Serosa membrane plays a key role in transferring vitellin polypeptides to the perivitelline fluid in insect embryos

In mid-embryogenesis, the stick insect Carausius morosus comes to be comprised of three distinct districts: the embryo proper, the yolk sac and the perivitelline fluid. A monolayered epithelium, the so-called serosa membrane, encloses the yolk sac and its content of vitellophages and large yolk granules. During embryonic development, the yolk sac declines gradually in protein concentration due to Vt polypeptides undergoing limited proteolysis to yield a number of Vt cleavage products of lower molecular weights. mAbs 1D1 and 5H11 are monoclonal antibodies raised against some of the Vt cleavage products generated by this process in the yolk sac. At the confocal microscope, antibody fluorescence is initially associated with a few yolk granules, while it is gradually displaced in the cytosolic spaces of the vitellophages. With the proceeding of embryonic development, label appears also in the serosa membrane in the form of clustered dots. At the ultrastructural level, gold particles are initially associated with the vitellophages that are labeled on a few yolk granules and in the cytosolic space flanking the yolk granules. Subsequently, the serosa cells become labeled on vesicles close to the yolk granules or just underneath the plasma membrane. Inside the serosa cells, label is also associated with granules budding from the Golgi apparatus, but never with the intercellular channels percolating the serosa membrane. These observations are interpreted as indicating that Vt cleavage products leak out from the yolk granules into the cytosolic spaces of the vitellophages and are eventually transferred to the perivitelline fluid via transcytosis through the serosa cells.     

Yolk utilization in stick insects entails the release of vitellin polypeptides into the perivitelline fluid

This study investigates the developmental fate of vitellin (Vt) polypeptides generated by limited proteolysis in an insect embryo. To this end, a number of polyclonal (pAb) and monoclonal antibodies (mAb) were raised against the yolk sac and the perivitelline fluid of late embryos of the stick insect Carausius morosus. Two dimensional immuno gel electrophoresis and Western blotting demonstrate that polypeptides resulting from Vt processing are present both in the yolk sac and the perivitelline fluid. At the confocal microscope, different labelling patterns were detected in the ooplasm depending on the stage of development attained by the embryo. At early developmental stages, label is associated with large unsegmented portions of the fluid ooplasm. During embryonic development, the fluid ooplasm is gradually transformed into yolk granules by intervention of vitellophages. Prior to dorsal closure, the yolk sac is separated from the perivitelline fluid by interposition of serosa cells (the so called serosa membrane). Several mAbs raised against the perivitelline fluid react specifically with this membrane suggesting that the release of Vt polypeptides from the yolk sac occurs by intracellular transit through the serosa cells. By immunocytochemistry, gold label appears associated with the cell surface and a number of vacuoles of the serosa membrane. These data are interpreted as suggesting that Vt polypeptides resulting from limited proteolysis in stick insect embryos are not exhaustively degraded within the yolk sac, but are instead transferred transcytotically to the perivitelline fluid through the serosa membrane.     

Chondroitin sulfate proteoglycan is a constituent of the basement membrane in the rat embryo parietal yolk sac

Summary In addition to containing Type IV collagen, laminin and entactin, basement membranes contain small amounts of proteoglycans substituted primarily with heparan sulfate chains. We have previously shown, however, that parietal yolk sacs in organ culture synthesize predominantly chondroitin sulfate proteoglycan. In the present study, we have used histochemical and immunohistochemical techniques coupled with chondroitinase ABC digestion to provide evidence for the presence of chondroitin sulfate proteoglycan in the basement membrane (Reichert's membrane) of the 14.5-day rat embryo parietal yolk sac. The results revealed numerous cuprolinic blue-positive filaments and granules, 20–30 nm in greater length or diameter, dispersed throughout the thickness of the basement membrane. Both structures were removed by preincubating freshly isolated parietal yolk sacs with chondroitinase ABC. A similar labeling pattern was also obtained with immunoelectron microscopy using gold-labeled monoclonal anti-bodies directed against the three major isomers of protein-bound chondroitin sulfate. In contrast, coarser cuprolinic blue granules, 40–100 nm in diameter, were neither sensitive to chondroitinase ABC digestion nor labeled by the monoclonal antibodies. These results thus indicate that Reichert's membrane contains chondroitin sulfate proteoglycan in addition to heparan sulfate proteoglycan.     

The yolk sac in late embryonic development of the stick insect Carausius morosus (Br.)

Differentiation of the yolk sac was examined ultrastructurally and cytochemically in late embryonic development of the stick insect Carausius morosus. During migration along the yolk sac, endodermal cells form a discontinuous cell epithelium, leaving wide intercellular channels between neighbouring cell clusters. Within the same cell cluster, cells are all joined by septate junctions. In the proximity of the proctodeum region, intercellular channels are filled with numerous cell debris which are shown to derive from vitellophages undergoing cell lysis. Yolk sacs resolved by gel electrophoresis are shown to release a number of vitellin polypeptides into the culture medium. These are equivalent in molecular weight to those present in the vitellophage yolk granules This observation is consistent with the evidence that the basement lamina may act as a course physical filter, retaining particles larger than colloidal thorium dioxide and allowing free percolation of peroxidase. Differentiating endodermal cells form a microvillar striated border along the apical plasma membrane. A number of vesicular criptae were frequently seen in these differentiating endodermal cells. Electron dense granules released by endodermal cells are suggested to play a role in vitellophage lysis and vitellin release from the enclosed yolk granules.     

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.     

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.     

半滑舌鳎胚胎发育中后期卵黄囊超微结构观察

在胚胎发育中期,半滑舌鳎胚胎由胚体、卵黄囊和卵周液构成.对半滑舌鳎胚胎发育中后期的卵黄囊进行超微结构观察.结果表明,卵黄囊是由卵黄囊膜和包裹其内的卵黄物质组成.在半滑舌鳎胚胎发育过程中,卵黄囊内的卵黄物质逐渐消耗产生低分子量的卵黄磷蛋白分裂小泡.分裂小泡转移到卵黄囊内部消黄细胞中,在消黄细胞的作用下分裂小泡转化成卵黄颗粒.随后卵黄颗粒在卵黄囊内表面聚集成囊状结构并转移运输到卵黄囊膜内部,最后把卵黄物质从卵黄囊膜转移并释放到卵周液中,为胚胎发育提供营养.     

The role of the fat body, midgut and ovary in vitellogenin production and vitellogenesis in the female tick, Dermacentor variabilis.

Polyclonal antibodies directed against D. variabilis vitellin were utilized for immunocytochemistry at the ultrastructural level. We localized vitellogenin (Vg) in rough endoplasmic reticulum cisternae, secretory granules and secreted products of fat body trophocytes and midgut vitellogenic cells from feeding and ovipositing females. Vg was localized in the oocyte Golgi bodies and in the yolk bodies of both feeding and ovipositing females. Uptake of exogenous Vg was indicated by the presence of immunospecific gold probe in coated pits and coated vesicles at the apical plasma membrane of oocytes from females in rapid engorgement and oviposition. In unmated females little detectable evidence of Vg uptake by developing oocytes suggests that mating and host detachment signal the beginning of vitellogenesis. We conclude that fat body trophocytes, midgut vitellogenic cells and oocytes are involved in the synthesis and/or processing of Vg and that feeding is the signal associated with the initiation of Vg synthesis and/or processing.     

The serosa of Manduca sexta (Insecta, Lepidoptera): ontogeny, secretory activity, structural changes, and functional considerations

In Manduca sexta, the blastoderm forms successively and becomes immediately cellularized as the cleavage energids reach the surface of the oocyte. Presumptive serosal cells are large and contain 2 or 4 large polyploid nuclei; presumptive embryonic cells are small and mononuclear. All parts of the blastoderm participate in the uptake and digestion of yolk material. About 10 h post-oviposition, the blastoderm breaks at the amnioserosal fold and the extraembryonic part closes above the germ band and constitutes the serosa (12 h post-oviposition, i.e. 10% development completed). At once, the serosa starts to secrete a cuticle consisting of an epi- and a lamellated endocuticle. Detachment of the serosal cuticle, 22h post-oviposition, is reminiscent of apolysis of larval cuticle. Thereafter, the serosa deposits a membranous structure, the serosal membrane. The sercretory process lasts from 23h to 44h post-oviposition. At first a fine granular layer, then an amorphous, spongy-like, fibrillar layer is secreted via microvilli. This persisting membrane is tough, rubbery and very elastic. It may serve to bolster the serosa during katatrepsis (48h post-oviposition) and later embryonic movements. After detachment of the serosal membrane, 44h post-oviposition, a distinct subcellular reorganization of the serosa takes place. The nuclei become still larger and more irregular. Uptake of yolk granules, but not of lipid droplets, ceases, although interaction of serosa and yolk cells are intense. Serosal cells include many mitochondria, large areas of rER, besides some sER, increasing amounts of lysosomal bodies and prominent Golgi complexes. Most conspicuous is the assembly of spindle-shaped, electron-lucent vesicles below the apical surface. These vesicles may contain metabolic products which are released into the peripheral space. The studies show that the serosa assumes changing functions during embryogenesis: digestion of yolk substances, synthesis of a serosal cuticle and a serosal membrane, which may have a protective function, and excretion.     

Vitellin of the sweet potato whitefly,Bemisia tabaci: Biochemical characterization and titer changes in the adult

SDS-PAGE of the sweet potato whitefly (Bemisia tabaci) egg extract showed one major band (approximately 190 kDa) and two minor bands (approximately 75 kDa and 67 kDa). A distinct 190 kDa band was also present in male extract. On SDS gels the vitellin band of the greenhouse whitefly (Trialeurodes vaporarium) was larger, about 220 kDa. The native molecular mass of sweet potato whitefly vitellin was estimated to be 375 kDa using 4–20% native pore-limiting gel electrophoresis. Its isoelectric point was estimated to be 7.3 using isoelectric focusing. Two-dimensional gel electrophoresis and densitometry were used to estimate vitellin subunit composition; the data suggest that the sweet potato whitefly vitellin is likely to be a 380 kDa native molecule formed by two 190 kDa subunits. The two minor bands (75 kDa and 67 kDa) may be breakdown products of the native vitellin. This conclusion was supported by a Western blot of an SDS-PAGE gel of partially degraded female and egg extracts, which showed that polyclonal antiserum raised against the 190 kDa polypeptide recognized the 75 kDa and 67 kDa bands. Seven hybridoma cell lines secreting monoclonal antibodies against the 190 kDa band were screened, and one of them (S1A2G9H2) was mass produced. The antibody recognized the 190 kDa band in a Western blot. All the screened monoclonal antibodies were female and egg-specific by ELISA and/or Western blot, suggesting that the 190 kDa band in male extract was not a vitellin. A sensitive ELISA was established that could detect as little as 1/40 of an egg equivalent of vitellin using the monoclonal antibody from S1A2G9H2. Profiles of female sweet potato whitefly reproductive activities (egg laying, amount of vitellin in the female, and total vitellin produced by a female) within 2 days after eclosion were determined. Arch. Insect Biochem. Physiol. 34:223–237, 1997. © 1997 Wiley-Liss, Inc.     

Proteolytic activity of Boophilus microplus Yolk pro-Cathepsin D (BYC) is coincident with cortical acidification during embryogenesis

In a previous report (Parasitology 116 (1998) 525) we isolated and characterized Boophilus Yolk pro-Cathepsin (BYC), an aspartic proteinase precursor from the eggs of the hard tick. The present study was designed to characterize the function of BYC in the consumption of vitellin (VT), the major yolk protein, during embryogenesis. Both purified BYC and total egg homogenate proteolytic activity showed a similar pH dependence profile with an acidic optimum. Purified BYC presented higher activity against VT as a substrate when compared to other proteins. The VT degradation pattern observed in vitro also showed a similar profile to that observed in vivo. Co-localization of BYC and acidic cortical yolk granules was performed by immunocytochemistry and confocal microscopy. Proton-pumping activity of yolk granules in vitro was higher in eggs collected 4 day after oviposition than in newly laid eggs. Taken together, our data suggest that BYC plays a major role in the degradation of VT and that its activity is controlled by acidification of yolk platelets localized at the cortical cytoplasm of the developing Boophilus microplus egg.     

Cells released in vitro from the embryonic yolk sac of the stick insect carausius morosus (BR.) (PHASMATODEA : HETERONEMIIDAE) may include embryonic hemocytes

The embryonic yolk sac and the adult dorsal vessel of the stick insect Carausius morosus (Br.) (Phasmatodea : Heteronemiidae) were shown to release a number of cells that appear morphologically similar to circulating adult hemocytes. Like adult hemocytes, these cells reacted positively when tested for both phenoloxidase activity and a monoclonal antibody specifically raised against a vitellin polypeptide. Based on this evidence, it is suggested that yolk sac-released cells behave as potential embryonic hemocytes. A model is thus proposed whereby the yolk sac might host a number of hemopoietic stem cells on their way to the dorsal vessel, and in so doing, it may temporally act as an embryonic hemopoietic organ.     

Location and identification of the collagen found in the 14.5-d rat embryo visceral yolk sac

The collagens associated with 14.5-d rat visceral yolk sacs were localized and identified by a variety of procedures. Morphological examination showed that both the visceral epithelium and mesothelium rested upon thin basement membranes, whereas the majority of the extracellular matrix consisted of a stroma containing occasional cells and abundant banded fibrils. Immunohistochemistry at the electron microscope level showed that the basement membranes specifically cross- reacted with antibodies directed against mouse basement membrane components, whereas the stroma specifically cross-reacted with antibodies directed against rat type I collagen. Extractions of acellular visceral yolk sacs and subsequent analyses showed that type I collagen components were prevalent. Furthermore, in vitro biosynthetic studies showed only the presence of type I procollagen components (or their conversion products) and alpha-fetoprotein. These findings, taken together with our previous studies on the 14.5-d rat parietal yolk sac, provide us with protein markers for studying the origin of cells in rat parietovisceral yolk sac carcinomas.     

Identification of Reissner's fiber-like glycoproteins in two species of freshwater planarians (Tricladida), by use of specific polyclonal and monoclonal antibodies

By using one polyclonal antiserum raised against bovine Reissner's fiber and seven monoclonal antibodies raised against bovine Reissner's fiber and against immunopurified bovine subcommissural organ glycoproteins, we have investigated two freshwater planarian species (Girardia tigrina, Schmidtea mediterranea) by light- and electron-microscopic immunocytochemistry. ELISA probes showed that the monoclonal antibodies recognized different, nonoverlapping, unrepeated, proteinaceous epitopes present in the same compounds of bovine Reissner's fiber. Cells immunoreactive to the polyclonal and monoclonal antibodies were found in the dorsal and ventral integument of both planarian species. Labeled cuboid epidermal cells bore cilia and displayed several types of secretory granules; they were covered by a film of immunoreactive material. Studies on adjacent thin and semithin sections revealed coexistence of label in the same regions and in the same cells when two different monoclonal antibodies were used. These results indicate that a secretory substance immunologically similar to the secretion of the vertebrate subcommissural organ is present in primitive tripoblasts such as planarians, suggesting that these secretions are ancient and well conserved in phylogeny.     

Confocal scanning laser microscopy of the follicular epithelium in ovarioles of the stick insect Carausius morosus

Ovarian follicles of the stick insect Carausius morosus were analyzed by confocal laser microscopy and immunocytochemistry with a view to studying cell polarity in the follicular epithelium. Such probes as anti-α-tubulin antibodies and Rh-phalloidin were employed to establish how the follicle cell cytoskeleton changes during ovarian development. Data show that α-tubulin prevails over the basal end, while F-actin appears more abundant along the apical end of the follicle cells. This finding was further corroborated by immunogold cytochemistry, showing that label along the basal end is primarily associated with microtubules, while that along the apical end is due to follicle cell microvilli interdigitating with the oocyte plasma membrane. A monoclonal antibody specifically raised against a vitellin polypeptide was used to investigate the role the follicular epithelium might play in relation to vitellogenin (Vg) uptake by the oocyte. Data show that under these conditions label is restricted to the intercellular channels of the follicular epithelium, thus providing further support to the notion that Vg enters the oocyte through the extracellular pathway leading from the basement lamina to the oocyte surface. By contrast, the use of a monoclonal antibody raised against a fat-body-derived protein of 85 kDa that is specifically sulfated within the follicle cells provides evidence for the existence of an alternative way of gaining access to the oocyte surface, that is by transcytosis through the follicular cell epithelium. These findings confirm our earlier observations on stick insect ovarioles whereby polarization in the follicular epithelium is primarily addressed to sustain a transcytotic vesicular traffic between opposite poles of the follicle cell of Vg toward the oocyte surface.     

Purification and characterization of the common yolk protein,vitellin, from the estuarine amphipod Leptocheirus plumulosus

Vitellin is a major yolk protein that plays a significant role in the embryonic development of crustacean embryos. This protein was rapidly purified from embryos of the estuarine amphipod, Leptocheirus plumulosus, by subjecting the crude protein homogenate to high affinity column chromatography. SDS-PAGE revealed a single band with an approximate molecular weight of 200,000 daltons. Vitellin was characterized by SDS-PAGE techniques and amino acid composition analysis. L. plumulosus vitellin is a lipoglycophosphoprotein with serine, glutamic acid/glutamine, alanine, and aspartic acid/asparagine accounting for almost 66% of all amino acid residues. Polyclonal antibodies were raised against L. plumulosus vitellin and antibody reactivity was verified by dot-blotting and immuno-fluorescence confocal microscopy. These antibodies are specific for purified vitellin and show little cross-reactivity with other embryonic proteins.     

Monoclonal antibodies as probes for processing of the mosquito yolk protein; a high-resolution immunolocalization of secretory and accumulative pathways

A library of monoclonal antibodies (mAB) directed against yolk polypeptides of the mosquito Aedes aegypti was utilized to visualize the secretory pathway of these polypeptides in the fat body and their accumulative pathway in developing oocytes. Single and double immunolabelling using mABs and colloidal gold of different sizes confirmed biochemical observation that 200 +/- 5 and 65 +/- 3 kDa polypeptides represent subunits of the yolk protein. This immunocytochemical analysis showed that, in trophocytes of the fat body, both the subunits of the yolk protein were routed simultaneously through the Golgi complex into secretory granules and were subsequently secreted. The yolk protein subunits were also directed together through all the steps of the accumulative pathway in the oocyte. Double immunogold labelling revealed that the subunits were present together during their binding to the oocyte membrane, transportation into and accumulation in the transitional yolk body, and, finally, crystallization in the mature yolk body. Electron microscopical immunocytochemistry also confirmed immunofluorescent data and showed that mABs directed against different steps in the biosynthetic processing of the yolk protein in the fat body, as well as in its accumulative pathway in oocytes.     

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.     

Murine cell surface glycoproteins: immunochemical analysis of a major differentiation alloantigen of phagocytic cells

Four independent monoclonal antibodies derived from spleen cells of rats immunized with mouse NIH/3T3 cells were found to precipitate an 80,000-dalton plasma membrane glycoprotein, identified as a polymorphic differentiation antigen of murine mesenchymal cells. The homology of the four immunoprecipitated polypeptides was proven by analysis of partial proteolytic cleavage products. The genetic polymorphism detected by the four antibodies was shown to reside in a single antigenic site by several criteria: (i) The expression of the antigenic determinant among different strains of mice; (ii) cross-inhibition of antibody binding; (iii) precipitation of partial proteolytic cleavage fragments of the 80,000-dalton glycoprotein; (iv) the kinetics of heat inactivation. These antibodies thus define a single polymorphic site of a major phagocytic cell surface glycoprotein and provide the basis for genetic and functional characterization of this glycoprotein.     

Localization of the proenzyme form of the vitellin-processing protease in Blattella germanica by affinity-purified antibodies

During Blattella germanica embryo development, the nutritive yolk protein vitellin is processed by a cysteine protease, which is activated proteolytically from a proprotease during acidification of yolk granules. A murine polyclonal antiserum was generated with the purified proprotease as the immunogen. The antiserum was made monospecific to proprotease by subtractive affinity chromatography using proprotease-free yolk proteins as ligand. The purified antibodies were employed to investigate the temporal and spatial expression of the proprotease during vitellogenesis and embryo development. Anti-proprotease-reactive peptides appeared in extracts of fat bodies and ovarian follicles of post-mating females, but not in fat bodies of males or the fat bodies or follicles of unmated females, suggesting that the proprotease is synthesized extraovarially. Use of the antibodies was extended to monitor the kinetics of proprotease disappearance during early embryo development. Arch. Insect Biochem. Physiol. 38:109–118, 1998. © 1998 Wiley-Liss, Inc.