Nucleolar size in parallel with ribosomal RNA synthesis at diapause termination in the eggs of Bombyx mori

The eggs of Bombyx mori, both in diapause and nondiapause, were subjected to cytological examination of nucleoli and measurement of RNA precursor incorporation (2 hours) into ribosomal RNA. In diapause eggs, the nucleoli were very small and the rate of ribosomal RNA synthesis was the lowest of the samples tested. Most cells in diapause possessed nuclei with one nucleolus. In contrast, the eggs activated from diapause by long chilling attained the largest size of nucleoli and the highest rate of ribosomal RNA synthesis. A significant proportion of the cell nuclei still had only one nucleolus at this stage. Three days after activation, the eggs exhibited intermediate levels in both the size of nucleoli and the rate of ribosomal RNA synthesis. At this stage, about half of the egg cell nuclei had two nucleoli.This paper is dedicated with respect and admiration to Professor D.F. Poulson, who has made many significant contributions on the genetics, embryology and cytology of Drosophila as well as other Insecta, in commemoration of his retirement     

Diapause development in Bombyx eggs in relation to ‘esterase A’ activity

Diapause is broken by hydrochloric acid treatment and also terminated by long chilling of eggs in the silkworm, Bombyx mori. One of esterases in silkworm eggs named ‘esterase A’ is closely related to diapause of this insect.Hydrochloric acid treatment of diapause eggs induced a prompt elevation of esterase A activity. The elevation was observed within 30 min after treatment. This HCl treatment effectively stimulated the eggs to hatch. This indicates that the increase of esterase A activity is correlated with an active resumption of morphogenesis.The question was examined of whether chilling also increases esterase A activity or not. It was found that chilling also caused an increase of esterase A activity. This increase occurred before the re-appearance of glycogen in eggs, which indicates the termination of diapause in this insect. In fact, the establishment of hatchability in chilled eggs was observed after esterase A activity has reached the maximum level. Thus the increase of esterase A activity could be regarded as associated with the termination of diapause per se but not with the subsequent process of post-diapause development. This change in esterase A activity was observed only in chilled diapause eggs and was not observed in diapause eggs without chilling and non-diapause eggs.These results suggest that the increase of esterase A activity during chilling may be a kind of activity that occurs during the diapause stage in preparation for resumption of morphogenesis or diapause development.     

Development of the large nucleolus in the oocytes of the copepod Acanthocyclops vernalis: an electron microscope study

A central feature of oogenesis in the copepod crustacean, Acanthocyclops vernalis, is the development of a very large nucleolus in the oocytes. This nucleolus appears to be the only source of rRNA for the oocyte, as no helper cells are present. Previous work has suggested that ribosomal DNA sequences other than those found at the morphological nucleolar organizers are participating in the elaboration of this nucleolus. It has been hypothesized that chromatin diminution, which occurs during early embryonic development, may involve the loss of these rDNA sequences, which are needed only for the production of ribosomes during oogenesis. The present study examines the development of the large oocyte nucleolus at the electron microscopic level. Nucleologenesis in A. vernalis was found to proceed through 5 stages. During the first 3 stages nucleolar morphology resembled that described in other organisms. In the last 2, however, nucleolar morphology changed radically and the nucleolus was seen to increase greatly in size while breaking up into multiple subunits. The subunits initially resemble active nucleoli, although in the last stage, synthesis appears to stop, as the nucleolus was found to consist only of dense areas containing ribosome-like particles. These observations are consistent with the hypothesis that diminuted DNA contains ribosomal RNA genes.     

Cellular DNA replication is independent of the synthesis or accumulation of ribosomal RNA

We have used an antibody against RNA polymerase I to investigate the role of rRNA synthesis and/or accumulation in the control of cell proliferation. The antibody was microinjected directly into the nuclei of quiescent Swiss 3T3 cells that were subsequently stimulated with serum. Under the experimental conditions used, the microinjection of the antibody against RNA polymerase I (RNA pol I) caused a 50–70% decrease in nucleolar RNA synthesis that lasted for at least 17 h, a >90% inhibition in the accumulation of nucleolar RNA, and a 70% inhibition in the accumulation of total cellular RNA. A control IgG, similarly microinjected into Swiss 3T3 cells had no inhibitory effect on either the synthesis or accumulation of nucleolar and cellular RNA. Despite the dramatic effect on the synthesis and accumulation of ribosomal RNA (rRNA) the antibody against RNA pol I was totally ineffective in inhibiting the entry into S phase of serum-stimulated Swiss 3T3 cells. Cells depleted of cellular RNA by metaphase arrest also entered S phase with subnormal amounts of cellular RNA. The results of these experiments clearly indicate that a normal rate of nucleolar RNA synthesis, and a normal rate of accumulation of total cellular RNA are not a prerequisite for the entry of cells into S phase.     

中华稻蝗长沙种群的生活史及其卵滞育的进化意义

中华稻蝗Oxya chinensis为重要的水稻害虫,在我国除青海、西藏、新疆、内蒙古等未见报道外,南起海南北至东北均有分布,在许多的分布区域1年发生1代。为探索中华稻蝗长沙种群的生活史及其季节适应特征,通过野外和实验室的研究,调查了其发生代数、孵化率和卵滞育率的季节变化及越冬卵的存活率。结果显示,中华稻蝗长沙种群为1年2代和1年1代混合发生:第1代卵产卵后大部分孵化为若虫而1年完成2代,但亦有19.4% -4.1%的卵不孵化而1年只能完成1世代。第1代成虫于6月上旬至8月上旬羽化,6月下旬至8月中旬产卵;第2代若虫于7月初开始孵化,9-10月羽化为成虫,10月上旬至11月下旬产卵。在室外自然条件下,中华稻蝗长沙种群6-8月(第1代)和10-11月(第2代)所产卵块均为部分滞育,滞育率为30%左右,皆无显著差异。然而,其卵滞育率在12月以后显著降低,仅为6.6%或以下,卵滞育快速地得以解除。因此,包括非滞育卵和滞育解除卵,中华稻蝗长沙种群的越冬卵皆以非滞育状态度过其后的寒冷季节。即使是遭遇长江流域2007年末至 2008年初异常寒冷的冬季,在长沙地区越冬后其卵的存活率亦在98%以上。非滞育状态的中华稻蝗长沙种群越冬卵完全能安全地越冬,其滞育的发生并非是为了提高其耐寒性而安全度过不适环境。并探讨了中华稻蝗长沙种群卵滞育的进化意义。     

A novel RNA helicase-like protein during early embryonic development in silkworm Bombyx mori: molecular characterization and intracellular localization

In order to understand the molecular mechanism of development during early embryogenesis in diapause and non-diapause of the silkworm, mRNA from diapause and non-diapause eggs was compared using the differential display technique. We cloned the full length of a cDNA encoding a novel RNA helicase-like (RHL) protein by the RACE method using a cDNA fragment which was one of the specific cDNAs in the non-diapause eggs. A BLAST search using the predicted amino acid sequence of RHL revealed a low homology (21–25% identity of its partial length) with that of the DEAD-box RNA helicase. Gene expression of the RHL gene of the diapause and non-diapause eggs was investigated by RT-PCR until 60 h after oviposition. Amplified RHL cDNA was observed through all the stages in the non-diapause eggs, while in the diapause eggs, cDNA was found in eggs 0–12 h after oviposition but disappeared 24–60 h after oviposition. When the diapause eggs were activated by HCl treatment after chilling at 4 °C for 6 days from 48 h after oviposition (artificial diapause termination), cDNA was observed from 12 h after HCl treatment. We also investigated the immunohistochemical distribution and localization of RHL in non-diapause eggs using anti-recombinant His-tag RHL antiserum. RHL was distributed in blastoderm cells and yolk cells and was localized in the nucleus and the cytosol of yolk cells. These data suggest that RHL has an important role in the early embryo of the silkworm.     

Ribosomal protein mRNAs increase dramatically during Xenopus development

The amount of messenger RNA per microgram of rRNA increases three- to fourfold during Xenopus early development. This increase is the same when measured by stimulation of in vitro protein synthesis or by poly(U) hybridization. The increase in mRNA per embryo therefore is approximately six- to eightfold since the ribosome content doubles between fertilization and the stage 41 tadpole. The amount of ribosomal protein mRNA, as assayed by in vitro protein synthesis, also increases dramatically during early development. This increase is much more pronounced than the general increase in mRNA content, i.e., there is a dramatic increase in the abundance as well as the amount of the ribosomal protein mRNA. Since ribosomal protein mRNAs are predominantly small mRNAs, the increase in ribosomal protein mRNA abundance contributes to the general decrease in the average size of pA⁺ RNA that occurs during early development in Xenopus.     

The Insect Growth Regulator Pyriproxyfen Terminates Egg Diapause in the Asian Tiger Mosquito,Aedes albopictus

The Asian tiger mosquito, Aedes albopictus, is a highly invasive mosquito species that transmits chikungunya and dengue. This species overwinters as diapausing eggs in temperate climates. Early diapause termination may be a beneficial strategy for winter mosquito control; however, a mechanism to terminate the diapause process using chemicals is not known. We tested the hypothesis that a hormonal imbalance caused by the administration of juvenile hormone analog would terminate egg diapause in A. albopictus. We tested the insect growth regulator pyriproxyfen on all developmental stages to identify a susceptible stage for diapause termination. We found that pyriproxyfen treatment of mosquito eggs terminated embryonic diapause. The highest rates of diapause termination were recorded in newly deposited (78.9%) and fully embryonated (74.7%) eggs at 0.1 and 1 ppm, respectively. Hatching was completed earlier in newly deposited eggs (25–30 days) compared to fully embryonated eggs (71–80 days). The combined mortality from premature diapause termination and ovicidal activity was 98.2% in newly deposited and >98.9% in fully embryonated eggs at 1 ppm. The control diapause eggs did not hatch under diapausing conditions. Pyriproxyfen exposure to larvae, pupae and adults did not prevent the females from ovipositing diapausing eggs. There was no effect of pyriproxyfen on diapausing egg embryonic developmental time. We also observed mortality in diapausing eggs laid by females exposed to pyriproxyfen immediately after blood feeding. There was no mortality in eggs laid by females that survived larval and pupal exposures. In conclusion, diapausing eggs were the more susceptible to pyriproxyfen diapause termination compared to other life stages. This is the first report of diapause termination in A. albopictus with a juvenile hormone analog. We believe our findings will be useful in developing a new control strategy against overwintering mosquito populations.     

Decreased number of nucleolar organizing regions in collagen lattice cultured fibroblasts

Dermal fibroblasts cultivated in tridimensional matrices (lattices) of collagen exhibit a very low metabolic activity, and a low protein synthesis in particular. We have previously shown that ribosomal RNA content and half-life were decreased in collagen lattice cultured fibroblasts when compared to monolayer cultured fibroblasts. In this study, we seeded fibroblasts in collagen lattices and investigated the influence of matrix on the number of nucleolar organizing regions. We found that fibroblasts in fully retracted lattices exhibited a significant decrease of 45 % (P < 0.001) in the number of nucleolar organizing regions when compared to monolayer cultured fibroblasts. This decrease was correlated to the decrease in ribosomal RNA content. These data suggest that extracellular matrix induces early alterations of synthesis and/or processing of ribosomal RNAs, explaining, at least partly, the resulting low metabolic activity.     

Involvement of ERK/MAPK in regulation of diapause intensity in the false melon beetle, Atrachya menetriesi

Embryonic diapause is commonly terminated by exposure to low temperature for a certain duration. Previous studies using the silkworm, Bombyx mori, showed that extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase family, was activated by cold exposure and regulated diapause termination. The involvement of ERK in regulation of diapause termination was investigated in the false melon beetle, Atrachya menetriesi. Embryonic diapause of this beetle is terminated both by cold exposure and by mercury. Phospho-ERK levels remained high during the pre-diapause period but decreased after the eggs entered diapause. Exposure to 7.5 degrees C, which was effective for diapause termination, increased phospho-ERK levels, and these levels were maintained under 7.5 degrees C at least for 100 d. Incubation at 25 degrees C after the eggs were kept at 7.5 degrees C for 20 d, which intensified diapause, decreased the phospho-ERK level. An insufficient cold treatment, i.e., incubation at 0 degrees C for diapause termination did not activate ERK. However, incubation at 0 degrees C after cold treatment at 7.5 degrees C, which is effective for diapause termination, induced high phospho-ERK levels. Moreover, mercury treatment also increased phospho-ERK. Therefore, changes in the phospho-ERK level correlated well with diapause intensity. The results suggest that ERK plays a key role in the regulation of embryonic diapause.     

Nucleolar ultrastructure in bovine nuclear transfer embryos

Nuclear transfer experiments in mammals have attempted to reprogram a donor nucleus to a state equivalent to the zygotic one. Reprogramming of the donor nucleus is, among other features, indicated by a synthesis of ribosomal RNA (rRNA). The initiation of rRNA synthesis is simultaneously reflected in nuclear morphology as a transformation of the nucleolus precursor body into a functional rRNA synthesising nucleolus with a characteristic ultrastructure. We examined nucleolar ultrastructure in bovine in vitro produced (control) embryos and in nuclear transfer embryos reconstructed from a MII phase (nonactivated) or S phase (activated) cytoplasts. Control embryos were fixed at the two-, four-, early eight- and late eight-cell stages; nuclear transfer embryos were fixed at 1 and 3 hr post fusion and at the two-, four-, and eight-cell stages. Control embryos possessed a nucleolar precursor body throughout all three cell cycles. In the eight-cell stage embryo, a primary vacuole appeared as an electron lucid area originating in the centre of the nucleolar precursor body. In nuclear transfer embryos reconstructed from nonactivated cytoplasts, the nuclear envelope was fragmented or completely broken down at 1 hr after fusion and, by 3 hr after fusion, it was restored again. At this time, the reticulated fibrillo-granular nucleolus had an almost round shape. The nucleolar precursor body seen in the two-cell stage nuclear transfer embryos consisted of intermingled filamentous components and secondary vacuoles. A nucleolar precursor body typical for the two-cell stage control embryos was never observed. None of the reconstructed embryos of this group reached the eight-cell stage. Nuclear transfer embryos reconstructed from activated cytoplasts, in contrast, exhibited a complete nuclear envelope at all time intervals after fusion. In the two-cell stage nuclear transfer embryo, the originally reticulated nucleolus of the donor blastomere had changed into a typical nucleolar precursor body consisting of a homogeneous fibrillar structure. A primary vacuole appeared in the four-cell stage nuclear transfer embryos, which was one cell cycle earlier than in control embryos. Only nuclear transfer embryos reconstructed from activated cytoplasts underwent complete remodelling of the nucleolus. The reorganisation of the donor nucleolar architecture into a functionally active nucleolus was observed as early as in the four-cell stage nuclear transfer embryo. These ultrastructural observations were correlated with our autoradiographic data on the initiation of RNA synthesis in nuclear transfer embryos.     

Diapause termination, post-diapause development and reproduction in the beet webworm, Loxostege sticticalis (Lepidoptera: Pyralidae)

Effects of photoperiod and cold exposure on diapause termination, post-diapause development and reproduction in Loxostege sticticalis were examined. Larvae were reared at diapause inducing condition (22 °C, L:D 12:12) consistently or transferred to long day photoperiod (L:D 16:8) and darkness (L:D 0:24) respectively, after entering into diapause. Diapause was terminated in approximately 40% of the larvae after 36 days, and no significant differences were observed between photoperiods, suggesting larval diapause was terminated spontaneously without being induced by photoperiods. Cold exposure significantly hastened diapause termination. The diapause termination incidence increased significantly with peaks of 98% at both 5 °C and 0 °C exposure for 30 days, as compared to 42% in controls not exposed to cold, while the mortality and number of days required for diapause termination decreased dramatically. The optimal low temperature exposure periods under 5 °C or 0 °C were 20 days and 30 days, showing a higher termination incidence and shorter time for diapause termination. This suggests that the low temperatures in winter play an important role in diapause termination under natural conditions. The threshold temperatures for post-diapause development in prepupae and pupae were 9.13 °C and 10.60 °C respectively, with corresponding accumulations of 125 and 200 degree-days. Adults that experienced larval diapause significantly delayed their first oviposition, oviposition period was prolonged, and the lifetime number of eggs laid was decreased, however both males and females have significantly longer longevity. The field validation of diapause termination, the degree-days model, and the relationship between diapause and migration in L. sticticalis were also discussed.     

RNA transcription and translation in sea urchin oocytes and eggs

The steady-state concentrations and absolute rates of synthesis of ribosomal RNA (rRNA) molecules were measured in oocytes, eggs, embryos, and larvae of the Hawaiian sea urchin Tripneustes gratilla. The steady-state concentration per genome of the RNA precursor sequences measured by hybridization to a cloned rDNA fragment was approximately 100- to 300-fold greater in the RNA obtained from oocytes and eggs than in the RNA extracted from embryos and larvae. Since the rate of processing of the rRNA precursor at different stages is not greatly different, the rates of rRNA synthesis must be considerably greater in oocytes than in embryo cells. The absolute rate of RNA synthesis in oocytes and embryos was determined from the incorporation of [³H]guanosine into cellular GTP pools and into both precursor and mature rRNA species. The data indicate an approximately 40-fold higher rate of rRNA synthesis in oocytes than that measured in embryos or previously in larvae (J. Griffith and T. Humphreys, 1979, Biochemistry18, 2178–2185). Together these results indicate that the ribosomal genes are transcribed much more rapidly during sea urchin oogenesis than during embryogenesis or larval stages.     

Enzymatic capacity for accumulation of polyol cryoprotectants changes during diapause development in the adult red firebug, Pyrrhocoris apterus

Abstract. Diapausing adults of the red firebug, Pyrrhocoris apterus, were maintained in the laboratory at constant conditions of 20 °C and short days (LD 12 : 12 h) for 5 months. Spontaneous termination of diapause is seen in 0.4% of adults. At different times of diapause development, groups of adults were exposed to an 8‐week gradual cold treatment ending at 0 °C. Ribitol and sorbitol contents remain very low at constant 20 °C and their rapid accumulation only occurrs at temperatures below 5 °C. The capacity to accumulate ribitol in response to low temperature stimulus remains relatively stable but the capacity to accumulate sorbitol decreases to zero during the 5 months of diapause development. Glycogen (whole‐body and fat‐body reserves) accumulates relatively rapidly during the early phase (up to 1–2 months) and slowly depletes during the late phase of diapause development under constant conditions. Upon cold treatment, part of the glycogen reserve was depleted. The activities of most enzymes involved in polyol metabolism (namely glycogen phosphorylase, glucose‐6‐P dehydrogenase, phosphofructokinase, aldose reductase, polyol dehydrogenase and ketose reductase) increase relatively rapidly during the early phase of diapause development and, in the late phase, they either become stable or slowly decrease. Cold treatment has either no effect or results in a moderate increase in activity when applied in the early phase of diapause development but results in a more or less obvious decrease of enzymatic activity when applied in the late phase.     

Modulatory effect of rRNA synthesis and ppUL83 nucleolar compartmentalization on human cytomegalovirus gene expression in vitro

The nucleolus is a nuclear domain involved in the biogenesis of ribosomes, as well as in many other important cellular regulatory activities, such as cell cycle control and mRNA processing. Many viruses, including herpesviruses, are known to exploit the nucleolar compartment during their replication cycle. In a previous study, we demonstrated the preferential targeting and accumulation of the human cytomegalovirus (HCMV) UL83 phosphoprotein (pp65) to the nucleolar compartment and, in particular, to the nucleolar matrix of lytically infected fibroblasts; such targeting was already evident at very early times after infection. Here we have investigated the possible effects of rRNA synthesis inhibition upon the development of HCMV lytic infection, by using either actinomycin D or cisplatin at low concentrations, that are known to selectively inhibit RNA polymerase I activity, whilst leaving RNA polymerase II function unaffected. Following the inhibition of rRNA synthesis by either of the agents used, we observed a significant redistribution of nucleolar proteins within the nucleoplasm and a simultaneous depletion of viral pp65 from the nucleolus; this effect was highly evident in both unextracted cells and in nuclear matrices in situ. Of particular interest, even a brief suppression of rRNA synthesis resulted in a very strong inhibition of the progression of HCMV infection, as was concluded from the absence of accumulation of HCMV major immediate‐early proteins within the nucleus of infected cells. These data suggest that a functional relationship might exist between rRNA synthesis, pp65 localization to the nucleolar matrix and the normal development of HCMV lytic infection. J. Cell. Biochem. 108: 415–423, 2009. © 2009 Wiley‐Liss, Inc.     

The plurennial life cycles of the European Tettigoniidae (Insecta: Orthoptera)

Summary The effect of drought on embryonic development and on hatching was studied in 13 European Tettigoniidae species. Drought can affect development in three different ways: (1) Embryonic development proceeds slower than if the eggs are in contact with water; (2) it stops (for final diapause) in an earlier embryonic stage; (3) it affects maintenance and termination of the initial embryonic diapause.In many Tettigoniidae species, the initial diapause is prolonged, and may last several years. Without draught stress, between 1 and 7 cold treatments in the laboratory, and with eggs of the Tettigonia-species between 1 and 6 winters in the field were necessary to enable all eggs to complete initial diapause. In Central European species, the number of eggs maintaining initial diapause significantly increased when the eggs had no contact with water at the time when they should recover from diapause. In contrast, termination of initial diapause in Tettigonia caudata from Greece, when the environment became favorable for growth again, was highest in that group of eggs that had lost most water in a preceding period of drought. The importance of the prolonged initial diapause for the survival of unpredictable adverse conditions is discussed.