Molecular Regulators of Embryonic Diapause and Cancer Diapause-like State.

Embryonic diapause is an enigmatic state of dormancy that interrupts the normally tight connection between developmental stages and time. This reproductive strategy and state of suspended development occurs in mice, bears, roe deer, and over 130 other mammals and favors the survival of newborns. Diapause arrests the embryo at the blastocyst stage, delaying the post-implantation development of the embryo. This months-long quiescence is reversible, in contrast to senescence that occurs in aging stem cells. Recent studies have revealed critical regulators of diapause. These findings are important since defects in the diapause state can cause a lack of regeneration and control of normal growth. Controlling this state may also have therapeutic applications since recent findings suggest that radiation and chemotherapy may lead some cancer cells to a protective diapause-like, reversible state. Interestingly, recent studies have shown the metabolic regulation of epigenetic modifications and the role of microRNAs in embryonic diapause. In this review, we discuss the molecular mechanism of diapause induction.

Progress in the characterization of insulin-like peptides in aphids: Immunohistochemical mapping of ILP4.

Aphids were the first animals described as photoperiodic due to their seasonal switch from viviparous parthenogenesis to sexual reproduction (cyclical parthenogenesis) caused by the shortening of the photoperiod in autumn. This switch produces a single sexual generation of oviparous females and males that mate and lay diapausing cold-resistant eggs that can overcome the unfavourable environmental conditions typical of winter in temperate regions. Previous studies have hinted at a possible implication of two insulin-like peptides (ILP1 and ILP4) in the aphid seasonal response, changing their expression levels between different photoperiodic conditions. Moreover, in situ localization of their transcripts in particular neurosecretory cells (NSCs) in the aphid brain supported the idea that these neuropeptides could correspond to the formerly called virginoparin, an uncharacterized factor originally proposed to be transported directly to the aphid embryos to promote their development as parthenogenetic individuals. To further investigate the fate of these ILPs, we raised a specific antiserum against one of them (ILP4) and mapped this neuropeptide by immunohistochemistry (IHC) in Acyrthosiphon pisum and Megoura viciae aphids. Coincident with in situ localization, our results show that ILP4 is synthesized in two groups (one in each brain hemisphere) of four neurosecretory cells in the pars intercerebralis (NSC group I) and then it is transported outside the brain to the corpora cardiaca. From there, three nerves (two laterals and one medial) transport it to the abdomen. Although no precise site of release has been found, the terminations of these nerves near the germaria would be compatible with the proposal of a direct connection between group I of NSCs and the reproductive system by localized release. In addition, we detected some collateral arborizations originating from the eight NSCs going to the pars lateralis, where clock neurons and some photoreceptors have been previously localized, suggesting a possible communication between the circadian and photoperiodic systems.

ROS and hypoxia signaling regulate periodic metabolic arousal during insect dormancy to coordinate glucose, amino acid, and lipid metabolism.

Metabolic suppression is a hallmark of animal dormancy that promotes overall energy savings. Some diapausing insects and some mammalian hibernators have regular cyclic patterns of substantial metabolic depression alternating with periodic arousal where metabolic rates increase dramatically. Previous studies, largely in mammalian hibernators, have shown that periodic arousal is driven by an increase in aerobic mitochondrial metabolism and that many molecules related to energy metabolism fluctuate predictably across periodic arousal cycles. However, it is still not clear how these rapid metabolic shifts are regulated. We first found that diapausing flesh fly pupae primarily use anaerobic glycolysis during metabolic depression but engage in aerobic respiration through the tricarboxylic acid cycle during periodic arousal. Diapausing pupae also clear anaerobic by-products and regenerate many metabolic intermediates depleted in metabolic depression during arousal, consistent with patterns in mammalian hibernators. We found that decreased levels of reactive oxygen species (ROS) induced metabolic arousal and elevated ROS extended the duration of metabolic depression. Our data suggest ROS regulates the timing of metabolic arousal by changing the activity of two critical metabolic enzymes, pyruvate dehydrogenase and carnitine palmitoyltransferase I by modulating the levels of hypoxia inducible transcription factor (HIF) and phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK). Our study shows that ROS signaling regulates periodic arousal in our insect diapasue system, suggesting the possible importance ROS for regulating other types of of metabolic cycles in dormancy as well.

Wnt/Beta-catenin/Esrrb signalling controls the tissue-scale reorganization and maintenance of the pluripotent lineage during murine embryonic diapause.

The epiblast, which provides the foundation of the future body, is actively reshaped during early embryogenesis, but the reshaping mechanisms are poorly understood. Here, using a 3D in vitro model of early epiblast development, we identify the canonical Wnt/ß-catenin pathway and its central downstream factor Esrrb as the key signalling cascade regulating the tissue-scale organization of the murine pluripotent lineage. Although in vivo the Wnt/ß-catenin/Esrrb circuit is dispensable for embryonic development before implantation, autocrine Wnt activity controls the morphogenesis and long-term maintenance of the epiblast when development is put on hold during diapause. During this phase, the progressive changes in the epiblast architecture and Wnt signalling response show that diapause is not a stasis but instead is a dynamic process with underlying mechanisms that can appear redundant during transient embryogenesis.

Effects of photoperiod and aging on the adult spermatogenesis of Polygonia c-aureum (Lepidoptera: Nymphalidae), in relation to adult diapause.

Adult spermatogenesis of Polygonia c-aureum was compared between non-diapausing and diapausing butterflies before overwintering. This butterfly has seasonal polyphenism, i.e., summer and autumnal forms. Summer form butterflies that emerged in summer reproduce shortly after emergence, while autumnal forms that emerged in autumn mate in spring. Immatures were reared under either a long photoperiod, which produced the summer form without diapause or under a short photoperiod, which produced the autumnal form with diapause. We found almost no differences in adult spermatogenesis between the two seasonal forms, indicating that adult spermatogenesis is not related to adult diapause. Although adult diapause in the autumnal form is maintained under short photoperiods and terminated under long photoperiods, such a photoperiod did not affect the spermatogenesis of the autumnal form. Our earlier studies indicate that relatively few eupyrene and apyrene sperm are produced after overwintering. Although apyrene spermatogenesis occurred in young adults, eupyrene spermatogenesis did in a small scale before overwintering. These results suggest strongly that male autumnal form butterflies prepare the sperm until overwintering, which had been formed during the larval, pupal and young adult stages.

Serpin7 controls egg diapause of migratory locust (Locusta migratoria) by regulating polyphenol oxidase.

Diapause is a state of arrested growth, which allows insects to adapt to diverse environments. Serine protease inhibitors (serpins) play an important role in various physiological processes, including blood coagulation, fibrinolysis, development, complement activation and extracellular matrix remodeling. We hypothesized that serpin may affect energy metabolism and thereby control diapause of migratory locust (Locusta migratoria) embryos by regulating protease cascades. A total of seven nonredundant serpin genes (named serpin1-serpin7) of L. migratoria were obtained through transcriptomic analysis. We further performed label-free proteomic sequencing and analysis of diapause and nondiapause eggs of L. migratoria, revealing significant differences in serpin7 expression. A significant reduction in diapause rate under the short photoperiod was observed in insects treated with serpin7 double-stranded RNA. Furthermore, knockdown of the serpin7 gene resulted in significant upregulation of the activity of polyphenol oxidase. We therefore propose that the observed serpin7 gene plays a crucial role in diapause, suggesting that control of energy metabolism may have potential as a future strategy for the reproductive control of insect pests.

The role of N6-methyladenosine modification on diapause in silkworm (Bombyx mori) strains that exhibit different voltinism.

N6-methyladenosine (m6 A) plays a key role in regulating gene expression in myriad organisms. Diapause is an important plastic phenotype that allows insects to survive under specific environmental conditions. However, the diapause molecular mechanism remains unknown. In this study, we analyzed the phylogenetics of genes related to the m6 A modification complex in the silkworm (Bombyx mori) based on identified sequences from other organisms. We detected the expression of these genes during different developmental phases from four strains with different voltinism. We also determined total m6 A content in cells treated with different diapause hormone concentrations or eggs exposed to hydrochloric acid. Our data revealed that m6 A-modification-related gene expression and m6 A content were greater in diapause-destinated compared to nondiapause-destined strains. Our findings suggest that m6 A modification may provide significant epigenetic regulation of diapause-related genes in the silkworm.

Do ovarian steroid hormones control the resumption of embryonic growth following the period of diapause in roe deer (Capreolus capreolus)?

Embryonic diapause in the European roe deer includes a period of five months from August to December in which embryonic development is extremely decelerated. Following exit from diapause, the embryo rapidly elongates and subsequently implants. In diapausing carnivores and marsupials, resumption of embryonic growth is regulated by ovarian steroid hormones. In the roe deer, the role of steroid hormones is not known to date. In the present study, progesterone (P4), estradiol-17ß (E2) and total estrogens (Etot) were determined in blood plasma and endometrium of roe deer shot in the course of regular huntings between September and December. Steroid hormone concentrations were correlated to the corresponding size of the embryo derived from ex vivo uterine flushing and to the date of sampling. The mean plasma concentrations of P4 (5.4 ± 0.2 ng/ml, mean ± SE, N = 87), E2 (24.3 ± 2.6 pg/ml, N = 86) and Etot (21.7 ± 2.6 pg/ml, N = 78) remained constant over the sampling period and were not correlated to embryonic size. Likewise, endometrial concentrations of P4 (66.1 ± 6.5 ng/g), E2 (284.0 ± 24.43 pg/g) and, Etot (440.9 ± 24.43 pg/g) showed no changes over time [corrected]. Therefore, it was concluded that ovarian steroid hormones do not play a determining role in resumption of embryonic growth following the period of diapause in the roe deer.

DEK terminates diapause by activation of quiescent cells in the crustacean Artemia.

To cope with harsh environments, the Artemia shrimp produces gastrula embryos in diapause, a state of obligate dormancy, having cellular quiescence and suppressed metabolism. The mechanism behind these cellular events remains largely unknown. Here, we study the regulation of cell quiescence using diapause embryos of Artemia We found that Artemia DEK (Ar-DEK), a nuclear factor protein, was down-regulated in the quiescent cells of diapause embryos and enriched in the activated cells of post-diapause embryos. Knockdown of Ar-DEK induced the production of diapause embryos whereas the control Artemia released free-swimming nuaplii. Our results indicate that Ar-DEK correlated with the termination of cellular quiescence via the increase in euchromatin and decrease in heterochromatin. The phenomena of quiescence have many implications beyond shrimp ecology. In cancer cells, for example, knockdown of DEK also induced a short period of cellular quiescence and increased resistance to environmental stress in MCF-7 and MKN45 cancer cell lines. Analysis of RNA sequences in Artemia and in MCF-7 revealed that the Wnt and AURKA signaling pathways were all down-regulated and the p53 signaling pathway was up-regulated upon inhibition of DEK expression. Our results provide insight into the functions of Ar-DEK in the activation of cellular quiescence during diapause formation in Artemia.

The potential role of Annexin 3 in diapause embryo restart of Artemia sinica and in response to stress of low temperature.

Annexins are highly conserved and ubiquitous in various somatic cell types. They are involved in membrane transport and a range of calcium-regulated activities on the cell membrane surface, including vesicular transport, membrane fusion in exocytosis, signal transduction, and formation of calcium channels. They also regulate inflammatory response, cell differentiation, and interaction between cytoskeletal proteins. In this study, for the first time, an ANX3 gene from Artemia sinica ( As-anx3) was cloned. The As-anx3 full-length complementary DNA comprises 1,024 bp and has a 948 bp open reading frame encoding a 315-amino-acid polypeptide with four ANX domains. The profiles of both As-ANX3 mRNA and protein expression exhibited peaks at the 0 hr stage and had the same significant downregulation trend throughout the post-diapause embryo development stage. The ERK1/2, the phosphorylation levels of ERK1/2, and cell cycle-related protein (CDK4) expressions were analyzed by western blot analysis. The results showed that CDK4 presented a significantly ascending trend from 0 and 40 hr, although the phosphorylation levels of ERK1/2 did not increase significantly. The transcriptional and protein expressions of As-ANX3 were highly upregulated when the temperature was lowered from 25 to 15°C, but the expressions showed a gradual downward trend when the temperature was further lowered to 5°C. These results indicated that As-ANX3 plays a crucial role in restarting diapause and low-temperature stress in A. sinica.

The chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) controls cellular quiescence by hyperpolarizing the cell membrane during diapause in the crustacean Artemia.

Cellular quiescence, a reversible state in which growth, proliferation, and other cellular activities are arrested, is important for self-renewal, differentiation, development, regeneration, and stress resistance. However, the physiological mechanisms underlying cellular quiescence remain largely unknown. In the present study, we used embryos of the crustacean Artemia in the diapause stage, in which these embryos remain quiescent for prolonged periods, as a model to explore the relationship between cell-membrane potential (Vmem) and quiescence. We found that Vmem is hyperpolarized and that the intracellular chloride concentration is high in diapause embryos, whereas Vmem is depolarized and intracellular chloride concentration is reduced in postdiapause embryos and during further embryonic development. We identified and characterized the chloride ion channel protein cystic fibrosis transmembrane conductance regulator (CFTR) of Artemia (Ar-CFTR) and found that its expression is silenced in quiescent cells of Artemia diapause embryos but remains constant in all other embryonic stages. Ar-CFTR knockdown and GlyH-101-mediated chemical inhibition of Ar-CFTR produced diapause embryos having a high Vmem and intracellular chloride concentration, whereas control Artemia embryos released free-swimming nauplius larvae. Transcriptome analysis of embryos at different developmental stages revealed that proliferation, differentiation, and metabolism are suppressed in diapause embryos and restored in postdiapause embryos. Combined with RNA sequencing (RNA-Seq) of GlyH-101-treated MCF-7 breast cancer cells, these analyses revealed that CFTR inhibition down-regulates the Wnt and Aurora Kinase A (AURKA) signaling pathways and up-regulates the p53 signaling pathway. Our findings provide insight into CFTR-mediated regulation of cellular quiescence and Vmem in the Artemia model.

Stress tolerance in diapausing embryos of Artemia franciscana is dependent on heat shock factor 1 (Hsf1).

Embryos of the crustacean, Artemia franciscana, may undergo oviparous development, forming encysted embryos (cysts) that are released from females and enter diapause, a state of suppressed metabolism and greatly enhanced stress tolerance. Diapause-destined embryos of A. franciscana synthesize three small heat shock proteins (sHsps), p26, ArHsp21 and ArHsp22, as well as artemin, a ferritin homologue, all lacking in embryos that develop directly into nauplii. Of these diapause-specific molecular chaperones, p26 and artemin are important contributors to the extraordinary stress tolerance of A. franciscana cysts, but how their synthesis is regulated is unknown. To address this issue, a cDNA for heat shock factor 1 (Hsf1), shown to encode a protein similar to Hsf1 from other organisms, was cloned from A. franciscana. Hsf1 was knocked down by RNA interference (RNAi) in nauplii and cysts of A. franciscana. Nauplii lacking Hsf1 died prematurely upon release from females, showing that this transcription factor is essential to the survival of nauplii. Diapause cysts with diminished amounts of Hsf1 were significantly less stress tolerant than cysts containing normal levels of Hsf1. Moreover, cysts deficient in Hsf1 possessed reduced amounts of p26, ArHsp21, ArHsp22 and artemin, revealing dependence on Hsf1 for expression of their genes and maximum stress tolerance. The results demonstrate an important role for Hsf1, likely in concert with other transcription factors, in the survival and growth of A. franciscana and in the developmentally regulated synthesis of proteins responsible for the stress tolerance of diapausing A. franciscana cysts.

Reactive oxygen species extend insect life span using components of the insulin-signaling pathway.

Reactive oxygen species (ROS) are well-known accelerants of aging, but, paradoxically, we show that physiological levels of ROS extend life span in pupae of the moth Helicoverpa armigera, resulting in the dormant state of diapause. This developmental switch appears to operate through a variant of the conventional insulin-signaling pathway, as evidenced by the facts that Akt, p-Akt, and PRMT1 are elevated by ROS, but not insulin, and that high levels of p-Akt fail to phosphorylate FoxO through PRMT1-mediated methylation. These results suggest a distinct signaling pathway culminating in the elevation of FoxO, which in turn promotes the extension of life span characteristic of diapause.

Inhibition of polyamine synthesis causes entry of the mouse blastocyst into embryonic diapause.

Embryonic diapause is a common reproductive strategy amongst mammals, requiring an intimate cross-talk between the endometrium and the blastocyst. To date, the precise molecular signals responsible are unknown in the mouse or any other mammal. Previous studies in the mink implicate polyamines as major regulators of the control of diapause. In the mouse, inhibiting the rate-limiting enzyme of polyamine synthesis, ornithine decarboxylase (ODC1) during early pregnancy largely prevents implantation, but the fate of the nonimplanted embryos is unknown. To determine whether polyamines control mouse embryonic diapause, we treated pregnant mice with an ODC1 inhibitor from d3.5 to d6.5 postcoitum. At d7.5, 72% of females had no signs of implantation whilst the remaining females exhibited disrupted placental formation and degenerate embryos. In the females with no implantation, we obtained viable blastocysts that had attenuated cell proliferation, indicating a state of diapause. When cultured in vitro, these exhibited trophoblast outgrowth, indicative of reactivation of embryogenesis. In contrast, direct culture of d3.5 blastocysts with an ODC1 inhibitor failed to cause entry into diapause. Examination of the polyamine pathway enzymes and a number of implantation factors indicated inhibition of ODC1 resulted in a uterine phenotype that resembled diapause, with some compensatory increases in crucial genes. Thus, we conclude that an absence or paucity of polyamines induces the uterine quiescence that causes entry of the blastocyst into embryonic diapause.

Embryo arrest and reactivation: potential candidates controlling embryonic diapause in the tammar wallaby and mink†.

Embryonic diapause is a period of developmental arrest which requires coordination of a molecular cross-talk between the endometrium and blastocyst to ensure a successful reactivation, but the exact mechanisms are undefined. The objectives of this study were to screen the tammar blastocyst for potential diapause control factors and to investigate the potential for members of the epidermal growth factor (EGF) family to coordinate reactivation. A select number of factors were also examined in the mink to determine whether their expression patterns were conserved across diapause species. The full-length sequences of the tammar genes of interest were first cloned to establish their level of sequence conservation with other mammals. The uterine expression of EGF family members EGF and heparin-binding EGF (HBEGF) and their receptors (EGFR and erb-b2 receptor tyrosine kinase 4 (ERBB4)) was determined by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. Both HBEGF and EGF were significantly upregulated at reactivation compared to diapause. In the blastocyst, the expression of the potential diapause factors Forkhead box class O family members (FOXO1, FOXO3, and FOXO4), tumor protein 53 (TP53), cyclin-dependent kinase inhibitor 1A (CDKN1A), and the EGF family were examined by RT-PCR and immunofluorescence. Nuclear (and hence active) FOXO expression was confirmed for the first time in a mammalian diapause blastocyst in both the tammar and the mink-CDKN1A was also expressed, but TP53 is not involved and EGFR was not detected in the blastocyst. These results indicate that the EGF family, FOXOs, and CDKN1A are promising candidates for the molecular control of embryonic diapause in mammals.

The evolution of an annual life cycle in killifish: adaptation to ephemeral aquatic environments through embryonic diapause.

An annual life cycle is characterized by growth, maturity, and reproduction condensed into a single, short season favourable to development, with production of embryos (seeds, cysts, or eggs) capable of surviving harsh conditions which juveniles or adults cannot tolerate. More typically associated with plants in desert environments, or temperate-zone insects exposed to freezing winters, the evolution of an annual life cycle in vertebrates is fairly novel. Killifish, small sexually dimorphic fishes in the Order Cyprinodontiformes, have adapted to seasonally ephemeral water bodies across much of Africa and South America through the independent evolution of an annual life history. These annual killifish produce hardy desiccation-resistant eggs that undergo diapause (developmental arrest) and remain buried in the soil for long periods when fish have perished due to the drying of their habitat. Killifish are found in aquatic habitats that span a continuum from permanent and stable to seasonal and variable, thus providing a useful system in which to piece together the evolutionary history of this life cycle using natural comparative variation. I first review adaptations for life in ephemeral aquatic environments in killifish, with particular emphasis on the evolution of embryonic diapause. I then bring together available evidence from a variety of approaches and provide a scenario for how this annual life cycle evolved. There are a number of features within Aplocheiloidei killifish including their inhabitation of marginal or edge aquatic habitat, their small size and rapid attainment of maturity, and egg properties that make them particularly well suited to the colonization of ephemeral waters.