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The yolk sac in late embryonic development of the stick insect Carausius morosus (Br)
Abstract
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.
... Le Bihan et al. (2004) De nombreuses études ont décrit les cathepsines et les phosphatases acides comme étant les enzymes responsables de la dégradation du vitellus dans les oeufs d'oiseaux (Gerhartz et al., 1997), de poissons (Sire et al., 1994, Kestemont et al., 1999, Martinez et al., 1999, Carnevali et al., 2001, d'amphibiens (Lemanski et Aldoroty, 1974, Fagotto et Maxfield, 1994b, Yoshizaki et Yonezawa, 1994, Komazaki et Hiruma, 1999, d'insectes (Fialho et al., 1999(Fialho et al., , 2002(Fialho et al., , 2005, d'échinodermes (Schuel et al., 1975, Mallya et al., 1992, et de polychètes (Bonnier et Baert, 1992 (Fagotto, 1990, Komazaki et Hiruma, 1999. La digestion de ces réserves dépend donc de la transmission de ces enzymes lysosomales au cours de l'ovogenèse de la mère à l'oeuf (Fausto et al., 1997). De plus, les réserves vitellines n'étant pas dégradées avant le début de l'embryogenèse, ceci implique l'existence de mécanismes régulant les activités des enzymes (Fagotto et Maxfield, 1994a). ...
The common cuttlefish Sepia officinalis makes large reproductive migrations in spring to mate and spawn in coastal waters. The eggs are laid in shallow waters and are therefore likely subjected to the anthropogenic contaminations leading to potential toxicity effects of metals during the embryonic development. In this study, cuttlefish eggs revealed efficient accumulation capacities following exposure to 9 dissolved radiotracers (110mAg, 241Am, 109Cd, 57Co, 134Cs, 203Hg, 54Mn, 210Pb and 65Zn) all along the development time. In this context, the eggshell played a key role in the accumulation process, as the greatest fraction of most of these elements remained associated with its glycoproteic components. The radiotracers distribution showed that the eggshell acts as an efficient shield against trace element penetration during organogenesis, i.e. during the first month of development. A selective permeability of the eggshell appeared during the last stages of the development, i.e when the embryo growth began until hatching. The trace element capacities to diffuse through the eggshell seemed to be dependent of their chemical properties and of the biological processes of the embryogenesis. Thus, metals could be accumulated in the embryo with various affinities (i.e. Ag >> Zn > Hg > Mn > Co ≈ Cd > 134Cs ≠ 241Am, Pb).
All along the development time, the exposure of the eggs to Ag, Cd and Cu (1, 0.5 and 250 µg.l-1, respectively) in experimental conditions showed no effect on the egg growth and did not induce abnormalities or mortality in embryos, confirming the protective role of the eggshell during organogenesis. However, during the last developmental stages, Ag and Cd reduced the acid phosphatase activities, which are involved in the digestive processes, whereas Cu acted as an activator. Moreover, phenoloxidase-like activity, which is here reported in the embryo for the first time, was modulated during the post-organogenesis phase, following exposure to dissolved Ag and Cu.
Despite the protective role of the eggshell during the organogenesis, trace elements would be accumulated in the embryo during the first developmental stages following their maternal transfer in the egg. In this context, radiotracers technique highlighted that the maternal transfer was a selective way, 1) with only Ag, Se and Zn being transferred following a dietary exposure of the female to 110mAg, 241Am, 109Cd, 60Co, 134Cs, 54Mn, 75Se and 65Zn, and 2) Se and Zn being incorporated in the vitellus whereas Ag was found in similar proportions in the vitellus and in the eggshell.
... Both AcP and cathepsins are localized in specialized organelles known as yolk platelets, which are modified lysosomes containing vitellin reserves, i.e. vitellogenin, phosvitin, lipovitellin, nucleic acids, polysaccharides, lectin, and growth factors (Fagotto, 1990(Fagotto, , 1995Komazaki and Hiruma, 1999). Therefore, the utilization of the yolk during embryogenesis implies that (i) the lysosomal enzymes are maternally transferred during oogenesis (Fausto et al., 1997) and (ii) the yolk platelets do not degrade their content until specific developmental stages are reached (Fagotto and Maxfield, 1994). Indeed, activation of AcP and cathepsins depends on egg fertilization (Fialho et al., 2002) and the stimulation of yolk platelets by acidification of these organelles. ...
Lacoue-Labarthe, T., Le Bihan, E., Borg, D., Koueta, N., and Bustamante, P. 2010. Acid phosphatase and cathepsin activity
in cuttlefish (Sepia officinalis) eggs: the effects of Ag, Cd, and Cu exposure. – ICES Journal of Marine Science, 67: 1517–1523.
Changes in the activity levels of acid phosphatase (AcP) and cathepsin during cuttlefish embryo development are described,
as are the effects of exposure to heavy metals. Enzyme activity kinetics appear to be linked to the developmental stage. The
activities of both enzymes increased during the final days of development, suggesting de novo production by the maturing embryo in the digestive gland. The effects of selected heavy metals, Ag (0.06, 1.2, 60, 1200 ng
l−1), Cd (31, 61, 305, 610 ng l−1), and Cu (0.23, 2.3, 23, 230 µg l−1), were assessed based on AcP and cathepsin activities at the end of embryonic development and on hatchling weight. Enzyme
activities were not impacted by Ag but were significantly inhibited by Cd, at all four concentrations for AcP and at 610 ng
l−1 for cathepsin. Cu (at 2.3 µg l−1) stimulated AcP activity. No cause–effect relationship was found between the effects of metals on the enzyme activities and
hatchling weight, suggesting that heavy metals could affect other physiological functions during embryogenesis.
... Both AcP and cathepsins are localized in specialized organelles known as yolk platelets, which are modified lysosomes containing vitellin reserves, i.e. vitellogenin, phosvitin, lipovitellin, nucleic acids, polysaccharides, lectin, and growth factors (Fagotto, 1990, 1995; Komazaki and Hiruma, 1999). Therefore, the utilization of the yolk during embryogenesis implies that (i) the lysosomal enzymes are maternally transferred during oogenesis (Fausto et al., 1997) and (ii) the yolk platelets do not degrade their content until specific developmental stages are reached (Fagotto and Maxfield, 1994). Indeed, activation of AcP and cathepsins depends on egg fertilization (Fialho et al., 2002) and the stimulation of yolk platelets by acidification of these organelles. ...
This paper describes the changes of the acid phosphatases (AcP) and cathepsin activities throughout the cuttlefish embryo development. The enzyme activity kinetics appeared to be linked with the respective developmental stage. Activities of both enzymes increased during the last days of development suggesting a de novo production of these proteins by the maturing embryo in the digestive gland. The effects of selected heavy metals, i.e. Ag (0.06, 1.2, 60, 1200 ng.l-1), Cd (31, 61, 305, 610 ng.l-1) and Cu (0.23, 2.3, 23, 230 µg.l-1), were assessed on the AcP and cathepsin activities at the end of embryonic development and on the hatchling's weight. Enzymatic activities were not impacted by Ag and were significantly inhibited by the four Cd concentrations for AcP and at 610 ng.l-1 for cathepsin. Cu stimulated AcP activity at 2.3 µg.l-1. No cause-consequence relationship was found between metal effect on the enzymatic activities and the reduction of hatchling weight, suggesting that heavy metals could affect other physiological functions during embryogenesis.
... s'apparentent à des lysosomes modifiés dans lesquels sont stockées les réserves vitellines, i.e. sous forme de vitellogenine, phosvitine, lipovitelline, ainsi que de petites quantités d'acides nucléiques, des polysaccharides acides, des lectines, et des facteurs de croissance (Fagotto, 1990, Komazaki et Hiruma, 1999. La digestion de ces réserves dépend donc de la transmission de ces enzymes lysosomales au cours de l'ovogenèse de la mère à l'oeuf (Fausto et al., 1997). De plus, les réserves vitellines n'étant pas dégradées avant le début de l'embryogenèse, ceci implique l'existence de mécanismes régulant les activités des enzymes (Fagotto et Maxfield, 1994a). ...
Au printemps, la seiche commune Sepia officinalis effectue des migrations du large vers la côte pour se reproduire et mourir. La ponte a lieu en zone côtière, sujette à la contamination métallique d'origine anthropique, ce qui pose la question de la toxicité des éléments traces sur le développement embryonnaire. Dans ce travail, l'exposition à neuf radiotraceurs (110mAg, 241Am, 109Cd, 57Co, 134Cs, 203Hg, 54Mn, 210Pb et 65Zn) durant toute la durée du développement révèle que l'œuf de seiche possède de grandes capacités de bioaccumulation des éléments traces. Dans ce contexte, la capsule glycoprotéique de l'œuf joue un rôle majeur en fixant la plus grande proportion de ces éléments. La distribution des radiotraceurs dans l'œuf montre que cette capsule constitue une barrière protectrice contre la pénétration des métaux depuis la ponte jusqu'à la fin de l'organogenèse. Cependant, lorsque la phase de croissance de l'embryon et de l'œuf commence, la capsule montre progressivement une perméabilité sélective aux éléments traces jusqu'à l'éclosion. La disposition des composants de la capsule à les laisser diffuser à travers les différentes enveloppes qui la composent semble dépendante des propriétés chimiques des éléments traces mais aussi des processus biologiques liés à l'embryogenèse. Ainsi, certains éléments pénètrent dans l'œuf et s'accumulent dans l'embryon avec une affinité variable (i.e. Ag >> Zn > Hg > Mn > Co ≈ Cd > 134Cs ≠ 241Am, Pb).
Des expositions sur le long terme à l'Ag, au Cd et au Cu dissous (1, 0,5 et 250 µg.l-1, respectivement) en condition expérimentale, n'ont aucun effet sur la croissance de l'œuf et n'induisent ni malformation, ni mortalité des embryons, confirmant l'effet protecteur de la capsule pendant les stades sensibles de l'embryogenèse. Néanmoins, pendant la période de croissance de l'embryon, l'Ag et le Cd semblent ralentir l'augmentation de l'activité des phosphatases acides, impliquée dans les processus digestifs alors que le Cu révèle un effet stimulateur. De même, l'activité de type phénoloxydase, impliquée dans les fonctions immunitaires des Invertébrés, décrite pour la première fois dans l'embryon de seiche, est modulée par l'Ag et le Cu, lors de la phase post-organogenèse.
Malgré le rôle protecteur de la capsule durant l'organogenèse, les éléments traces peuvent s'accumuler dans les embryons dès les premiers stades de développement du fait de leur apport par transfert maternel. Dans ce contexte, l'utilisation des radiotraceurs montre que le transfert de la mère à l'œuf est sélectif, 1) seuls l'Ag, le Se et le Zn étant transférés suite à une exposition de la mère par de la nourriture marquée au 110mAg, à l'241Am, au 109Cd, au 60Co, au 134Cs, au 54Mn, au 75Se et au 65Zn et 2) le Se et du Zn étant stockés dans le vitellus de l'œuf alors que l'Ag se retrouve à la fois dans le vitellus et la capsule.
... These are multimeric proteins of maternal origin that are degraded by a process of limited proteolysis to metabolically sustain the embryo [13,27]. To accomplish this goal, vitellins are first confined within cells, the socalled vitellophages [5], that invade the yolk fluid mass from the egg periphery and partition it into a number of large yolk granules [6]. Maternally derived proteolytic enzymes are also sorted out within these granules and gradually activated by acidification [4,11,21,22]. ...
During embryonic development, insect vitellins (Vt) are degraded by limited proteolysis to yield a number of lower-molecular weight polypeptides. The aim of the present study was to identify these polypeptides in the embryo and to verify how they relate to Vt polypeptides deposited in the oocyte during vitellogenesis. To this end a panel of poly- and monoclonal antibodies (Pab, Mab) was raised against Vt polypeptides and employed by immunoelectrophoresis and immunoblotting on embryos belonging to different developmental stages. Through this approach three major staining patterns were observed. First, Mab 4 reacts with both polypeptides B1 and E20, suggesting that polypeptide B1 is gradually trimmed to yield polypeptide E20 in late embryos. Second, Mab 12 is specific for polypeptide A3 which is retained unchanged throughout embryogenesis. Third, Pab anti-A2 and Mab 13 show that polypeptide A2 is processed to yield polypeptide E9 through limited proteolysis. In conclusion, the staining patterns reported in this study show that Vt polypeptides in developing embryos of the stick insect Carausius morosus undergo at least two major processing events concerning polypeptides B1 and A2.
... The underlying vitellophages are huge polygonal cells comprised of numerous tightly clustered yolk granules (Fig. 1A). The origin of these cells and the morphological changes they undergo during development have already been described elsewhere and will not be reiterated here (Fausto et al. 1994(Fausto et al. , 1997. When fractured along the external surface, the gelified perivitelline fluid appears to be made of numerous particles of about 2 mm in mean diameter (Fig. 1B). ...
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.
... We have studied yolk utilisation in embryos of the stick insect Carausius morosus and found that yolk granules in this species are reconstituted anew with the beginning of embryonic development (Fausto et al. 1994). By this time, the fluid ooplasm is gradually transformed into a collection of progressively smaller yolk granules due to invasion of secondary vitellophages from the egg periphery (Fausto et al. 1997). On their way into the fluid ooplasm these macrophagic cells engulf yolk by bulk endocytosis and partition it into newly formed yolk granules. ...
Newly laid eggs of stick insects comprise a unique fluid ooplasm that is gradually partitioned into a number of yolk granules by invasion of secondary vitellophages. This study aimed at establishing how yolk granules become acidified in the course of embryonic development. Data show that acidified yolk granules are rather scarce and randomly distributed in vitellophages of early embryos, while they tend to increase gradually in number as development proceeds to completion. Yolk granule acidification is progressively more inhibited in the presence of increasing concentrations of chloroquine, monensin and bafilomycin. A pro-protease was identified cytochemically and by immunoblotting in yolk extracts of progressively more advanced embryos. A specific monoclonal antibody raised against this pro-protease helped to demonstrate that it is gradually processed to yield a lower molecular weight polypeptide as development proceeds to completion. This latter polypeptide was identified as a protease using electrophoresis in polyacrylamide gels containing yolk extracts. Simultaneous administration of a fluorescent substrate for cysteine protease and an acidotropic probe produced superimposable labelling patterns, suggesting that only acidified yolk granules possess a proteolytic activity. On the other hand, yolk granules probed simultaneously for acidification and latent pro-protease yielded labelling patterns partially superimposed. Pro-protease labelling is gradually lost as yolk granules are progressively more acidified during development. Distinct labelling patterns were also obtained in vitellophages processed for the simultaneous detection of pro-protease and protease, suggesting that the two activities are expressed by different yolk granule populations, and that one is gradually converted into the other as time goes by.
... Newly laid eggs of the stick insect C. morosus are characterized by a fluid yolk mass gradually partitioned into a number of yolk granules during embryonic development (Fausto et al., 1994). Partitioning occurs by virtue of the phagocytic activity of vitellophages invading the ooplasm from the egg periphery (Fausto et al., 1997). Vitellophages were examined by confocal fluorescence microscopy, following exposure to anti-vitellin polypeptide antibodies. ...
Vitellin polypeptides are proteolytically processed in ovarian follicles and embryos of the stick insect Carausius morosus. Data show that vitellin polypeptide A(3) of 54kDa is processed to yield polypeptide A(3)(*) of about 48kDa upon completion of ovarian development, whereas vitellin polypeptide A(2) of 90kDa yields polypeptide E(9) during embryonic development. As vitellin polypeptides are processed, polypeptides A(3)(*) and E(9) are transferred from the yolk granules to the cytosolic space of the vitellophages and start to express a ubiquitin reactivity. At the confocal microscope, anti-ubiquitin antibodies label specifically numerous small yolk granules and the cytosolic space of vitellophages. During embryonic development, ubiquitin carrying granules undergo acidification in much the same way as larger yolk granules. However, only these latter organelles are capable of converting a latent cysteine pro-protease into an active yolk protease upon acidification of their luminal space. These data are interpreted as indicating that ubiquitin-like polypeptides are restricted to small granules throughout ovarian and embryonic development, and that vitellin cleavage products are ubiquitinated following acidification of large yolk granules and transfer to the cytosolic space of the vitellophages.
Developing embryos of the stick insect Carausius morosus were examined ultrastructurally with a view to studying vitellophage invasion of the yolk mass during and after germ band formation. Newly laid eggs in C.morosus have a unique yolk fluid compartment surrounded by a narrow fringe of cytoplasm comprising several small yolk granules. Vitellophages originate mainly from a thin layer of stem cells, the so-called yolk cell membrane, interposed between the germ band and the yolk mass. Throughout development, a thin basal lamina separates the yolk cell membrane from the overlying embryo.
Vitellophages extend from the yolk cell membrane with long cytoplasmic processes or filopodia to invade the central yolk mass. Along their route of entrance, filopodia engulf portions of the yolk mass and sequester it into membrane-bounded granules. As this process continues, the yolk mass is gradually partitioned into a number of yolk granules inside the vitellophages.
Later in development, the yolk cell membrane is gradually replaced by the endodermal cells that emerge from the anterior and posterior embryonic rudiments. From this stage of development onwards, vitellophages remain attached to the basal lamina through long filopodia extending between the endodermal cells. Yolk confined in different vitellophagic cells appears heterogeneous both in density and texture, suggesting that yolk degradation may be spatially differentiated.
This review focuses on the current status of knowledge regarding the process of vitellogenin (Vg) uptake and vitellin (Vt) storage in insect oocytes. We also consider the overall morphology of the yolk sac as an embryonic organ allowing for both temporal and spatial differentiation of yolk degradation to provide the primary food supply for the embryo. In this context we describe the evidence that demonstrates the occurrence of Vt polypeptide processing and how it may be affected by maternally derived proteases stored in the yolk granules and by the ubiquitin–proteasome system in the cytosol. The extent of Vt polypeptide processing induced by these proteases will be correlated with the structural modifications affecting yolk granules and vitellophages in developing insect embryos. To accomplish these goals the ultrastructural and cytochemical composition of yolk granules during vitellogenesis and embryogenesis will be reviewed. Last but not the least, our current understanding of the role played by acidification of yolk granules in the activation of maternally derived proteases will also be examined.
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.
Knowledge of the chemistry and biology of yolk proteins has expanded explosively in the past decade and promises to be equally as volatile in the present one. Some old issues have been partially resolved but new issues are arising with the application of new technology.
Yolk spheres in tick eggs contain a latent procathepsin L, which is activated in vivo, in parallel with yolk degradation, and in vitro by acid treatment (Fagotto, F., Arch. Insect Biochem. Physiol., 1990b in press). Mature cathepsin L hydrolyzes vitellin at acidic pH (Fagotto, F., Arch. Insect Biochem. Physiol., 1990a in press). Here, yolk spheres' pH has been estimated using acridine orange. In the early development, all yolk spheres are neutral, then an increasing number acidify, until hatching, where general acidification seems to occur. This fits well with vitellin utilized slowly during embryogenesis, more intensely at hatching (Chinzei, Y. and I. Yano, Experientia 41, 948, 1985), and can be related to sequential degradation of individual spheres observed during embryonic development, then extensive yolk liquefaction in the larva. Different yolk spheres populations have been separaded on Percoll density gradients. In freshly laid eggs, yolk spheres are dense, neutral, undegrated and contain exclusively the precursor of cathepsin L. In later stages, yolk spheres are progressively recovered in lower density fractions, displaying acidic interior and cytological signs of degradation. They cosediment with mature cathepsin L. It is concluded that acidification initiates yolk degradation through procathepsin L activation.
We report a phenomenon previously unknown for oviparous animals; in Aedes aegypti mosquitoes a serine carboxypeptidase is synthesized extraovarially and then internalized by oocytes. The cDNA encoding mosquito vitellogenic carboxypeptidase (VCP) was cloned and sequenced. The VCP cDNA hybridizes to a 1.5-kilobase mRNA present only in the fat body of vitellogenic females. The deduced amino acid sequence of VCP shares significant homology with members of the serine carboxypeptidase family. Binding assays using a serine protease inhibitor, [3H]diisopropyl fluorophosphate, showed that VCP is activated in eggs at the onset of embryonic development. Activation of VCP is associated with the reduction in its size from 53 kDa (inactive proenzyme) to 48 kDa (active enzyme). The active, 48-kDa, form of VCP is maximally present at the middle of embryonic development and disappears by the end.
In crude extracts of eggs of the soft tick Ornithodoros moubata, maximum degradation of vitellin is at pH 3-3.5, whereas no proteolysis is detected at neutral or weakly acidic pHs. Acidic proteolysis is maintained at high level throughout embryonic development, and rapidly decreases in the larva, during the high phase of yolk degradation. Proteinase, acid phosphatase, and N-acetylglucosaminidase are localized within the yolk spheres; these can be considered as lysosomal-like organelles containing both substrate (vitellin) and the degradative machinery. Proteolytic activity has been essentially attributed to a cathepsin L-like enzyme through substrate specificity and inhibitors. The molecular weight is 37,000 to 39,000 as shown using gelatin-containing SDS-PAGE activity gels. At neutral pH the enzyme binds to vitellin, as demonstrated by gel filtration and PAGE under nondenaturing conditions. Acid proteinase activity at pH 5-6 is undetectable both with proteins and synthetic substrates, but is strongly increased after preincubation at pH 3-4. Activation at low pH could be important in the regulation of yolk degradation.
Vitellogenin (Vg), the hemolymph precursor to the major yolk protein in mosquitoes, is synthesized in the fat body of blood-fed females. Mosquito Vg consists of two subunits with Mr = 200,000 and 66,000. Here, we demonstrate that both the Vg subunits are first synthesized as a single precursor. The identity of this Vg precursor was confirmed by immunoprecipitation with subunit-specific monoclonal antibodies. In cell-free translation of fat body poly (A)+ RNA, the Vg precursor had Mr = 224,000 which increased to 240,000 in the presence of canine pancreatic microsomal membranes. A precursor with Mr = 250,000 was immunoprecipitated in microsomal fractions isolated from rat bodies. With in vitro pulse labeling, the 250-kDa precursor could be detected in homogenates of fat bodies from blood-fed mosquitoes only during the first few hours accumulation of the Vg precursor was achieved by an in vitro stimulation of Vg synthesis in previtellogenic fat bodies cultured with an insect hormone, 20-hydroxyecdysone. The 250-kDa precursor was glycosylated and to a much lesser degree phosphorylated. Treatment of fat bodies with tunicamycin yielded the precursor with Mr = 226,000 which was neither glycosylated nor phosphorylated. The reduction in molecular mass of the 250-kDa Vg precursor and of both mature Vg subunits combined was similar after digestion with endoglycosidase H, indicating that glycosylation is completed prior to cleavage of the Vg precursor. In vitro pulse-chase experiments revealed rapid proteolytic cleavage of the 250-kDa precursor to two polypeptides with Mr = 190,000 and 62,000 which transformed into mature Vg subunits of 200- and 66-kDa as the last step prior to Vg secretion. This last step in Vg processing was inhibited by an ionophore, monensin, and therefore occurred in the Golgi complex. Sulfation as an additional, previously unknown, modification of mosquito Vg was revealed by the incorporation of sodium [35S]sulfate into both Vg subunits. Since sulfation of Vg was predominantly blocked by monensin, the final maturation of Vg subunits in the Golgi complex is, at least in part, due to this modification.
A yolk protein, egg-specific protein, synthesized and accumulated in the developing ovaries of Bombyx mori serves not only as the nutritive source for embryogenesis but also for the reorganization of the yolk system through limited degradation. Using the purified egg-specific protein as a substrate, a protease responsible for its limited hydrolysis was identified in embryonating eggs and purified to homogeneity. The protease had an apparent molecular mass of 30,500 with one subunit of 29,000 daltons. It hydrolyzes synthetic substrates at carbonyl bonds of Arg or Lys residues, and the hydrolysis is strongly inhibited by diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, and leupeptin, suggesting that it is a trypsin-like protease. The protease shows an extremely high degree (over 2,000-fold) of specificity for egg-specific protein compared to other yolk proteins. Intact egg-specific protein is cleaved into three fragments in two steps; the first releases a 8.7-kDa peptide as an end product and a 55-kDa peptide intermediate, and in the second the intermediate is cleaved into 36- and 17.2-kDa peptides. By relating the NH2-terminal amino acid sequences of these peptides to the sequence of the intact egg-specific protein, the protease was shown to cleave first at a Lys-Asn site and secondly at Arg-Asp. Proteolytic activity abruptly appears mid-way in embryogenesis and increases steeply during completion of larval differentiation.
A cysteine protease that initiates degradation of vitellin (Vt) in the orthopteran Blattella germanica, and its proprotease precursor, were purified from yolk and partially characterized. The protease, purified 300-fold, contains three peptides of Mr 27,000, 29,000, and 31,000. A comparison of the purified enzyme's action pattern on Vt in vivo and in vitro confirmed its role in Vt processing. Protease-deficient yolk (day 0 postovulation) contained peptides of Mr 35,500, 37,000, 39,000, and 41,000, which were absent from yolk with protease activity. These were replaced by three peptides of approximately Mr 29,000, at days 2-3, the same time in development that protease expression and acidification of yolk granules occur (Nordin, J. H., Beaudoin, E. L., and Liu, X. (1991) Arch. Insect Biochem. Physiol. 18, 177-192). Acidification of purified proprotease converted it to three peptides of approximately Mr 29, 000 with cysteine protease activity. This conversion also required participation of a cysteine protease. Activated proprotease had the same pH activity profile, susceptibility to inhibitors, and cathepsin classification (L) as the protease. These results indicate that the Vt-processing protease is derived from a proprotease, which is activated in vivo by a developmentally regulated decrease in intragranular pH.
Within the digestive tract, the ingested food is broken down into simpler molecules by the action of enzymes and thus rendered absorbable through the gut wall. The midgut is composed of a long straight tube which often contains blind pouches (caeca). It is the main site of production of digestive enzymes and of absorption. In many insect species, it secretes a peritrophic membrane which is permeable to the digestive enzymes and to the products of digestion. In most insects, the structure of the midgut seems to be rather uniform, and thus the same cells are both secretory and absorptive. However, ultrastructural and cytochemical studies have shown a variety of differences which are related sometimes to cycles of cellular activities. In other cases, the midgut exhibits a clear division into functional segments or a mixture of different kinds of specialized cells may be present. Physiological experiments combined with ultrastructural and cytochemical investigations have sometimes clearly established the functions of these segments, or cells. It was studies of this type that showed the important role of the midgut epithelium in ionic regulation and mineral accumulation in insects belonging to several different orders. The transfer of fluid and ions through the wall of the midgut sometimes leads to curious arrangements that bring into contact a midgut segment with the Malpighian tubules, and concurrently certain specialized gut cells lose their digestive function in order to undertake an absorptive role.
The frontispiece of William Harvey’s “De Generatione Animalium” (Concerning the Generation of Animals) published in 1651 shows the hand of Jove holding an egg, or rather an eggshell, out of which have emerged a wide variety of animals: a child, a dolphin, a spider and so on. Worked into the design is the motto “Ex ovo omnia”—everything comes from an egg. The egg is surely one of nature’s most remarkable and versatile inventions. It is a compact self-contained capsule containing everything necessary for the creation of a new life-be it a slug, a worm, a moth, a shark, an ostrich or a turtle.
Malaria, dengue and other mosquito-borne infections are among the most devastating diseases (57, 144). The maintenance and dispersal of mosquito-borne disease depends upon the successful reproduction of the mosquito. The cornerstone of the reproductive cycle is vitellogenesis involving massive production of yolk protein precursors and their accumulation in developing oocytes. In anautogeneous mosquitoes, vitellogenesis is dependent on the availability of a blood meal and, as a consequence, is linked to transmission of pathogens. Pathogens acquired transovarially or from an infected host during an initial blood meal are transmitted during subsequent blood meals. Therefore, elucidation of vitellogenesis and other aspects of mosquito reproductive physiology is critical for the successful development of novel strategies in vector and disease management.
In eggs of the cockroach Blattella germanica, vitellin (Vt) utilization by the embryo is initiated at day 4 postovulation by the proteolytic processing of its three subunits to a specific set of peptides. A report from our laboratory (Nordin et al.: Archives of Insect Biochemistry and Physiology 15:119, 1990) described a yolk proteinase, activated at days 3–4, which processes the Vt. Further investigation of this event has focused on the yolk granules. Granules from eggs 4–6 days postovulation contained a significant subpopulation which accumulated high concentrations of the dye acridine orange (AO), a fluorescent probe of vesicle acidification, while those from eggs 0–3 days postovulation did not. AO accumulation was caused by proton translocation and was not due to dye binding or a Donnan equilibrium. The temporal correlation of granule acidification with Vt processing suggests a role for this event in yolk proteinase activation in B. germanica. This hypothesis was supported by the finding that incubation of yolk from freshly ovulated eggs in vitro at pH of 5 and below resulted in Vt processsing. Yolk granules of the blowfly Phormia regina also became acidified but this occurred in the oocyte prior to egg deposition.
1.1. Adult females of the stick insect Carausius morosus were injected with either [35S]methionine or [3H]leucine and eggs laid during the following 2 weeks collected for further analysis.2.2. After the injection, radioactivity started to appear in newly laid eggs with a time-lag of 9 days, reached a maximum incorporation on day 10 and then levelled off, due to dilution of the injected tracer.3.3. Newly laid eggs from injected females were analyzed by gel electrophoresis and the incorporated radioactivity revealed fluorographically.4.4. Data showed that vitellin polypeptides were the only egg constituent to become labelled under these conditions.5.5. In addition, light microscope autoradiography proved that the radioactivity incorporated in vivo into ovarian follicles and eggs was restricted to the forming yolk spheres or to the central ooplasm, respectively.6.6. Radioactive embryos were allowed to develop and the polypeptides appearing during development identified fluorographically on SDS gels.7.7. To establish whether these newly appearing polypeptides were due to de novo synthesis or to fragments of pre-existing vitellin polypeptides, embryos were also cultured in vitro in the presence of the above radioactive amino acids.8.8. These conditions allowed newly synthesized proteins to be identified in both native and SDS gels and to be distinguished from the vitellin polypeptides labelled in vivo.
The embryonic cytodifferentiation of Clitumnus midgut occurs very late when compared to that of other tissues in the embryo. It proceeds from hemolymph towards the yolk, first at the level of the muscular–connective tissue sheath, by the appearance of myofilaments in external–then internal–muscle fibers. In the gut epithelium, cytodifferentiation begins with the appearance of infoldings of the basal membranes of the cells. Then, microvilli and continuous junctions form at the apices of the cells. Microvilli appear in crypts, which seem to represent localized dilatations of intercellular spaces. At the level of these crypts, continuous junctions are formed somewhat later than are microvilli. This midgut differentiation coincides with deposition of the third embryonic (first larval) cuticle, and with a high titer of ecdysteroids.
The acid hydrolases of Drosophila are of maternal origin and appear subjected to differentiated control during embryogenesis. The enzymes are found associated with yolk granules. This association decreases during embryogenesis, in parallel with yolk degradation. As suggested before (Medina et al. Arch. Biochem. Biophys., 263, 355–363) the acid proteinase seems to be involved in the degradation of the yolk protein. The developmental profile of activity of the proteinase fits rather well with its involvement in the degradation of yolk granules. We have isolated intermediates of degradation of these subcellular structures. The intermediates have acid hydrolase activity and decrease in buoyant density during embryogenesis, in parallel with yolk degradation. The electron microscopic analysis has revealed that they are morphologically heterogenuous. A population of yolk granules appears to store mitochondria in their interior. The mitochondrial marker cytochrome oxidase is detected in density gradients associated with the intermediates of degradation, also supporting the storage of mitochondria in yolk granules in early development. The fact that the acid hydrolases are of maternal origin suggests that they have a role during embryogenesis. We propose that acid hydrolase(s) are involved in yolk degradation.
Developing embryos of the stick insect Carausius morosus were examined ultrastructurally with a view to studying vitellophage invasion of the yolk mass during and after germ band formation. Newly laid eggs in C.morosus have a unique yolk fluid compartment surrounded by a narrow fringe of cytoplasm comprising several small yolk granules. Vitellophages originate mainly from a thin layer of stem cells, the so-called yolk cell membrane, interposed between the germ band and the yolk mass. Throughout development, a thin basal lamina separates the yolk cell membrane from the overlying embryo.
Vitellophages extend from the yolk cell membrane with long cytoplasmic processes or filopodia to invade the central yolk mass. Along their route of entrance, filopodia engulf portions of the yolk mass and sequester it into membrane-bounded granules. As this process continues, the yolk mass is gradually partitioned into a number of yolk granules inside the vitellophages.
Later in development, the yolk cell membrane is gradually replaced by the endodermal cells that emerge from the anterior and posterior embryonic rudiments. From this stage of development onwards, vitellophages remain attached to the basal lamina through long filopodia extending between the endodermal cells. Yolk confined in different vitellophagic cells appears heterogeneous both in density and texture, suggesting that yolk degradation may be spatially differentiated.
Two vitellins of the stick insect Carausius morosus are utilized progressively during embryonic development. The relative titre of each vitellin was determined as a function of the developmental time using rocket immunoelectrophoresis with the specific anti-vitellin serum. The vitellins differ in the rate of utilization, one declining faster than the other. The pattern of vitellin in embryos differing in developmental time was demonstrated by polyacrylamide gel electrophoresis and subjected to immunoblotting using an anti-vitellin serum. Polypeptides E20, E9 and E5 that appeared de-novo during embryonic development were identified as fragments of the vitellins originally accumulated in newly laid eggs. Embryos differing in developmental stages were exposed to [35S]-methionine in vitro. Polypeptides labelled in vitro differed in molecular weight from those reactive to the anti-vitellin serum. Based on the above observations, it is concluded that vitellin degradation in C. morosus entails a stepwise degradation of vitellin polypeptides leading to the appearance of several derivatives of smaller molecular masses.
The metabolic fate of yolk proteins was followed by examining changes in subunit structure and immunochemical properties during embryogenesis in Bombyx mori. SDS-polyacrylamide gel electrophoresis of extracts from newly laid eggs showed eight major polypeptides that corresponded to the subunits of three yolk proteins, vitellin (Vtn), egg-specific protein (ESP) and the 30 kDa proteins. During embryogenesis, the peptides corresponding to Vtn and ESP decreased, and four new peptides (96, 55, 46 and 36 kDa) appeared at specific stages, but they disappeared suddenly before larval hatching. Vtn and ESP peptides stained with dyes specific for lipids (Sudan black), for sugars (Schiff's PAS reagent) and for phosphate (methyl green), but the newly appearing peptides stained only with Schiff's PAS reagent. Using a specific antiserum bound-protein A affinity column, two of the new peptides (55 and 36 kDa) were shown to be derived from ESP, and the other two peptides (96 and 46 kDa) from Vtn, suggesting that limited proteolysis of ESP and Vtn occurs during embryogenesis. In contrast, the 30 kDa proteins remained almost unchanged throughout the egg stage.
Ovarian growth in stick insects (Phasmatodea) was examined ultrastructurally and cytochemically with a view to studying: (1) the kinetics of oocyte growth and the staging characteristics of ovarian follicles undergoing vitellogenesis; (2) the endocytic capability of the growing oocyte, including the post-endocytic fate of the vitellins sequestered by the oocyte during vitellogenesis; (3) the differentiation of the follicular epithelium in relation to the appearance of intercellular spaces and the extracellular release of a follicle cell product. These structural observations were interpreted in relation to the nature and kinetics of the vitellin processing in follicles undergoing vitellogenesis.
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.
A cysteine proteinase with properties similar to mammalian cathepsins has been found in silkmoth eggs. Enzyme activity in the ovary increased abruptly 5–6 days after larval-pupal ecdysis. Activity in the mature ovary is quite high, and high activity is maintained throughout embryonic development. In a previous paper, we reported that the enzyme was synthesized in the ovary. To define the site of synthesis in more detail, follicle cells were isolated from ovaries and cultured in a medium containing 14C-amino acids. Isolated follicle cells synthesized and secreted cysteine proteinase into the medium. Immunohistochemistry also showed that the major site of synthesis is the follicle cells. Cysteine proteinase is able to degrade vitellin (Vn) and egg specific protein (ESP) in vitro in a profile similar to that found in vivo. The results are discussed in relation to the degradation mechanism of yolk proteins during early embryonic development.
The origin and development of the vitellophags are described throughout embryogenesis of the African migratory locust, Locusta migratoria migratorioides (Orthoptera : Acrididae). Primary vitellophags are formed by the intravitelline segregation of energids that remain within the yolk, while the other energids migrate toward the superficial cytoplasm. Secondary vitellophags are formed by the backward postmitotic migration into the yolk of certain blastodermal nuclei. A short time afterward, the 2 kinds of energids become indistinguishable from each other. During the first part of embryogenesis, i.e. before katatrepsis, the number of yolk nuclei increases fourfold; after early mitoses, the chromatin does not condense to form chromosomes and mitoses are never seen (the multiplication process remains unknown). The yolk nuclei, which are most numerous near the embryo, become highly hypertrophied, probably owing to an endomitotic polyploidy; their nucleoli are multiplied and their chromatin, which appears very dense in early stages, gradually disperses within the nucleoplasm. The peripheral cytoplasm spreads between yolk inclusions. These storage bodies are finally enmeshed into huge polyhedral yolk cells; this yolk cleavage begins as soon as the end of gastrulation but is completed in the anterior yolk core only before the onset of katatrepsis.During dorsal closure of the embryo, the 2 anterior-thirds of the yolk become quite disordered in relation to the serosal resorption and about one-fourth of the vitellophag population degenerates. Most of the other yolk cells migrate centrifugally and then are temporarily organized into a thick pseudo-epithelium underneath the midgut wall.
Hydrolytic activity of (BApNA) by crude egg extracts exhibited two peaks during the latter period of embryogenesis of the silkworm, Bombyx mori. The first peak appeared at the time of marked degradation of vitellin, and consisted of two proteases. The two proteases were purified from day 8 eggs to homogeneity by DEAE-cellulose, Sephadex G-75 and native-gel electrophoresis. One enzyme had a molecular mass of 30,000 Da and the other 24,000 Da. The NH2-terminal amino acid sequences were different from one another and only the 30 k-protease showed the sequence common to the trypsin family. Both enzymes showed similar properties in Km values and optimum pH, but different temperature dependence. Inhibition spectrum and substrate specificity suggest that these enzymes belong to the trypsin-like serine protease family.The purified proteases cleaved two yolk proteins, vitellin and egg-specific protein, into small peptides through limited hydrolysis, but could not attack the 30 kDa proteins which are the second major yolk proteins of silkworms. Degradation of vitellin was characterized by the preferential hydrolysis of a 178 kDa large subunit. From the biochemical properties and developmental changes, these two proteases appear to be responsible for the degradation of vitellin in eggs undergoing embryogenesis.
Cathepsin L-like proteinase found in the eggs of the tick Ornithodoros moubata is latent during embryogenesis, but can be activated by acid treatment. In crude extracts as well as in partially purified fractions, activation requires reducing conditions and is inhibited by leupeptin, which indicates that it is mediated by a thiol proteinase, probably by the cathepsin L itself. Latency disappears in vivo at the time of the acute phase of yolk digestion, which takes place during late embryonic development and larval life. When egg cathepsin L is localized through its gelatinolytic activity on SDS-PAGE, the activated enzyme migrates as lower Mr bands than the latent form. Disappearance of the higher Mr bands corresponding to the latent form is directly related to appearance of the lower Mr bands characteristic of the active one; transition from one pattern to the other and enzymatic activation are in perfect agreement with regard to kinetics and sensitivity to inhibitors. The same pattern change occurs in vivo, parallel to latency removal and intense yolk degradation. These results strongly suggest that egg cathepsin L is stored in the yolk as a proenzyme which is activated by partial proteolysis at low pH.
Fertilized eggs are the crossroads of development. They represent both the origin and the biological goal of each individual organism. Eggs provide in two ways for the embryo they enclose: They contain developmental instructions given by the mother to direct the initial phases of embryogenesis (see Chapters 11–13), and they are usually provisioned with nutritive substances to support the embryo until it can obtain its own food. This chapter concentrates on a portion of the second aspect of oogenesis—how storage materials come to reside in insect eggs.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Vitellogenin receptors (VgRs) play a critical role in egg development of oviparous animals by mediating endocytosis of the major yolk protein precursor, vitellogenin. A modification of the method for extracting the mosquito (Aedes aegypti) VgR from ovary membranes resulted in an 11-fold higher yield and 56-fold increase in relative purity of the VgR, in turn permitting purification, antibody production, and microsequencing. A Kd of 15 nM was estimated from binding assays for the enriched VgR, indicating a very high affinity for its ligand. Immunoprecipitation of [14C]VgR using anti-VgR polyclonal antibodies followed by SDS-PAGE under reducing conditions and fluorography demonstrated that the 205 kDa VgR does not consist of subunits held together with disulfide bonds. However, an immunoblot of the native VgR suggests that it exists as an approximately 390 kDa noncovalent homodimer in its native state. Immunoblot assays confirmed that the VgR is present only in ovarian tissue. A quantitative immunoassay of VgR extracts showed that VgR was present in previtellogenic ovaries on the day of emergence, increasing from 2 ng to more than 10 ng per ovary by day 5. After initiation of vitellogenesis and onset of Vg uptake, VgR quantity increased rapidly between 8 and 24 h after a blood meal, then began to decline between 24 and 36 h. Immunocytochemistry confirmed the presence of substantial amounts of the VgR in 4-day-old previtellogenic oocytes. In both previtellogenic and vitellogenic ovaries, the VgR was present only in the oocyte, primarily in the cortex.
The Science of Entomology
- W S Rosemer
- J G Stoffolano
Rosemer, W.S. and Stoffolano, J.G. 1994. The Science of
Entomology. Wm. C. Brown Communications, Inc., USA.
Limited degradation of vitellin and egg specific protein in Bornbyx mori
- L S Indrasith
- T Eurusawa
- M Shikata
- O Yamashita
Indrasith, L.S., Eurusawa, T., Shikata, M. and Yamashita, O. 1987.
Limited degradation of vitellin and egg specific protein in
Bornbyx mori. Insect Biochem., 20, 725-734.
Vitellogenesis in Insects
- Postlethwait