Diapause Embryos Research Articles

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.

Complexity of the heat-soluble LEA proteome in Artemia species

The brine shrimp Artemia is a well known stress tolerant invertebrate found on most continents. Under certain conditions females produce cysts (encysted gastrulae) that enter diapause, a state of obligate dormancy. During developmental formation of diapause embryos several different types of stress proteins accumulate in large amounts, including the late embryogenesis abundant (LEA) proteins. In this study we used a combination of heterologous group 3 LEA antibodies to demonstrate that the heat-soluble proteome of the cysts contains up to 12 distinct putative group 3 LEA proteins that complement the group 1 LEA proteins found previously. Most antibody-positive, heat-soluble proteins were larger than 50kDa although antibody positive proteins of 20–38kDa were also detected. Both nuclei and mitochondria had distinct complements of the putative group 3 LEA proteins. A few small group 3 LEA proteins were induced by cycles of hydration–dehydration along with one protein of about 62kDa. The expression of group 3 LEA proteins, unlike members of group 1, was not restricted to encysted diapause embryos. Three to five putative group 3 LEA proteins were expressed in gravid females and in larvae. Cysts of different species from various geographic locations had distinct complements of group 3 LEA proteins suggesting rapid evolution of the LEA proteins or differences in the type of group 3 Lea genes expressed. Our results demonstrate the potential importance of group 3 LEA proteins in embryos and other life cycle stages of this animal extremophile.

Characterization of the cell morphology differences between pre‐diapause and diapause embryos of the killifish Austrofundulus limnaeus

Austrofundulus limnaeus is a freshwater annual killifish from Venezuela that produces embryos capable of entering three distinct stages of diapause. Diapause II is a state of developmental arrest that can last over 100 days and confers extreme anoxia tolerance. Diapause I and III are inducible while II is obligate. Diapause II occurs in ideal conditions (25oC) whereas incubation at 30oC induces a bypass of diapause II. Diapause II provides an opportunity to study an arrest of development in a vertebrate animal without exposing the organism to compounding stresses. Currently, little is known about the molecular mechanisms that induce and maintain this developmentally arrested state. We hypothesize that down regulation of gene expression is central to inducing diapause II. Our aim is to use assays that are principally based upon immunohistochemistry to characterize at the cellular level differences in cell morphology between pre‐DII and DII embryos. We are looking at changes in chromatin modifications, which are known to be involved in the repression or activation of developmental genes, as well as changes in mitotic cell division. Preliminary results show a change in the signal of H3K27 chromatin modifiers as the animal transitions between stages of pre‐DII and DII. This research is leading to a greater molecular understanding of the remarkable phenomenon of obligate developmental arrest in a vertebrate embryo.

Deubiquitinating enzyme BAP1 is involved in the formation and maintenance of the diapause embryos of Artemia

The modification of proteins by ubiquitination and deubiquitination plays an important role in various cellular processes. BRCA1-associated protein-1 (BAP1) is a deubiquitinating enzyme whose function in the control of the cell cycle requires both its deubiquitinating activity and nuclear localization. In the present study, a ubiquitin carboxyl-terminal hydrolase belonging to the BAP1 family was identified and characterized from Artemia parthenogenetica, a member of a family of brine shrimp that, under certain conditions, produce and release diapause embryos in which cell division and turnover of macromolecules are arrested. Western blot analysis and in vitro enzyme activity assay revealed ArBAP1 to be a cytoplasmic protein with typical ubiquitin hydrolase activity. Northern blot analysis revealed that ArBAP1 was abundant in the abdomen of Artemia producing diapause-destined embryos. Furthermore, by in situ hybridization, ArBAP1 was located exclusively in the embryos. In vivo knockdown of ArBAP1 by RNA interference resulted in the formation of embryos with split shells and abortive nauplii. The present findings suggest that ArBAP1, the first reported cytoplasmic BAP1, participates in the formation of diapause embryos and plays an important role in the control of cell cycle arrest in these encysted embryos.

Thermostable proteins in the diapausing eggs of Brachionus manjavacas (Rotifera)

Diapausing embryos (resting eggs) from brachionid rotifers are able to withstand desiccation and thermal stress. Resting eggs can remain viable for decades, and develop normally once placed in a permissive environment that allows for hatching, growth and development. The exact mechanisms of resistance are not known, although several molecules have been suggested to confer protection during desiccation and thermal stress. In this study, we have identified by mass spectrometry two thermostable proteins, LEA (late embryogenesis abundant) and VTG (vitellogenin-like), found exclusively in the resting eggs of Brachionus manjavacas. This is the first observation that LEA proteins may play a role in thermostability and the first report of a VTG-like protein in the phylum Rotifera. These proteins exhibited increased expression in rotifer resting eggs when compared to amictic females. Our data suggest the existence of alternate pathways of desiccation and thermal resistance in brachionid rotifers.

Involvement of Cyclin K Posttranscriptional Regulation in the Formation of Artemia Diapause Cysts

Background Artemia eggs tend to develop ovoviviparously to yield nauplius larvae in good rearing conditions; while under adverse situations, they tend to develop oviparously and encysted diapause embryos are formed instead. However, the intrinsic mechanisms regulating this process are not well understood.Principal FindingThis study has characterized the function of cyclin K, a regulatory subunit of the positive transcription elongation factor b (P-TEFb) in the two different developmental pathways of Artemia. In the diapause-destined embryo, Western blots showed that the cyclin K protein was down-regulated as the embryo entered dormancy and reverted to relatively high levels of expression once development resumed, consistent with the fluctuations in phosphorylation of position 2 serines (Ser2) in the C-terminal domain (CTD) of the largest subunit (Rpb1) of RNA polymerase II (RNAP II). Interestingly, the cyclin K transcript levels remained constant during this process. In vitro translation data indicated that the template activity of cyclin K mRNA stored in the postdiapause cyst was repressed. In addition, in vivo knockdown of cyclin K in developing embryos by RNA interference eliminated phosphorylation of the CTD Ser2 of RNAP II and induced apoptosis by inhibiting the extracellular signal-regulated kinase (ERK) survival signaling pathway.Conclusions/SignificanceTaken together, these findings reveal a role for cyclin K in regulating RNAP II activity during diapause embryo development, which involves the post-transcriptional regulation of cyclin K. In addition, a further role was identified for cyclin K in regulating the control of cell survival during embryogenesis through ERK signaling pathways.

Characterization and Processing of Superoxide Dismutase-Fused Vitellogenin in the Diapause Embryo Formation: A Special Developmental Pathway in the Brine Shrimp, Artemia parthenogenetica1

To withstand environmental stress, Artemia release diapause cysts via an oviparous pathway instead of producing swimming nauplius larvae by the ovoviviparous pathway. Encased in such a cyst, the embryos at diapause can survive for many years. Vitellogenin (Vtg), the precursor of vitellins, the main yolk proteins, is crucial for embryonic development. This study compares vitellogenesis between oviparity and ovoviviparity, the two reproductive modes occurring in A. parthenogenetica. A Vtg gene was cloned, based on N-terminal amino acid sequence analysis, PCR amplification, and cDNA library construction and screening, and was found to consist of 6778 bp with a 6657 bp open reading frame encoding 2219 amino acids. From the deduced primary structure, Artemia vitellogenin (ArVtg) was found to possess six copies of the consensus cleavage site, R-X-X-R, and to contain a superoxide dismutase (SOD)-like domain at the N-terminus. This is an unusual finding for crustacean Vtg proteins, having been reported only in one previous crustacean, Daphnia magna. Using Northern blot analysis and in situ hybridization, ArVtg gene expression was observed at early stages of vitellogenesis in the connective tissue located in the cephalothorax, with trace expression in the ovary. Western blot analysis and several N-terminal sequences revealed that ArVtg was cleaved at each consensus cleavage site and that more than 10 subunits were formed during posttranslational processing in ovarian maturation. Of these, only the SOD-containing subunits (∼90 and 60 kDa) showed different profiles between the oviparous and ovoviviparous pathways. This suggests that these high concentration components have an important function for the encysted diapaused embryos during long-term cell-cycle arrest, which has remained unknown up until now.

Polyamines Are Implicated in the Emergence of the Embryo from Obligate Diapause

Embryonic diapause is a poorly understood phenomenon of reversible arrest of embryo development prior to implantation. In many carnivores, such as the mink (Neovison vison), obligate diapause characterizes each gestation. Embryo reactivation is controlled by the uterus by mechanisms that remain elusive. Because polyamines are essential regulators of cell proliferation and growth, it was hypothesized that they trigger embryo reactivation. To test this, mated mink females were treated with α-difluoromethylornithine, an inhibitor of ornithine decarboxylase 1, the rate-limiting enzyme in polyamine biosynthesis, or saline as a control during the first 5 d of reactivation. This treatment induced polyamine deprivation with the consequence of rearrest in embryo cell proliferation. A mink trophoblast cell line in vitro subjected to α-difluoromethylornithine treatment likewise displayed an arrest in cell proliferation, morphological changes, and intracellular translocation of ornithine decarboxylase 1 protein. The arrest in embryo development deferred implantation for a period consistent with the length of treatment. Successful implantation and parturition ensued. We conclude that polyamine deprivation brought about a reversible rearrest of embryo development, which returned the mink embryo to diapause and induced a second delay in embryo implantation. The results are the first demonstration of a factor essential to reactivation of embryos in obligate diapause.

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 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.

Late Embryogenesis Abundant (LEA) Proteins in Nondesiccated, Encysted, and Diapausing Embryos of Rotifers1

Two genes encoding for late embryogenesis abundant proteins (LEAs) are expressed in encysted diapausing embryos (or resting eggs) of rotifers (Brachionus plicatilis O.F. Müller) and females forming them. The two genes (bpa-leaa and bpa-leab) share approximately 50% of their nucleotides sequence, and bpa-leaa is more than twofold longer than bpa-leab. The deduced amino acid sequences show high abundance of alanine, glycine, lysine, and glutamic acid; a hydropathy index of lower than one; and a relatively high (81-82%) predicted probability of forming alpha-helices in their secondary structure, all of which are characteristic features of LEAs. The predicted molecular masses of bpa-LEAA ( approximately 67 kDa) and bpa-LEAB ( approximately 27 kDa) are similar to the molecular mass determined by Western-blot analyses, suggesting a low probability of posttranslational modifications. In silico analysis reveals that the two LEAs resemble group 3 LEAs based on the repeats for 11mer motifs, although they also display several putative amino acids typical of the 20mer motif of group 1 LEAs. The rotifer LEAs do not contain a predicted target sequence and are more likely localized in the cytosol. LEAs were expressed in resting eggs and females producing them, but not in other female forms or males. LEA transcripts and proteins are degraded during hatching, suggesting that LEAs are developmentally programmed during resting egg formation and hatching. LEAs probably equip the resting eggs to withstand desiccation if that occurs during dormancy. The present study expands our knowledge about the biological pathways associated with formation of rotifer resting eggs and also demonstrates the occurrence of LEAs in dormant, nondesiccated, encysted animal embryos.

Decoupling development and energy flow during embryonic diapause in the cricket,Allonemobius socius

Respiration rate increases 6.3-fold during 15 days of post-oviposition development in embryos of the Southern ground cricket, Allonemobius socius. This ontogenetic increase in metabolism of non-diapause insects is blocked during diapause, such that metabolic rate is only 36% of the rate measured for 15 days developing embryos. Surprisingly, however, there is not an acute metabolic depression during diapause entry at the point when developmental ceases (4-5 days post-oviposition), as measured by blockage of morphological change and DNA proliferation. The results indicate a decoupling of developmental arrest from metabolism. Both non-diapause and diapause embryos have unusually high [AMP]:[ATP] ratios and low [ATP]:[ADP] ratios during early embryogenesis, which suggests embryos may have experienced hypoxia as a result of an insect chorion that limits water loss but may restrict oxygen diffusion. The similar adenylate profiles for these two developmental states indicate the atypical energy status is not a specific feature of diapause. In addition embryos at day 3 have high levels of lactate that decrease as development proceeds up to day 7. Calorimetric-respirometric (CR) ratios of -353 (day 3) to -333 (day 7) kJ mol(-1) O2 are consistent with embryos that are aerobically recovering from hypoxia, but are inconsistent with an ongoing anaerobic contribution to metabolism. Superfusing 3-day embryos with O2 enriched air (40% O2) forces these metabolic indicators toward a more aerobic poise, but only partially. Taken together these biochemical data indicate the metabolic poise of A. socius is only partly explained by hypoxia in early development, and that the atypical set points are also intrinsic features of this ontogenetic period in the life cycle.

Parameters influencing derivation of embryonic stem cells from murine embryos

The derivation of ES cells is poorly understood and varies in efficiency between different strains of mice. We have investigated potential differences between embryos of permissive and recalcitrant strains during diapause and ES cell derivation. We found that in diapause embryos of the recalcitrant C57BL/6 and CBA strains, the epiblast failed to expand during the primary explant phase of ES cell derivation, whereas in the permissive 129 strain, it expanded dramatically. Epiblasts from the recalcitrant strains could be expanded by reducing Erk activation. Isolation of 129 epiblasts facilitated very efficient derivation of ES cell lines in serum- and feeder-free conditions, but reduction of Erk activity was required for derivation of ES cells from isolated C57BL/6 or CBA epiblasts. The results suggest that the discrepancy in ES cell derivation efficiency is not attributable merely to variable prodifferentiative effects of the extra-embryonic lineages but also to an intrinsic variability within the epiblast to maintain pluripotency.

ArHsp22, a developmentally regulated small heat shock protein produced in diapause‐destined Artemia embryos, is stress inducible in adults

Diapause embryos of the crustacean Artemia franciscana exhibit extreme stress tolerance, a property thought to involve molecular chaperones known as small heat shock proteins. To further explore this idea, the structure, function and synthesis of ArHsp22, an Artemia small heat shock protein, were characterized. ArHsp22 contains amino-terminal WXDPF motifs, an alpha-crystallin domain with a highly conserved arginine, and a carboxy-terminal I/VXI/V motif, all typical of small heat shock proteins. ArHsp22 formed large oligomers and exhibited molecular chaperone activity in vitro, protecting citrate synthase and insulin from denaturation. Quantitative PCR and immunoprobing of western blots revealed that ArHsp22 synthesis is restricted to diapause-destined Artemia embryos and that the protein is degraded during post-diapause development. ArHsp22 was observed in cyst nuclei, a location shared by p26 but not ArHsp21, which are two other diapause-specific Artemia small heat shock proteins. ArHsp22 production was enhanced by thermal stress, but only in adults, thus representing the first crustacean small heat shock protein whose synthesis is known to be both developmentally regulated and stress inducible. The results demonstrate that expression of the gene for ArHsp22 is modulated by multiple cues that vary with life history stage. Such findings are of importance in understanding diapause maintenance in Artemia embryos and the survival of adult animals experiencing environmental insult.

Bioenergetic profiles of isolated mitochondria from diapausing embryos of the annual killifish Austrofundulus limnaeus

The annual killifish, Austrofundulus limnaeus, occupy ephemeral pond habitats by producing diapausing embryos which can reduce their oxygen consumption rate by up 90% compared to developing embryos. Mitochondria isolated from whole embryos collected at 10 dpf, diapause II, post‐diapause, diapause III, post‐hatch larvae, and adult liver exhibited oxygen consumption rates that increased with developmental stage in the presence of glutamate or succinate. Measurements of electron transport chain activity reflected life‐stage specific regulation. Minimum ETC activity was observed during diapause II: complex I activity was 12% of adult, complex II was 25% of adult, and complex IV activity was 20% of adult. Proton leak was significantly greater during diapause III and elevated in diapause II and larvae relative to 10 dpf and adult in the presence of glutamate, whereas no significant difference in life stage‐specific proton leak was observed in the presence of succinate. P:O ratios in the presence of glutamate or pyruvate and succinate were between 2 and 2.5 and between 1 and 1.2 respectively for diapause III embryos and adults. Overall, these data agree with whole‐embryo oxygen consumption rates previously measured and suggest a coordinated suppression of the mitochondrial electron transport chain and oxidative capacity during diapause. Supported by NSF grant IBN‐0344578 to JEP.

The Encysted Dormant Embryo Proteome of Artemia sinica

The possibility of the brine shrimp Artemia to produce dormant embryo (cysts) in diapause is a key feature in its life history. In the present study, we obtained a proteomic reference map for the diapause embryo of Artemia sinica using two-dimensional gel electrophoresis with a pH range of 4-7 and a molecular weight range of 10-100 kDa. Approximately 233 proteins were detected, and 60 of them were analyzed by capillary liquid chromatography tandem mass spectrometry (LC-MS/MS). Of these, 39 spots representing 33 unique proteins were identified, which are categorized into functional groups, including cell defense, cell structure, metabolism, protein synthesis, proteolysis, and other processes. This reference map will contribute toward understanding the state of the diapause embryo and lay the basis and serve as a useful tool for further profound studies in the proteomics of Artemia at different developmental stages and physiological conditions.

Extreme anoxia tolerance in embryos of the annual killifishAustrofundulus limnaeus: insights from a metabolomics analysis

The annual killifish Austrofundulus limnaeus survives in ephemeral pond habitats by producing drought-tolerant diapausing embryos. These embryos probably experience oxygen deprivation as part of their normal developmental environment. We assessed the anoxia tolerance of A. limnaeus embryos across the duration of embryonic development. Embryos develop a substantial tolerance to anoxia during early development, which peaks during diapause II. This extreme tolerance of anoxia is retained during the first 4 days of post-diapause II development and is then lost. Metabolism during anoxia appears to be supported mainly by production of lactate, with alanine and succinate production contributing to a lesser degree. Anoxic embryos also accumulate large quantities of gamma-aminobutyrate (GABA), a potential protector of neural function. It appears that the suite of characters associated with normal development and entry into diapause II in this species prepares the embryos for long-term survival in anoxia even while the embryos are exposed to aerobic conditions. This is the first report of such extreme anoxia tolerance in a vertebrate embryo, and introduces a new model for the study of anoxia tolerance in vertebrates.

Salinity tolerance in diapausing embryos of the annual killifish Austrofundulus limnaeus 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) H(2)O(-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.

Induction of mictic females in the rotiferBrachionus: oocytes of amictic females respond individually to population‐density signal only during oogenesis shortly before oviposition

Summary1. One at a time during the reproductive period of amictic females, oocytes fill with yolk and undergo a mitotic maturation division (oogenesis), are oviposited as single cells, and then develop parthenogenetically into females. Sexual reproduction inBrachionusand several other genera is initiated when amictic females are crowded and oviposit some eggs induced to differentiate into mictic females. Mictic females produce haploid eggs that can develop parthenogentically into males or be fertilised and develop into diapausing embryos called resting eggs.2. This study examines the time when oocytes in amictic females respond to maternal population density. Is the fate of all oocytes in the germarium irreversibly determined during the early postnatal life of the mother, or is each oocyte labile until just before oviposition? In the former case, the probability of an amictic female producing a mictic daughter at any time throughout her reproductive period would reflect the population density she experienced while young and not that at the time she oviposited an egg.3. Amictic females of two clones of a Florida strain ofB. calycifloruswere cultured singly from birth at a low or high density (in a large or small volume) until about halfway through their reproductive period and then switched (experimental treatment), or not (control treatment), to the other density condition. The results indicate that the female fate of an oocyte is determined by maternal population density during oogenesis. Eggs oviposited soon after transfer from low to high density had the same, or a higher, probability of becoming mictic females compared with those produced by control females kept at the high density; eggs oviposited after transfer from the high to the low density had the same low probability of becoming mictic females as those produced by control females kept at the low density.4. Control females kept at the high density were less likely to produce mictic daughters as they aged. This decline is not because of a decreased propensity of older females to respond to crowding, as older females responded maximally when transferred from a low to a high population density.5. As oocytes in amictic females respond to maternal population density only during oogenesis, there is a negligible lag between the population‐density signal in the environment and the commitment to sexual reproduction. This minimises the obligatory two‐generation lag between this signal and production of resting eggs, and thus reduces the possibility that crowding will lead to food limitation before production of these eggs.

Caspase activity during cell stasis: avoidance of apoptosis in an invertebrate extremophile,Artemia franciscana

Evaluation of apoptotic processes downstream of the mitochondrion reveals caspase-9- and low levels of caspase-3-like activities in partly purified extracts of Artemia franciscana embryos. However, in contrast to experiments with extracts of human hepatoma cells, cytochrome c fails to activate caspase-3 or -9 in extracts from A. franciscana. Furthermore, caspase-9 activity is sensitive to exogenous calcium. The addition of 5 mM calcium leads to a 4.86 +/- 0.19 fold (SD) (n = 3) increase in activity, which is fully prevented with 150 mM KCl. As with mammalian systems, high ATP (>1.25 mM) suppresses caspase activity in A. franciscana extracts. A strong inhibition of caspase-9 activity was also found by GTP. Comparison of GTP-induced inhibition of caspase-9 at 0 and 2.5 mM MgCl(2) indicates that free (nonchelated) GTP is likely to be the inhibitory form. The strongest inhibition among all nucleotides tested was with ADP. Inhibition by ADP in the presence of Mg(2+) is 60-fold greater in diapause embryos than in postdiapause embryos. Because ADP does not change appreciably in concentration between the two physiological states, it is likely that this differential sensitivity to Mg(2+)-ADP is important in avoiding caspase activation during diapause. Finally, mixtures of nucleotides that mimic physiological concentrations in postdiapause and diapause states underscore the depressive action of these regulators on caspase-9 during diapause. Our biochemical characterization of caspase-like activity in A. franciscana extracts reveals that multiple mechanisms are in place to reduce the probability of apoptosis under conditions of energy limitation in this embryo.

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.