七星瓢虫的卵黄发生:植入雄虫的卵巢中卵黄原蛋白的合成

为了证实七星瓢虫的卵巢能合成卵黄原蛋白, 并查明雄虫体内是否具有卵黄发生所必需的激素环境, 我们将刚羽化雌虫的一侧卵巢或数个卵巢管植入雄虫体内.移植的卵巢或卵巢管在雄虫体内能够发育, 其卵母细胞能沉积卵黄, 一部分可达成熟.体外培养证明移植的卵巢可合成卵黄原蛋白, 但受体雄虫的脂肪体不合成卵黄原蛋白, 而且其血淋巴中也不存在这种蛋白.用保幼激素类似物ZR-512处理受体雄虫, 可促进移植卵巢的发育, 但不能诱导其脂肪体合成卵黄原蛋白.此结果表明, 象大多数昆虫一样, 七星瓢虫的卵黄发生的性二型现象表现在激素的靶组织——脂肪体, 而不是激素本身.     

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%。     

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。     

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。     

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.     

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。     

天蚕卵黄发生的激素调控

报告了蜕皮激素和保幼激素对天蚕Antheraea yamamai卵黄发生的调控作用。当单独以20－羟基蜕皮酮或保幼激素类似物methoprene处理,以及同时用这两种激素处理天蚕蛹时,蛹期脂肪体和血淋巴中卵黄原蛋白（Vg)含量明显高于对照,即二对Vg的合成起促进作用。然而,卵巢中卵黄蛋白（Vt)含量则因激素种类而异,以保幼激素处理时明显低于对照,以20－羟基蜕皮酮处理则反之,即前抑制卵巢对Vg的摄取,而后则起促进作用。离体培养脂肪体并以激素处理的结果表明,20－羟基蜕皮酮和methoprene均能促进Vg合成,但前作用更。综合考虑上述结果可以认为蜕皮激素对该蚕的卵黄发生起主要调控作用。     

七星瓢虫的卵黄发生:卵黄原蛋白的发生和取食代饲料的影响

1.用凝胶电泳和免疫扩散法研究了七星瓢虫成虫脂肪体、血淋巴和卵巢中总蛋白和卵黄原蛋白的含量变化和相互关系。查明七星瓢虫和某些被研究过的昆虫一样,卵黄原蛋白在脂肪体内合成,释放到血淋巴,然后被发育的卵母细胞摄取。 2.系统观察了七星瓢虫成虫血淋巴中卵黄原蛋白和产卵的关系。在适温下取食蚜虫的成虫多数在羽化后四天血淋巴中出现卵黄原蛋白,十天后开始产卵,如食料适宜,在整个产卵期,血淋巴中卵黄原蛋白的水平较高。 3.对比了取食不同饲料的个体中脂肪体和卵巢鲜重的变化。对取食代饲料的产卵与不产卵个体的脂肪体、血淋巴、卵巢进行了分析比较。讨论了取食代饲料的部分个体不产卵的原因。 4.保幼激素类似物ZR-512促进卵黄原蛋白的合成,使取食代饲料不产卵个体的血淋巴中卵黄原蛋白的含量明显提高。     

蝗虫微孢子虫对东亚飞蝗卵黄原蛋白含量的影响

采用免疫学方法,对东亚飞蝗Locusta migratoria manilensis感染蝗虫微孢子虫Nosema locustae后体内卵黄蛋白含量的变化进行了研究。结果表明,感病蝗虫与对照健虫相比,卵黄发生有严重障碍,脂肪体和卵巢中卵黄原蛋白或卵黄蛋白含量极低,导致感病雌虫丧失产卵能力。脂肪体中卵黄原蛋白含量最高峰健虫为18.7 mg/mL,而病虫只有4.7 mg/mL;血淋巴中卵黄原蛋白含量最高峰健虫为7.6 mg/mL,而病虫只有2.6 mg/mL;卵巢中卵黄蛋白含量最高峰健虫为73.4 mg/mL,而病虫只有4.9 mg/mL。     

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.     

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     

HORMONAL CONTROL OF THE VITELLOGENESIS IN THE JAPANESE OAK SILKWORM, ANTHERAEA YAMAMAZ (LEPIDOPTERA: SATURNIIDAE)

Abstract Effects of ecdysteroid and juvenile hormone (JH) on vitellogenesis of the Japanese oak silkworm, Antheraea yamami are reported in this article. After topical treatment with 20-hydroxyecdysone alone or JH analog (i.e. methoprene) alone and combined treatment with these two chemicals, vitellogenin (Vg) titers in the fat body and haemolymph at the pupal stage were mostly higher than those of the control, indicating that both ecdysteroid and JH exerted a promoting effect on the synthesis of Vg. In contrast, the Vg uptake was markedly inhibited by JH while stimulating effect of the ecdysteroid could be shown that vitellin (Vt) titer in the ovary was lower after methoprene treatments, but higher after 20-hydroxyecdyson treatments. Meanwhile, effects of these two hormones on Vg synthesis in the fat body were also tested with the incubation in vitro with Grace medium containing H-leucine and the hormones. The results demonstrated that Vg synthesis was stimulated after treating with methoprene alone or 20-hydroxyecdysone alone and combined treating with these two chemicals, and particularly ecdysteroid had more marked positive effect. To comprehensively concluded our results, it could be regarded that ecdysteroid play the main role in the regulation of vitellogenesis for the Japanese oak silkworm.     

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.     

Biosynthesis of mosquito vitellogenin

Vitellogenin (Vg), the hemolymph precursor to the major yolk protein in mosquitoes, is synthesized in the fat body of blood-fed females. Mosquito Vg consists of two subunits with Mr = 200,000 and 66,000. Here, we demonstrate that both the Vg subunits are first synthesized as a single precursor. The identity of this Vg precursor was confirmed by immunoprecipitation with subunit-specific monoclonal antibodies. In cell-free translation of fat body poly (A)+ RNA, the Vg precursor had Mr = 224,000 which increased to 240,000 in the presence of canine pancreatic microsomal membranes. A precursor with Mr = 250,000 was immunoprecipitated in microsomal fractions isolated from rat bodies. With in vitro pulse labeling, the 250-kDa precursor could be detected in homogenates of fat bodies from blood-fed mosquitoes only during the first few hours accumulation of the Vg precursor was achieved by an in vitro stimulation of Vg synthesis in previtellogenic fat bodies cultured with an insect hormone, 20-hydroxyecdysone. The 250-kDa precursor was glycosylated and to a much lesser degree phosphorylated. Treatment of fat bodies with tunicamycin yielded the precursor with Mr = 226,000 which was neither glycosylated nor phosphorylated. The reduction in molecular mass of the 250-kDa Vg precursor and of both mature Vg subunits combined was similar after digestion with endoglycosidase H, indicating that glycosylation is completed prior to cleavage of the Vg precursor. In vitro pulse-chase experiments revealed rapid proteolytic cleavage of the 250-kDa precursor to two polypeptides with Mr = 190,000 and 62,000 which transformed into mature Vg subunits of 200- and 66-kDa as the last step prior to Vg secretion. This last step in Vg processing was inhibited by an ionophore, monensin, and therefore occurred in the Golgi complex. Sulfation as an additional, previously unknown, modification of mosquito Vg was revealed by the incorporation of sodium [35S]sulfate into both Vg subunits. Since sulfation of Vg was predominantly blocked by monensin, the final maturation of Vg subunits in the Golgi complex is, at least in part, due to this modification.