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。     

Processing of pro-vitellogenin in insect fat body: a role for high-mannose oligosaccharide

Several discrete events were resolved in the processing of vitellogenin in Blattella germanica. Using tunicamycin to inhibit the synthesis of high-mannose oligosaccharide, a high molecular weight pro-vitellogenin peptide (apo-proVG, Mr 215,000) was identified in fat body. Dosages of tunicamycin which inhibited glycosylation of vitellogenin by 98% inhibited its synthesis by as much as 59%, yet led to an intracellular accumulation of apo-proVG. Reversibility and dose dependency of these effects on vitellogenin synthesis, glycosylation, proteolytic processing, and secretion were demonstrated. In control insects, glycosylation of apo-proVG yielded a Mr 240,000 pro-vitellogenin peptide (proVG). FITC-Concanavalin A bound to purified proVG but not to apo-proVG, thus confirming an absence of high-mannose oligosaccharide in the apo-protein. Following its glycosylation, proVG was processed rapidly in fat body to Mr 160,000 (VG160) and Mr 102,000 (VG102) peptides which subsequently were secreted into hemolymph. After uptake into developing oocytes, the VG160 peptide was processed further prior to chorionation, yielding subunits of Mr 95,000 and 50,000. Uniqueness of the peptides of mature vitellin (Mr 102,000, 95,000, and 50,000) was indicated by comparison of the CNBr fragments of each purified subunit. Staining of CNBr fragments with FITC-Concanavalin A also indicated that high-mannose oligosaccharides are attached at one or more sites within each vitellin subunit. Resolution of the substructure of this insect vitellin and identification of events involved in the processing and secretion of its fat body apo-protein provide a basis for further study of the assembly and transport of vitellogenin, its packaging in eggs, and utilization during embryogenesis.     

Oosorption in the stink bug,Plautia crossota stali: induction and vitellogenin dynamics

Oosorption, resorption of developing oocytes in the ovary, in P. c. stali is characterized by changes in appearance of oocytes from opaque greyish green or orange to transparent, degeneration of yolk granules and disappearance of oocyte contents. Starvation and virginity were indicated to be factors that induce oosorption. SDS PAGE/Western blotting analysis using anti-vitellogenin antiserum detected two major and many minor bands in haemolymph samples. Egg extracts showed a more complicated set of positive bands in the same analysis. Yolk protein, vitellin, therefore, seemed to be formed after complicated processing of vitellogenin following its uptake by the oocytes. In starved, oosorption-induced females, vitellogenin concentration in the haemolymph was lower than that of fed females, and Western blotting failed to detect either oosorption-specific or ovary-specific peptide fragments in haemolymph samples collected from those females. These results suggest that once oosorption was induced vitellogenin/vitellin in oocytes was degraded rapidly and released into the haemolymph in the form of amino acids or small peptides too small to be recognized by the anti-vitellogenin antiserum.     

Inhibition of (3H)vitellogenin uptake by isolated amphibian oocytes injected with cytoplasm from progesterone-treated mature oocytes.

Fully grown meiotically immature (germinal vesicle stage) amphibian oocytes incorporate radioactive protein ([³H]vitellogenin) following in vitro culture. In vitro exposure of such oocytes to exogenous progesterone induces germinal vesicle breakdown and inhibits incorporation of vitellogenin. In the present studies, we have investigated the effects of cytoplasm taken from mature and immature oocytes on incorporation of vitellogenin and nuclear breakdown following microinjection of this material into immature oocytes. Vitellogenin incorporation was markedly suppressed in oocytes which underwent nuclear breakdown following injection with cytoplasm from mature oocytes. Incorporation of vitellogenin into oocytes which did not mature after injection with cytoplasm taken from mature oocytes resembled that seen in oocytes injected with immature cytoplasm. The degree of suppression of vitellogenin incorporation following cytoplasmic injections was similar to that seen in uninjected oocytes treated with progesterone. Oocytes injected with cytoplasm obtained from immature oocytes did not undergo either nuclear breakdown or changes in vitellogenin incorporation. The results suggest that cytoplasm obtained from mature oocytes contains a factor(s) which alters directly or indirectly the capacity of the oocyte cell membrane to incorporate vitellogenin. Enucleated immature oocytes also incorporated [³H]vitellogenin, and injection of such oocytes with mature, but not immature, oocyte cytoplasm suppressed vitellogenin incorporation. Suppressive effects of injected cytoplasm thus appear to be mediated through physiological changes in the recipient oocyte cytoplasm rather than the nuclear component.     

Patterns of haemolymph vitellogenin and ovarian vitellin in the German cockroach, and the role of Juvenile Hormone

Abstract. The concentrations of fat body and haemolymph vitellogenin and ovarian vitellin during the first gonadotropic cycle of the cockroach Blattella germanica (L.) (Dictyoptera, Blattellidae) have been studied. For these purposes, a polyclonal antibody against B. germanica vitellogenin and vitellin has been obtained, and an ELISA to quantify these proteins has been developed. Ovarian vitellin levels follow a pattern which parallels those of basal oocyte growth and Juvenile Hormone production by the corpora allata. This suggests that Juvenile Hormone regulates vitellogenin uptake into oocytes. Fat body and haemolymph vitellogenin levels give cyclic and parallel patterns. However, the cycle of Juvenile Hormone appears delayed with respect to that of vitellogenin. We suggest that the production of Juvenile Hormone, although cyclic in profile, does not modulate alone the cycle of vitellogenin. At least a supplementary mechanism, apparently independent of Juvenile Hormone, may be involved in the decline of vitellogenin production at the end of the vitellogenic cycle.     

Heterosynthetic origin of the major yolk protein,vitellin, in a nereid,Perinereis cultrifera (polychaete annelid)

• 1.1. An examination of proteins synthesized by Perinereis cultrifera oocytes incubated in vitro with [³H]leucine clearly shows that these cells are not capable of synthesizing the main yolk protein previously identified in this worm.
• 2.2. In addition, the detection of radiolabelled vitellin in oocytes after in vitro incubation of an oocyte-coelomocyte cell mixture in presence of [³H]leucine strongly suggests that the coelomocytes, free cells in the coelomic cavity, synthesize and secrete a vitellin precursor, vitellogenin, that is subsequently taken up by the oocytes.
• 3.3. Two native proteins differing in mol. wt but reacting with anti-vitellin antibodies have been identified in coelomocyte incubation medium. Also found in the coelomic fluid, they have been designated VG1 (M_r = 530,000) and VG2 (M_r = 320,000).
• 4.4. The two vitellogenins consist of a single type of polypeptide of M_r = 176,000 and are incorporated in the oocytes where they are apparently observed under a single molecular form corresponding to VG1, the highest mol. wt protein similar in size to the initial form of vitellin (VI, 530,000).
• 5.5. From these data, it seems likely that VG2 is a monomeric molecule that is taken up by the oocytes as a dimer of VG1.
• 6.6. We conclude that P. cultrifera accumulates vitellin heterosynthetically and that vitellogenin is produced by the coelomocytes. Moreover, a single polypeptide similar in size to the polypeptidic component of secreted vitellogenin has been detected in the coelomocytes.
• 7.7. Since this polypeptide has been identified previously as the single intraoocytic precursor of the four lower mol. wt products that make up the mature form of vitellin (V5), it appears that P. cultrifera exhibits for vitellogenin a processing pathway in which cleavage of the precursor occurs only after uptake by the oocyte.

     

Vitellogenesis in Acanthoscelides obtectus (Coleoptera: Bruchidae). I—Oocyte development and vitellogenin in a European strain

Oocyte development was studied in a European strain of the bean weevil, Acanthoscelides obtectus. In the experimental system used, oocyte growth begins after several hours of imaginal life. The lengths of the terminal and penultimate oocytes were measured and correlations between these measurements showed that the latter stopped growing when the former was in the phase of accelerated growth.

Vitellogenin could be immunologically detected in the hemolymph of sexually mature females and was undetectable in males, larvae and in females immediately after imaginal exuviation. The moment of vitellogenin appearance in young females varies from one insect to another.

It appears that no specific external stimulus is required to initiate the synthesis and incorporation of the vitellin protein in this European strain.     

Identification and characterization of vitellin in a hermophrodite shrimp,Pandalus kessleri

• 1.1. A female specific protein (FSP, vitellogenin) in hemolymph and its related ovarian protein (vitellin) of Pandalus kessleri were studied by means of electrophoretical and immunological procedures.
• 2.2. The vitellin was purified from vitellogenic ovaries using hydroxylapatite, DEAE cellulose and Sepharose 6B columns, consecutively.
• 3.3. The vitellin had a molecular weight of approximately 560 kD and was composed of two subunits, 81 and 110 kD, respectively.
• 4.4. The vitellogenin concentrations in the hemolymph increased as vitellogenesis in the ovarian oocytes advanced and dropped markedly after the release of mature eggs.

     

Hepatopancreas is the likely organ of vitellogenin synthesis in the freshwater prawn, Macrobrachium rosenbergii

The objective of the present study was to investigate the source of vitellogenin in the freshwater prawn, Macrobrachium rosenbergii. Ovarian development of M. rosenbergii was classified into five stages (stage I-V). Vitellin/vitellogenin was detected in the ovary and the hepatopancreas in different stages by native-PAGE and Western blotting. Two and three subunits of vitellin were observed in the ovary at the early- (I-II), mid- and late- (III-V) stages, respectively. The subunit of vitellogenin was not detected in the hepatopancreas at different stages of prawns. Hepatopancreas had positive immunocytological staining (against vitellin antibody) in different ovarian stages of prawn. Only vitellogenic oocyte but not previtellogenic oocytes and follicle cells had a positive immunocytological staining. Hepatopancreas could synthesize radiolabeled immunoreactive proteins after incubation with radiolabeled glycine on the basis of immunoprecipitation (against vitellin antiserum). Therefore, it is concluded that hepatopancreas is the most likely organ to synthesize vitellogenin in the freshwater prawn, M. rosenbergii.     

The vitellogenin of Leucophaea maderae: Synthesis as a large phosphorylated precursor

Phosphorylation of vitellogenin (yolk protein precursor) and vitellin (major yolk protein) polypeptides of Leucophaea maderae was studied by [³²P]ortho phosphate labeling and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography. The vitellogenin molecule was isolated from the hemolymph and fat body by antibody precipitation and high-performance liquid chromatography (HPLC), and shown to consist of at least five polypeptides (“subunits”) which had apparent molecular masses of 155, 112, 95, 92 and 54 kD. Labeling studies with ³²P showed that the covalently attached phosphorus was distributed in an uneven fashion among the five polypeptides. The two heavily-phosphorylated polypeptides, 112 and 54 kD, corresponded to the large and small, mature vitellin subunits. Quantitative SDS-PAGE analysis of long-term ³²P-labeled vitellin showed that these large and small “subunits” contained 55 and 30%, respectively, of the total radioactivity.When fat body was pulse-labeled with ³²P we found a heavily-phosphorylated intracellular 215 kD polypeptide which was precipitable with anti-vitellogenin. The synthesis of this intracellular precursorform of vitellogenin (pre-Vg) was under absolute juvenile hormone control. In vitro³²P pulse-chase experiments showed that pre-Vg was proteolytically processed within the fat body into some (or possibly all) of the mature vitellogenin subnits. Furthermore, peptide mapping confirmed that all of the phosphorylated vitellogenin subunits were derived from pre-Vg. Since previous studies have shown that phosphoserine residues account for essentially all of the covalently-attached phosphorus of the native vitellogenin molecule, we speculate that the asymmetric pattern of vitellogenin and vitellin subunit-phosphorylation is due to an uneven distribution of phosphoserine residues along the initial pre-Vg polypeptide chain. Finally, we conclude that phosphorylation of vitellogenin occurred post-translationally in the fat body endoplasmic reticulum because we could identify ³²P-labeled pre-Vg in purified microsomal vesicles but not in nascent vitellogenin polypeptide chains attached to vitellogenin polyribosomes.     

In vitro study of selective endocytosis of vitellogenin by locust oocytes

Summary Oocytes ofLocusta migratoria selectively sequester the female-specific protein vitellogenin at a high rate. In vitro studies using in vivo radiolabelled vitellogenin show that uptake rates increase proportionally with time and temperature (with a sharp change between 10° and 15°C). They also depend on oocyte size and vitellogenin concentration. The uptake process is saturable. Vitellogenin is taken up more specifically than vitellin. Bovine serum albumin, casein and male locust haemolymph proteins do not inhibit endocytosis of vitellogenin. However, several metabolic inhibitors and phosphorylated compounds, such as phosvitin or phosphohexoses, are potent inhibitors of vitelogenin incorporation.     

蜜蜂卵黄原蛋白的作用

卵黄蛋白不仅为胚胎发生提供营养物质,它在生物体内还具有其他的生物学功能。昆虫卵黄蛋白是昆虫卵内的营养储备,它的前体主要来源于脂肪体的雌性特异血蛋白——卵黄原蛋白(Vg),它是近年来昆虫生理学和生化学最活跃的领域之一,文章介绍蜜蜂卵黄原蛋白在蜜蜂生殖过程中的作用,以及与蜜蜂社会性生活及寿命的关系。     

Heart contains two substrates (Mr = 40,000 and 41,000) for pertussis toxin-catalyzed ADP-ribosylation that co-purify with Ns

Two peptides (Mr = 40,000 and 41,000) in membranes of rabbit heart are radiolabeled when the membranes are incubated in the presence of activated pertussis toxin and [32P]NAD+. The 41,000-Mr peptide appears to be the alpha subunit of the inhibitory regulatory protein of adenylate cyclase, Ni. The 40,000-Mr substrate for pertussis toxin in the heart was investigated. Purification of the stimulatory regulatory protein of adenylate cyclase, Ns, results in the co-purification of the alpha subunits of both Ns and Ni, the putative beta- (Mr = 35,000) and gamma- (Mr approximately equal to 15,000) subunits of Ns and Ni, and the additional 40,000-Mr peptide that is ADP-ribosylated by pertussis toxin. This 40,000-Mr substrate for pertussis toxin action appears to be a major N-protein of mammalian heart.     

The relationship between nutrition,vitellogenin, vitellin and ovarian development in the housefly, Musca domestica L.

The effect of adult nutrition on oögenesis during the first gonotropic cycle was studied in three strains of the housefly, Musca domestica. Two of the strains were anautogenous and the third was autogenous. In these strains, three subunits (51, 43 and 42 kdaltons) of vitellogenin and vitellin were electrophoretically identical using SDS-PAGE electrophoresis for haemolymph proteins of vitellogenic females and for egg extracts. Each developmental stage of the ovary in individual females flies of both autogenous and anautogenous strains fed on either sugar or protein clearly reflected the appearance of electrophoretic bands for vitellogenin and vitellin. Using immunological analysis, a very small amount of vitellogenin was detectable in the haemolymph of previtellogenic flies. The highest level of vitellogenin appeared in the haemolymph at the middle of vitellogenic phase and reached about 25% of the total haemolymph protein. There were differences in vitellogenin concentration in females with mature eggs between the two anautogenous strains: vitellogenin was not detectable in one strain, and the other showed 30% of the maximal level.     

Vitellogenin synthesis in the ovary of scallop,Patinopecten yessoensis: Control by estradiol-17 beta and the central nervous system

Elucidation of a profile of scallop vitellin formation associated with oogenesis and its endocrine control, and identification of a vitellogenin synthesizing site were immunologically undertaken by using anti-scallop Vn serum. Vn content increased during ovarian growth and accounted for more than 80% of the water soluble protein of the ovary at the mature stage. In vivo injection of estradiol-17 beta (E(2)) resulted in an increase in Vn content in the ovary. In vitro accumulation of Vn in the ovarian tissue was promoted with E2 and a vitellogenesis promoting factor (VPF) from cerebral plus pedal ganglion which was heat stable, less than MW 10,000 and trypsin/chymotrypsin resistant. Estrogen receptor (ER)-like immunoreactivity was found in the growing oocyte and the auxiliary cell in close contact with growing oocytes, in which Vn immunoreactivity was also found. It is suggested that the vitellogenin synthesis occurred inside the ovary, especially in the auxiliary cell, and is controlled by E2 and VPF via ER.     

Role of juvenile hormone in the synthesis and sequestration of vitellogenins in the red cotton stainer, Dysdercus koenigii (Heteroptera: Pyrrhocoridae)

Investigations were carried out to determine the role of juvenile hormone (JH) and 20-hydroxy ecdysone in the synthesis and uptake of vitellogenins, which were earlier identified, purified and characterised, in Dysdercus koenigii. The concentration(s) of vitellogenin(s) in fat body, haemolymph and that of vitellin(s) in ovary were significantly lower after chemical allatectomy at eclosion. In addition, at 70 h after emergence, chemical allatectomy reduced ovarian vitellin concentration, but vitellogenin levels remained normal in the fat body and haemolymph. The haemolymph vitellogenins were not incorporated into oocytes in such insects. Administration of JH-III at 20 h after allatectomy restored vitellogenin levels in the fat body and haemolymph, but the ovary failed to incorporate the available vitellogenins from haemolymph in such insects. However, when JH-III was administered twice, one at 20 h and then at 70 h after allatectomy, vitellogenin concentrations in fat body and haemolymph and also vitellin concentrations in ovary approached control levels. It is suggested that JH has two separate roles, one in vitellogenin synthesis and the other in uptake. 20-hydroxy ecdysone had no apparent role in either vitellogenin synthesis or uptake in D. koenigii.     

Yolk sphere formation is initiated in oocytes before development of patency in follicles of the moth,Plodia interpunctella

We describe a provitellogenic stage, a previously unrecognized stage of follicle development in moths, and show that oocytes begin yolk sphere formation prior to the development of patency by the follicular epithelium. The vitellogenic activities of follicles from pharate adult femalePlodia interpunctella (Hübner) were determined by visualizing the subunits of vitellin (YP1 and YP3) and the follicular epithelium yolk protein (YP2 and YP4) using monospecific antisera to each subunit to immunolabel whole-mounted ovaries or ultrathin sections. At 92 h after pupation, yolk spheres that contained only YP2 began to proliferate in the oocytes. The inter-follicular epithelial cell spaces were closed at 92 h making vitellogenin inaccessible to the oocyte, and consequently, the vitellin subunits were not observed in the yolk spheres. YP2 uptake most likely occurred across the brush border from the follicular epithelial cells to the oocyte at this time. At 105 h, the inter-follicular epithelial cell spaces appeared closed yet trace amounts of labeling for vitellin were observed in the spaces and also in the yolk spheres along with YP2. Equivalent labeling for all four YPs in yolk spheres was finally observed at 112 h after pupation when the follicular epithelium had become patent. These data indicate that the provitellogenic stage is an extended transition period between the previtellogenic and vitellogenic stages that lasts for approximately 13 h, and it is marked at the beginning by YP2 yolk sphere formation in the oocyte and at the end by patency in the follicular epithelium.     

Vitellogenesis in Xenopus laevis and chicken: cognate ligands and oocyte receptors. The binding site for vitellogenin is located on lipovitellin I

Vitellogenesis is the process of yolk formation in rapidly growing oocytes of oviparous species. The transport of yolk precursor proteins from the blood plasma into the oocyte is achieved by receptor-mediated endocytosis. Although the Xenopus oocyte is one of the prime experimental systems for expression of foreign genes and their products, the receptor for the main vitellogenic protein, vitellogenin, from this extensively utilized cell has not been identified. Here we have applied ligand and immunoblotting to visualize the Xenopus laevis oocyte receptor for vitellogenin as a protein with an apparent Mr of 115,000 in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. The receptor from the amphibian oocyte also recognizes chicken vitellogenin, and vice versa; furthermore, the two receptor proteins are immunologically related as revealed by Western blotting with anti-chicken vitellogenin receptor antibodies. The receptors from both species bind the lipovitellin moiety of vitellogenin, as revealed by ligand blotting with radiolabeled lipovitellin polypeptides as well as by a novel reverse ligand blotting procedure utilizing nitrocellulose-immobilized ligand. Since vitellogenins of chicken and Xenopus have been shown to be structurally similar and evolutionarily related (Nardelli, D., van het Schip, F. D., Gerber-Huber, S., Haefliger, J.-A., Gruber, M., AB, G., and Wahli, W. (1987) J. Biol. Chem. 262, 15377-15383), it appears that conservation of key structural elements required for efficient vitellogenesis extends from the ligands to their receptors on the oocyte plasma membrane.     

Vitellogenin Receptors From Lobster Oocyte Membrane: Solubilization and Characterization by a Solid Phase Binding Assay

Summary

A solid phase binding assay was developed to study the vitellogenin binding sites from solubilized Homarus americanus oocyte membrane. Different detergents (SDS, CHAPS, DOC) were tested and DOC (sodium deoxycholate) was found to be the most effective agent. The solid phase binding assay involves an adsorption of solubilized membranes in wells of microtitration plates. Enzyme labelling of the ligand was realized by coupling glutaraldehyde treated peroxidase with purified vitellin. Scatchard analysis after competition experiments in different conditions (time and temperature) revealed an apparent equilibrium dissociation constant (Kd) close to 70 nM, reached after one hour incubation at 37°C. Binding activity of oocyte membranes is maximal at the beginning of vitellogenesis and decreases in older oocytes.     

Mosquito cathepsin B-like protease involved in embryonic degradation of vitellin is produced as a latent extraovarian precursor

Here we report identification of a novel member of the thiol protease superfamily in the yellow fever mosquito, Aedes aegypti. It is synthesized and secreted as a latent proenzyme in a sex-, stage-, and tissue-specific manner by the fat body, an insect metabolic tissue, of female mosquitoes during vitellogenesis in response to blood feeding. The secreted, hemolymph form of the enzyme is a large molecule, likely a hexamer, consisting of 44-kDa subunits. The deduced amino acid sequence of this 44-kDa precursor shares high similarity with cathepsin B but not with other mammalian cathepsins. We have named this mosquito enzyme vitellogenic cathepsin B (VCB). VCB decreases to 42 kDa after internalization by oocytes. In mature yolk bodies, VCB is located in the matrix surrounding the crystalline yolk protein, vitellin. At the onset of embryogenesis, VCB is further processed to 33 kDa. The embryo extract containing the 33-kDa VCB is active toward benzoyloxycarbonyl-Arg-Arg-para-nitroanilide, a cathepsin B-specific substrate, and degrades vitellogenin, the vitellin precursor. Both of these enzymatic activities are prevented by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), a thiol protease inhibitor. Furthermore, addition of the anti-VCB antibody to the embryonic extract prevented cleavage of vitellogenin, strongly indicating that the activated VCB is involved in embryonic degradation of vitellin.