Temperature-dependent activation of ERK/MAPK in yolk cells and its role in embryonic diapause termination in the silkworm Bombyx mori

The silkworm Bombyx mori requires 2-3 months of low temperature (5 degrees C) to terminate embryonic diapause. The molecular mechanisms, however, are unknown. Extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) is temperature-dependently activated in the yolk cells of diapausing eggs after 45 days at 5 degrees C, coincident with the acquisition of developmental competence of the embryos at 25 degrees C. Yolk cell granulation and dissociation also begin in diapause eggs incubated at 5 degrees C for 45 days. We used dechorionated egg culture as a model system of diapause termination and observed that both yolk cell dissociation and embryonic development are inhibited by MAPK-ERK kinase (MEK) inhibitor U0126. Therefore, we suggest that ERK in yolk cells has a role in regulating changes in yolk morphology and termination of embryonic diapause in B. mori.     

Studies on embryonic diapause in thepnd mutant of the silkworm,Bombyx mori

Summary Two-dimensional gel electrophoresis has been used to analyse patterns of proteins synthesized in the eggs from theBombyx mutantpnd, whose homozygous embryo never enters diapause owing to a genetic defect. At the middle to late stage of gastrulation the diapause type of the heterozygous embryo, derived from a homozygouspnd female mated to a wild-type male, synthesizes eight proteins which are not detected in the homozygouspnd embryo. To examine the relationship between embryonic diapause and the appearance of the heterozygote-specific proteins, the pattern of proteins synthesized in the heterozygotes of the diapause type was compared with that in heterozygotes which were artificially altered so that they would continue development. Only one of the eight heterozygote-specific proteins was constitutively synthesized according to the embryonic genome, irrespective of their developmental state, whereas appearance of the remaining seven proteins was exclusively dependent on their developmental nature. This finding strongly suggests that the unique protein might result from the expression of thepnd ⁺ gene, and the other proteins might be synthesized along with diapause initiation in the heterozygotes. The possible role of the putativepnd ⁺ gene-specific protein at the onset of embryonic diapause is discussed in relation to the action of the diapause factor, which predetermines embryonic diapause by affecting the developing oocytes.     

Studies on the embryonic diapause of thepnd mutant of the silkworm,Bombyx mori

Summary The changing pattern in free amino acids following the embryonic development in the non-diapause and diapause eggs of thepnd mutant of theBombyx silkworm was studied. In the diapause eggs, heterozygous for thepnd gene, the levels of most of the amino acids increased concomitantly with the substantial decrease in oxygen consumption. Among the amino acids, alanine was the only amino acid that showed a large accumulation. The accumulation could be induced experimentally in the non-diapause eggs, homozygous for thepnd gene, by reducing the oxygen supply. In contrast, it was prevented in the diapause eggs by increasing the oxygen supply. From these results, it is suggested that the alanine accumulation is the consequence of anaerobic metabolism in the eggs during diapause. The possible significance of the alanine accumulation is discussed in relation to the anaerobic carbohydrate metabolism associated with the embryonic diapause in thepnd mutant.     

Isolation of the diapause hormone from the silkworm,Bombyx mori

The diapause hormone (DH) secreted from the suboesophageal ganglion is responsible for the embryonic diapause in the silkworm, Bombyx mori. It was extracted from two million mated male adult heads. At least two species of DH were separated from the extracts (a complex lipid fraction) by gel permeation chromatography with Sephadex LH-20 by organic eluants. One of them was further purified by repeating gel permeation column chromatography with Merckogel^® Type OR 6000 in the cold and dark to give finally a single peak with high DH activity. This preparation seems to be chemically pure, and its molecular weight is chromatographically estimated to be between 2000 and 4000. Other characteristics of DH are also presented. Biological activity of the final DH preparation is discussed with reference to other insect hormones such as juvenile hormone and α-ecdysone.     

An esterase in relation to yolk cell lysis at diapause termination in the silkworm,Bombyx mori

Esterase A is one of the esterase isozymes in eggs of Bombyx mori. The effect of this esterase A on the yolk cells of diapause eggs was examined with a hanging-drop culture in order to discover the mechanism of diapause termination in silkworm eggs.The culture of yolk cells in diapause eggs shows spherical forms with dark fine grains in the central parts, large translucent granules in the outer parts, and a membrane on the exterior. When such yolk cells were incubated with yolk materials of acid-treated or diapause-terminated eggs, they were damaged and cell lysis occurred. This suggested that substance(s) causing the cell lysis were present in diapause-terminated eggs. When esterase A separated electrophoretically from non-diapause eggs and diapause-terminated eggs was added to hanging-drop cultures of yolk cells of diapause eggs, the yolk cells were also greatly affected. That is, a part of the yolk cell membrane was dissolved or disappeared, and the central dark fine grains diffused over the cell causing the whole cell to become dark. A few cells lost almost all of their contents and collapsed. Other esterase fractions and fractions without esterase activity in the electrophoresis exerted little effect on the yolk cells. Furthermore, a parallelism between esterase activity to hydrolyse 2-naphthyl acetate as substrate and the lytic activity on the yolk cell membrane was observed in this esterase A fraction from different sources.From these results it is highly probable that the substance responsible for cell lysis is the esterase A enzyme itself. Diapause termination of silkworm eggs is discussed in relation to the lysis of yolk cells.     

A time-interval activation of esterase A4 by cold: Relation to the termination of embryonic diapause in the silkworm Bombyx mori

The inactive esterase A4 (Ease A4) purified from the diapausing eggs of the silkworm, Bombyx mori, was chilled in vitro. The enzyme activity was very low during the early chilling period and it was suddenly elevated at a certain time of the chilling (2 weeks or less after chilling), depending upon when the chilling period began, and was followed by a rapid fall. The sudden elevation of the Ease A4 activity in vitro is equivalent to that observed in vivo and is coincident with the chilling period, the latter being indispensable for diapause termination.Data are also presented that suggest that the cold-induced activation of the Ease A4 may result from an autonomous structural change of the enzyme molecule which proceeds gradually in the cold.     

Neuroendocrine control of diapause hormone secretion in the silkworm,Bombyx mori

To clarify the control mechanism of diapause hormone (DH) secretion in the silkworm Bombyx mori a series of anatomical and pharmacological experiments were carried out. The arrangement of 'diapause' and 'non-diapause' eggs in the ovarioles of the moths was determined by the coloration method to estimate the accumulation of 3-hydroxykynurenine in the eggs. The females destined to lay non-diapause eggs (non-diapause producers) had diapause eggs in their ovaries if their subesophageal ganglions (Sg) had been surgically removed at 2days after larval-pupal ecdysis or later. In contrast when the surgical extirpation extended to the brain and the corpora cardiaca (CC)-corpora allata (CA) complex in addition to the Sg, the non-diapause producers had no diapause eggs. When the Sg was removed from the females destined to lay diapause eggs (diapause-producers), diapause eggs appeared in response to the treatment at 2days after larval-pupal ecdysis, but the appearance of diapause eggs was delayed by 2days when the brain-CC-CA complex was included among the organs removed. These observations suggested that DH is produced in Sg and transferred to the CC-CA complex, and that the secretion of DH from the complex is suppressed in non-diapause producers. The pattern of diapause and non-diapause eggs induced by the transection of the subesophageal connective in diapause and non-diapause producers suggested a regenerative and secretory capacity of the neurosecretory cells after the operation. The appearance of diapause eggs in non-diapause producers with transected protocerebrum of the brain confirmed that there was an inhibitory center in the protocerebrum. Changes in parts of the ovarioles containing diapause and non-diapause eggs with time of injection of gamma-aminobutyric acid (GABA) and picrotoxin suggested that a GABAergic inhibitory mechanism in DH secretion may be active in non-diapause producers but inactive in diapause producers throughout the pupal stage.     

Oxygen consumption in relation to sorbitol utilization at the termination of diapause in eggs of the silkworm,Bombyx mori

Rates of oxygen consumption were followed throughout the entire period of diapause in eggs of Bombyx mori. In non-diapause eggs at 25 degrees C, O(2) uptake was divisible into three phases, corresponding to morphogenetic processes. In diapause eggs at 25 degrees C, O(2) uptake showed a peak (100 &mgr;l/g eggs/h) at 1 day and then suddenly dropped to reach a level of 8-10 &mgr;l/g eggs/h at 10 days and thereafter. To break diapause, eggs were exposed to 5 degrees C for varying periods. When O(2) uptake was measured at 5 degrees C, it remained at 6 &mgr;l/g eggs/h. When eggs were chilled for increasing periods and O(2) uptake was measured immediately after warming to 25 degrees C, the rates increased after a lag phase. In HCl-treated eggs, O(2) uptake increased immediately after acid-treatment. In all cases, highly increasing O(2) uptake at 25 degrees C coincided with termination of diapause. These results were discussed in relation to sorbitol utilization at the termination of diapause.     

Differential gene expression during early embryonic development in diapause and non-diapause eggs of multivoltine silkworm Bombyx mori

Quantification of the differential expression of metabolic enzyme and heat-shock protein genes (Hsp) during early embryogenesis in diapause and non-diapause eggs of the silkworm B. mori was carried out by semi-quantitative RT-PCR. Data analysis revealed that, the phosphofructokinase (PFK) expression started at a higher level in the early stage (6 h after oviposition) in non-diapause eggs, while in diapause induced eggs, it started at a lower level. However, the PFK gene expression in diapause eggs was comparatively higher than in non-diapause eggs. PFK facilitates use of carbohydrate reserves. The lower level of PFK gene expression in the early stage of diapause induced eggs but comparatively higher level of expression than in non-diapause eggs is due to enzyme inactivation via protein phosphorylation during early embryogenesis followed by de-phosphorylation in later stage. The sorbitol dehydrogenase-2 (SDH-2) gene was down regulated in diapause induced eggs up to 24 h and its expression levels in diapause induced eggs coincided with that of PFK gene at 48h in non-diapause eggs. During carbohydrate metabolism, there is an initial temporary accumulation of sorbitol which acts as protectant. The down regulation of SDH-2 gene during the first 24 hours in diapause induced eggs was due to the requirement of sorbitol as protectant. However, since the diapause process culminates by 48 h, the SDH-2 gene expression increased and coincided with that of PFK gene expression. The trehalase (Tre) gene expression was at a lower level in diapause induced eggs compared to non-diapausing eggs. The induction of Tre activity is to regulate uptake and use of sugar by the tissues. The non-diapause eggs revealed maximum expression of GPase gene with major fluctuations as well as an overall higher expression compared to diapause induced eggs. The diapause process requires less energy source which reflects lower activity of the gene. Heat shock protein (Hsp) genes (Hsp20.4, 40, 70, and 90) revealed differential levels of expression in both the eggs at all stages of embryonic development. The present study thus provides an overview of the differential expression levels of metabolic enzyme and Hsp genes in non-diapause and diapause induced eggs of multivoltine silkworm B. mori within 48 h after oviposition, confirming the major role of in early embryogenesis.     

The early embryonic mitosis in normal and cooled eggs of the silkworm,Bombyx mori

Early embryonic mitosis of the silkworm, Bombyx mori, was morphologically studied in the normal eggs and in the eggs treated by low temperature (?10°C). The first embryonic mitosis is observed in the eggs at 120 to 150 minutes after deposition at 26°C. After egg and sperm pronuclei unite, a spindle is formed in each of the pronuclei independently. At metaphase and anaphase paternal and maternal chromosomes are in separate groups on a spindle (gonomeric) and karyogamy takes place at telophase when they reach the poles. The second embryonic mitosis is shown in the eggs at 180 to 210 minutes after deposition. The division of two nuclei is not synchronous in the silkworm, and the mitosis is not gonomeric. In the eggs treated by low temperature, spindle fibers are not observed at all at ?10°C, and chromosomes, which form two deeply stained masses of irregular shape, are seen in the less stained area of spindle shape. When the eggs are returned to 26°C, some eggs go into normal gonomeric division, while some form two small and compact spindles, which seem to be derived from each of the pronuclei. It was observed that these compact spindles are able to continue mitosis.     

家蚕滞育卵与非滞育卵中几种关键酶活性的比较

家蚕Bombyx mori是卵滞育的昆虫, 在滞育期间无形态变化, 也不存在器官发育和组织分化, 然而其生理代谢过程仍在进行。为进一步研究家蚕滞育的机制, 本研究测定了家蚕滞育卵、 即时浸酸处理的滞育卵及非滞育卵在胚胎发育过程中的超氧化物歧化酶(superoxide dismutase, SOD, EC 1.15.1.1)、 过氧化氢酶(catalase, CAT, EC 1.11.1.6)、 丙酮酸激酶(pyruvate kinase, PK, EC 2.7.1.40)、 乙酰胆碱酯酶(acetylcholine esterase, AchE, EC 3.1.1.7)和乳酸脱氢酶(lactate dehydrogenase, LDH, EC 1.1.1.28) 的活性变化。结果表明： 处理后1-7 d, 即时浸酸处理的滞育卵, SOD活性由56 517.00 U/g提高到81 986.94 U/g, CAT活性由14.98 U/g提高到106.90 U/g, PK活性由25.19 U/g提高到181.70 U/g, AChE活性由17.88 U/g提高到287.86 U/g, 而LDH活性由169.96 U/g下降到122.82 U/g。 而在非滞育卵中, SOD活性由86 417.99 U/g下降到66 024.19 U/g, LDH活性由169.07 U/g下降到135.02 U/g; CAT活性由1.47 U/g提高到44.37 U/g, PK活性由20.56 U/g提高到92.09 U/g, AChE活性由21.40 U/g提高到99.17 U/g。在滞育卵中, SOD和AChE活性较稳定; CAT活性随发育上升, 而LDH活性随发育而下降; PK活性在胚胎发育的前 4 d呈上升趋势, 随后基本保持稳定。通过了解家蚕滞育卵、 非滞育卵与即时浸酸卵的相关酶活性在胚胎发育过程中存在的变化, 有助于进一步揭示家蚕滞育的机理。     

Gene expression profiling between embryonic and larval stages of the silkworm, Bombyx mori

To elucidate the molecular mechanisms associated with metamorphic phenomenon relating to Bombyx mori, an important organism in the sericulture industry, we identified genes that are expressed in the different developmental stages, specifically the embryonic (ES) and larval (LS) stages of B. mori. Of 8230 high-quality ESTs from two full-length enriched cDNA libraries, 3442 of the ES ESTs were coalesced into 1325 clusters, while 4788 were coalesced into 927 clusters. The functional classification of these ESTs based on Gene Ontology showed that the types of genes that are associated with oxidoreductase activity, enzyme inhibition, and larval development were highly observed in LS, whereas the types of genes that are involved in nucleotide binding, enzyme activity, and protein transport activity were highly observed in ES. In addition, when the gene expression profile between ES and LS was examined by counting the EST frequencies in each library, 69 genes were identified as being either up- or down-regulated in the larval stage compared to the embryonic stage (P>0.99) and this was confirmed by semi-quantitative RT-PCR. The results show that genes involved in proteolysis and peptidolysis, and lipid and carbohydrate metabolism were dramatically up-regulated in LS, while those related to protein metabolism, DNA/RNA, and coenzymes were highly down-expressed. In particular, a GO analysis of these genes revealed that genes that are involved in hydrolase activity were observed to be highly expressed in amount as well as diversity in LS, while those involved in nucleic acid binding were highly expressed in ES. These data may contribute to elucidating genetic events that distinguish the developmental stage and to our understanding of the metamorphosis of B. mori.     

Effect of diapause hormone on cyclic nucleotide metabolism in developing ovaries of the silkworm, Bombyx mori

1. The content of cyclic AMP and cyclic GMP in ovaries of the silkworm, Bombyx mori, changed differently depending on development. 2. The extirpation of suboesophageal ganglion caused cyclic AMP to decrease and cyclic GMP to increase at different developmental stages. 3. The reversed changes in the cyclic nucleotides were brought about by implantation of the ganglion or by injection of the diapause hormone preparation. 4. No change in both cyclic nucleotides was induced in ovaries during the first 24 hr after an injection of the hormone, but the clear effects appeared after a lag phase of a few days.     

Gene expression profiling in the silkworm, Bombyx mori, during early embryonic development

We prepared a cDNA library for a microarray from eggs of the silkworm, Bombyx mori, at the germ-band formation (24 hours after fertilization) stage. Using a microarray constructed with 2,445 ESTs, we screened gene expression profiles during germ-band formation at six specific time points in the early embryonic stages (from the unfertilized egg to the formation of abdominal leg appendages), and determined 241 of these cDNAs to represent genes that were expressed differentially during the germ-band formation stage. These differentially expressed genes grouped into two clusters. In the early and late clusters, 203 and 38 genes were upregulated, respectively. In the upregulated clusters, we isolated several genes that were associated with development and cell communication, including egalitarian, RAD23b, innexin 2, and senescence-associated protein. Northern blot hybridization revealed that the expression patterns of 14 genes had changed in each of the stages. In this study, we assessed changes in the levels of gene expression in relation to the germ-band formation stages in whole Bombyx embryos.