For uptake of yolk precursors,epithelial cell-oocyte gap junctional communication is required by insects representing six different orders

For uptake of vitellogenin protein into nascent yolk spheres, communication through open gap junction channels between the follicle epithelium and oocyte is required by six different insects representing six different orders. It was recently shown in the hemipteran, Oncopeltus fasciatus, that endocytic uptake of yolk protein resulting in the formation of nascent yolk spheres depended upon an intact epithelium communicating with the oocyte through patent gap junctions. Following treatment with octanol, which down-regulated gap junctions below the level of dye coupling, vitellogenin uptake was terminated. Yet, for another hemipteran, Dysdercus intermedius, it has been shown that yolk spheres can form even when all epithelial cells have been stripped from the oocyte. To determine if the mechanism seen in Oncopeltus is present in other insects, we utilized the same techniques to study nascent yolk sphere production in a dipteran, Drosophila melanogaster, a lepidopteran, Actias luna, a hymenopteran, Xylocopa virginica, a coleopteran, Tenebrio molitor and an orthopteran, Acheta domesticus. In each of these, when gap junctions were down-regulated yolk uptake quickly stopped. That six different insects from six different orders all required a gap junctionally transmitted chemical signal of epithelial cell origin suggests that this mechanism is widespread throughout the insects.     

大阪鲫鱼两种卵黄蛋白免疫细胞化学的研究

以电泳提纯的卵黄脂磷蛋白和卵黄蛋白Ｌ制备兔抗两种蛋白的抗血清,采用ＰＡＰ法对性腺成熟雌性大阪鲫鱼的肝细胞和卵母细胞进行两种蛋白免疫细胞化学位研究。肝细胞的粗面内质网上有强烈的卵黄脂磷蛋白的阳性反应,特别是在线粒体的基质中也发现卵黄脂磷蛋白的阳性反应,而另外一种类似于卵黄高磷蛋白的卵黄蛋白－卵黄蛋白Ｌ在肝细胞的粗面内质网和线粒体均呈现阴性反应,提示卵黄脂磷蛋白的前体物质存在于肝细胞的粗面内质网和线粒     

Trans-order yolk protein can stimulate endocytic activity in Drosophila oocytes

Vitellogenins (Vgs) and mature yolk from some non-Dipteran insects can be recognized by Drosophila melanogaster oocyte Vg receptors and incorporated via receptor-mediated endocytosis into nascent yolk spheres (NYS). It had previously been assumed that only Vgs of Drosophila or other Dipterans could be so endocytosed. Drosophila ovarian follicles from 4-day old females were incubated in the presence of physiological salt solution (PSS) containing some fluorescent TexasRed-Dextran (Dex-red) or PSS-Dex-red in which either female hemolymph, or vitellin (mature yolk) from lysed oocytes was present from any of the following: (1) Drosophila (Diptera); (2) Oncopeltus (milkweed bug, Hemiptera); (3) Acteaus (luna moth, Saturniidae Lepidoptera); (4) Papilio (swallowtail butterfly, Papilionidae Lepidoptera); or (5) Xylocopa (carpenter bee, Hymenoptera). Under incubation conditions, any NYS would become fluorescent due to non-specific fluid-phase uptake. Ovarian follicles incubated in PSS-DexRed alone or in PSS with hemolymph from males did not carry out endocytosis detectable by this technique, but all other treatments listed above did.     

Oocyte growth in the sheepshead minnow: Uptake of exogenous proteins by vitellogenic oocytes

The structure of the vitellogenic follicle of the sheepshead minnow, Cyprinodon variegatus, is described. Follicles enlarge primarily by protein yolk accumulation (vitellogenesis) and subsequently increase in size by hydration. This study uses the electron-dense tracer, horseradish peroxidase, and a larger heterologous protein,Xenopus laevis [³H]vitellogenin, to follow the fate of exogenous proteins from the maternal circulation to yolk spheres of the growing oocyte. Materials appear to leave the perifollicular capillaries via an interendothelial route, traverse the theca and the patent intercellular channels of the follicular epithelium and the pore canals of the vitelline envelope. At the oocyte surface they are incorporated via micropinocytosis and translocated to growing yolk spheres in the peripheral ooplasm. In contrast to other studies on oocyte growth in teleosts which suggest that yolk is an autosynthetic product, this study substantiates the importance of heterosynthetic processes during oocyte growth in C. Variegatus.     

Development of Vitellogenin-ELISA,an In Vivo Bioassay,and Identification of Two Vitellogenesis-Inhibiting Hormones of the Tiger Shrimp <Emphasis Type="BoldItalic">Penaeus monodon</Emphasis>

A sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of vitellogenin in the hemolymph of Penaeus monodon is reported. Lipovitellin from the mature ovary was purified using hydroxylapatite column chromatography and used as the standard protein, which was serially diluted from 3336 to 6.51 μg and gave a linear plot. Sensitivity results showed ELISA was insensitive to samples that did not contain vitellogenin or lipovitellin. Specificity results showed the degree of the discrimination of the assay between positive samples (having vitellogenin or lipovitellin) and negative samples (devoid of vitellogenin or lipovitellin). Reproducibility studies showed that the intraassay coefficient of variation was 5.1% (n= 11) and the interassay coefficient of variation was 5.15% (n= 13). Separation of X-organ sinus gland peptides using a reversed phase column gave a total of 37 fractions. Two fractions were found to reduce the hemolymph vitellogenin concentration in a time-dependent manner and could be identified as vitellogenesis-inhibiting hormones I and II. Received February 2, 2001; accepted June 24, 2001     

PROTEIN UPTAKE IN THE OOCYTES OF THE CECROPIA MOTH

The formation of yolk spheres in the oocyte of the cecropia moth, Hyalophora cecropia (L.), is known immunologically to result largely from uptake of a sex-limited blood protein. Recent electron microscope analyses of insect and other animal oocytes have demonstrated fine structural configurations consistent with uptake of proteins by pinocytosis. An electron microscope analysis of the cecropia ovary confirms the presence of similar structural modifications. With the exception of two apparently amorphous layers, the basement lamella on the outer surface of the follicular epithelium and the vitelline membrane on the inner, there is free access of blood to the oocyte surface between follicle cells. Dense material is found in the interfollicular cell space and adsorbed to the outer surface of the much folded oocyte membrane. Pits in the oocyte membrane and vesicles immediately under it are lined with the same dense material not unlike the yolk spheres in appearance. Introduction of ferritin into the blood of a developing cecropia moth and its localization adsorbed to the surface of the oocyte, and within the vesicles and yolk spheres of the oocyte cortex, is experimental evidence that the structural modifications of the oocyte cortex represent stages in the pinocytosis of blood proteins which arrive at the oocyte surface largely by an intercellular route. Small tubules attached to the yolk spheres are provisionally interpreted as a manifestation of oocyte-synthesized protein being contributed to the yolk spheres.     

Xenopus lipovitellin 1 is a Zn2+- and Cd2+-binding protein

This report discusses the identification of a Zn²⁺- and Cd²⁺-binding protein of Xenopus laevis that is abundant in vitellogenic oocytes and in embryos from fertilization to stage 46. Oocyte or embryo homogenates were fractionated by SDS-PAGE, blotted onto nitrocellulose, and probed with ⁶⁵Zn²⁺ or ¹⁰⁹Cd²⁺. The resulting autoradiograms showed binding of both radio-nuclides to a protein, designated pCdZn. Freon extraction of oocyte and embryo homogenates showed pCdZn to be a yolk protein. When pCdZn was isolated from oocyte homogenates by ammonium sulfate precipitation, delipidation, and chromatography, it co-purified with lipovitellin 1. The amino acid composition of pCdZn closely resembled the reported composition of lipovitellin 1 and the molecular weight of purified pCdZn (～115 kD) corresponded to reported values for lipovitellin 1 (111–121 kD). Amino acid sequence analyses of five peptides derived from pCdZn yielded 94% identity to the reported sequence of lipovitellin 1, deduced from the DNA sequence of the Xenopus vitellogenin A2 precursor gene. Based on these findings, pCdZn was identified as lipovitellin 1. This study suggests that lipovitellin 1 is the major storage protein for zinc in mature oocytes and developing embryos of Xenopus laevis. © 1995 wiley-Liss, Inc.     

中华稻蝗卵子卵黄发生期超微结构研究

利用透射电镜研究了中华稻蝗Oxya chinesis卵子发生中卵黄发生期的超微结构.卯黄发生初期,滤泡上皮细胞胞质内出现大量粗面内质网及线粒体等细胞器,可能与为卵母细胞提供营养有关.卵黄发生期卵母细胞胞质内卵黄球逐渐增多,它也许有多种来源.观察到环形片层结构,并讨论了其可能功能.     

The role of an epithelial occlusion zone in the termination of vitellogenesis in Hyalophora cecropia ovarian follicles.

At the end of vitellogenesis, the follicular epithelium of Hyalophora cecropia follicles forms an occlusion zone that can halt the access of horseradish peroxidase to the oocyte surface in living follicles, and of lanthanum nitrate in fixed preparations. It is proposed that this barrier is responsible for terminating the uptake of blood proteins by the oocyte. Although three types of interfollicle cell junctions were observed, only tight junctions appeared to be responsible for the observed impermeability. Sodium dodecyl sulfate-acrylamide gel electrophoresis of [³H]leucinelabeled proteins revealed no change in the protein synthetic pattern during the transformation of follicles from vitellogenesis to the subsequent terminal growth period; in addition, pinocytotic figures continued to be formed in the postvitellogenic oocyte. These findings suggest that the epithelial secretion which the oocyte is known to deposit in yolk during vitellogenesis continues to be sequestered in the absence of blood proteins after occlusion zone formation. The proposal explains the origin of a layer of membrane-limited bodies which occupy the cortex of the oocyte in mature silkworm eggs, and which differ markedly in appearance from the protein yolk spheres assembled earlier.     

Adsorptive endocytosis of vitellogenin,lipophorin, and microvitellogenin during yolk formation in Hyalophora

Yolk in Hyalophora cecropia is a mixture of proteins that are derived from the extracellular medium. We have measured for five of these proteins the number of moles deposited in each egg, the molarity of their precursors in the hemolymph at a midpoint in vitellogenesis (day 18 of adult development), and the degree to which they are concentrated by the oocyte, relative to inulin. The proteins were isolated by gel permeation and ion exchange chromatography and used to generate antibodies in rabbits. Preliminary studies established that yolk proteins are essentially quantitatively extractable in media suitable for measuring antigen concentrations by precipitation with antibodies and that yolk and hemolymph forms of the five proteins have, effectively, the same antibody-binding specificities as the isolated standards. Content per egg was about 900 pmol for vitellogenin, 600 pmol for microvitellogenin, and 300 pmol for lipophorin. By contrast, two hemolymph storage hexamers, arylphorin and a flavoprotein, occurred at less than 3 pmol per egg. In principle, yolk precursors are taken in both as solutes in the fluid phase of the endocytotic vesicles and as ligands adsorbed to vesicle membranes. Measurements of inulin uptake indicated that fluid phase endocytosis could account for only 4% of vitellogenin, 1% of microvitellogenin, and 15% of lipophorin in the yolk, when hemolymph precursors are at their day 18 concentrations. By the same comparison, arylphorin and flavoprotein appear to be excluded from the yolk, relative to inulin.     

Vitellogenin-derived yolk proteins of white perch,Morone americana: purification,characterization, and vitellogenin-receptor binding

The objectives of this study were to 1) purify and characterize vitellogenin-derived yolk proteins of white perch (Morone americana), 2) develop a nonisotopic receptor binding assay for vitellogenin, and 3) identify the yolk protein domains of vitellogenin recognized by the ovarian vitellogenin receptor. Four yolk proteins derived from vitellogenin (YP1, YP2 monomer [YP2m] and dimer [YP2d], and YP3) were isolated from ovaries of vitellogenic perch by selective precipitation, ion exchange chromatography, and gel filtration. The apparent molecular masses of purified YP1, YP2m, and YP2d after gel filtration were 310 kDa, 17 kDa, and 27 kDa, respectively. YP3 appeared in SDS-PAGE as a approximately 20-kDa band plus some diffuse smaller bands that could be visualized by staining for phosphoprotein with Coomassie Brilliant Blue complexed with aluminum nitrate. Immunological and biochemical characteristics of YP1, YP2s, and YP3 identified them as white perch lipovitellin, beta'-components, and phosvitin, respectively. A novel receptor-binding assay for vitellogenin was developed based on digoxigenin (DIG)-labeled vitellogenin tracer binding to ovarian membrane proteins immobilized in 96-well plates. Lipovitellin from white perch and vitellogenin from perch and other teleosts effectively displaced specifically bound DIG-vitellogenin in the assay, but phosvitin and the beta'-component could not, demonstrating for the first time that the lipovitellin domain of teleost vitellogenin mediates its binding to the oocyte receptor. Lipovitellin was less effective than vitellogenin in this regard, suggesting that the remaining yolk protein domains of vitellogenin may interact with its lipovitellin domain to facilitate binding of vitellogenin to its receptor.     

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.     

Distribution of incorporated and synthesized protein among cell fractions of Xenopus oocytes

Developing oocytes of Xenopus laevis were isolated, pulsed for 10 minutes with either vitellogenin-³H, ³²P or a mixture of l-leucine-³H and ³²P_i, and subsequently incubated for various lengths of time in unlabeled medium. Homogenates were then prepared and centrifuged on 20–60% sucrose gradients.Vitellogenin-³H, ³²P was found to associate initially with membranous material, but within 45 min more than half the label was associated with the yolk platelets. Since it takes at least 60–120 min for vitellogenin to be converted into lipovitellin and phosvitin, this transformation must occur within the platelets rather than at the oocyte surface or within pinosomes.Eighteen hours after a pulse with l-leucine-³H and ³²P_i, neither the yolk nor the mitochondria fraction became significantly labeled; this indicates that the macromolecular components of these structures are not synthesized or phosphorylated by the oocyte to any great extent during vitellogenesis. Instead, various components within the membranous and “soluble” regions became labeled.     

Goldsinny wrasse (Ctenolabrus rupestris) is an extreme vtgAa-type pelagophil teleost

During oocyte maturation in the goldsinny wrasse (Ctenolabrus rupestris) extensive proteolysis of yolk proteins generates a large pool of free amino acids that drive hydration of the pelagic egg. By cloning hepatic vitellogenins (vtg) and using mass spectrometry, N-terminal microsequencing, and Western-immunoblotting to identify the yolk proteins (Yp), we show that multiple forms of vitellogenin mRNAs (vtgAa, vtgAb, and vtgC) are expressed in the liver, but only a single major class of the Yps derived from vtgAa predominates in the oocytes. Some Yps derived from vtgAb and vtgC appear also to be incorporated in the oocytes and eggs, but only at background levels. During oocyte hydration the vtgAa-derived lipovitellin heavy chain (LvH-Aa) and its cleavage variants are completely degraded leaving only a processed lipovitellin light chain (LvL-Aa) fragment as the major yolk protein for embryonic development. The maturational cleavage site of the LvL-Aa is identified as two amino acids downstream from the conserved Tyr(1168) of VtgAa in Atlantic halibut. In addition, although a beta'-component (approximately 18 kDa) is present in the oocytes, it is not fully degraded during the hydration process.     

The crystalline yolk-platelet proteins and their soluble plasma precursor in an amphibian, Xenopus laevis

A single lipophosphoprotein complex, vitellogenin, was isolated and purified from the plasma of oestrogen-stimulated female toads by preparative ultracentrifugation and chromatography on TEAE-cellulose (triethylaminoethylcellulose). The protein contains 12% lipid, 1.5% phosphorus, 1.6% calcium and smaller amounts of carbohydrates and biliverdin. In amino acid composition it is identical with total yolk-platelet protein. The platelet protein, however, is fractionated on TEAE-cellulose into two components, a high-molecular-weight lipovitellin and a smaller phosvitin. Analyses of the soluble plasma vitellogenin suggest that it is a complex of two phosvitin molecules covalently bound to one lipovitellin dimer, and that it is the immediate precursor of the yolk proteins, into which it is converted by a molecular rearrangement. Uptake of vitellogenin from the plasma into the growing oocyte, and its subsequent crystallization as a yolk platelet, appear to be enhanced by gonadotrophic hormones.     

The specificity of yolk protein uptake in cyclorrhaphan diptera is conserved through evolution

Summary Yolk proteins are transported from the hemolymph into the oocytes of insects during vitellogenesis by receptor-mediated endocytosis. Since other hemolymph proteins, both native and foreign, are not accumulated in the oocyte, the process of uptake is selective for yolk proteins. Peptide domains within the yolk proteins must therefore be involved in receptor recognition. With the longterm aim of identifying these domains and to open the possibility of understanding the molecular basis of receptor-mediated endocytosis of yolk proteins, we began investigating how well this mechanism has been conserved in evolution. We studied the uptake of yolk proteins from 13 different Drosophila species and five other dipteran species, namely, Calliphora erythrocephala, Sarcophaga argyrostoma, Musca domestica, Lucilia servicata, and Protophormia terrae-novae, into the ovaries of Drosophila melanogaster and Drosophila funebris. The results from these experiments showed that in all cases the foreign yolk proteins were taken up by the host ovaries, indicating that the mechanism and peptide domains of the yolk proteins involved in recognition of the receptor have been well conserved in dipteran evolution. Offprint requests to: M. Bownes     

Incorporation de la vitellogénine tritiée dans les ovocytes du Crustacé Amphipode Orchestia gammarellus (Pallas) / Incorporation of tritiated vitellogenin into the oocytes of the crustacean amphipod Orchestia gammarellus (Pallus)

Summary

During the secondary vitellogenesis the oocytes of Orchestia gammarellus accumulate yolk spheres and lipid droplets. We studied the uptake of tritiated vitellogenin by the oocyte and its accumulation in the yolk spheres.     

THE ROUTE OF ENTRY AND LOCALIZATION OF BLOOD PROTEINS IN THE OOCYTES OF SATURNIID MOTHS

The oocytes of saturniid moths take up proteins selectively from the blood. The distribution of blood proteins in the ovary during protein uptake was investigated by staining 2 µ sections of freeze-dried ovaries with fluorescein-labeled antibodies. The results indicate that blood proteins occur primarily in the intercellular spaces of the follicle cell layer, in association with a brush border at the surface of the oocyte, and within the oocyte in the yolk spheres. That proteins derived from the blood are associated with the yolk spheres was confirmed by isolating these bodies and showing that lysis, which can be induced by any of a number of mechanical means, causes them to release immunologically defined proteins known to be derived from the blood. That the level of blood proteins in the cytoplasm is low relatively to that in the yolk spheres was confirmed by the observation that the yellow pigments associated with several blood proteins, although conspicuous in the yolk spheres, are not visible in the translucent layer of centrifuged oocytes. From these and previous physiological observations, it is proposed that blood proteins reach the surface of the oocyte by an intercellular route, that they combine with some component of the brush border, and that they are transformed into yolk spheres by a process akin to pinocytosis.     

Vitellogenesis in Varroa jacobsoni,a parasite of honey bees

Reproduction in Varroa jacobsoni occurs only in cells of the capped honey bee brood. Female mites were sampled at different times after cell sealing and ovaries containing a vitellogenic oocyte of the first gonocycle were examined under an electron microscope. It was found that the cytoplasmic connection between the lyrate organ and the oocyte persists far into the vitellogenic growth phase. In addition, a large amount of yolk material is taken up from the haemolymph. All ultrastructural features characteristic of vitellogenesis, such as microvilli, coated pits, vesicles and growing yolk platelets, are present. If more than four Varroa females live in an overcrowded brood cell, they appear to be in stress conditions and their vitellogenic oocytes may become atretic. Alterations typical for oocyte degradation and oosorption were observed in such situations.