The role of the fat body, midgut and ovary in vitellogenin production and vitellogenesis in the female tick, Dermacentor variabilis.

Polyclonal antibodies directed against D. variabilis vitellin were utilized for immunocytochemistry at the ultrastructural level. We localized vitellogenin (Vg) in rough endoplasmic reticulum cisternae, secretory granules and secreted products of fat body trophocytes and midgut vitellogenic cells from feeding and ovipositing females. Vg was localized in the oocyte Golgi bodies and in the yolk bodies of both feeding and ovipositing females. Uptake of exogenous Vg was indicated by the presence of immunospecific gold probe in coated pits and coated vesicles at the apical plasma membrane of oocytes from females in rapid engorgement and oviposition. In unmated females little detectable evidence of Vg uptake by developing oocytes suggests that mating and host detachment signal the beginning of vitellogenesis. We conclude that fat body trophocytes, midgut vitellogenic cells and oocytes are involved in the synthesis and/or processing of Vg and that feeding is the signal associated with the initiation of Vg synthesis and/or processing.     

Vitellogenin mRNA in locust fat body: coordinate induction of two genes by a juvenile hormone analog

Levels of vitellogenin (Vg) mRNA in Locusta migratoria fat body were determined as indicators of gene expression induced by the juvenile hormone analog methoprene. After injection of methoprene into juvenile hormone-deprived locusts, excised fat bodies were cultured with [3H]leucine for immunochemical assay of Vg synthesis, and RNA was assayed for Vg mRNA content by hybridization with probes from the previously cloned locust Vg genes A and B. In general, the rise in Vg mRNA paralleled the rise in Vg synthesis. During the primary response to methoprene (in female locusts in which the corpora allata had been destroyed immediately after emergence), Vg mRNA was first detected after 18-24 hr and accumulated rapidly between 36 and 48 hr. The secondary response (in locusts allatectomized during vitellogenesis and kept until Vg disappeared) was accelerated, as Vg mRNA was detectable at 12 hr and titers rose steeply after 18 hr. When Vg synthesis was prematurely induced by injection of methoprene into fifth-stage female larvae, the kinetics of mRNA accumulation were similar to those of primary stimulation in the adult. After allatectomy of vitellogenic females, fat body Vg mRNA decayed with a half-life of about 24 hr, roughly paralleling the decline in Vg synthesis. Assays with the two Vg probes showed coordinate accumulation of gene A and gene B messages under all conditions tested: during primary and secondary stimulation in adult females and in the low-level response obtained by treating male larvae with methoprene.     

七星瓢虫的卵黄发生:保幼激素类似物对卵黄原蛋白合成的调节

本文用脂肪体体外培养方法,研究了取食天然食物和基础人工饲料的七星瓢虫雌虫中卵黄原蛋白、其他分泌蛋白和RNA合成的发育期变化,以及保幼激素类似物ZR-512的调节作用。结果表明:(1)取食蚜虫的雌虫脂肪体羽化后3天即开始合成卵黄原蛋白。11天时合成急剧上升,13天到达最高峰。脂肪体RNA的合成随发育天数而逐渐上升,第9天出现高峰。(2)取食基础人工饲料的雌虫脂肪体合成卵黄原蛋白的能力很弱;在羽化后20天内一直停留在极低的水平,所合成的卵黄原蛋白仅为取食蚜虫时合成高峰的3%。其他分泌蛋白的合成被抑制的程度小得多。脂肪体的RNA合成也一直比较低。(3)取食基础人工饲料的雌虫点滴或喂食ZR-512后,卵黄原蛋白的合成在高峰期比对照组分别增加44倍和67倍。而其他分泌蛋白的合成仅比对照组提高近3倍,表明保幼激素对卵黄原蛋白合成有特别明显的促进作用。激素处理后脂肪体RNA的合成比对照组提高6—7倍,证明保幼激素作用于转录水平。     

七星瓢虫的卵黄发生:脂肪体与卵巢中卵黄原蛋白的合成与分泌

本文利用[³H]亮氨酸参入及特异性抗体沉淀等方法,研究了七星瓢虫体外培养的脂肪体中卵黄原蛋白合成与分泌的动力学,以及不同发育期脂肪体与卵巢中卵黄原蛋白合成的定量变化。脂肪体中卵黄原蛋白的合成与分泌在培养1—4小时内直线上升,到6小时稍下降。保留在脂肪体内的卵黄原蛋白缓慢积累,但一直水平很低。卵黄原蛋白合成的最初30分钟,分泌速率较慢,60%以上的卵黄原蛋白保留在脂肪体内。1小时后分泌速率加快,70%以上的卵黄原蛋白被分泌,保留的卵黄原蛋白在4小时中逐渐被释放。在4小时,被分泌的卵黄原蛋白超过80%,最高可达92%。 在雌虫发育过程中,脂肪体中卵黄原蛋白合成的高峰在羽化后11—15天,所合成的卵黄原蛋白占整个发育期合成总量的80%。在合成高峰期分泌的卵黄原蛋白高达90%以上,但在发育的早期和晚期分泌的卵黄原蛋白仅占30%或稍多。 卵黄发生前的卵巢就开始合成卵黄原蛋白,但卵巢中卵黄原蛋白的合成高峰期与脂肪体中大致相同。与脂肪体相反,卵巢合成的卵黄原蛋白大部分保留在卵巢内。在卵黄发生盛期,卵巢合成的卵黄原蛋白为脂肪体合成的卵黄原蛋白的20%。     

Fat body is the site of vitellogenin synthesis in the soft tick,Ornithodoros moubata

Summary Vitellogenin (Vg) synthesis in cultured tissues was analysed biochemically in a soft tick,Ornithodoros moubata. Nine tissue fractions dissected from reproductive females were incubated in vitro in a specially designed Ringer containing³⁵S-methionine. The synthesis of total protein and Vg was assayed by the radioactivity incorporated into precipitates with trichloroacetic acid and antivitellin (Vn)-serum, respectively. Fat body was the most active tissue in Vg synthesis, which comprised 46% of the Vg synthesis by all tissues and 42% of total protein synthesis by fat body. Protein synthesized by the fat body and precipitated with anti-Vn-serum was shown by electrophoresis and fluorography, to consist of six radioactive polypeptides corresponding to the components of Vg. Vg synthesized in cultured fat body was first accumulated in the tissue and secreted into the medium during incubation. Some tissues other than fat body showed low Vg synthesis (in each, less than 12% of total protein synthesis) which, however, may be due to contamination by fat body cells as seen with the scanning electron microscope (SEM). SEM also showed that fat body cells in the active stage of Vg synthesis expanded about 10-fold in length. Immunohistochemical analysis showed a very strong reaction with anti-Vn-IgG in the cytoplasm of fat body from reproductive females. Fat body from unfed females and other tissues including midgut, did not show any specific fluorescence. A positive reaction was obtained with developing oocytes. These results indicate that the fat body is the only site of Vg synthesis in this tick.Abbreviations Vg vitellogenin - Vn vitellin - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - SEM scanning electron microscopy - TCA trichloroacetic acid     

Regulation of vitellogenin synthesis by juvenile hormone analogue in Coccinella septempunctata

Vitellogenesis in the lady beetle, Coccinella septempunctata, is controlled by juvenile hormone (JH). When immature females were reared on an artificial diet, they were characterized by hypertrophy of the fat bodies and slow development of the ovaries. Vitellogenin (Vg) synthesis in the fat body remained at a very low level throughout adult development. RNA synthesis also stayed at a relatively low level. Treatment with hydroprene induced vitellogenesis in these non-fecund females. Stimulation in Vg synthesis in hormone-treated females was demonstrated both in vivo and in fat body culture. Synthesis of fat body RNA also increased after hormone treatment. Fat body RNA from hormone-treated females directed the synthesis of Vg polypeptides both in Xenopus oocytes and in a reticulocyte lysate. Thus the induction of Vg synthesis by JH involves an increase in the level of translatable Vg mRNA.     

Juvenile hormone analog and injection effects on locust hemolymph protein synthesis

In adult female Locusta migratoria, at about day 8 after eclosion, when vitellogenin (Vg) is first produced as a result of induction by juvenile hormone (JH), the intensity of hemolymph protein electrophoretic bands at about 75 kDa and 20 kDa increases sharply, suggesting that JH may induce additional proteins. A major component of the elevated protein is persistent storage protein (PSP; subunit 74 kDa). Administration of the JH analog, methoprene, to precocene-treated adult locusts was followed by a rise in hemolymph levels of PSP but not in apolipophorin III (19 kDa), identified immunochemically and electrophoretically. The synthesis of PSP in adult fat body was confirmed by incorporation of [³H]leucine. At 48 h after treatment with methoprene, Vg synthesis was induced in females (as previously observed) and synthesis of PSP in both sexes was elevated above controls, while synthesis of apolipophorin III was not stimulated. We conclude that in adult locust fat body the synthesis of several proteins responds in different ways to the JH analog: Vg (and a 21 kDa protein described elsewhere) is induced de novo solely in females; PSP (and a 19 kDa protein described elsewhere) is stimulated in both sexes but is not fully JH-dependent; apolipophorin III is not stimulated. In these experiments, methoprene was administered both by injection in mineral oil and topically in acetone. After injection of mineral oil as a vector control, incorporation into secreted proteins was stimulated at 24 h, presumably due to a wound effect; topical application of acetone avoids this effect and is a preferred route for administration of JH analog. © 1992 Wiley-Liss, Inc.     

七星瓢虫卵黄原蛋白基因的表达mRNA的体外转译

七星瓢虫成熟雌虫脂肪体总RNA和poly(A)~+RNA中可转译mRNA的水平约为雄虫和不成熟雌虫的两倍,其中所含的卵黄原蛋白mRNA可在体外转译系统中指导卵黄原蛋白多肽的合成。 雌虫取食人工饲料时,其脂肪体RNA中可转译mRNA的水平很低,不能指导卵黄原蛋白多肽的合成。保幼激素类似物能诱导可转译卵黄原蛋白mRNA的出现。     

Inhibition of vitellogenin synthesis in Apis mellifera workers by a juvenile hormone analogue,pyriproxyfen

Insect juvenile hormone (JH) has been related to modulation of vitellogenin (Vg) synthesis, a protein produced by fat body cells, secreted in haemolymph and sequestered by developing oocytes. A stimulatory JH action has been described for the majority of species studied thus far. In some insects, however, Vg synthesis has been inhibited or unaffected by JH. The aim of this study was to re-examine the action of JH on Vg synthesis in Apis mellifera workers, since contrasting effects of this hormone were described. Newly emerged worker bees were treated with different doses of pyriproxyfen (PPN), a potent JH analogue. Vg and total protein were quantified in haemolymph samples of newly emerged up to 6-day-old worker bees. Protein synthesis activity of fat body cultured in vitro and ultrastructure of fat body cells were also examined. High doses (1.25, 2.5, 5 and 10 &mgr;g) of PPN inhibited the onset and accumulation of Vg in the haemolymph of young worker bees in a dose-dependent fashion. This inhibition was not a result of fat body cell degeneration or death, as illustrated by fat body cells ultrastructure analysis, but by impairing Vg synthesis, as demonstrated by in vitro culture of fat body cells. Low doses (0.001, 0.01 and 0.1 &mgr;g) neither affected the normal synthesis and secretion of Vg into the haemolymph nor caused an early onset of Vg in treated bees (which could be interpreted as a JH-activating effect), as shown by Vg quantification at 24-h intervals. The results suggest that a low JH titre in honey bee workers permits the onset and accumulation of Vg in haemolymph, whereas high JH levels turn off Vg synthesis.     

家蝇的卵黄发生及其激素调节

用5—15%SDS-PAGE分析表明,家蝇Musce domestica viaina卵黄蛋白由三个亚基组成,其亚基分子量分别为58KD、50KD、48KD.火箭免疫电泳的结果表明,脂肪体、血淋巴和卵巢内卵黄原蛋白的变化具有密切的相关性,卵黄原蛋白在体内最早出现在羽化后30小时左右,然后迅速增加,在羽化后48小时,脂肪体和血淋巴中卵黄原蛋白含量达到最大值,卵巢开始沉积卵黄蛋白在羽化后30小时,到产卵前达到最大值,脂肪体在离体培养条件下,通过测定³H-亮氨酸掺入卵黄原蛋白的量,对不同发育时期家蝇脂肪体合成卵黄原蛋白的能力及激素的调节作用进行了研究,结果表明,羽化12小时后,合成能力迅速上升,48小时时形成高峰,60小时后迅速下跌直至产卵,其合成能力一直维持在低水平,产卵后合成能力又迅速回升,激素处理结果表明,保幼激素可以促进卵黄发生前期和后期家蝇脂肪体的卵黄原蛋白合成,20-羟基蜕皮酮可以大幅度促进卵黄发生期家蝇脂肪体的卵黄原蛋白合成.当二种激素共同处理时,对卵黄发生前期和卵黄发生期的家蝇脂肪体有协同促进作用,而对卵黄发生后期的脂肪体没有这种作用.本文还对家蝇卵黄发生过程中脂肪体、血淋巴和卵巢三者之间的关系及家蝇卵黄发生的激素调节进行了讨论.     

七星瓢虫卵黄原蛋白基因的表达:保幼激素类似物对mRNA的诱导

在七星瓢虫(Coccinella septempunctata)中,保幼激素调控脂肪体中卵黄原蛋白基因的表达。蛋白质合成实验证明,保幼激素类似物大幅度地促进取食人工饲料的雌虫脂肪体中卵黄原蛋白的合成。保幼激素类似物的作用有高度选择性,使卵黄原蛋白占总蛋白的百分比提高12倍。取食人工饲料的雌虫中,脂肪体RNA含量及其转译活性均极低,转译产物中不存在卵黄原蛋白多肽。保幼激素类似物能显著提高脂肪体RNA的含量及其中可转译mRNA的水平。处理后的雌虫,象蚜虫饲养的成熟雌虫一样,其脂肪体RNA能在体外转译系统中指导卵黄原蛋白多肽的合成,并在变性琼脂糖凝胶电泳上显示一条高分子量的带(约5100核苷酸),初步鉴定为卵黄原蛋白mRNA。由此证明,保幼激素类似物能诱导卵黄原蛋白mRNA的出现和积累。     

Juvenile hormone-dependent vitellogenin synthesis in Locusta migratoria fat body: Inducibility related to sex and stage

Juvenile hormone (JH)-dependent vitellogenin (Vg) synthesis in the fat body of Locusta migratoria is normally limited to sexually mature adult females. As a step toward examining the basis of this limitation, we have tested female and male locusts in a series of stages after the third larval molt for inducibility of Vg synthesis by the synthetic JH analog, methoprene. We find that in the fourth and fifth larval instars fat body of both sexes can be induced to produce Vg, but in the adult stage females respond strongly while no more than trace amounts can be induced in males. Quantitative assays show relative responsiveness in the order: adult female > fifth instar female > fifth instar male ? adult male. During the fifth instar of both sexes, maximal vitellogenic response was obtained in midinstar. After the larval-adult ecdysis, female fat body was unresponsive during the first 4 days, then responsiveness increased and by Day 8 after ecdysis fat bodies were fully as competent to produce Vg as at Day 14, the usual maximum of the first vitellogenic cycle due to endogenous JH. Larval and adult female fat bodies implanted into male larvae are competent for Vg synthesis after metamorphosis, so that the differences between adult male and female cannot be imposed by the male milieu intérieur during the larval-adult molt. In male and female precocious adults, produced by treatment of fourth instars with precocene, fat body responded to methoprene as in normal adults. We conclude that factors intrinsic to the fat body cells, determined early in development, are responsible for differential gene programing in males and females, which is partially expressed by the fifth instar but fully manifest only after a molt in the absence of JH.     

Lipophorin as a yolk protein precursor in the mosquito, Aedes aegypti

We examined expression of the lipophorin (Lp) gene, lipophorin (Lp) synthesis and secretion in the mosquito fat body, as well as dynamic changes in levels of this lipoprotein in the hemolymph and ovaries, during the first vitellogenic cycle of females of the yellow fever mosquito, Aedes aegypti. Lipophorin was purified by potassium bromide (KBr) density gradient ultracentrifugation and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Polyclonal antibodies were produced against individual Lp apoproteins, apolipoprotein-I (apoLp-I) and apolipoprotein-II (apoLp-II), with molecular weights of 240 and 75 kDa, respectively. We report here that in the mosquito A. aegypti, Lp was synthesized by the fat body, with a low level of the Lp gene expression and protein synthesis being maintained in pre- and postvitellogenic females. Following a blood meal, the Lp gene expression and protein synthesis were significantly upregulated. Our findings showed that the fat body levels of Lp mRNA and the rate of Lp secretion by this tissue reached their maximum at 18 h post-blood meal (PMB). 20-Hydroxyecdysone was responsible for an increase in the Lp gene expression and Lp protein synthesis in the mosquito fat body. Finally, the immunocytochemical localization of Lp showed that in vitellogenic female mosquitoes, this protein was accumulated by developing oocytes where it was deposited in yolk granules.     

昆虫卵黄发生研究进展

昆虫卵黄发生研究进展李乾君,龚和,管致和（中国科学院动物研究所北京１０００８０）（北京农业大学植保系北京１０００９４）昆虫卵的成熟一般分为三个时期－－卵黄发生前期（Ｐｒｅｖｉｔｅｌｌｏｇｅｎｉｃｓｔａｇｅ）、卵黄发生期（ｖｉｔｅｌｌｏｇｅｎｉｃｓｔａ...     

Effect of ecdysone agonists on vitellogenesis and the expression of EcR and USP in codling moth (Cydia pomonella)

The effects of tebufenozide and methoxyfenozide on vitellogenin (Vg) synthesis/release in the fat body, translocation in hemolymph, uptake by the ovary, and the expression of the ecdysone receptor (EcR) and its heterodimer partner, ultraspiracle protein (USP) in fat body, were investigated in Cydia pomonella. The results indicated that both ecdysone agonists significantly increased the Vg level in the adult hemolymph when the moths were exposed to agonist-treated surfaces. However, these agonists did not affect Vg release from the fat body nor Vg deposition in the first batch oocytes. Western blot analysis revealed that the expression of EcR and USP was significantly increased in tebufenozide- and methoxyfenozide-treated samples compared to the control, suggesting that ecdysone agonists regulated the Vg synthesis via the EcR and USP proteins complex.     

七星瓢虫的卵黄发生:体外培养的脂肪体中卵黄原蛋白的合成

为了深入研究七星瓢虫的卵黄发生及其激素调节机理,我们建立了脂肪体的体外培养方法,证明了脂肪体在体外培养条件下能够正常进行卵黄原蛋白的合成与分泌。在体外合成的卵黄原蛋白与体内合成的具有相同的电泳迁移率、免疫学特性和部分水解肽谱。放射性氨基酸在体外参人卵黄原蛋白的动力学与在体内的相似。用脂肪体的体外培养方法,结合放射免疫沉淀、SAS-聚丙烯酰胺凝胶电泳、放射自显影等技术,研究了不同发育期脂肪体的合成能力,以及取食人工饲料的雌虫中保幼激素类似物对卵黄原蛋白合成的促进作用。     

Effects of precocenes on vitellogenesis in the adult female tick,Ornithodoros moubata (Acari: Argasidae)

Vitellogenin (Vg) concentrations in the hemolymph and ovarian development were studied inOrnithodoros moubata after treatment with precocenes 1 (P1) and 2 (P2). Precocene was dissolved in acetone or DMSO and topically applied to the dorsal surface of ticks: (1) at adult ecdysis; (2) 24 h before engorgement; (3) immediately after engorgement; and (4) 24 h after engorgement. Subsequently, P1 and P2 were dissolved in olive oil and injected through the gonopore into the body cavity 24 h after engorgement. Vitellogenin concentration was measured on days 5 and 10 after engorgement and ovarian development was scored on day 10, 20 or 30. Oviposition was also recorded and the average weight of eggs laid by females was determined. No differences in concentration of Vg in the hemolymph occurred between the control ticks and ticks treated topically or by injection with P1 and P2. Precocene did not suppress the synthesis of Vg inO. moubata. However, oviposition was reduced in ticks that survived repeated treatment with high doses of P2 dissolved in acetone.     

Lipophorin as a yolk protein precursor in the mosquito, Aedes aegypti

We examined expression of the lipophorin (Lp) gene, lipophorin (Lp) synthesis and secretion in the mosquito fat body, as well as dynamic changes in levels of this lipoprotein in the hemolymph and ovaries, during the first vitellogenic cycle of females of the yellow fever mosquito, Aedes aegypti. Lipophorin was purified by potassium bromide (KBr) density gradient ultracentrifugation and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Polyclonal antibodies were produced against individual Lp apoproteins, apolipoprotein-I (apoLp-I) and apolipoprotein-II (apoLp-II), with molecular weights of 240 and 75 kDa, respectively. We report here that in the mosquito A. aegypti, Lp was synthesized by the fat body, with a low level of the Lp gene expression and protein synthesis being maintained in pre- and postvitellogenic females. Following a blood meal, the Lp gene expression and protein synthesis were significantly upregulated. Our findings showed that the fat body levels of Lp mRNA and the rate of Lp secretion by this tissue reached their maximum at 18 h post-blood meal (PMB). 20-Hydroxyecdysone was responsible for an increase in the Lp gene expression and Lp protein synthesis in the mosquito fat body. Finally, the immunocytochemical localization of Lp showed that in vitellogenic female mosquitoes, this protein was accumulated by developing oocytes where it was deposited in yolk granules.     

Full-length sequence, regulation and developmental studies of a second vitellogenin gene from the American dog tick, Dermacentor variabilis

Vitellogenin (Vg) is the precursor of vitellin (Vn) which is the major yolk protein in eggs. In a previous report, we isolated and characterized the first Vg message from the American dog tick Dermacentor variabilis. In the current study, we describe a second Vg gene from the same tick. The Vg2 cDNA is 5956 nucleotides with a 5775 nt open reading frame coding for 1925 amino acids. The conceptual amino acid translation contains a 16-residues putative signal peptide, N-terminal lipid binding domain and C-terminal von Willebrand factor type D domain present in all known Vgs. Moreover, the amino acid sequence shows a typical GLCG domain and several RXXR cleavage sites present in most isolated Vgs. Tryptic digest-mass fingerprinting of Vg and Vn recognized 11 fragments that exist in the amino acid translation of DvVg2 cDNA. Injection of virgin females with 20 hydroxyecdysone induced DvVg2 expression, vitellogenesis and oviposition. Using RT-PCR, DvVg2 expression was detected only in tick females after mating and feeding to repletion. Northern blot analysis showed that DvVg2 is expressed in fat body and gut cells of vitellogenic females but not in the ovary. DvVg2 expression was not detected in adult fed or unfed males. The characteristics that distinguish Vg from other similar tick storage proteins like the carrier protein, CP (another hemelipoglycoprotein) are discussed.