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Embryonic and adult stem cells enable development and regeneration. Embryonic cells, like adult stem cells, can enter dormancy as part of their lifecycle. Recent evidence suggests that this cellular transition to dormancy requires active rewiring of metabolism. The dormancy-induced metabolic switches in embryonic and adult stem cells are explored here. [ABSTRACT FROM AUTHOR]

An alarming number of large mammalian species with low reproduction rates is threatened with extinction. As basic knowledge of reproductive physiology is currently lacking in many species, increasing the understanding of reproductive physiology is imperative and includes the development of novel artificial reproduction technologies. Despite the relatively comprehensive knowledge on molecular mechanisms underlying reproduction in livestock species such as cattle, pregnancy failures are likewise far from understood. Contrary to other wildlife species, the European roe deer (Capreolus capreolus) displays a remarkably high pregnancy rate. In parts, cattle and roe deer exhibit comparable features of preimplantation embryo development. Therefore, understanding the high fertility rate in the roe deer holds a great potential for cross-species knowledge gain. As the only known species among the artiodactylae, the roe deer displays a long period of embryonic diapause. The preimplantation blastocyst reaches a diameter of 1 mm only at around 4 months compared to around 13 days post estrus in cattle. The expanded blastocyst survives in a uterine microenvironment that contains a unique set of yet unidentified factors that allow embryonic stem cells to proliferate at low pace without impairing their developmental potential. Upon reactivation, intimate embryo-maternal communication comparable to those reported in cattle is thought to occur. In this review, current knowledge, parallels and differences of reproductive physiology in cattle and roe deer are reviewed. The roe deer is proposed as a unique model species to (1) enhance our knowledge of fertility processes, (2) define factors that support embryo survival for an extended period, (3) advance knowledge on embryonic stem cells, and (4) unravel potential implications for the development of novel strategies for artificial reproductive technologies. [ABSTRACT FROM AUTHOR]

Blue-breasted quail has been recognized as a potential model animal. The aim of this study is to investigate the low-temperature-induced embryonic diapause in blue-breasted quail. To this end, the early embryonic staging in blue-breasted quail was briefly described and various incubation temperatures were tested. While the embryonic diapause in early blue-breasted quail embryos can be induced when the eggs were stored at 21°C, a lower temperature such as 16°C yielded a significantly better hatchability (P = 0.0231). Additionally, prolonged storage duration from 3, 7 to 14 d significantly reduced the hatchability (P < 0.0001). Visual examination on the unhatched eggs revealed that reduced hatchability in prolonged storage was significantly correlated with embryonic mortality during the first half of incubation period (R2 = 0.9999, P = 0.0055). High-throughput RNA sequencing with de novo assembly showed that a gene network cluster consisted of ND4, ND5, ND6 , and COX3 , which are components of mitochondrial respiratory complexes, was down-regulated in the cold-stored embryos, while a stress-responsive gene network cluster consisted of JUN, ATF3 , and DUSP1 was up-regulated. Accordingly, cell death in the blastoderm was significantly increased as the storage duration prolonged from 3 to 10 d. Taken together, our study provided basic information on the temperature-induced embryonic diapause in blue-breasted quail. Furthermore, transcriptomic analysis sheds light for the molecular basis on how blastoderm cells respond to the prolonged cold-stress and stay diapause. [ABSTRACT FROM AUTHOR]

• LY294002 inhibited phosphorylation of GSK-3β and Akt in Bombyx eggs. • Changes in Akt phosphorylation were different between diapause and developing eggs. • Changes in bombyxin-Z1 gene expression coincided with Akt phosphorylation. • Bombyxin/Akt signaling is related to Bombyx embryonic diapause process. Our previous study showed that phosphorylation of glycogen synthase kinase (GSK)-3β is related to the embryonic diapause process in Bombyx. However, the upstream signaling pathway was not clearly understood. In the present study, we examined bombyxin/Akt signaling in relation to the embryonic diapause process of B. mori. Results showed that GSK-3β phosphorylation stimulated by dechorionation was blocked by LY294002, a specific phosphatidylinositol 3-kinase (PI3K) inhibitor, indicating involvement of PI3K in GSK-3β phosphorylation in dechorionated eggs. Direct determination of Akt phosphorylation showed that dechorionation stimulated Akt phosphorylation. The Akt phosphorylation was blocked by LY294002. Temporal changes in Akt phosphorylation showed that different changing patterns exist between diapause and developing eggs. Relatively higher phosphorylation levels of Akt were detected between days 3 and 5 after oviposition in non-diapause eggs compared to those at the same stages in diapause eggs. Upon treatment with HCl, which prevents diapause initiation, Akt phosphorylation levels exhibited a later and much broader peak compared to diapause eggs. Examination of expression levels of the bombyxin-Z1 gene showed that in diapause eggs, a major peak occurred 1 day after oviposition, and its level then sharply decreased on day 2. However, in both non-diapause and HCl-treated eggs, a major broad peak was detected between days 1 and 4 after oviposition. These temporal changes in bombyxin-Z1 gene expression levels during embryonic stages coincided with changes in Akt phosphorylation, indicating that bombyxin-Z1 is likely an upstream signaling component for Akt phosphorylation. Taken together, our results indicated that PI3K/Akt is an upstream signaling pathway for GSK-3β phosphorylation and is associated with the diapause process of B. mori eggs. To our knowledge, this is the first study to demonstrate the potential correlation between bombyxin/Akt signaling and the embryonic diapause process. [ABSTRACT FROM AUTHOR]

Abstract Embryonic diapause occurs in over 130 species of mammals. In embryonic diapause, there is a reversible cessation phase in embryo development and subsequent reactivation and continuation of development with no adverse effects. This allows animals to prolong gestation and give birth to offspring at a more favorable time from a survival perspective. Embryonic diapause is divided into two types: facultative diapause which is induced by physiological conditions and obligate diapause which is induced by season of the year. The process of embryonic diapause includes three phases: onset, maintenance, and reactivation. With facultative diapause the suckling stimulus promotes secretion of prolactin, which suppresses development and secretion activity of the corpus luteum (CL). The decrease of progesterone, secreted by CL, subsequently results in embryos in the diapause state. The embryonic diapause that occurs in mink is of the obligate type and is regulated by photoperiod. The short daylight (<12 h) before the vernal equinox induces an increase in the release of melatonin from the pineal gland. The secretion of prolactin is suppressed and the production of progesterone from CL is reduced. During these phases, many others factors that regulate embryonic and uterine status, hormonal profile, polyamines, and uterine secretion, have been monitored. In recent times, the understanding of the role of different molecular events in embryonic diapause has been advanced. The present review summarizes the research advances related to embryonic diapause to enhance the understanding of this phenomenon and to be considered when there are future research endeavors related to embryonic diapause. [ABSTRACT FROM AUTHOR]

Proper control of the cellular processes requires a variety of regulatory proteins that are involved in the cell cycle, proliferation and apoptosis. Cyclin-dependent kinase inhibitor (CKI) negatively regulates transcription and arrests the cell cycle in G 1 phase. KIP2 is a member of CKI family, which could inhibit proliferation by tight-binding with several cyclin-CDK complexes. During the embryonic development of the brine shrimp, Artemia sinica , KIP2 plays a key role in the cell cycle regulation, but the specific mechanisms remain unknown. Herein, the 1023 bp full-length cDNA of kip2 from A. sinica was cloned. The mRNA expression patterns of As-kip2 , As-carp-1 in different development stages and pattern of As-kip2 under environmental stresses were investigated. In situ hybridization of As-kip2 mRNA and immunofluorescence of As- CARP-1 protein showed no tissue or organ specificity. Furthermore, western blotting showed the expressions levels of As -KIP2, As -E2F1, As -p53, As -cyclin E, As -SODD protein, and pattern of As- KIP2 under environmental stresses. Our research revealed that As -KIP2 plays crucial role in the restarting process of diapause embryo in Artemia sinica . [ABSTRACT FROM AUTHOR]

[Display omitted] • The expression levels of BmINR and BmAC6 are significantly higher in NDEPs compared to DEPs. • Knockdown of BmINR upregulates the 3-hydroxykynurenine synthesis pathway and results in a change in the diapause phenotype. • Overexpression of BmINR or BmAC6 highly upregulates the expression levels of key genes related to carbohydrate metabolism. • BmINR and BmAC6 play a crucial role in regulating embryonic diapause via the insulin/IGF signaling pathway. Diapause of the silkworm (Bombyx mori) is an important ecological adaptation strategy regulated by multiple signaling pathways. As an evolutionarily conserved signaling pathway, the insulin/IGF signaling (IIS) pathway is essential in regulating lifespan, energy accumulation, and stress resistance in diapause insects. However, the regulatory mechanism of IIS on diapause in B. mori is still not fully understood. To investigate the role of the IIS pathway in regulating diapause, we first analyzed the transcription levels of the insulin receptor (BmINR) and its downstream gene adenylate cyclase 6 (BmAC6). The diapause-terminated eggs of a bivoltine strain QiuFeng (V2-QF) were incubated at 25 °C in natural room light for preparing diapause egg producers (DEPs) and at 17 °C in total darkness for preparing non-diapause egg producers (NDEPs), respectively. Then we investigated the effects of BmINR and BmAC6 on diapause phenotype and expression of diapause-related genes by RNA interference (RNAi) and overexpression techniques. The results showed that the mRNA expression levels of BmINR and BmAC6 in the head and ovary of NDEPs were higher than those in DEPs during the early and middle pupal stages. Furthermore, when BmINR was knocked down in the NDEPs, approximately 14.43% of eggs were in light red color and subsequently changed into gray-purple color after 48 h post-oviposition, then stayed in a diapause state. On the other hand, overexpression of BmINR or BmAC6 via recombinant baculoviruses did not cause any obvious phenotypic alterations in NDEPs, but it upregulated the expression of genes related to carbohydrate metabolism, which provides energy for embryonic growth and development. Therefore, it can be concluded that BmINR and BmAC6 genes regulate embryonic diapause in bivoltine B. mori. [ABSTRACT FROM AUTHOR]

DNA parentage testing using microsatellite markers confirmed that a Thoroughbred foal conceived from a mare that was bred with two different stallions at an interval of 22 days in successive normal estrus cycles was not sired by the last mated stallion. The results of the stallions were confirmed by retesting DNA parentage test and video recording during the mating sessions. Clinical examinations excluded the absence of an embryo in the uterus after the first mating but confirmed the presence of twin embryos after the last mating, despite confirmation of only one ovulation. One of the twin embryos was crushed by hand, and the other embryo was normally delivered at 332 days after the last mating (354 days after the first mating). Furthermore, to identify similar cases in Thoroughbred mares, we retrospectively reviewed all breeding records over the previous 16 years in Japan and identified three similar cases among 7,787 foaled mares by stallion change. Because the mating interval was approximately one estrus cycle in these cases, pregnancy seemed unlikely to have resulted from prolonged survival of the sperm. Embryonic diapause characterized by delayed implantation of an embryo after the first mating should be strongly considered for proper identification in Thoroughbred lines. [ABSTRACT FROM AUTHOR]

To better understand the molecular mechanisms of diapause initiation, we used the sensitive cDNA subtraction (selective amplification via biotin- and restriction-mediated enrichment) method and isolated a novel gene expressed abundantly in diapause eggs of the silkworm, Bombyx mori, which encodes a homolog of the human oxidation resistance 1 (OXR1) protein. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting analyses confirmed that BmOXR1 mRNA and its 140-kDa protein were differentially expressed in diapause eggs compared to non-diapause eggs. OXR1 double-stranded RNA (dsRNA) was injected into diapause-destined eggs before the cellular blastoderm stage, and 4days later, when untreated eggs reached the diapause stage, the OXR1 protein disappeared; however, these eggs remained in diapause, suggesting that BmOXR1 is not essential for diapause initiation and/or maintenance. To further investigate the in vivo function of BmOXR1 apart from its role in diapause, we overexpressed BmOXR1 in Drosophila melanogaster. The fruit fly male adult life-span was significantly extended in the 50%-survival time when adults were reared on diets both with and without H2O2 solution under 25°C incubation. These results suggest that BmOXR1 functions in D. melanogaster via a possible antioxidant effect. As BmOXR1 was expressed mainly in the nuclei of D. melanogaster cells, the mechanism underlying its antioxidation effect appears to be different from that in humans where it is expressed mainly in the mitochondria. Taken together, these results suggest that BmOXR1 might serve as an antioxidant regulator during the early diapause stage. [ABSTRACT FROM AUTHOR]

Abstract: To understand the molecular mechanisms of how 5°C-incubation activates mRNA expression of Hsp70a and Samui genes in Bombyx mori diapause eggs, we first searched the 5′-upstream regions of the Hsp70a and Samui genes for heat shock elements (HSEs) and found two regions [Hsp70aHSE-1 (−95 to −58) and -2 (−145 to −121), and SamuiHSE-1 (−84 to −55) and -2 (−304 to −290)] corresponding to HSEs (repeats of nGAAn and/or nTTCn). We cloned four cDNAs encoding heat shock factor (HSF)-a2 (627 amino acids), -b (685 aa), -c (682 aa) and -d (705 aa), which were produced by alternative splicing. When we exposed diapause eggs to 5°C beginning at 2 day post-oviposition to break diapause, HSFd mRNA only increased after chilling for 6–8 days, a pattern very similar to those of Hsp70a and Samui mRNAs. To examine further whether HSFd binds to the respective HSEs, we carried out a gel shift assay using HSFd protein expressed in a cell-free system and the isolated HSEs; migration of the respective digoxigenin(DIG)-labeled HSE-1 and -2 of Hsp70a and Samui was retarded by addition of HSFd; the retarded bands disappeared after addition of the corresponding unlabeled HSE-1 and -2 as competitors, but were not affected by addition of the respective mutated unlabeled HSE-1 and -2. These results indicated that HSFd protein binds to the respective HSEs and may activate mRNA expression of Hsp70a and Samui genes upon exposure of diapause eggs to 5°C. [Copyright &y& Elsevier]

Abstract: Phosphorylation of the translational repressor 4E-binding protein (4E-BP) plays a critical role in regulating the overall translation levels in cells. In the present study, we investigated 4E-BP phosphorylation of Bombyx mori eggs by an immunoblot analysis of a conserved phospho-specific antibody to 4E-BP and demonstrated its role during embryonic development. When HCl treatment was applied to diapause-destined eggs at 20h after oviposition, a dramatic increase in the phosphorylation of 4E-BP occurred 5min after treatment with HCl, and high phosphorylation levels were maintained throughout embryonic stage in HCl-treated eggs compared to those in diapause (control) eggs. When HCl treatment was applied to diapause eggs on day 10 after oviposition, no dramatic activation in 4E-BP phosphorylation occurred, indicating stage-specific effects of HCl treatment. In both non-diapause eggs and eggs whose diapause had been terminated by chilling of diapausing eggs at 5°C for 70 days and then were transferred to 25°C, high phosphorylation levels of 4E-BP were also detected. Moreover, 4E-BP phosphorylation dramatically increased when dechorionated eggs were incubated in medium. The addition of rapamycin, a specific inhibitor of mammalian target of rapamycin (TOR) signaling, and LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor, but not the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor, U0126, dose-dependently inhibited 4E-BP phosphorylation in dechorionated eggs, indicating that PI3K/TOR signaling is an upstream signaling event involved in 4E-BP phosphorylation. Examination of 4E-BP gene expression levels showed no differences between treatments with HCl and water in the first hour after treatment, indicating that changes in phosphorylation of 4E-BP upon HCl treatment are mainly regulated at the post-transcriptional level. In addition, MAPK pathways and glycogen synthase kinase (GSK)-3β phosphorylation were not significantly affected in the first hour after HCl treatment. These results demonstrate that the rapid phosphorylation of 4E-BP is an early signaling event in embryonic development in the eggs whose diapause initiation was prevented by HCl treatment, thus being involved in the embryonic development of B. mori. [Copyright &y& Elsevier]

Abstract: We previously cloned a cDNA for sorbitol dehydrogenase (SDH1) from Bombyx mori. In the present study we cloned two additional cDNAs encoding SDHs (designated as SDH2a and SDH2b). The amino acid sequences of SDH2ab were almost the same and had higher similarity to the SDHs of other organisms than to B. mori SDH1. The SDH2ab and SDH1genes were located in tandem within about 40kbp on chromosome 21. SDH2ab mRNAs increased after exposing diapause eggs to 5°C for 40 days, beginning at 2 days post-oviposition, to break diapause. However, they were at very low levels in diapausing eggs incubated at 25°C continuously from oviposition. These changes in expression pattern of SDH2ab mRNA were almost the same as that of SDH1 mRNA. To understand whether SDH1 and SDH2 were responsible for the SDH activity seen in diapause eggs exposed to 5°C for more than 60 days, we expressed a His-tagged SDH2a fusion protein in Escherichia coli and examined its enzymatic parameters. The maximum activity of SDH2a observed at pH 8.4∼9.0, and the Km value for sorbitol was 12.6mM, similar to the kinetic properties of other SDHs. Due to the significantly higher similarity between SDH2a and b, they were thought to have similar kinetic properties. Therefore, we purified SDH from B. mori diapause-terminated eggs exposed to 5°C for 300 days which showed higher SDH activity using two-step affinity chromatography. The highly purified SDH showed a higher Km value (125mM) for sorbitol, being similar to the value (136mM) determined previously from Eadie-Hofstee plots using egg crude extract as an enzyme source; additionally, the plots showed one slope indicating one Km value. Moreover, in silico analysis indicated that no SDH genes other than SDH1 and 2ab are present in B. mori genomic DNA. These results suggest that SDH1 activity may be responsible for the majority of the increased SDH activity seen in diapause eggs after acclimation to 5°C rather than SDH2ab. Further, the relative sequence divergence among these genes is consistent with the idea/hypothesis that the original SDH gene was first duplicated into SDH1 and SDH2, and then SDH2 was duplicated into the SDH2a and SDH2b genes. [Copyright &y& Elsevier]

Abstract: Bombyx mori eggs enter diapause immediately after completion of mesoderm segregation. HCl treatment of approximately 24-hour-old eggs (germband formation stage) is well known to be the most effective procedure to prevent entry into diapause, although the molecular mechanism remains unclear. In this study, we examined the protein composition of diapausing and nondiapausing eggs after various HCl treatments known to prevent or break diapause and found that proteins of approximately 11 and 8kDa disappeared immediately after HCl treatment. Partial amino acid sequences of these proteins indicated that they were members of the chorion class A protein L12 family synthesized in follicle cells. Under the hypothesis that the chorion provides a barrier to oxygen, dechorionation of diapausing eggs induces resumption of embryonic development. Hence, to test this and other hypotheses about the function of these proteins, we used 20% SDS-PAGE with Coomassie Brilliant Blue staining to trace their disappearance from embryos and eggshells after treatment with HCl under different conditions and on polyvoltine, univoltine, and bivoltine silkworm races. Even when 10-day-old diapausing eggs were treated with HCl, which did not break diapause, the 11 and 8kDa proteins disappeared. Our results suggest that disappearance of these proteins is not directly associated with preventing entry into or breaking a diapause state. Nevertheless, our results cannot completely rule out the possibility that the 11 and 8kDa proteins function to block permeability of O2 during the period when HCl treatment is physiologically effective to prevent diapause so that after the diapause system is established within the egg, even removing the 11 and 8kDa proteins may not affect to prevent diapause. We also discuss the role of these proteins in choriogenesis. [ABSTRACT FROM AUTHOR]

Abstract: In an effort to understand whether heat shock protein 70 (Hsp70) participates in the environmental 5°C signal reception/transduction toward breaking embryonic diapause of the silkworm Bombyx mori, we isolated a cDNA for Hsp70a and examined the expression of Hsp70a mRNA in B. mori diapause and nondiapause eggs by quantitative real-time PCR. Hsp70a mRNA gradually increased in diapause eggs continuously kept at 25°C after oviposition to maintain diapause. When diapause eggs were exposed to the diapause-terminating condition of 5°C beginning at 2 days post-oviposition, Hsp70a mRNA increased beginning at 5 days post-cold treatment. Even in nondiapause eggs, Hsp70a mRNA increased slightly with exposure to 5°C. These results suggest that Hsp70a is involved in reception/transduction of the diapause-terminating (5°C) signal via gene activation. The expression patterns of Hsp70a mRNA are discussed in relation to those of the cold-response gene Samui. [Copyright &y& Elsevier]

Abstract: With initiation of diapause, glycogen is converted into sorbitol and glycerol in eggs of the silkworm Bombyx mori. At diapause termination promoted by incubation at 5 °C, sorbitol and glycerol are utilized. Although sorbitol utilization is triggered by NAD-sorbitol dehydrogenase induced by acclimation to 5 °C, the initial enzyme utilizing glycerol remains unclear. In this study, we detected glycerol kinase activity in diapause-terminated eggs and then characterized its properties; maximal activity was seen at pH 8.5–9.0, and Km values for glycerol and ATP were 0.32 and 0.095 mM, respectively. In diapause eggs continuously kept at 25 °C, the activity was almost negligible. However, activity was seen after chilling for 60 days and thereafter increased when the eggs were exposed to 5 °C after 2 days post-oviposition, indicating that glycerol kinase is a rate-limiting enzyme in glycerol utilization. We then cloned cDNAs encoding glycerol kinase-1, -2 and -3 from B. mori. Only gene expression of glycerol kinase-3 was up-regulated in diapause eggs exposed to 5 °C, indicating that glycerol kinase activity is regulated via transcription of the glycerol kinase-3 gene. [Copyright &y& Elsevier]

Abstract: Glycogen synthase kinase-3 (GSK-3) is a multifunctional protein kinase that plays important roles in regulating both glycogen synthesis and protein synthesis. In the present study, we investigated GSK-3β phosphorylation of silkworm eggs by immunoblotting with a conserved phospho-specific antibody to GSK-3β. Results showed that the temporal changes in GSK-3β phosphorylation were closely related to changes in glycogen levels previously reported by other researchers. In diapause eggs, an abrupt decrease in phosphorylation of GSK-3β was found with the onset of diapause, and phosphorylation level of GSK-3β reached a minimum level within 1 week after oviposition. However, when diapause eggs were incubated at 25°C for 15 days and then transferred to 5°C, a great increase in GSK-3β phosphorylation was observed 5 days after transfer to 5°C and high levels were maintained throughout the chilling period. In both non-diapause eggs and eggs whose diapause initiation was prevented by HCl, levels of GSK-3β phosphorylation appeared to remain relatively high for several days and then greatly decreased 2 or 3 days before hatching. Moreover, GSK-3β phosphorylation dramatically increased when dechorionated eggs were incubated in medium. The addition of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor, U0126, did not inhibit GSK-3β phosphorylation in dechorionated eggs, although U0126 dose-dependently inhibited ERK phosphorylation. This result showed that ERK phosphorylation is not involved in upstream signaling for GSK-3β phosphorylation and that there may be two distinct signaling pathways involved in diapause processing in Bombyx mori eggs. [Copyright &y& Elsevier]

Abstract: Incubation of northern snake-necked turtle (Chelodina rugosa) eggs and subsequent sale of hatchlings for the pet industry has the potential to provide culturally suitable employment for indigenous communities in northern Australia. Developmental arrest in response to egg inundation is unique to C. rugosa. Eggs can be stored under water for up to 10 weeks without appreciable impact on egg or embryo survival, allowing the transport and sale of eggs into niche markets without high levels of mortality, and permitting eggs to accumulate in diapause until there are sufficient numbers to incubate as batches. Eggs that are not inundated or inundated for short periods experience similar survival rates to eggs inundated for lengthy periods. Incubation temperature influences embryo survival and development period in C. rugosa. Embryonic survival is greatest at 26 °C, steadily declining as temperature increases to 32 °C. A similar increase in incubation temperature decreases incubation period by approximately 40 days, however almost half of this variation is attributed to the increase in incubation temperature from 26 to 28 °C. Hatchling growth in C. rugosa is characterized by two phases. There is an initial phase of relatively slow growth under the partial influence of initial egg size and incubation duration, followed by a second phase of relatively rapid growth under the partial influence of water temperature and mass at hatching. Post-hatching survival is negatively correlated with duration of egg inundation and water temperature. Evidence suggests that inundation of C. rugosa eggs for 6 weeks, incubation of embryos at 28 °C and raising hatchlings in 28 °C water will yield the best overall outcomes. [Copyright &y& Elsevier]

Abstract: To understand the hormonal control of embryonic diapause, juvenile hormone analogs (JHAs), methoprene and hydroprene, and 20-hydroxyecdysone (20E) were applied onto diapause eggs of Locusta migratoria and Oxya yezoensis. These insects enter diapause at the mid-stage of embryogenesis prior to blastokinesis. Topical application of JHAs significantly facilitated diapause termination in both species but JHA-treated embryos underwent abnormal morphogenesis, pigmentation and sclerotization without dorsal closure. The Locusta eggs immersed in the 20E solution for 24h terminated diapause in a dose-dependent manner. We also investigated phosphorylation of extracellular signal-regulated kinase (ERK), a member of mitogen-activated protein kinase (MAPK), during diapause-terminating process of Locusta migratoria and found that ERK was activated either by cold exposure or JHA treatment. The possible involvement of the hormones and ERK in embryonic diapause and the possibility of ecdysteroids synthesis by prothoracic glands of diapause embryo were proposed. [Copyright &y& Elsevier]

Abstract: A prediction for the onset of a summer diapause in the eggs of the redlegged earth mite, Halotydeus destructor, was developed for Australia. In this species diapause eggs pass the summer in the cadavers of adult female mites. Adult female mites were collected for several weeks from pastures in spring at 18 sites in south-western Australia and dissected to determine the timing of the production of diapause eggs. Some sites were sampled for several years between 1990 and 1997. A model was developed to predict the time for onset of diapause. The week at which 90% of eggs were in diapause was predicted best by daylength (80.1% of the variability), then by duration of the long-term plant growing season (10.4%, of variability), leaving 9.5% due to other factors. A single chemical spray in spring 2 weeks before the production of 90% diapause eggs resulted in 99% fewer mites present in autumn 7–8 months later at three sites. The timing of the spring spray was the factor leading to successful control. This model was tested at 17 sites across the whole geographical distribution of the redlegged earth mite in Australia between 1998 and 2001. The observed week of 90% diapause was within 1 week of the predicted week on 81% of occasions, and 2 weeks earlier on 15% of occasions. A database was created for the predicted date of onset of 90% diapause for the whole distribution of the redlegged earth mite in southern Australia on a 10km2 grid. Australian farmers are using this for timing a spring spray to control mites in the following autumn. [Copyright &y& Elsevier]

In the Ibaraki population (Japan) of Locusta migratoria, adult locusts produce diapause eggs under short-day (SD) conditions and non-diapause eggs under long-day (LD) conditions. The identity and titre of ecdysteroids in the ovaries and eggs from LD and SD adult females were investigated by RIA/HPLC. Maternal ecdysteroids accumulated in the developing ovaries represented about 90% polar conjugates, 5% free ecdysteroids and 5% non-hydrolyzable metabolites. Before oviposition the quantity of ecdysteroids reached 29.8±1.85 ng 20-hydroxyecdysone equiv. per mg tissue ovaries from LD females and 13.1±3.55 ng 20E equiv./mg in ovaries from SD females. The sum of RIA-positive materials in newly laid eggs was more than three times higher in non-diapause eggs than in diapause eggs. Ecdysteroids present in egg extracts comprised about 85% polar conjugates, 5% free ecdysteroids and 10% non-hydrolyzable metabolites. On the other hand, after diapause termination the amount of ecdysteroids increased drastically. Also, the composition of ecdysteroids differed from that observed during diapause and became comparable to that of non-diapause eggs. The significant differences in the ecdysteroids between non-diapause and diapause eggs may suggest the possible involvement of these compounds in the control of embryonic diapause of this locust. [ABSTRACT FROM AUTHOR]

We report a possible case of extended gestation in the koala, Phascolarctos cinereus. Birth of a pouch young was first observed 127 days after the removal of the male from a multi-female colony at Taronga Zoo. No other males were present at that time or had access to the facility. Head measurements and other growth data collected at the time of detection and over the period of pouch life indicates the time from removal of the male and the date of birth to be between 50 and 77 days. DNA fingerprinting using microsatellite loci unambiguously assigned paternity of the pouch young to this male.These observations suggest either an extended period of gestation of at least 50 days, or activation of a dormant blastocyst from the previous breeding season, as the female entered the period of seasonal oestrus. [Copyright &y& Elsevier]

Mink embryonic diapause occurs when embryos, at the blastocyst stage, enter a state of a reversible arrest in development and metabolism. Some ovarian factors are required because ovariectomy leads to prevention of implantation in mink. Mechanisms regulating this process, however, remain largely unknown. To explore ovarian modifications associated with emergence of embryonic diapause in mink, there was comparison of transcriptomes after embryonic activation to when there was embryonic diapause using RNA-sequencing. A library of 655 differentially expressed genes (DEGs) of all assembled 33,656 genes was generated. Among these, 558 genes were annotated with 106 genes being expressed to a greater extent in ovaries during embryonic diapause, whereas 452 genes were more abundantly expressed in ovaries after embryonic activation. The major categories of genes with differential transcript abundances include metabolic pathways, metabolism of tryptophan, tyrosine and vitamin B6, oxidoreductase activity, calcium signaling pathway, steroid biosynthesis and lysosome. The APOE and APOA1 hub genes identified through the protein-protein interaction (PPI) analysis have important functions in cholesterol transport and steroidogenesis. Transcript abundances associated with 39 genes were investigated using RT-qPCR procedures to confirm RNA-sequencing data. Of 29 mRNA transcripts, 26 were validated using RNA-sequencing, whereas three of ten indistinguishable genes determined using RNA-sequencing were confirmed. Most of these verified DEGs are involved in the prolactin signaling pathway, formation of functional corpora lutea, and steroid synthesis, suggesting these biological processes are implicated in embryonic reactivation. Overall, results provide new insights into ovarian signaling at the time of emergence of the blastocyst from diapause in mink. [ABSTRACT FROM AUTHOR]

• PKC-dependent phosphorylation was examined in different silkworm eggs. • A higher level was found in developing eggs compared to diapause eggs. • A higher PKC activity was detected in developing eggs compared to diapause eggs. • PKC signaling appears to be related to Bombyx embryonic development. In the present study, we investigated the expression of protein kinase C (PKC) signaling during the embryonic diapause process of Bombyx mori. PKC activity, determined using an antibody to phosphorylated substrates of PKC, was found to be significantly higher in developing eggs as compared to that of diapause eggs. In eggs whose diapause initiation was prevented by HCl, non-diapause eggs, and eggs in which diapause had been terminated by chilling of diapausing eggs at 5 °C for 70 days and then were transferred to 25 °C, PKC-dependent phosphorylation levels of multiple proteins showed dramatic stage-dependent increases compared to those of diapause eggs. Higher protein levels of PKC were also detected in developing eggs as compared to those of diapause eggs. Determination of PKC enzymatic activity during the middle embryonic stage showed higher PKC activity in developing eggs compared to diapause eggs, thus further confirming differential regulation of PKC signaling during the embryonic diapause process. Examination of temporal changes in mRNA expression levels of classical PKC (cPKC) and atypical PKC (aPKC) showed no difference between diapause and HCl-treated eggs. These results demonstrated that differential expressions of PKC signaling between diapause and developing eggs are related to the embryonic diapause process of B. mori. [ABSTRACT FROM AUTHOR]