抑卵激素对家蝇卵巢周期性发育的调控

抑卵激素是调控家蝇Musca dorncstica vicina卵巢周期性发育的关键因子之一。在家蝇中,当第一个周期的卵母细胞处于卵黄发生期或卵黄发生后期时,其第二个周期的卵母细胞的发育不进入卵黄发生期。本文建立了家蝇抑卵激素的生物测定方法,即用一对卵巢提取物注射1头羽化后12h家蝇,并在羽化后60h观察卵母细胞的发育及卵黄蛋白的沉积情况。抑卵激素的作用首先是延缓了卵母细胞在卵黄发生前期的发育;其次,抑卵激素抑制脂肪体中卵黄蛋白的合成,导致血淋巴中卵黄蛋白含量的下降,从而抑制了卵母细胞的发育。抑卵激素并不抑制卵母细胞对卵黄原蛋白的摄取。卵发育神经激素可以颉抗抑卵激素的抑制作用。抑卵激素无种属特异性。     

龟纹瓢虫卵黄蛋白的分子特性及发生动态

研究了龟纹瓢虫Propylea japonica (Thunberg) 卵黄蛋白的基本特性以及卵黄发生过程中卵黄蛋白的动态变化。PAGE和SDS-PAGE实验表明,龟纹瓢虫卵黄蛋白分子量为294.81±40.70 kD,并由分子量分别为144.68±0.03 kD和51.23±0.27 kD的两种亚基组成。对卵黄蛋白的氨基酸组成和含量分析发现,其必需氨基酸总量占57.48％,略高于非必需氨基酸,其中谷氨酸（Glu）含量最高,为15.26％;色氨酸（Trp）和蛋氨酸（Met）含量较低,分别为0.50％和0.11％。采用间接竞争ELISA法,系统测定了龟纹瓢虫成虫期脂肪体、血淋巴和卵巢中卵黄蛋白的动态变化,结果表明：脂肪体是卵黄原蛋白合成的场所,卵黄原蛋白的合成始于羽化后第2天;脂肪体、血淋巴和卵巢中卵黄原蛋白的滴度在羽化后第4天开始迅速上升,至成虫期的第8天左右达到高峰期。     

家蝇卵巢摄取卵黄蛋白的机理

在家蝇Musca domestica viaina 的卵黄发生过程中,卵母细胞摄取卵黄原蛋白与滤泡开放是相关的。观察不同发育时期的家蝇滤泡结果表明,在摄取活动最旺盛的时期也就是卵黄发生的顶盛时期,其滤泡开放程度最大,而在卵黄发生前期和后期基本上没有摄取活动,此时的滤泡上皮细胞间不开放。卵巢体外培养的激素处理表明,JH可以促进滤泡开放。家蝇卵巢微粒体制备物的Na⁺-K⁺ATP酶活力在卵巢发育过程中存在着动态变化。羽化后24小时时有一定的酶活性,随着卵黄发生的进行,酶活力逐渐增加,到羽化48小时时酶活力最高,然后又开始下降,到羽化72小时时已经很小。羽化32小时的家蝇点滴或注躬 JH之后,测得的卵巢微粒体制备物的Na⁺-K⁺ATP酶活力比正常羽化36小时的高,羽化44小时的家蝇点滴和注射JH之后,测得酶活力比正常羽化48小时的低。羽化36小时和48小时的家蝇卵巢微粒体制备物与JH共同作用后,其Na⁺-K⁺ATP酶的活力分别增加2.95倍和3.50倍,羽化48小时的家蝇卵巢在含有JH的培养液中培养启,其匀浆液的酶活性为对照组的1.26倍。 由此我们可以推测在家蝇的卵黄发生过程中,JH通过促进滤泡开放和增加卵巢微粒体制备物Na⁺-K⁺ATP酶的活力,从而调控卵母细胞对卵黄蛋白的摄取。     

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。     

早熟素II对家蝇卵黄发生的影响

本实验通过卵巢发育分级的解剖观察、可溶性蛋白质和核酸的定量测定、火箭免疫电泳定量测定卵黄原蛋白及激素处理等方法,研究了早熟素对家蝇(Muscadomestica vicina)卵黄发生的影响。试验结果表明用20ug早熟素处理每头刚羽化家蝇时,家蝇卵黄发生处于不完全抑制状态,其卵黄发生过程比对照组“延迟”约12小时。处理后48小时,血淋巴中卵黄原蛋白的滴度为lo.5ug/ul,接近对照组,而其卵巢鲜重和发育等级明显低于对照组,这种不完全抑制状态表明卵母细胞对卵黄原蛋白的吸收作用受到抑制。当用高剂量100ug早熟素11处理每头刚羽化家蝇时,血淋巴中卵黄原蛋白滴度、卵巢鲜重及其发育均受到明显的抑制,这种抑制效应能自然恢复。 当早熟素11和保幼激素(JH-III)、20-羟基蜕皮酮共同处理时,保幼激素具有明显的去抑制作用,可使血淋巴中卵黄蛋白浓度成倍增加,20-羟基蜕皮酮的去抑制效应不明显。本文还对早熟素作用于双翅目昆虫的方式作了讨论。     

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: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.     

家蝇卵巢中保幼激素结合蛋白的研究(英语)

本文运用活性炭结合试验测定了家蝇卵巢中保幼激素结合蛋白(JHBP)的含量.卵巢中JHBP对JHⅢ有较高的结合活性,其结合常数为2.1×10~(-8)M,~3H保幼激素Ⅲ(~3H-JH)和JHBP的结合可被未标记的JH Ⅲ抑制,但保幼激素的类似物ZR 512或ZR515均无抑制效应.卵巢中JHBP的含量在家蝇羽化后48小时达最大值,是羽化后60小时、72小时家蝇卵巢中JHBP含量的6.5倍和15.5倍.羽化后24小时、36小时的家蝇卵巢无JHBP的结合活性.若刚羽化的家蝇用JHⅢ处理,36小时后,JHBP则可检测到.本实验的结果表明卵巢中JHBP浓度的变动可能和JH对家蝇卵黄发生的作用有关.     

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。     

家蝇卵巢摄取卵黄蛋白的机理

在家蝇Ｍｕｓｃａ　ｄｏｍｅｓｔｉｃａ　ｖｉａｉｎａ的印黄发生过程中,卵母细胞摄取卵黄原蛋白与滤沟开放是相关的。观察不同发育时期的家蝇滤泡结果表明,在摄取活动最旺盛的时期也就是卵黄发生的顶盛时期,其滤泡开放程度最大,而在卵黄发生前期和后期基本上没有摄取活动,此时的滤泡上皮细胞间不开放。卵巢体外培养的激素处理表明,ＪＨ可以促进滤泡开放。家蝇卵巢微粒体制备物的Ｎａ＋－Ｋ＋ＡＴＰ酶活力在卵巢发育过程中存在着动态变化。羽化后２４小时时有一定的酶活性,随着卵黄发生的进行,酶活力逐渐增加,到现化４８小时时酶活力最高,然后又开始下降,到弱化７２小时时已经很小。羽化３２小时的家蝇点滴或注射ＪＨ之后,测得的卵巢微粒体制备物的Ｎａ＋－Ｋ＋ＡＴＰ酶活力比正常羽化３６小时的高,羽化率４４小时的家蝇点滴和注射ＪＨ之后,测得酶活力比正常羽化４８小时的低．羽化３６小时和４８小时的家蝇卵巢微粒体制备物与ＪＨ共同作用后,其Ｎａ＋－Ｋ＋ＡＴＰ酶的活力分别增加２．９５倍和３．５０倍,羽化４８小时的家蝇卵巢在含有ＪＨ的培养液中培养后,其匀浆液的酶活性为对照组的１．２６倍。由此我们可以推测在家蝇的卵黄发生过程中,ＪＨ通过促进滤泡开放和增加卵巢微粒体制     

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.     

Regulation of vitellogenesis in the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae)

Studies were undertaken to investigate vitellogenesis and its regulation in female adults of the fall armyworm, Spodoptera frugiperda. A single female-specific protein, likely to be the S. frugiperda vitellogenin (Vg), appeared approximately 5 h after adult eclosion in the hemolymph of virgin females. The concentration of the protein increased with age as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed. A protein with the same relative molecular mass was also present in egg extracts, but absent from hemolymph samples from male moths. The relative molecular mass of the designated S. frugiperda Vg was determined as 164.5+/-2.5 kDa. Vitellogenic oocytes became visible 36-48 h after emergence and egg deposition began on day 3 of adult life. Vg could not be detected in the hemolymph of females decapitated directly after eclosion. When decapitated virgin females were injected with the JH-mimic methoprene (MP), the level of Vg was comparable to that in non-decapitated moths, indicating that vitellogenesis in S. frugiperda depends on juvenile hormone (JH). However, the number of vitellogenic oocytes was somewhat lower than in non-decapitated virgin females. Injection of 20-hydroxyecdysone (20E) promoted Vg production to a similar extent in decapitated female moths, but in contrast to methoprene injection, treatment with 20E never resulted in the production of vitellogenic oocytes. In vitro cultivated ovaries of adult females dissected directly after eclosion produced lower amounts of ecdysteroids than those isolated on day 1 after emergence. Our results suggest a crucial role for 20E in the induction of vitellogenesis in the noctuid S. frugiperda, while JH seems to be essential for the continued uptake of Vg by developing oocytes and may trigger 20E biosynthesis in the ovary.     

Vitellogenesis in the hematophagous Dipetalogaster maxima (Hemiptera: Reduviidae), a vector of Chagas' disease

Oocyte extracts of anautogenous Dipetalogaster maxima were chromatographed on an ion-exchange column in order to purify vitellin (Vt), the main insect yolk protein precursor. Purified Vt (Mr ~443 kDa) was composed of four subunits with approximate molecular weights of 174, 170, 50, and 44 kDa. Polyclonal anti-Vt antibody, which cross-reacted equally with fat body extracts and hemolymph vitellogenin (Vg), was used to measure the kinetics of Vg expression in the fat body and the levels in hemolymph. In addition, morphological and immunohistochemical changes that took place in the ovary during vitellogenesis were analyzed. The study was performed between 2 and 8 days post-ecdysis and between 2 and 25 days post-blood feeding. During the post-ecdysis period, D. maxima showed decreased synthesis of Vg and concomitantly, low levels of Vg in hemolymph (4.5 x 10(-3) microg/microl at day 4). After a blood meal, Vg synthesis in the fat body and its levels in hemolymph increased significantly, reaching an average of 19.5 microg/microl at day 20. The biochemical changes observed in the fat body and hemolymph were consistent with the histological and immunohistochemical finds. These studies showed noticeable remodeling of tissue after blood feeding.     

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.     

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.     

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。