Multiple vitellogenins (Vgs) in mosquitofish (Gambusia affinis): identification and characterization of three functional Vg genes and their circulating and yolk protein products

The objectives of this study were to characterize multiple forms of vitellogenin (Vg) in mosquitofish (Gambusia affinis) and to discover the fate of each Vg during its processing into product yolk proteins. Two Vg preparations, with apparent masses of 600 kDa (600 Vg) and 400 kDa (400 Vg), were isolated from the plasma of fish treated with estradiol-17beta (E(2)) by various chromatographic procedures. Immunological analyses verified the presence of two different Vg proteins (600 VgA and 600 VgB) in the 600 Vg preparation and of a single protein in the 400 Vg preparation. Three major yolk proteins (Yps) with apparent masses of 560, 400, and 28 kDa were observed in extracts of ovarian follicles from vitellogenic females. Immunological analyses demonstrated that the 400 Vg underwent no change in native mass after being incorporated into oocytes. The 600 Vgs gave rise to a 28 kDa beta'-component and a native 560 kDa Yp, which was heterodimeric in structure, consisting of two types of complexes between phosvitin (Pv) and lipovitellin (Lv) heavy- and light-chains. Full-length cDNAs encoding the 600 VgA, 600 VgB, and 400 Vg were isolated from a liver cDNA library of E(2) treated fish. Similar to the zebrafish vg3 gene, the 400 Vg cDNA lacked a Pv domain and was classified as an incomplete or phosvitinless (C-type) Vg. The deduced primary structures of 600 VgA and 600 VgB were complete, and these were categorized as type A and type B Vgs, respectively, according to our recent classification scheme. This is the first report on the characterization of three functional Vg genes and their circulating and yolk protein products in any vertebrate species.     

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。     

Demonstration of two forms of vitellogenin in serum of estradiol-17 beta-treated Indian major carp, Labeo rohita.

Vitellogenin (Vg) synthesis was induced in the male and non-vitellogenic female Rohu, the Indian major carp, by estradiol-17 beta(E2) where effect was more in female. A crude preparation of Vg was isolated in the second peak after gel filtration on Ultrogel AcA 34 from the sera of vitellogenic female Rohu and E2-treated male and female Rohu. Estimation of alkali-labile phosphorus was shown to be used as an index of Vg. Native-PAGE analysis has revealed the presence of two forms of Vg (Vg1: 430,000 dalton and Vg2:240,000 dalton) in Vg fraction obtained after gel filtration as well as in the sera of E2-treated male and female Rohu. Immunological cross-reaction studies between antiserum to yolk protein and Vg fractions as well as the sera from E2-treated male and female Rohu further indicates the presence of two precipitin lines (not clearly visible as the two lines fused to form a thick line) suggesting the occurrence of two forms of Vg in the Rohu.     

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.     

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...     

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.     

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.     

Cypermethrin induction of vitellogenesis and ovarian development in unfed adult female Ornithodoros moubata (Acari: Argasidae)

Summary

Vitellogenesis in ticks is known to be induced by engorgement and mating. In this paper, the synthetic pyrethroid cypermethrin (CyM) is shown to induce production of yolk protein precursor, vitellogenin (Vg), and ovarian development in unengorged mated adult female Ornithodoros moubata. The levels of Vg found in the hemolymph and ovarian development induced by CyM were dose-dependent. i.e., CyM doses of more than 0.2 and 1.0 μg/tick were needed for significant increase of Vg titer in the hemolymph and yolk deposition in oocytes, respectively. Immunological and electrophoretical analyses of Vg and Vitellin (Vn) induced by CyM were identical with those induced by engorgement. Vg titer induced by CyM in unengorged females followed approximately the same time course as that in the normal engorged females. However, Vg titer induced by CyM continued to increase after day 8 and reached a maximum (95 μg/μ1) on day 10 after treatment, while Vg titer induced by engorgement decreased again after reaching a maximum (60 μg/μ1) on day 6, correlated with yolk Vn deposition in oocytes. Ovarian development induced by even high doses (10 or 20 μg/tick) of CyM was slow compared to normal development stimulated by engorgement. Oviposition was not observed in females treated with CyM.     

Vitellogenin synthesis,processing and hormonal regulation in the tick,Ornithodoros parkeri (Acari:Argasidae)

This study was undertaken to determine the processing of vitellogenin (Vg) and the role of juvenile hormone (JH) in the regulation of vitellogenesis in the tick Ornithodoros parkeri. Ticks usually require a blood meal to induce vitellogenesis. However, we have shown that a pyrethroid, cypermethrin (CyM), can stimulate Vg synthesis in unfed Ornithodoros moubata females. Vg concentration and synthesis were analyzed by SDS-PAGE spotting-scanning and fluorography using [³⁵S]-methionine. Although unfed females show high titers of Vg in the hemolymph, this is not due to new synthesis. Vg synthesis stimulated by engorgement increases beginning on day 2 after engorgement and reaches a maximum level on day 8. Vg is synthesized in the fat body, secreted into the hemolymph and then processed and incorporated into the ovaries as vitellin. JH I, II and III, methoprene (JHA), and CyM were topically applied to unfed females and Vg synthesis analyzed on day 5 by fluorography. JH and JHA did not stimulate Vg synthesis. CyM stimulated Vg synthesis but not ovarian development. These preliminary results indicate that JH does not function in the regulation of vitellogenin synthesis in this species.     

Site of vitellogenin synthesis determined from a cDNA encoding a vitellogenin fragment in the freshwater giant prawn, Macrobrachium rosenbergii.

Vitellogenesis is an important part of reproductive process in crustaceans, and the process is characterized by the synthesis and accumulation of yolk protein in the developing oocytes. The yolk proteins in crustaceans mainly consist of vitellogenin (Vg) and vitellin (Vn), which are respectively present in extra-oocyte tissues and intra-oocytes. The site and the process of yolk protein synthesis in crustaceans are still controversial. The synthesis site of Vg in a crustacean species, Macrobrachium rosenbergii, is determined by immunological and immunohistochemical techniques, and molecular cloning of a cDNA encoding the primary structure of Vn in this study. The hepatopancrease is clearly shown to be the synthesis site of Vg in this species. The length of Vg mRNA was estimated as about 6 kb from Northern blotting analysis. The partial primary structure of Vg gene is presented, and the post-translational processing are further discussed. For the first time, the partial primary structure of Vg gene and the synthesis site of Vg approached by molecular cloning in crustaceans are presented.     

Sequence and the developmental and tissue-specific regulation of the first complete vitellogenin messenger RNA from ticks responsible for heme sequestration

The first full-length mRNA for vitellogenin (Vg) from ticks was sequenced. This also represents the first complete sequence of Vg from the Chelicerata and of a heme binding Vg. The Vg cDNA from the American dog tick, Dermacentor variabilis was 5744nt in length (GenBank Accession number AY885250), which coded for a protein of 1843 aa with a calculated molecular weight of 208 kD. This protein had an 18 aa signal sequence, a single RXXR cleavage signal that would generate two subunits (49.5 and 157K in molecular weight) and lipoprotein N-terminal and carboxy von Willebrand factor type D domains. Tryptic digest MS analysis of vitellin protein confirmed the function of the cDNA as the tick yolk protein. Apparently, vitellin in D. variabilis is oligomeric (possibly dimeric) and is comprised of a mixture of the uncleaved monomer and subunits that were predicted from the single RXXR cleavage signal. The highly conserved GL/ICG motif close to the C-terminus in insect Vg genes was different in the tick Vg message, i.e., GLCS. This variant was also present in a partial sequence of Vg from Boophilus microplus. Phylogenic analysis showed that the full length Vg cDNA from D. variabilis and the partial cDNA from B. microplus were distinct from insects and Crustacea. The Vg message was not found in whole body RNA from unfed or fed males or in unfed and partially fed (virgin) females as determined by Northern blotting. The message was found in replete (mated) pre-ovipositional females, increased to higher levels in ovipositing females and was absent after egg laying was complete. The endocrine regulation of the Vg mRNA is discussed. The tissue sources of the Vg message are both the gut and fat body. Tryptic digest MS fingerprinting suggests that a second Vg mRNA might be present in the American dog tick, which needs further study.     

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。     

Juvenile hormone regulation of vitellogenin synthesis in the red flour beetle,Tribolium castaneum

To elucidate the endocrine regulation of vitellogenin (Vg) synthesis in the red flour beetle, Tribolium castaneum, the titers of juvenile hormone (JH) and ecdysteroids in the whole body of female beetles were measured and compared with Vg mRNA levels. Juvenile hormone levels remained high while the ecdysteroid levels declined steadily during 1–5 days post adult emergence (PAE). The Vg mRNA levels began to increase by the end of 3rd day PAE and peaked by the 4th–5th day PAE. Gene expression profiling by microarray and quantitative real-time PCR analyses of RNA isolated from 1 to 5 days PAE beetles revealed that the genes coding for proteins involved in JH biosynthesis and action, but not those involved in 20-hydroxyecdysone (20E) biosynthesis and action had similar expression patterns as the genes coding for Vg. RNA interference (RNAi)-aided knock-down in the expression of these genes showed that both JH and 20E were required for Vg gene expression. However, Vg mRNA was induced by the application of JH III but not by the injection of 20E into the previtellogenic females. These data suggest that JH is required for Vg synthesis in the fat body and 20E influences Vg synthesis through its action on oocyte maturation.     

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.     

Determination and distribution of a female-specific protein in the brown planthopper, Nilaparvata lugens Stal (Homoptera: Delphacidae)

Electrophoretic analyses of hemolymph and body or ovary homogenates from reproducing females, males, and 5th instar nymphs of the brown planthopper, Nilaparvata lugens Stal, revealed a protein band of 175 kDa in females. An immunoblot test using antibody against this protein showed a positive reaction with a 175 kDa protein from female body or ovary homogenates. It is likely that this protein in hemolymph is vitellogenin (Vg). Distribution of Vg was determined by immunofluorescence and immunogold labeling techniques. The results showed that the positive immunofluorescence reactions were present in yolk particles, the intercellular space of follicle cells, hemolymph, and the epithelial plug of ovarioles. In addition, the yeast-like symbiotes (YLS) in mycetocytes of adults and various nymphal instars as well as those free in hemolymph or entering oocytes also exhibited a positive reaction. Electron micrographs showed that immunogold particles were found most in yolk mass and YLS over other tissues. Especially the YLS in various developmental stages all contained immunogold particles, implying that the symbiote is somewhat related with production of the female-specific protein.     

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。     

Molecular analysis of nutritional and hormonal regulation of female reproduction in the red flour beetle, Tribolium castaneum

Female reproduction includes maturation of oocytes and the synthesis of yolk proteins (vitellogenin, Vg) in the fat body and their deposition into the oocytes. Our recent studies showed that juvenile hormone (JH) regulates Vg synthesis and 20-hydroxyecdysone (20E) regulates oocyte maturation in the red flour beetle (Tribolium castaneum). Here, we report on the role of nutritional signaling on vitellogenesis and oogenesis. Comparison of gene expression between fed and starved beetles by microarray analysis showed the up-regulation of genes involved in energy homeostasis and down-regulation of genes involved in egg production in the starved beetles. The RNA interference (RNAi) aided knock-down in the expression of genes involved in insulin and TOR signaling pathways showed that both these signaling pathways play key roles in Vg synthesis and oocyte maturation. Starvation of female beetles resulted in a block in Vg synthesis but not in the progression of primary oocyte development to the resting stage. Feeding after starvation induced Vg synthesis and the progression of primary oocytes from the resting stage to the mature stage. However, in the beetles where JH or 20E synthesis or action was blocked by RNAi, both Vg synthesis and oocyte maturation were affected suggesting that both these hormones (JH and 20E) and nutritional signaling and their cross-talk regulate vitellogenesis and oogenesis.     

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。