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.     

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.     

Immunocytochemical location of vitellin in the egg of the silkworm,Bombyx mori,during early developmental stages

Summary Vitellin was purified from eggs of the silkworm,Bombyx mori, by a new method in which vitellin was extracted from isolated yolk granules. The purified vitellin had a molecular weight of 540,000. An antibody against purified vitellin was prepared in rabbits. It reacted with the hemolymph vitellogenin as well as with purified vitellin, but not with other proteins in the hemolymph or in the extract from yolk granules. The anti-vitellin IgG was used to immunocytochemically locate vitellin in theBombyx non-diapause egg during early developmental stages. In the egg, just after oviposition, vitellin was located in internal yolk granules and in small yolk granules of the periplasm. During the early developmental stages studied, vitellin was not metabolized uniformly throughout the egg. The vitellin of the internal yolk granules located at the posterior-dorsal part and of the small peripheral yolk granules was utilized in 16 h and 2 days, respectively, after oviposition. A thin, very vitellin-poor layer was located between the periplasm and the vitellin-rich interior in the newly laid egg. it was always in close contact with the periphery where blastoderm and germ-band cells developed.     

The vitellin-processing protease of Blattella germanica is derived from a pro-protease of maternal origin

Proteolytic processing of vitellin in Blattella germanica embryos is accomplished by activation of a yolk-borne cysteine protease (Mr 29 000) derived from a pro-protease precursor of Mr 40 000 (Liu et al., 1997). In the present study, fat body, ovaries and embryos of different developmental stages were examined immuno-cytochemically with purified murine anti-proprotease antibodies (Liu, 1995) to determine the intracellular location of the pro-protease. Proenzyme was detected in discrete secretory granules of the fat body and in large lysosome-like vesicles of both the follicle cell cytoplasm and the cortical ooplasm of previtellogenic ovarian follicles. In vitellogenic oocytes, coated pits and vesicles are scantily labelled for proprotease and no clear gold pattern could be discerned over the yolk granules. During embryonic development, pro-protease is associated with some, but not all, yolk granules. In newlyovulated eggs (day 0), pro-protease is either distributed over the entire granule or confined to some internal vesicles. As development proceeds, it becomes associated with almost every yolk granule and restricted to the superficial layer. By day 6, pro-protease is evident over all yolk granules but the intensity of reaction has greatly diminished, due probably to conversion of the pro-protease to the mature enzyme. Yolk granules are flanked along their margin by vesicles that are stained after zinc-osmium fixation. This observation suggests that the pro-protease may be transferred between yolk granules via vesicular shuttling. B. germanica embryos of different developmental stages were also exposed to [(3)H]-DAMP. Data show that autoradiographic grains are not evenly distributed among closely adjacent yolk granules within vitellophagic cells, a result consistent with the known slight temporal asynchrony of the acidification event.     

Vitellogenin of the cockroach,Leucophaea maderae: nucleotide sequence,structure and analysis of processing in the fat body and oocytes

A cDNA encoding vitellogenin (Vg) of the cockroach, Leucophaea maderae was cloned and sequenced. The deduced amino acid sequence consisting of 1913 residues (including 15 residues for a putative signal peptide) was obtained. Amino-terminal sequence analysis demonstrated that the pro-Vg was cleaved into four polypeptide 'subunits' following the three consensus RXXR cleavage site sequences, which were secreted as four Vg polypeptides (apparent molecular weights = 112-, 100-, 92- and 55-kD), sequestered, and deposited in the egg as four respective vitellin (Vn) polypeptides. There was, however, an additional 90-kD Vn polypeptide existed in the egg. We show that this polypeptide is a processed product from 92-kD Vn polypeptide. Northern blot analysis of poly (A)+ RNA reveals that mRNA coding for Vg is present only in the female fat body cells but neither in the ovary nor in the male fat body cells. The deduced amino acid sequence contained a serine-rich stretch at the C-terminal region. This stretch occurred also in Vgs of Periplaneta americana (Vg1 and Vg2) and Blattella germanica. The Vg of L. maderae had 26% and 31% homology with those of P. americana (Vg1 and Vg2) and B. germanica, respectively. Phylogenetic analysis (neighbour-joining) was made using four cockroach Vgs and the tree was compared with other molecular and conventional phylogenetic trees.     

Proteome mining for novel IgE-binding proteins from the German cockroach (Blattella germanica) and allergen profiling of patients

Although cockroaches are known to produce allergens that can cause IgE-mediated hypersensitivity reactions, including perennial rhinitis and asthma, the various cockroach allergens have not yet been fully studied. Many proteins from the German cockroach show high IgE reactivity, but have never been comprehensively characterized. To identify these potential allergens, proteins were separated by 2-DE and IgE-binding proteins were analyzed by nanoLC-MS/MS or N-terminal sequencing analysis. Using a combination of proteomic techniques and bioinformatic allergen database analysis, we identified a total of ten new B. germanica IgE-binding proteins. Of these, aldolase, arginine kinase, enolase, Hsp70, triosephosphate isomerase, and vitellogenin have been reported as allergens in species other than B. germanica. Analysis of the Food Allergy Research and Resource Program allergen database indicated that arginine kinase, enolase, and triosephosphate isomerase showed significant potential cross-reactivity with other related allergens. This study revealed that vitellogenin is an important novel B. germanica allergen. Personalized profiling and reactivity of IgE Abs against the panel of IgE-binding proteins varied between cockroach-allergic individuals. These findings make it possible to monitor the individual IgE reactivity profile of each patient and facilitate personalized immunotherapies for German cockroach allergy disorders.     

A 15,000-Mr protein proteolytically derived from vitellogenin within oocyte of Perinereis cultrifera (polychaete annelid). Identification, purification and partial characterization

It has been shown previously in our laboratory that, in Perinereis cultrifera, the four mature vitellin subunits (Mr 98,000, 22,000, 20,000, 16,000) are proteolytically derived within the oocyte from a single extraoocytic precursor, vitellogenin, with an apparent Mr (176,000) higher by 20,000 than the sum of the Mr of the four end products. In this report, it is shown that a 15,000-Mr protein, designated as P15, not only accumulates in maturing oocytes but also originates from outside these cells similarly to vitellin. Moreover in vivo labelling experiments indicate that the appearance of P15 occurs after vitellogenin enters the oocyte, concurrently with the appearance of the lower-Mr fragments characteristic of vitellin. From these data, it is concluded that P15 most likely represents a vitellogenin-derived protein which is generated within the oocytes during the transformation of vitellogenin into vitellin. This conclusion is further supported by the additional finding that P15 immunologically cross-reacts with vitellogenin but not with mature vitellin. P15 has been purified to homogeneity from the soluble protein fraction of submature oocytes and partially characterized. The 15,000-Mr protein exists in a monomeric form with a pI of about 7.7. Unlike vitellin, P15 does not contain significant amounts of lipid or carbohydrate and has a low absorbance at 280 nm. The amino acid composition of the purified protein is also presented.     

Characterization of vitellin protein in the twospotted spider mite, Tetranychus urticae (Acari: Tetranychidae)

In mites, vitellogenin synthesis, regulation and uptake by the oocytes as vitellin remain practically unknown. Although a partial sequence of the gene is now available, no previous studies have been conducted that describe the native vitellin protein in mites. The objective of this study was to characterize vitellin in the twospotted spider mite, Tetranychus urticae. The native twospotted spider mite vitellin migrated as a single major band with a molecular weight of 476 ± 14.5 kDa as compared to 590 ± 25.5 kDa for vitellin from the American dog tick, Dermacentor variabilis. However, isoelectric focusing analysis of native spider mite vitellin showed five bands with pI values slightly acidic to neutral (pH 5.8, 6.2, 6.7, 7.0 and 7.2), as is the case for insect and tick vitellins. Reducing conditions (SDS-PAGE) also revealed multiple subunits ranging from 290.9 to 3.6 kDa and was similar to that found in D. variabilis. Spider mite vitellin weakly bound lipids and carbohydrates compared to the tick. Unlike D. variabilis, the spider mite egg yolk protein does not bind heme. The significance of non-heme binding in mites is discussed.     

褐飞虱卵黄蛋白的分离及其生化特性

电泳结合不同染色方法证实, 褐飞虱Nilaparvata lugens (Stål)卵黄蛋白为一种糖脂结合蛋白,其分子量约为314 kD,由148 kD、124.5 kD和39.6 kD 3个亚基组成。免疫反应证明,卵黄蛋白只存在于生育期的雌性褐飞虱成虫体内。褐飞虱卵黄蛋白具有种的特异性,其免疫血清与白背飞虱的卵黄蛋白无交叉反应。     

Development of a quantitative enzyme-linked immunosorbent assay for vitellin in the mysid Neomysis integer (Crustacea: Mysidacea)

Mysid crustaceans have been put forward by several regulatory bodies as suitable test organisms to screen and test the potential effects of environmental endocrine disruptors. Despite the well-established use of mysid reproductive endpoints such as fecundity, egg development time, and time to first brood release in standard toxicity testing, little information exists on the hormonal regulation of these processes. Control of vitellogenesis is being studied intensively because yolk is an excellent model for studying mechanisms of hormonal control, and vitellogenesis can be chemically disrupted. Yolk protein or vitellin is a major source of nourishment during embryonic development of ovigorous egg-laying invertebrates. The accumulation of vitellin during oocyte development is vital for the production of viable offspring. In this context, we developed a competitive enzyme-linked immunosorbent assay (ELISA) for vitellin of the estuarine mysid Neomysis integer. Mysid vitellin was isolated using gel filtration, and the purified vitellin was used to raise polyclonal antibodies. The ELISA was sensitive within a working range of 4 to 500 ng vitellin/mL. Serial dilutions of whole body homogenates from female N. integer and the vitellin standard showed parallel binding curves, validating the specificity of the ELISA. The intra- and interassay coefficients of variation were 8.2% and 13.8%, respectively. Mysid vitellin concentrations were determined from ovigorous females and eggs at different developmental stages. The availability of a quantitative mysid vitellin ELISA should stimulate further studies on the basic biology of this process in mysids. Furthermore, it could provide a means to better understand and predict chemically induced reproductive effects in mysids.     

Mating structure and inbreeding and outbreeding depression in the rare plant Gentianella germanica (Gentianaceae)

Isolation and small size of populations as a result of habitat destruction and fragmentation may negatively affect plant fitness through pollinator limitation and increased levels of inbreeding. To increase genetic variation in small populations of rare plants artificial gene flow has been suggested as a management tool. We investigated whether pollinator limitation and inbreeding depression could reduce fitness in Gentianella germanica, an endangered biennial of increasingly fragmented calcareous grasslands in Central Europe. We experimentally excluded pollinators and generated progenies by hand-pollinating flowers with pollen from different distances. G. germanica was highly selfing. Pollinator exclusion strongly reduced seed set, indicating that pollinator limitation could potentially reduce plant fitness. Germination rate as well as number of leaves and rosette size of progeny from 10-m crosses was higher than that of progeny from open pollinations, self-, 1-m, and interpopulation crosses. After 6 mo of growth differences in the number of surviving plants persisted, whereas differences in plant size did not. The results suggest that inbreeding depression may reduce plant performance in G. germanica. Outbreeding depression in the performance of progeny from interpopulation crosses indicates that caution is necessary in using artificial interpopulation gene flow as a management tool.     

Postendocytic Provitellin Processing in the Growing Oocyte of the Short Horned Grasshopper, Oxyajaponicajaponica (Orthoptera: Acrididae)

Polyclonal antibodies made against 86 kDa (86 k), 80 kDa (80 k) and 54 kDa (54 k) vitellins of Oxya japonica japonica are used for Western blotting. Anti‐80k vitellin antibody is cross‐reacted with a 95 kDa (95 k) vitellin. While 95 k vitellin is present both in the female hemolymph and in the oocyte, 80 k vitellin is detected only in the oocyte and the laid egg. In the growing oocytes, as 95 k vitellin is faded out gradually, 80 k vitellin is accumulated increasingly, indicating postendocytic processing of 95 k vitellin brings 80 k vitellin. Further conforming the hypothesis, partial digestion of 95 k vitellin with pepsin and α‐chymotrypsin makes several protein bands of molecular weight around 80 kDa. Thus, the 95k vitellin may have a cleavage site (s) to produce 80 k vitellin which forms fairly stable tertiary structure. In the reduced condition (20 mM glutathion), both 95 k and 80 k vitellins were digested throughly by endogenous proteinase at pH 4. Both 86 k and 54 k vitellins, respectively, show no apparent molecular weight changes in the growing oocyte and in the hemolymph.     

Purification and partial characterization of vitellin from the eggs of the hard tick,Dermacentor variabilis

The major yolk proteins were purified from the eggs of the hard tick, Dermacentor variabilis using gel filtration and ion exchange chromatography. Two vitellin proteins were identified and designated vitellin A (480 kilodaltons; kDa) and vitellin B (370 kDa). The isolectric points were pH 6.1 and 6.25, respectively. The absorption maxima for both proteins were 280 and 400 nm. The buoyant density of vitellin A was 1.281 g/ml and vitellin B 1.278 g/ml. The vitellins were hemoglycolipoproteins as indicated by selective staining of polyacrylamide gels, carbohydrate analyses and lipid analyses. Under reducing conditions (SDS-PAGE), vitellin A had eight major polypeptides at 135, 110, 98, 80, 67, 50, 45, and 35 kDa. Vitellin B was identical to vitellin A with the addition of a 93 kDa subunit. The only carbohydrate detectable in the proteins was mannose. The neutral lipids detected in both proteins were cholesteryl esters, triglycerides, free fatty acids and their methyl esters, and cholestrol. The only detectable phospholipid in both proteins was phosphatidylethanolamine. The purified vitellins were immunologically identical to female hemolymph proteins but not to host hemoglobin. Antivitellin antibodies to vitellin were used to identify possible locations of vitellogenin in the organs of ovipositing females.     

RAPD variation in relation to population size and plant fitness in the rare Gentianella germanica (Gentianaceae)

We investigated the distribution of genetic variation and the relationship between population size and genetic variation in the rare plant Gentianella germanica using RAPD (random amplified polymorphic DNA) profiles. Plants for the analysis were grown from seeds sampled from 72 parent plants in 11 G. germanica populations of different size (40-5000 fruiting individuals). In large populations, seeds were sampled from parents in two spatially distinct subpopulations comparable in area to the total area covered by small populations. Analysis of molecular variance revealed significant genetic variation among populations (P <0.001), while genetic variation among subpopulations was marginally significant (P <0.06). Average molecular variance within subpopulations in large populations did not differ significantly from whole-population values. There was a positive correlation between genetic variation and population size (P <0.01). Genetic variation was also positively correlated with the number of seeds per plant in the field (P <0.02) and the number of flowers per planted seed in a common garden experiment (P <0.051). We conclude that gene flow among natural populations is very limited and that reduced plant fitness in small populations of G. germanica most likely has genetic causes. Management should aim to increase the size of small populations to minimize further loss of genetic variation. Because a large proportion of genetic variation is among populations, even small populations are worth preserving.     

Identification of yolk platelet-associated hydrolases in the oocytes of Rhodnius prolixus.

The yolk platelets from Rhodnius prolixus, a blood-sucking bug, are composed mostly of vitellin and here are shown to contain at least two hydrolytic enzymes, a phosphatase and a cathepsin D-like proteinase. Both the proteinase and the phosphatase have an acid pH optimum. No hydrolytic activity was observed under alkaline or neutral conditions. Among several proteinase inhibitors tested, only pepstatin could abolish vitellin breakdown in vitro. The proteinase appears to be bound to the yolk platelet membranes. The phosphatase activity, using p-nitrophenyl phosphate as substrate, was enhanced after disruption of the platelet membrane by Triton X-100. This activity could be inhibited by tartrate but not by p-cloromercuribenzoate.     

Purification of vitellin from the ovary of Chinese mitten-handed crab (Eriocheir sinensis) and development of an antivitellin ELISA

Vitellin was purified from ovaries of mature female Chinese mitten-handed crab (Eriocheir sinensis) using gel filtration chromatography. Analysis by native PAGE showed the vitellin had a native molecular mass of 520 kDa, while denaturing SDS-PAGE revealed two subunits of 97 and 74 kDa. Purified vitellin was used to raise polyclonal antisera, with which an enzyme-linked immunosorbent assay (ELISA) was developed. The ELISA was sensitive and could effectively detect vitellin in the range of 7.8-500 ng. Furthermore, vitellin levels in various developmental stages of oogenesis were measured with the ELISA assay. The results indicated that levels of vitellin increased significantly from 0.22 mg/ovary at Stage II to 360.31 mg/ovary at Stage IV.     

Proteolytic processing of the vitellogenin precursor in the boll weevil,Anthonomus grandis

The soluble proteins of the eggs of the coleopteran insect Anthonomus grandis Boheman, the cotton boll weevil, consist almost entirely of two vitellin types with M_rs of 160,000 and 47,000. We sequenced their N-terminal ends and one internal cyanogen bromide fragment of the large vitellin and compared these sequences with the deduced amino acid sequence from the vitellogenin gene. The results suggest that both the boll weevil vitellin proteins are products of the proteolytic cleavage of a single precursor protein. The smaller 47,000 M vitellin protein is derived from the N-terminal portion of the precursor adjacent to an 18 amino acid signal peptide. The cleavage site between the large and small vitellins at amino acid 362 is adjacent to a pentapeptide sequence containing two pairs of arginine residues. Comparison of the boll weevil sequences with limited known sequences from the single 180,000 M_r honey bee protein show that the honey bee vitellin N-terminal exhibits sequence homology to the N-terminal of the 47,000 M_r boll weevil vitellin. Treatment of the vitellins with an N-glycosidase results in a decrease in molecular weight of both proteins, from 47,000 to 39,000 and from 160,000 to 145,000, indicating that about 10–15% of the molecular weight of each vitellin consists of N-linked carbohydrate. The molecular weight of the deglycosylated large vitellin is smaller than that predicted from the gene sequence, indicating possible further proteolytic processing at the C-terminal of that protein. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Species-specific oocyte proteins as molecular markers for Lineus taxonomy

The yolk proteins from the eggs of five species of thenemertean genus Lineus were analysed and theresulting data were used to define L. longissimus, L. sanguineus, L. lacteus, L. ruber, and L. viridis as distinct taxa (species to date). All fivespecies have at least one large (80–60 kDa) and onemedium sized (45–30 kDa) vitellin subunit, but thereare significant differences in the number and sizes ofthe molecular subunits. The large and medium sizedvitellin components of all the species areglycosylated, except for the 145 kDa protein of L. sanguineus. Rabbit antibodies to the L. lacteus vitellin subunits cross-reacted withthe vitellin subunits in the eggs of the otherspecies. The vitellin components of these five speciesof nemertean are very similar. Two species, L.ruber and L. viridis, lay their eggsin gelatinous masses, and the electrophoretic patternsof the jelly proteins show that the physicalconsistency of the jelly depends on molecular weightsof the components. The egg mass of L. viridis contains smaller proteins than the egg massof L. ruber. The repeatablespecies-specific patterns of vitellin componentsprovide a useful complement to the usual taxonomiccriteria.     

Atypical vitellins in ponerine ants (Formicidae: Hymenoptera)

Higher hymenopteran vitellogenin/vitellins have been characterized as containing one large apoprotein. We show that in the ant subfamily Ponerinae, species in the tribes Odontomachini, Platythyrini, and Amblyoponini, also have a vitellin with this simple structure, containing a single apoprotein of 180-190kDa. Species in tribes Ponerini and Ectatommini, however, have vitellins containing multiple subunits. The size and number of the subunits varies, with differences even among species within the same genus. This is the first report of diversity in vitellogenin structure in the higher Hymenoptera. Vitellin and vitellogenin in Harpegnathos saltator (Ponerini) contain two large subunits of about 165kDa and two small subunits of about 45 and 43kDa. N-terminal sequence analysis suggests that provitellogenin is cleaved at two different sites, producing two large and two small subunits differing slightly in size. Diversity of vitellin types in Ponerini and Ectatommini is similar to that found in the more primitive tenthredinoid sawflies (Hymenoptera, Symphyta), and may indicate polyphyly in the Ponerinae. Insect vitellogenins and yolk proteins show considerably more diversity than originally believed, and the possibility of the functional significance of these differences should be considered.     

Correlation of yolk phosphatase expression with the programmed proteolysis of vitellin in Blattella germanica during embryonic development

Proteolytic processing of the vitellin in Blattella germanica eggs occurs 4 days postovulation and is correlated with both the onset of its utilization and the major portion of the embryo's growth. Yolk phosphatase is also expressed coincident with this event, and some aspects of its activation have been investigated. The enzyme is absent from the ooplasm (yolk) during the first 2 days following ovulation but increases approximately 20-fold in specific activity between days 3 and 4, when assayed at pH 3.9 or 4.8 and 9-fold at pH 6.5. No activation is observed for yolk-bound α-mannosidase, its specific activity remains elevated through the first 6 days following ovulation. This suggests that expression of the phosphatase is regulated independently of that of α-mannosidase in the yolk. Yolk with active phosphatase can dephosphorylate native vitellin, delipidated vitellin, and phosphocasein. Sucrose density gradient centrifugation of yolk obtained from eggs 4 days postovulation, revealed that phosphatase activity cosediments with material which reacts with antivitellin antibodies, while α-mannosidase and β-N-acetyl glucosaminidase are found near the top of the gradient. Oothecae derived from crossing certain translocational heterozygote males and wild-type females contain some eggs with severely depressed levels of yolk phosphatase in which embryos do not grow. Vitellin in these eggs fails to undergo proteolytic processing as late as day 5 postovulation and retains the subunit composition of freshly ovulated vitellin.