间越短对葡萄冬芽自然休眠的诱导作用越强.总呼吸速率达到峰值是葡萄冬芽休眠诱导期结束的标志.在自然休眠诱导期间,葡萄冬芽磷酸戊糖途径运行活性和容量占总呼吸的比例迅速上升,其中自然条件的葡萄冬芽分别由16.0%和20.1%上升至22.3%和26.0%.自然低温是诱导葡萄冬芽底物氧化水平上呼吸途径发生变化的主导因素,短日照起促进作用.在葡萄冬芽自然休眠诱导期间,交替途径运行活性和容量占总呼吸的比例迅速上升,其中自然条件葡萄冬芽分别由19.4%和27.3%上升至38.2%和46.8%.自然低温和短日照均可诱导葡萄冬芽电子传递链水平上呼吸途径发生变化.     

胚胎干细胞周期调控的研究进展

胚胎干细胞（embryonic stem cells,Escs）具有自我复制和多潜能分化的特性。相对于体细胞,胚胎干细胞的细胞周期调控非常特别。比如,G1期较短;P53、RB等调控细胞周期“检验点”的蛋白分子功能“异常”等。胚胎干细胞细胞周期调控的研究对于研究胚胎干细胞的自我复制和多潜能性,都具有重要指导意义。该文将重点比较体细胞和胚胎干细胞在细胞周期调控方面的差异,并对近年来有关小鼠和人胚胎干细胞的细胞周期调控的研究进展进行介绍。     

Salinity tolerance in diapausing embryos of the annual killifish <Emphasis Type="Italic">Austrofundulus limnaeus</Emphasis> is supported by exceptionally low water and ion permeability

The annual killifish Austrofundulus limnaeus inhabits rainwater pools in the Maracaibo basin of Venezuela. This species persists in ephemeral habitats by producing diapausing embryos that are resistant to the stresses imposed by the drying of their aquatic habitat. Embryos of A. limnaeus are likely exposed to a highly variable osmotic environment during development, but their tolerance of osmotic stress has not been characterized. We investigated the capacity of these embryos to survive in hypersaline environments and evaluated the possible mechanisms used to support osmoregulation. Diapausing embryos of A. limnaeus defend their internal osmolality of around 290 mOsmol kg⁻¹ H₂O⁻¹ against salt stress as high as 50 ppt salinity. We find that diapausing embryos of A. limnaeus have a permeability to water that is orders of magnitude lower than other teleost fish embryos. The activity of ion motive ATPases that may be important in the extrusion of ions via mitochondrial rich cells do not appear to be playing a large role in osmoregulation of A. limnaeus embryos. We conclude that for the duration of embryonic development the unique properties of the enveloping cell layer of A. limnaeus embryos acts as a permeability barrier to water and ions and supports osmoregulation in this species in response to a broad range of osmotic environments. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cell cycle control and cancer chemotherapy

As detailed information accumulates about how cell cycle events are regulated, we can expect new opportunities for application to cancer therapy. The altered expression of oncogenes and tumor suppressor genes that commonly occurs in human cancers may impair the ability of the cells to respond to metabolic perturbations or stress. Impaired cell cycle regulation would make cells vulnerable to pharmacologic intervention by drug regimens tailored to the defects existing in particular tumors. Recent findings that may become applicable to therapy are reviewed, and the possible form of new therapeutic stratagems is considered.