Selective endocytosis,in vitro,by ovarian follicles from Hyalophora cecropia

Ovarian follicles of Hyalophora cecropia, incubated in vitro with isolated and radiolabelled hemolymph and yolk proteins, provided a satisfactory model of in situ vitellogenesis. Uptake of proteins was specific. The follicles accumulated vitellogenin and microvitellin at constant rates for 6 hr, depositing them in the protein yolk spheres of the oocyte. Uptake of these two proteins was saturable by high concentrations of homologous protein and inhibited by p-dinitrophenol. In contrast, two other abundant hemolymph proteins, arylphorin and flavoprotein, were taken up at lower rates, and become concentrated primarily in the basement lamina of the follicle. Their accumulation was not saturable and not inhibited by p-dinitrophenol. The two yolk precursors were accumulated only by follicles at stages known to be vitellogenic, and the rates of uptake were shown to approximate the rates of accumulation of these proteins in situ. The uptake of vitellogenin, but not microvitellin, was enhanced 2- to 3-fold by hemolymph ultrafiltrates. Vitellin from mature eggs was not distinguishable from vitellogenin by the endocytotic apparatus. Finally, endocytotic uptake was not affected by inhibition of protein synthesis. This finding supports the concept of membrane and receptor recycling in yolk formation, and argues against an essential role of the follicle cell product paravitellogenin in the mechanism of hemolymph protein uptake.     

Electrophoretic patterns of yolk proteins throughout ovarian development and their relationship to vitellogenin in the rainbow trout,Oncorhynchus mykiss

• 1.1. Electrophoretic patterns of yolk proteins were investigated throughout ovarian development and their relationship to vitellogenin determined in a pulse-chase experiment with ³H-vitellogenin.
• 2.2. Using a radioimmunoassay for vitellogenin, vitellogenin/yolk protein products of vitellogenin were detected in follicles throughout ovarian development and in ovulated eggs.
• 3.3. The majority of yolk proteins in follicles measuring less than 1.0 mm in diameter appeared to be derived from sources other than vitellogenin. In contrast, in the larger follicles all of the major yolk proteins detected were derived from vitellogenin.
• 4.4. Pulse-chase with ³H-vitellogenin revealed that all of the major yolk proteins in 3.0 mm follicles were derived from vitellogenin. The major peptides eluted with molecular masses of 110 and 30 kDa under non-reducing conditions (these are very likely to represent lipovitellin 1 and lipovitellin 2), and 88, 22, 16, and 12 kDa under reducing conditions.
• 5.5. There were no apparent differences in the major yolk proteins in ovulated eggs compared to those in vitellogenic follicles, indicating that no extensive proteolysis of these proteins had occurred during maturation and/or ovulation.

     

Nonvitellogenic female sterile mutants and the regulation of vitellogenesis in Drosophila melanogaster.

The genetic and endocrine regulation of vitellogenesis was investigated by studying 18 female sterile mutations that disrupt the development of normal vitellogenic follicles. Applications of exogenous juvenile hormone analog and reciprocal ovarian transplants between flies of different genotypes were employed to accomplish our first two objectives: to find (1) whether the mutation blocked development of the ovary directly, and (2) whether the mutation altered the hormonal milieu. In 15 of the mutants the developmental defect was localized to the ovary, but in the other 3 the ovary was competent to respond to a permissive environment. The internal milieu of these three mutants (ap⁴, fs(3)A1, fs(2)A18) was unable to provoke normal development in wild-type ovaries, suggesting that these mutations cause endocrine defects. Our third objective was to find whether an endocrine organ was itself defective in any of these mutants. The corpus allatum from two of the mutants was unable to provoke vitellogenesis in isolated wild-type abdomens, but corpora allata from wild-type females or from other mutants were able to promote maturation of ovarian follicles in isolated abdomens. Our fourth objective was to find whether any of the mutants were able to produce yolk proteins. Immunoelectrophoresis of fly hemolymph demonstrated that in all mutants tested vitellogenins were found in the blood. These experiments permit four main conclusions. First, they identify the first Drosophila mutants in which an endocrine gland is shown to be intrinsically defective during adulthood. Second, they show that the production of morphologically normal late previtellogenic follicles is not required for the induction of vitellogenin synthesis and secretion. Third, they show that juvenile hormone can cause ovarian follicles to sequester yolk in mutant flies. And finally, they show that mutants with defective corpora allata still synthesize and secrete vitellogenin. Taken together, these conclusions suggest that in Drosophila melanogaster the uptake of vitellogenin into follicles depends upon the availability of juvenile hormone, but that the synthesis and secretion of vitellogenin are independent of both normal ovaries and totally normal corpora allata.     

Properties of two follicle proteins and their possible role for vitellogenesis in the African Locust

Summary Insoluble proteins from the maturing follicle ofLocusta migratoria were analyzed by SDS-PAGE. A reproducible pattern of low molecular weight proteins was observed. Five of these proteins did not correspond to yolk or haemolymph proteins. At least two of these show marked quantitative changes during oocyte development. By in vitro incubation of follicles and fat body with a labelled precursor, and by the identification of the labelled polypeptides by SDS-PAGE, we could demonstrate that these two proteins are synthesized only during the time of vitellogenin uptake. This protein is probably a follicle product necessary for yolk formation. The other protein might be necessary for vitelline membrane and/or chorion formation.     

Studies on the carbohydrate moiety of vitellogenin from the tobacco hornworm,Manduca sexta

Vitellogenin can be isolated in large quantities from the hemolymph of the tobacco hornworm, Manduca sexta by a combination of KBr density gradient ultracentrifugation, gel permeation and cation exchange chromatography. Glycopeptides generated by exhaustive pronase digestion of vitellogenin were separated by gel permeation chromatography on a Bio-Gel P-6 column. The major glycopeptide was shown to bind to concanavalin A-Sepharose but not to lentil lectin-Sepharose and was sensitive to treatment with endo-β-N-acetylglucosaminidase H. Analysis of the glycopeptide by high field proton NMR spectroscopy revealed that the primary structure is of the high mannose class containing nine mannose and two N-acetylglucosamine units. These results suggested that N-glycosylation of insect proteins, as in mammals, yeasts and plants, involves the en bloc transfer of an oligosaccharide containing the unit (Glu)₃(Man)₉(GlcNAc)₂ from a lipid intermediate to an asparagine residue followed by removal of glucose residues. Processing to more complex structures does not seem to occur in M. sexta vitellogenin. In vitro uptake with isolated M. sexta follicles showed that deglycosylation had no significant effect on uptake of ¹²⁵I-labeled vitellogenin.     

THE DEPENDENCE OF CECROPIA YOLK FORMATION IN VITRO ON SPECIFIC BLOOD PROTEINS

The capacity of cecropia vitellogenic follicles to form yolk during short-term in vitro incubation in female blood was analyzed by labeling with fluorescein-conjugated serum globulin, tritiated cecropia blood proteins, or tritiated amino acid. As judged by fluorescence microscopy or autoradiography, yolk formation during 3–8 hr in vitro was similar in rate and in protein uptake specificity to that observed in vivo. When follicles were incubated in cecropia male blood, 6% gamma globulin, or cecropia saline, the yolk produced was markedly inferior in quality and quantity to that generated in female blood. Purified preparations of vitellogenin, the primary female blood protein deposited in the yolk, were equivalent to whole female blood in supporting yolk formation; this protein seems, therefore, to have a specific stimulatory role. An enhancement of the rate of pinocytosis at the oocyte surface by vitellogenin is postulated.     

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.     

Studies on binding and uptake of vitellogenin by follicles of the tobacco hornworm,Manduca sexta

An in vitro system for the uptake of ¹²⁵l-vitellogenin (VG) or vitellin into isolated follicles of the tobacco hornworm, Manduca sexta, is described. After incubation with ¹²⁵l-VG, follicles were disrupted and the internal yolk contents separated from the follicle membranes. The results showed that ¹²⁵l-VG was associated principally with the membranes (92%) after incubation at 4°C. However, at 27°C, ¹²⁵l-VG was mainly in the yolk (92%). Furthermore, trypsin treatment removed approximately 70% of VG bound to the follicles at 4°C. Labeled VG was shown to bind to sonicated follicle membranes with high specificity and affinity (K_D ? 1.3 × 10^?8 M). This binding was sensitive to pH and calcium concentration. The total binding sites were estimated at 4 × 10¹⁴ sites/g of membrane protein. Competition studies showed that binding of ¹²⁵l-VG to follicle membranes was blocked by excess unlabeled vitellin and deglycosylated vitellogenin but not by lipophorin (the major hemolymph lipoprotein), microvitellogenin, a female-specific protein (M_r ～ 31,000) found in both hemolymph and eggs, and the smaller vitellogenin subunit, apovitellogenin-II (M_r ～ 45,000). These results suggest that selective uptake of M. sexta VG from the hemolymph involves binding to specific receptors located on the follicle membranes.     

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.     

Yolk synthesis in ovarian follicles ofDrosophila

Summary The autonomous synthesis of yolk proteins in ovarian follicles ofDrosophila melanogaster was analyzed. Vitellogenic follicles were labelled with³⁵S-methionine in vitro and the newly synthesized yolk proteins were separated by SDS-polyacrylamide gel electrophoresis. Possible contamination of the follicle preparations caused by adhering fat body cells could be excluded by culturing follicles in males prior to labelling in vitro. When labelled follicles were cut at the nurse cell/oocyte border the three yolk proteins (YP1, YP2, YP3) were found only in posterior fragments containing ooplasm and follicle cells, whereas two radioactive protein bands (A and B) were detected in nurse cells (anterior fragments). The yolk proteins of these five bands were characterized by peptide mapping. Band A protein, migrating a little more slowly than YP2, is closely related to both YP1 and YP2 while band B contains a yolk protein which is very similar to YP3. Hence, the nurse cells have been identified as a site of vitellogenin synthesis within the ovary ofDrosophila.Supported by the Deutsche Forschungsgemeinschaft, SFB 46     

Juvenile hormone induced ovarian uptake of a female-specific blood protein in Oncopeltus fasciatus

An adult female-specific blood protein was demonstrated in Oncopeltus by gel electrophoresis. This protein is the major band in soluble yolk fractions. It is also present at substantial concentrations in the haemolymph of starved and diapausing adult females. Thus, the failure of the ovaries to form yolk under these conditions is characterized by an inability to remove vitellogenin from the blood. Application of a juvenile hormone analog (JHA) restored protein yolk deposition in starved and diapausing adult females. Whereas other blood proteins decreased no more than two-fold upon JHA treatment, the vitellogenin concentration decreased 20-fold in starved females. The vitellogenin concentration in the blood of diapausing females was not significantly affected by JHA, apparently because synthesis kept pace with ovarian uptake in this case.     

Evidence for the involvement of transglutaminase in the uptake of vitellogenin by Xenopuslaevis oöcytes

The yolk protein, vitellogenin, is sequestered by the developing oöcyte by receptor-mediated endocytosis, the process by which cells bind and internalize extracellular proteins. Endocytosis of a variety of proteins follows a similar pathway, whereby internalization of receptor-bound ligand takes place over clathrin-coated regions of the cell membrane. The protein crosslinking enzyme, transglutaminase, has been reported to be essential for the receptor-mediated endocytosis of insulin and α₂-macroglobulin. In this study, the presence of transglutaminase activity was demonstrated in the $\text{Xenopus}\text{laevis}$ ovary and was effectively inhibited by poly L-lysine, an inhibitor of vitellogenin uptake, and dansylcadaverine, a known inhibitor of transglutaminase activity. Two other less poteint inhibitors of transglutaminase, methylamine and bacitracin produced partial inhibition of the ovarian enzyme. Furthermore, dansylcadaverine and methylamine were found to inhibit the appearance of vitellogenin in the yolk platelets of the oöcyte.     

Lipophorin as a yolk precursor in Hyalophora cecropia: uptake kinetics and competition with vitellogenin

Vitellogenic follicles of Hyalophora cecropia were incubated in metabolically radiolabeled, high-density lipophorin isolated from pharate adult hemolymph by KBr density gradient centrifugation. The follicles transferred this probe from the incubation medium to the cortical yolk spheres in the oocyte by an energy-dependent and saturable mechanism. Vitellogenin and high-density lipophorin competed with each other for uptake, and are therefore concentrated by the follicle with a common mechanism. Microvitellin and lipophorin, in contrast, did not compete for uptake. The K(uptake) for the accumulation of high-density lipophorin was substantially higher than the value estimated earlier for vitellogenin (133 microM vs. 18 microM). This relationship helps explain why the shared concentrating mechanism does not deplete the lipid transport capacity of the hemolymph, and how a low vitellogenin: lipophorin molar ratio in the hemolymph yields a high ratio in the mature egg.     

Antigenic and electrophoretic variants of vitellogenin in Oncopeltus blood and their control by juvenile hormone

Antigenic analysis of adult female-specific blood and yolk proteins in Oncopeltus demonstrated an incomplete vitellogenin (A), which appears in the blood prior to yolk deposition and is later modified or joined by an antigenically complete molecule (AB). Vitellogenin AB is antigenically indistinguishable from the major yolk protein of mature eggs, though the electrophoretic mobilities of the two differ in 6% acrylamide gels. Vitellogenin A alone appears in the blood of adult females in which the corpora allata are known to be inactive, i.e., during starvation or photoperiodically induced diapause. Stimulation of these females with a juvenile hormone analog restores yolk deposition, and also induces the appearance of AB in the blood. While juvenile hormone is needed for the termination of diapause and the maturation of vitellogenin in this species, diapause begins with the vitellogenin-producing mechanism already partially assembled.     

Extracellular concentrating of proteins in the cecropia moth follicle

Yolk proteins, derived from the blood, are incorporated into the oocytes of insects and certain vertebrates by pinocytosis, but reach the oocyte surface only after penetrating the surrounding follicular epithelium via intercellular channels. In an investigation of the events occurring in these intrafollicular spaces, the dense extracellular material present between the follicle cells and in the oocyte's brush border was extracted from vitellogenic cecropia moth follicles by soaking in physiological saline. Quantitative immunochemical determination of several eluted blood proteins revealed that these components had been more concentrated in the extracellular spaces than in the blood. The average concentration factors were 2.5 for the predominant yolk protein, vitellogenin, and 4.5 for the carotenoid protein. Since injected foreign proteins were also accumulated in the spaces, the concentrating mechanism seemed to act on all available proteins. However, in vitro inhibition of yolk formation with dinitrophenol resulted in a selective increase in the amount of extracellular vitellogenin in follicles which had been previously exposed to a medium low in this protein, suggesting accumulation of a factor with a specific affinity for it. Furthermore under certain conditions vitellogenin was more readily released from the concentrate than was the carotenoid protein. These results indicate that, despite apparent lack of discrimination in the binding of blood proteins in the spaces, extracellular interactions may contribute to the selectivity known to occur during vitellogenesis.     

Vitellogenin and vitellin from the blowfly Calliphora vicina: Occurrence,purification and antigenic characterization

The occurrence and purification of vitellogenin and vitellin from Calliphora vicina Rob.-Dev. (= C. erythrocephala (Meig.)) are described together with the preparation of specific anti-vitellogenin antibodies. C. vicina vitellogenin and vitellin were purified from ovaries and eggs respectively; both proteins contain two polypeptide subunits identical to the dominating polypeptides in the growing oocytes. The polypeptides show molecular weights of 52,000 and 48,500 respectively, and are associated with carbohydrate and lipid. Polypeptides of similar size could be identified in haemolymph from yolk-depositing females, but were absent in ovariectomized females. The anti-bodies specifically precipitated the vitellogenin polypeptides from fat body homogenates of females depositing yolk or from the purified vitellogenin. Therefore, these antibodies were judged suitable for use in a study on the ultrastructural localization of vitellogenin in fat body cells (Thomsenet al., 1980).     

Development of monoclonal antibodies for monitoring Aedes atropalpus vitellogenesis

Monoclonal antibodies to a mixture of Aedes atropalpus and A. aegypti soluble yolk proteins were produced by hybridomas between the fusion of P3X63.653 myeloma cells and splenocytes of immunized BALB/c mice. Ascites fluid collected from mice innoculated with cloned hybridoma cells contained high specificity and affinity to the soluble yolk proteins of both Aedes species. Seven different hybridoma lines produced antibodies with specificity to both A. atropalpus and A. aegypti and one cell line produced antibodies monospecific to A. aegypti soluble yolk proteins. Monoclonal antibodies specific to A. atropalpus vitellin and vitellogenin were characterized by a combination of gel electrophoresis, western blotting and immunohistochemical staining. An indirect double antibody sandwich enzyme-linked immunosorbent assay was developed using a mixture of the seven hybridoma antibodies to A. atropalpus vitellin for monitoring vitellogenin levels in individual mosquito haemolymph samples. With this procedure, the peak period of vitellogenin synthesis in A. atropalpus was found to be 18 to 30 h after adult eclosion.     

家蝇卵巢在体外培育中摄取卵黄蛋白

本文报道家蝇Musca domestica卵巢在体外培养条件下,摄取异硫氰萤光素标记的家蝇卵黄蛋白的特点。用Grace's培养液标记蛋白浓度为2mg/m1,在27℃条件下培养2小时,卵巢摄取量依赖于培养液中卵黄蛋白浓度和温度,摄取高峰在羽化后48小时,正值卵母细胞发育阶段进入6-8时期。培养液中加入JHIII,能促进摄取,JHIIl的浓度和摄取量无明显相关性。乌本苷、牛血清蛋白和叠氮钠显著抑制卵巢的摄取活动。     

Vitellogenesis in the cockroach Nauphoeta cinerea: Separation of two classes of ovarian binding sites and calcium effects on binding and uptake

Two classes of vitellogenin binding sites with K_d-values of 7.3 nM and 290 nM were observed in follicle-membrane preparations of the cockroach Nauphoeta cinerea using a membrane-binding assay at pH 8. Separation of follicle cells and basal laminae from oocyte membranes prior to binding studies showed that the fraction consisting of follicle cells and basal laminae (FC/BL) contained high-affinity binding sites for vitellogenin (K_d=16.6 nM), whereas loweraffinity binding sites (K_d=200 nM) were found in the oocyte membrane fraction. The concentration of Ca²⁺ had a distinct effect on vitellogenin binding and uptake: maximal binding to the oocyte membrane fraction was observed at 0.3 mM Ca²⁺ and to the FC/BL fraction at 10 mM, whereas uptake of vitellogenin by oocytes in vitro was highest at 4 mM Ca²⁺. The calcium ionophore A23187 decreased vitellogenin uptake. This effect of A23187 could be counteracted by the calcium chelator Quin2. A hypothetical model for the uptake of vitellogenin into follicles of Nauphoeta cinerea is suggested.