Cysteine proprotease colocalizes with vitellogenin in compound granules of the cockroach fat body

A cysteine proprotease has been identified in developing embryos of the cockroach Blattella germanica and found to be a maternally encoded gene product that is transferred endocytically to the oocyte. The present study aims at establishing how this maternally derived proprotease is synthesized, packaged, and secreted during vitellogenesis. To this end, proprotease was localized immunocytochemically in the fat body of postmating females and its localization compared with that of vitellogenin over the same developmental periods. Fat bodies in cockroaches are comprised of two different cell types: trophocytes and bacteriocytes. Data show that proprotease and vitellogenin come to colocalize in compound granules of the fat body trophocytes. While synthesis of vitellogenin can be traced back to granules resulting from the coalescence of Golgi-derived vesicles in the trophocyte cytoplasm, proprotease appears to be localized predominantly on the cytolysosomes of both trophocytes and bacteriocytes. When probed with an anti-proprotease antiserum, bacteria are also positively labeled, regardless of whether they are segregated inside the cytolysosomes or free in the bacteriocyte cytoplasm. Since vitellogenin and proprotease colocalize within the same cell organelle, it is assumed that Golgi-derived vesicles, which contain vitellogenin, may fuse with cytolysosomes bearing proprotease to yield compound secretory granules. To account for the present observations, the origin and role of proprotease are discussed in relation to the turnover of bacteria in the fat body and to the requirements of endosymbiosis.     

Comparison of Yolk Production in Seven Pyralid Moth Species

Summary

The yolk proteins of six pyralid moths were analyzed and compared with the yolk proteins of Plodia interpunctella (Hübner). When cross-reacted in an Ouchterlony double immunodiffusion with antiserum raised to either total yolk proteins or purified vitellin from P. tnterpunctella, the yolk proteins of Anagasta kuehniella (Zeller), Cadra cautella (Walker), C. figulilella (Gregson), and Ephestia elutella (Hübner), closely related members of the subfamily Phycitinae, showed strong precipitation lines that consisted of four major yolk polypeptides (YPs). The yolk proteins from Amyelois transitella (Walker) were only weakly reactive, whereas yolk proteins from Galleria mel-lonella (L.) were not precipitated by either antiserum. Abdominal body walls (containing primarily fat body) from late pharate adult females were incubated in vitro and they secreted two major polypeptides that had molecular masses similar to the vitellogenins (YP1 and YP3) from P. interpunctella. In addition, ovarioles from late pharate adult females were incubated in vitro, and they secreted two major polypeptides that had molecular masses similar to YP2 and YP4 from P. interpunctella. When late pharate adult females were injected with ³⁵S-Met, the hemolymph of all species contained vitellogins that were secreted by their respective body walls in vitro. Ovarioles from injected females contained many labeled polypeptides, but there were four major bands that corresponded consistently to the vitellogenins secreted from the fat body and the two major polypeptides secreted from the ovarioles. These data show that the production of the major YPs in these closely related pyralid species is very similar, and that there is considerable conservation of immunological characters of yolk proteins in the subfamily Phycitinae.     

Distribution and accumulation of storage protein-1 in ovary of Hyphantria cunea Drury

Storage protein-1 (SP-1) is a major storage protein found in the hemolymph and fat body of Hyphantria cunea. In this study, the uptake and accumulation of SP-1 into the ovary of H. cunea was investigated using biochemical and immunocytochemical methods. SP-1 in H. cunea has a high methionine content (4.6%) but is not female-specific, like other high methionine storage proteins. In the 6-day-old pupal ovary, SP-1 was detectable in trace amounts but accumulated to significant levels toward the end of the pupal stage. After adult emergence, SP-1 rapidly decreased in the ovarian follicles and remained low in the egg. This suggest that SP-1 is either extensively modified or degraded, causing a loss of its antigenic property in the ovary after adult emergence. During vitellogenesis, SP-1 is present in the hemolymph and penetrates through the tunica propria to reach the perioocytic space. From there, SP-1 is incorporated into yolk bodies. These results clearly show that SP-1 is taken up by the developing oocyte. Its disappearance suggests that SP-1 might be an amino acid reservoir for providing precursors for egg formation, in contrast to yolk proteins, which are utilized during postembryonic development. Arch. Insect Biochem. Physiol. 37:115–128, 1998. © 1998 Wiley-Liss, Inc.     

Expression of the Helicoverpa cathepsin B-like proteinase during embryonic development

Cathepsin B-like proteinase from Helicoverpa armigera (HCB) was proposed as being involved in the degradation of yolk proteins during embryonic development. Recombinant HCB was expressed as a fusion protein with GST in Escherichia coli BL21 on the basis of its cDNA and purified to homogeneity. The fusion protein was cleaved with thrombin to generate a soluble protease with a mass of 37 kDa. A polyclonal antiserum against this recombinant protein, raised in the rabbit, recognized three isoforms of HCB in an ovary homogenate of this insect. Expression of this enzyme during embryonic development was studied using immunoblotting, immunohistochemistry and activity assay. It was found that HCB was expressed during embryonic development and that its proteolytic activity was detected from embryonic developmental eggs. The fact that HCB activity is observed in ovaries and developing eggs suggested that the enzyme had already been activated before embryonic development. Immunohistochemistry indicated that the enzyme was located in follicular cells, the sphere of yolk granules, and the fat bodies of female adult. These lines of evidence suggested strongly that HCB takes part in the degradation of yolk proteins during the development of embryo.     

A Possible Extra-Ovarian Site for Synthesis of Lipovitellin During Vitellogenesis in Artemia sp. (Crustacea; Anostraca)

Summary

Lipovitellin samples, extracted from yolk platelets of cysts, were applied to SDS-PAGE. A female specific antiserum was raised against the high molecular weight apoprotein lipovitellin alpha-1 (LV-α1) of the lipovitellin complex. This anti-LV-α1 was used in the peroxidase-anti-peroxidase staining method with frontal paraffin sections (of 4μm) of whole embedded Artemia. Females were studied during a complete vitellogenic cycle. The presence of exogenous yolk precursors in the fat storage cells of the thoracopods of female Artemia was demonstrated. The amount of the female specific yolk polypeptides and the number of positively stained cells changes during the vitellogenic cycle. In vitro experiments with ³⁵S-radiolabelled methionine show the synthesis of lipovitellin-like substances in the fat storage cells of vitellogenic females.     

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.     

Localization and functional role of a 41 kDa collagenase/gelatinase activity expressed in the sea urchin embryo

The egg storage compartment of the sea urchin embryo was investigated for a protein destined for export to the extracellular matrices. Using an antiserum prepared against a 41 kDa collagenase/gelatinase localized to the extraembryonic matrices (the hyaline layer and basal lamina), the egg storage compartment was mapped for this antigen. Indirect immunofluorescence analysis revealed the 41 kDa collagenase/gelatinase in the cortical granules as well as a second compartment which was dispersed throughout the egg cytoplasm. High resolution immunogold labeling defined this cytoplasmic compartment as the yolk granule organelle. Gelatin substrate gel zymography revealed the presence of a 41 kDa gelatin cleavage activity in purified yolk granules. These results suggest a role for yolk granules in regulated protein export and challenge the traditional view of this organelle as a benign storage compartment for nutrients. In additional experiments, embryos grown in the presence of the 41 kDa cleavage activity or the anti-41 kDa antiserum had severely delayed gut formation and spicule elongation. These results demonstrate a requirement for defined levels of the 41 kDa activity in the extracellular matrices of the developing embryo.     

Biochemical and immunocytochemical analysis of vitellogenesis in the olive fruit fly Dacus (bactrocera) oleae (Diptera: Tephritidae)

Synthesis and selective accumulation of the major yolk proteins in the developing oocytes of the species Dacus oleae (Diptera: Tephritidae) was studied biochemically and by immunoelectron microscopy. In the hemolymph of adult females, two yolk proteins precursors (or vitellogenins) have been detected. They each exhibit a similar molecular weight and isoelectric point to their respective mature yolk proteins (or vitellins), while electrophoretic analysis of their synthetic profile shows that their levels in the hemolymph increase rapidly during development. Immunogold electron microscopy of ovarian sections, revealed that the hemolymph vitellogenins reach the oocyte through enlarged inter-follicular spaces and demonstrated vitellogenin synthesis by the follicle cells of the vitellogenic follicles. The newly synthesized vitellogenins follow a distinct secretory pathway into these cells as compared to other components being synthesized at the same time (e.g. the vitelline envelope proteins), since they were found in secretory vesicles that appeared to be differentiated from those destined to participate in the vitelline envelope. The vitellogenin-containing vesicles exocytose their contents directionally into the follicle cell/vitelline envelope boundary, and subsequently the vitellogenins diffuse among the gaps of the forming vitelline envelope and reach the oocyte plasma membrane. Their internalization by the oocyte includes the formation of an endocytic complex consisting of coated pits, coated vesicles, endosomes, transitional yolk bodies, and finally mature yolk bodies, in which the storage of the vitellins and other yolk proteins occur. These results are discussed in relation to data obtained from other Dipteran species.     

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     

Storage proteins of the fall webworm,Hyphantria cunea drury

Two storage proteins, storage protein-1 (SP1) and storage protein-2 (SP2), were found in hemolymph and fat body during the development of Hyphantria cunea, the fall webworm. Both storage proteins show similiar quantitative changes during development in males and females; however, SP1 is more abundant. The hemolymph of last instar larvae contains high concentrations of the storage proteins. However, following pupation, the storage proteins accumulate in fat bodies. SP1 peaks in the hemolymph of males and females late in last instar larvae (8-day-old 7th instar larvae). SP1 has a native molecular weight of 460,000 and consists of six identical subunits (Mr = 76,700), while SP2 has a molecular weight of 450,000 and is composed of two different subunits (Mr = 74,100 and 72,400). Both SP1 and SP2 are hexamers and are phosphorylated glycolipoproteins. The pl values of SP1 and SP2 were determined to be 5.70 and 5.50, respectively. Antibodies raised against SP1 react positively with vitellogenin and ovary extract, as well as with proteins in the hemolymph from last instar larvae and proteins in pupal fat bodies. Storage protein synthesis starts in fat bodies of a 4-day-old 7th instar larvae and in female peaks at 6–8 days of the 7th instar.     

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.     

Onset of vitellogenin production and vitellogenesis,and their relationship to changes in the midgut epithelium and oocytes in the tickDermacentor variabilis

InDermacentor variabilis (Say), the onset of vitellogenin production and vitellogenesis (up-take of vitellogenin into oocytes) began during the rapid-engorgement feeding period. Mating was required for both vitellogenin production and vitellogenesis to complete the tick's life cycle. Complete immunological identity, as measured by Ouchterlony's double diffusion test, existed between vitellogenin from the fat body, midgut and hemolymph, and vitellin from the ovaries and eggs. Antivitellin antibody did not react with host hemoglobin nor with fat body, midgut, and ovary extracts from feeding females prior to rapid engorgement, feeding unmated females, or unfed or fed males. Some unmated females fed for 13 days and then hand-detached from the host eventually began oviposition after going through a preoviposition period. In these ticks, organ extracts from the midgut, fat body and ovary reacted with antivitellin antibody. The presence or absence of presumed vitellogenic cells in the midgut and yolk bodies in oocytes corresponded with the presence or absence of vitellogenin and vitellogenesis as measured by Ouchterlony's test. Presumed vitellogenic cells increased in size during the preoviposition period. These cells reached their greatest size during the time when the most eggs were being produced, and then declined in size toward the end of oviposition. Vitellogenin was deposited directly into developing yolk bodies in oocytes and was not processed through lysosomes. Feeding was the process that initiated the formation of eggshell cuticle. Detachment from the host was required for the initiation of oviposition.     

Vitellogenin content in fat body and ovary homogenates of workers and queens of Melipona quadrifasciata anthidioides during vitellogenesis

Abstract Vitellogenin (Vg) is an egg yolk protein that is produced primarily in the fat body of most female insects. In the advanced social structure of eusocial honeybees, the presence of the queen inhibits egg maturation in the workers’ ovaries. However in the stingless bee Melipona quadrifasciata, the workers always develop ovaries and lay a certain amount of eggs while provisioning the brood cells with larval food during what is known as the worker nurse phase. The present work is a comparative study of the presence of Vg in homogenates of the fat bodies and ovaries of the nurse workers, and the virgin and physogastric queens of M. quadrifasciata. The presence of Vg was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting using Apis mellifera anti‐egg antibody. Vg was not detected in the fat bodies or ovaries of the workers, but it was found in the ovaries of virgin and physogastric queens and in the fat body of physogastric queens. The results are discussed, taking into account the reproductive state of the individuals and the other possible roles of Vg, such as a storage protein for metoabolism of other organs.     

Morphological specializations of the yolk sac for yolk processing in embryonic corn snakes (Pantherophis guttatus: Colubridae)

Non‐avian reptiles commonly are assumed to be like birds in their overall patterns of development. However, colubrid corn snakes (Pantherophis guttatus ) have mechanisms of yolk cellularization and processing that are entirely different from the avian pattern. In birds, a vascular “yolk sac” surrounds and digests the liquid yolk. In contrast, in corn snakes, the yolk material is converted into vascularized cords of yolk‐filled cells. In this study, we used stereomicroscopy, histology, and scanning electron microscopy to analyze this unusual developmental pattern in corn snakes. Our observations reveal that the yolk sac cavity is invaded by endodermal cells that proliferate, absorb yolk spheres, and form aggregates of interconnected cells within the liquid yolk mass. As development proceeds, small blood vessels arise from the yolk sac omphalopleure, penetrate into the yolk mass, and become tightly encased in the endodermal cells. The entire vitellus ultimately becomes converted into a mass of vascularized, “spaghetti‐like” strands of yolk‐laden cells. The resulting arrangement allows yolk to be digested intracellularly and yolk products to be transported to the developing embryo. Indirect evidence for this pattern in other species raises the possibility that it is ancestral for squamates and quite possibly Reptilia in general.     

A morphologic,cytochemical, and chromatographic analysis of lipid yolk formation in the oocytes of the dog

The oocytes of carnivorous mammals are distinguished by the presence of large amounts of a lipid, yolk like material. In the oocytes of the dog, lipid yolk formation marks one of the earliest indications of occyte maturation. In early primary oocytes, the yolk bodies are scattered within the ooplasm, while in later stages they are in discrete clusters. Lipid yolk material appears to be formed by at least two mechanisms. Throughout most of oogenesis the oocyte contains scattered dense granular bodies that become vacuolated by droplets of lipid material and may be transformed, by this process, into lipid yolk bodies. These granular bodies are highly reactive for acid phosphatase and are positive for glycoprotein with the PA-CA-methenamine technique. In addition, other glycoprotiein-rich yolk bodies appear to arise from many of the small dictyosomes. In secondary follicles these two mechanisms often appear to act conjointly with the dense vacuolated granules coalesing with the larger yolk bodies. Small yolk bodies are intensely reactive for glycoprotein, becoming less reactive as they enlarge and mature. The developing yolk bodies are often associated with the acid phosphatase-positive granules. The peripheral portions of the larger yolk bodies are faintly reactive for both acid phosphatase and glycoprotein. All reactivity is lost in mature yolk bodies. Thin layer chromatography of the total lipids extracted from isolated oocytes reveals a pattern that is consistent among dogs of the same and of different breeds. The most abundant lipid fraction from each dog oocyte extraction stains strongly for glycolipid.     

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.     

Proteolytic processing of Blattella germanica vitellin during early embryo development

In the eggs of the cockroach Blattella germanica, vitellin (Vt) utilization is initiated 4 days postovulation by the proteolytic processing of its three subunits. These reactions yield a specific set of peptides that are consumed by the developing embryo. A yolk proteinase activity, believed central to this processing event, has been investigated. First expressed at day 3 postovulation, just prior to Vt's processing, its specific activity with synthetic substrates increased four-fold to 18-fold through day 6. In addition, a mixing experiment showed that these proteinases(s) can also process Vt's large subunits in vitro. A relationship between Vt processing and proteinase specific activity was also noted with two B. germanica translocation heterozygotes, which displayed differences in the extent of Vt processing. One group of eggs (group A) failed to process any Vt subunit. A second group (B) processed the M_r 102,000 subunit but not the M_r 95,000. A third group (C) processed their Vt normally. Proteinase specific activities in the yolk of translocant's eggs at day 6 mirrored the extent of processing, being highest in group C eggs and effectively absent from the yolk of group A eggs. Eggs defective in Vt processing also contained arrested embryos. It is concluded that the yolk proteinase activity described here participates in Vt processing at day 4 postovulation. Microscopic examination of yolk obtained from eggs of wild type females showed that, as processing began in vivo (day 4), the yolk granules also underwent an abrupt decrease in size from diameters of 15–30 μm to 3–10 μm. Yolk granules of those translocant's eggs that were defective in Vt processing did not undergo this size decrease, suggesting that granule reorganization and Vt proteolysis may be linked functionally.     

Effects of ablation of the pars intercerebralis on ecdysteroid quantities and yolk protein expression in the blowfly Protophormia terraenovae

Quantities of ecdysteroid are compared in the haemolymph and ovaries of the blowfly Protophormia terraenovae Robineau‐Desvoidy (Diptera: Calliphoridae) under reproductive (LD 18 : 6 h at 25 °C) and diapause (LD 12 : 12 h at 20 °C) conditions. The effects of ablation of the pars intercerebralis or ovaries on ecdysteroid quantities and of ablation of the pars intercerebralis on yolk protein expression are examined. Under reproductive conditions, the levels of ecdysteroid in vitellogenic females are high, although the levels in previtellogenic females and females with mature ovaries are low. Under diapause conditions, there are low quantities of ecdysteroid in both the haemolymph and ovaries. Ecdysteroid titres in the haemolymph are not significantly affected by the removal of the ovaries, suggesting that tissues other than the ovaries are also involved in the production of ecdysteroids. Reproductive females in which the pars intercerebralis of the brain is experimentally ablated have ecdysteroid levels that are not significantly different from sham‐operated or intact females. However, yolk protein expression in the fat body is suppressed after removal of the pars intercerebralis. These results suggest that the suppression of ecdysteroid levels in the haemolymph and ovaries is associated with reproductive diapause, and that the pars intercerebralis could play a role in yolk protein synthesis without mediating ecdysteroid production.