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Proteolytic processing ofBlattella germanica vitellin during early embryo development
Abstract
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 Mr 102,000 subunit but not the Mr 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.
... Yolk granules contain proteases that degrade their constituent proteins (5)(6)(7)(8)(9)(10). Cysteine (6,7,11,12) aspartyl (10), and serine (13,14) proteases have been identified. ...
... Previous work demonstrated that Vt utilization in Blattella germanica eggs is initiated at days 3-4 postovulation by proteolytic processing to a distinct set of peptides, which are then accessed by the embryo during development (9,15,16). Similar processing events are characteristic of Vt utilization in other insect species also (see Ref. 9, and references therein). ...
... Previous work demonstrated that Vt utilization in Blattella germanica eggs is initiated at days 3-4 postovulation by proteolytic processing to a distinct set of peptides, which are then accessed by the embryo during development (9,15,16). Similar processing events are characteristic of Vt utilization in other insect species also (see Ref. 9, and references therein). ...
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.
... To accomplish this goal, oocytes are endowed with the capability of sequestering a yolk precursor or vitellogenin (Vg) from the maternal blood stream through a process of receptor-mediated endocytosis (Raikhel 1984;Telfer and Pan 1988;Raikhel and Dhadialla 1992;Schneider 1996). The sequestered Vg is deposited in yolk granules as vitellin (Vt) (Giorgi et al. 1993a;Snigirevskaya et al. 1997aSnigirevskaya et al. , 1997b, and eventually degraded by a process of limited proteolysis to yield a number of lower molecular weight polypeptides with the onset of development (Indrasith et al. 1987;Masetti and Giorgi 1989;Nordin et al. 1990). As this process occurs, yolk granules are structurally modified and progressively reduced in size. ...
... An actual causal link between these two events is clearly indicated by the observation that yolk granule acidification in stick insect embryos coincides temporally with the onset of Vt polypeptide processing (Masetti and Giorgi 1989). In embryos of B. germanica, loss of enzyme activity has been shown to cause arrest of Vt utilisation and impairment of embryonic development (Nordin et al. 1990). ...
... It has been recurrently observed that acidified yolk granules are gradually reduced in size during yolk utilisation (Nordin et al. 1990). However, acidification studied in isolated yolk granules leads inevitably to some loss of spatial information, and this prevents a clear demonstration as to how size reduction may be achieved in vivo. ...
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.
... Concerning the importance of yolk protein for embryo growth, Nordin et al. (1990) reported that improper Vg processing in B. germanica affect embryo development. They have shown that there is a functional relation between the size of the Vg polypeptide that is processed and the presence of protease activity in the egg sacs that are laid (Nordin et al., 1990). ...
... Concerning the importance of yolk protein for embryo growth, Nordin et al. (1990) reported that improper Vg processing in B. germanica affect embryo development. They have shown that there is a functional relation between the size of the Vg polypeptide that is processed and the presence of protease activity in the egg sacs that are laid (Nordin et al., 1990). It has been proposed that Vg-programmed proteolysis during embryogenesis in insects may release maternal ecdysteroids with hormonal activity to initiate certain embryonic events (Lagueux et al., 1984;Bownes et al., 1988). ...
Vitellogenin (Vg), a yolk protein precursor, is the primary egg nutrient source involved in insect reproduction and embryo development. The Cotton Boll weevil (CBW) Anthonomus grandis Boheman, the most important cotton pest in Americas, accumulates large amounts of Vg during reproduction. However, the precise role of this protein during embryo development of this insect remains unknown. Herein, we investigated the effects of vitellogenin (AgraVg) knockdown on the egg-laying and egg viability in A. grandis females, and also characterized morphologically the unviable eggs. AgraVg transcripts were found during all developmental stages of A. grandis, with highest abundance in females. Silencing of AgraVg culminated in a significant reduction in transcript amount, around 90%. Despite this transcriptional reduction, egg-laying was not affected in dsRNA-treated females but almost 100% of the eggs lost their viability. Eggs from dsRNA-treated females showed aberrant embryos phenotype suggesting interference at different stages of embryonic development. Unlike for other insects, the AgraVg knockdown did not affect the egg-laying ability of A. grandis but only hampered A. grandis reproduction by perturbing embryo development. We concluded that the Vg protein is essential for A. grandis reproduction and a good candidate to bio-engineer resistance to this important cotton pest.
... With onset of development, Vt is gradually degraded to provide the growing embryo with an adequate food supply (Yamashita and Indrasith 1988). In recent years, a large body of evidence has clearly demonstrated that Vt undergoes a specific, developmentally regulated proteolysis during embryogenesis (Zhu et al. 1986;Masetti and Giorgi 1989;Nordin et al. 1990; Giorgi et al. 1999) and that the proteolytic enzymes required for this event are maternally derived gene products, stored in the oocyte's yolk granules as inactive, latent proproteases (Fagotto 1990;Cho et al. 1991Cho et al. , 1999Takahashi et al. 1993;Liu et al. 1997). Proteolytic cleavage of Vt is thought to be developmentally regulated by differential acidification of the yolk granules through activation of specific ion proton channels (reviews : Fagotto 1995;Takahashi et al. 1996). ...
... First, Vg may act as a potential inhibitor of premature proprotease conversion, and this may help maintain the proprotease in that form throughout vitellogenesis (Kucera and Turner 1981). Second, enzyme activation may be delayed until proprotease and Vg are fully dissociated from each other upon acidification of the yolk granules (Nordin et al. 1990). This possibility has been verified in B. germanica embryos since acidification was found to cause dissemination of proprotease to all yolk granules occupying the yolk sac of the developmentally advanced embryo (Giorgi et al. 1997). ...
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.
... Here, we have focused on cathepsin B-and L-like cysteine proteinases as possible ovarian proteinases involved in follicular atresia, simply because these two enzymes are commonly found in insects or other arthropod eggs (Yamamoto and Takahashi, 1993;Takahashi et al., 1996). For instance, cathepsin L-like proteinases are reported in the eggs of Bombyx mori (Kageyama and Takahashi, 1990;Yamamoto et al., 1994a), Blattela germanica (Liu et al., 1996), and tick Ornithodoros moubata (Fagotto, 1990a), and B-like forms in the eggs of Drosophila melanogaster (Medina et al., 1988), B. germanica (Nordin et al., 1990), Musca domestica (Ribolla et al., 1993), and Antheraea pernyi . In mosquitoes, too, intensive work on A. aegypti showed that a cathepsin B-like proteinase (vitellogenic cathepsin B; VCB), together with another ovarian proteinase, a serine carboxypeptidase (vitellogenic carboxypeptidase; VCP), are uniquely synthesized extraovarially in the fat body and then incorporated into the developing oocytes together with vitellogenin (Hays and Raikhel, 1990;Cho et al., 1991Cho et al., , 1999. ...
... Five pairs of mature ovaries were dissected out from females 5 days after feeding on half-diluted sheep blood, briefly rinsed with PBS (Sigma), and then stored in 100 l of PBS. Collected ovaries were first subjected to three cycles of freeze and thaw at Ϫ70°C and 20°C (Nordin et al., 1990), mixed with 100 l of extraction buffer (PBS, pH 7.0, 4 mM EDTA, 0.2% Triton X-100), and were homogenized with a hand homogenizer (NS-310E, Microtec Co. Ltd). To determine the optimum assay conditions for pH and preincubation period, 10 l of the total 200 l homogenate (equivalent to 1 ⁄4 of a pair of ovaries), was preincubated at 37°C for 5-40 min after being mixed with 80 l of a reaction medium composed of 0.125 M sodium formate (pH 2.5-3.5), ...
Within developing ovaries of many insects, some developing follicles or oocytes usually degenerate (follicular atresia or oosorption), while the others may continue to grow to maturity, thus maintaining the balance between the number of eggs and reproductive circumstances such as available nutrients. To help clarify the phenomenon of follicular atresia during ovarian development, we examined cysteine proteinases stored in mosquito Culex pipiens pallens ovaries. First, analysis using synthesized substrates showed that cathepsin B- and L-like proteinases gradually accumulated in the developing ovaries after a blood meal, which required more than 10 min of preincubation under acidic conditions to reach their maximum activities. However, homogenates of degenerating follicles 3 days after feeding showed proteolytic activities without acid treatment, suggesting that the proteinases had already been activated, while the extract of normally developing follicles collected from the same ovaries required more than 10 min of acid preincubation to reach the optimum activities, suggesting that the enzymes remained as inactive forms. Chemical and immunohistochemical analyses showed that more proteinases are located in the cytoplasm, rather than being associated with yolk granules. Ovarian proteinases, which are believed to become activated at the onset of embryogenesis, should also be activated during oogenesis, presumably to enhance oosorption.
... Insect embryos develop by utilizing a store of maternally derived proteins or vitellins (Nordin et al. 1990;Giorgi et al. 1999). These proteins are deposited in the oocyte during ovarian development as high molecular weight precursors or vitellogenins (Vg) by a process of receptor-mediated endocytosis (Raikhel 1984;Sappington et al. 1995;Mellman 1996;Snigirevskaya et al. 1997). ...
... cellular boundary interposed between the dorsal yolk sac and the surrounding perivitelline fluid. A number of studies have clearly demonstrated that vitellin (Vt) polypeptides of many oviparous species undergo limited proteolysis to yield a number of Vt cleavage products of lower molecular weight (Storella et al. 1985;Indrasith et al. 1987;Masetti & Giorgi 1989;Scott & Lennarz 1989;Nordin et al. 1990;Masetti et al. 1998). However, at present it is still unknown whether Vt polypeptides may be completely degraded within the yolk sac or transferred to other embryonic districts to be fully degraded (Scott et al. 1990;Cervello et al. 1994;Mayne & Robinson 1998). ...
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.
... It has been shown in a number of arthropod groups that, during embryonic development, yolk proteins are degraded by proteases to amino acids and low-molecular-weight peptides (McGregor and Loughton, 1974; Garesse et al., 1980; Ezquieta and Vallejo, 1985; Perona and Vallejo, 1985; Purcell et al., 1988; Fagotto, 1990; Nordin et al., 1990; Masetti et al., 1998). We speculate that during embryogenesis of the blue crab, proteases and lipases in vitellophages hydrolyze lipovitellin to amino acids and fatty acids, which the cuboidal cells then use to assemble LpI. ...
... Moreover , LpI does not react with antibodies to LpII (Tom et al., 1993). It has been shown in a number of arthropod groups that, during embryonic development, yolk proteins are degraded by proteases to amino acids and low-molecular-weight peptides (McGregor and Loughton, 1974; Garesse et al., 1980; Ezquieta and Vallejo, 1985; Perona and Vallejo, 1985; Purcell et al., 1988; Fagotto, 1990; Nordin et al., 1990; Masetti et al., 1998 ). We speculate that during embryogenesis of the blue crab, proteases and lipases in vitellophages hydrolyze lipovitellin to amino acids and fatty acids, which the cuboidal cells then use to assemble LpI. ...
An important lipoprotein in the hemolymph of crustaceans is LpI. It transports lipid to peripheral tissues and also has a role in crustacean immune recognition. We employed a monoclonal antibody specific for the LpI peptide to demonstrate by ELISA, western blot and immunohistochemistry the appearance of LpI during development of Callinectes sapidus, the blue crab. LpI was first found in stage 5 embryos and appeared to be synthesized by lateral basophilic cuboidal cells that demonstrated cytoplasmic immunoreactivity for LpI at their interface with the yolk mass. The embryonic cuboidal cells bore a strong cytologic resemblance to the hepatopancreas cells of later stages (zoea, megalopae, adults), which were also immunoreactive for LpI.
... Esta hipótese é corroborada por estudos realizados com diversos modelos como carrapato -O. moubata [30] e R. microplus [3], barata -Blatella germânica [74], ouriço do mar -Strongilocentrotus purpuratus e Lytechinus pictus [62] e sapo -Xenopus laevis [32,33]. Em todos eles, foi constatado que no início do desenvolvimento o pH dos grânulos de vitelo é neutro ou ligeiramente ácido e durante a embriogênese ocorre uma acidificação regulada dos grânulos, que desencadeia a degradação do vitelo. ...
Background: Ticks are distributed worldwide, with impacts on human and animal health. The cattle tick Rhipicephalus (Boophilus) microplus is the main parasite that affects livestock in tropical and subtropical regions of the world, causing large economical losses. Tick control methods are based on the application of chemical acaricides, which has resulted in selection of resistant ticks and a potential risk of environmental pollution and food contamination. Vaccines have showed to be a feasible tick control method that offers a cost-effective, environmental friendly alternative to chemical control. However, more than ten years after the commercialization of the first vaccine against ticks, the identification of tick-protective antigens remains a limiting step in the development of an efficient formulation that would avoid the use of chemical acaricides. So, the study of parasite biology and understanding physiological mechanisms could be a good strategy to find new targets for an efficient vaccine. Review: It was reviewed the main insights about the reproductive process in ticks, emphasizing the hormonal control of vitellogenesis and enzymes involved in vitellin processing during embryogenesis. The processes of vitellogenesis and embryogenesis have been studied in various organisms, particularly in cockroaches, flies and ticks. Although the roles of 20-hydroxyecdysone (20E) and juvenile hormone have been well characterized for vitellogenesis in insects, we know much less about the hormonal control of vitellogenesis in ticks. Initially, it was hypothesized that juvenile hormone was involved in tick vitellogenin-synthesis. However, more critical studies uncovered no evidence for the occurrence of juvenile hormone or juvenile hormone-like molecules in several tick species. Current research shows that in ticks, it appears that ecdysteroids, and not juvenile hormone, regulate the expression of the vitellogenin gene and the synthesis and release of vitellogenin protein into the hemolymph. In general, the carbohydrate, lipid and amino acid composition of tick vitellogenin is similar to that of insect vitellogenin. Once in the hemolymph, oocytes uptake vitellogenin through receptor-mediated endocytosys. However, there are different strategies to control vitellogenin synthesis and uptake by ovary in ixodide ticks. In the oocytes, vitellogenin is partially processed in the endosomal compartment and then stored as vitellin, the main reserve of protein for embryo development, in specialized organelles, the yolk granules. Embryo development depends on the availability of yolk material stored into oocytes. So, the characterization of molecules involved in vitellogenesis and embryo development contribute to a better understanding of the tick parasite physiology. During embryogesesis, acidic enzymes are responsible for the availability of this material and embryo nutrition. The Vitellin-Degrading Cysteine Endopeptidase (VTDCE), Boophilus Yolk Pro-Cathepsin (BYC) and Tick Heme Binding Aspartic Proteinase (THAP) are enzymes involved in vitellin hydrolysis in R. microplus eggs. These enzymes are produced by gut and fat body and transported through the hemolymph to be internalized into the oocytes and then play their role in tick embryo nutrition. As VTDCE, BYC and THAP are involved in an important physiological process, their potential as targets in an anti-tick vaccine is an attractive research topic. With this objective, various enzymes have been tested in native or recombinant forms as candidate immunogens to a multiantigenic anti-tick vaccine. Conclusion: Significant advancements have been made in recent years on understanding the tick reproductive process, and some molecules that can be possible targets for development of new tick control strategies have been characterized.
... Nusbaum (1886) observed numerous large energids within the cockroach yolk mass, and supposed that these vitellophages digested the stored nutrients. Despite this early observation, the functional significance of these energids during cockroach embryogenesis has only relatively recently been clarified (Storella et al, 1985;Purcell et al, 1988a, b;Nordin et al, 1990Nordin et al, , 1991. None of these workers, however, paid sufficient attention to the fact that symbiotic bacteria are also present in the cockroach embryos and that these are involved in the different phases of embryo development (Büchner, 1965), and that in the terminal stages of ontogenesis they may be found in the bacteriocytes of the fat body. ...
In this paper we describe the behaviour of the symbiotic bacteria of Blattella germanica and Periplaneta americana during embryo development using transmission and scanning electron microscopy. In Blattella germanica, the bacteria are transferred by endocytosis to the egg cell and are sited in its peripheral cytoplasm; thence they begin to internalize in the yolk, using cytoskeletic structures which are probably only synthesized at this particular phase of their migration. In the 6–7‐day‐old embryo, the bacteria are in close contact with the vitellophages scattered in the yolk, where some of them appear to be in a degenerative phase. In Periplaneta americana, after the internalization of the bacteria, there is the formation of the mycetome which appears to be made up of a syncytial envelope, probably formed by the vitellophages, that contains a ball of symbionts of considerable number. During development, there is a progressive decline in the bacterial population, caused by complex lytic processes which take place inside the mycetome. In embryos aged more than 17 days, the mycetome tends to regress, while there are many bacteriocytes present. A hypothesis for interpreting this phenomenon is that the embryo may use the bacteria as nutrients. It cannot, however, be excluded that the formation of the mycetome may be the expression of a cellular immune process.
... In the last decade, a number of studies have examined yolk metabolism in relation to the presence of endopeptidase in insect eggs and embryos (Perona & Vallejo, 1985;Fagotto 1990a). It is well known by now that acid hydrolases play a major role in yolk degradation during embryonic development of such diverse insect species as Drosophila melanogaster (Medina et al., 1988), Artemia saliņa (Perona & Vallejo, 1985), Blattella germanica (Nordin et al., 1990), Musca domestica (Ribolla et al., 1993) 1990). Being of maternal origin, all these enzymes are compartmentalized inside the yolk granules of the newly laid eggs (Medina & Vallejo, 1989). ...
Two distinct proteolytic activities were demonstrated in developing ovarian follicles of the stick insect Carausius morosus using aldolase as a substrate. One has an optimum activity around pH 5 and it is strongly inhibited by pepstatin A. Because of these properties, it is classifiable as an aspartic proteinase. The second one has an alkaline pH optimum and it is inhibited by aprotinin. Based on these properties it is classifiable as a serine, trypsin‐like, proteinase. This latter activity has also been visualized as a negatively stained protein fraction of about 26 kD on polyacrylamide gels containing casein as substrate. While the aspartic proteinase is predominantly associated with follicles approaching chorionogenesis, the serine proteinase is restricted to immature follicles and disappears prior to completion of ovarian development.
... Esta hipótese é corroborada por estudos realizados com diversos modelos como carrapato -O. moubata [30] e R. microplus [3], barata -Blatella germânica [74], ouriço do mar -Strongilocentrotus purpuratus e Lytechinus pictus [62] e sapo -Xenopus laevis [32,33]. Em todos eles, foi constatado que no início do desenvolvimento o pH dos grânulos de vitelo é neutro ou ligeiramente ácido e durante a embriogênese ocorre uma acidificação regulada dos grânulos, que desencadeia a degradação do vitelo. ...
Background: Ticks are distributed worldwide, with impacts on human and animal health. The cattle tick Rhipicephalus (Boophilus) microplus is the main parasite that affects livestock in tropical and subtropical regions of the world, causing large economical losses. Tick control methods are based on the application of chemical acaricides, which has resulted in selection of resistant ticks and a potential risk of environmental pollution and food contamination. Vaccines have showed to be a feasible tick control method that offers a cost-effective, environmental friendly alternative to chemical control. However, more than ten years after the commercialization of the first vaccine against ticks, the identification of tick-protective antigens remains a limiting step in the development of an efficient formulation that would avoid the use of chemical acaricides. So, the study of parasite biology and understanding physiological mechanisms could be a good strategy to find new targets for an efficient vaccine. Review: It was reviewed the main insights about the reproductive process in ticks, emphasizing the hormonal control of vitellogenesis and enzymes involved in vitellin processing during embryogenesis. The processes of vitellogenesis and embryogenesis have been studied in various organisms, particularly in cockroaches, flies and ticks. Although the roles of 20-hydroxyecdysone (20E) and juvenile hormone have been well characterized for vitellogenesis in insects, we know much less about the hormonal control of vitellogenesis in ticks. Initially, it was hypothesized that juvenile hormone was involved in tick vitellogenin-synthesis. However, more critical studies uncovered no evidence for the occurrence of juvenile hormone or juvenile hormone-like molecules in several tick species. Current research shows that in ticks, it appears that ecdysteroids, and not juvenile hormone, regulate the expression of the vitellogenin gene and the synthesis and release of vitellogenin protein into the hemolymph. In general, the carbohydrate, lipid and amino acid composition of tick vitellogenin is similar to that of insect vitellogenin. Once in the hemolymph, oocytes uptake vitellogenin through receptor-mediated endocytosys. However, there are different strategies to control vitellogenin synthesis and uptake by ovary in ixodide ticks. In the oocytes, vitellogenin is partially processed in the endosomal compartment and then stored as vitellin, the main reserve of protein for embryo development, in specialized organelles, the yolk granules. Embryo development depends on the availability of yolk material stored into oocytes. So, the characterization of molecules involved in vitellogenesis and embryo development contribute to a better understanding of the tick parasite physiology. During embryogesesis, acidic enzymes are responsible for the availability of this material and embryo nutrition. The Vitellin-Degrading Cysteine Endopeptidase (VTDCE), Boophilus Yolk Pro-Cathepsin (BYC) and Tick Heme Binding Aspartic Proteinase (THAP) are enzymes involved in vitellin hydrolysis in R. microplus eggs. These enzymes are produced by gut and fat body and transported through the hemolymph to be internalized into the oocytes and then play their role in tick embryo nutrition. As VTDCE, BYC and THAP are involved in an important physiological process, their potential as targets in an anti-tick vaccine is an attractive research topic. With this objective, various enzymes have been tested in native or recombinant forms as candidate immunogens to a multiantigenic anti-tick vaccine. Conclusion: Significant advancements have been made in recent years on understanding the tick reproductive process, and some molecules that can be possible targets for development of new tick control strategies have been characterized.
... In free living insects, embryo develops by consuming the maternally derived nutrients accumulated in vitellins [21], [22]. These reserves of nutrients, currently named yolk, are deposited in oocytes during the vitellogenesis. ...
Aphidius ervi (Hymenoptera: Braconidae) is an entomophagous parasitoid known to be an effective parasitoid of several aphid species of economic importance. A reduction of its production cost during mass rearing for inundative release is needed to improve its use in biological control of pests. In these contexts, a careful analysis of its entire development phases within its host is needed. This paper shows that this parasitoid has some characteristics in its embryological development rather complex and different from most other reported insects, which can be phylogenetically very close. First, its yolkless egg allows a high fecundity of the female but force them to hatch from the egg shell rapidly to the host hemocoel. An early cellularisation allowing a rapid differentiation of a serosa membrane seems to confirm this hypothesis. The serosa wraps the developing embryo until the first instar larva stage and invades the host tissues by microvilli projections and form a placenta like structure able to divert host resources and allowing nutrition and respiration of embryo. Such interspecific invasion, at the cellular level, recalls mammal's trophoblasts that anchors maternal uterine wall and underlines the high adaptation of A. ervi to develop in the host body.
... First, Vg may act as a potential inhibitor, maintaining the enzyme as a zymogen throughout vitellogenesis, as suggested in studies on the moth, Hemileuca oliviae and the cockroach, Blattella germanica (Kucera and Turner, 1981; Yin et al. 2001). Second, enzyme activation may be delayed until pro-protease and Vg are fully dissociated from each other due to acidification of the yolk granules (Nordin et al. 1990). Finally, a physiological inhibitor may be part of this complex, acting to control enzyme activity (Kucera and Turner, 1981). ...
The tick Rhipicephalus (Boophilus) microplus is an important parasite of cattle in many areas of the tropics. Characterization of molecules involved in mechanisms such as vitellogenesis and embryo development may contribute to a better understanding of this parasite's physiology. The vitellin-degrading cysteine endopeptidase (VTDCE) is the most active enzyme involved in vitellin hydrolysis in R. microplus eggs. Here we show an association between VTDCE and vitellin in an additional site, apart from the active site. Our data also demonstrate cysteine endopeptidase activity in different tissues such as ovary, gut, fat body, salivary gland and female haemolymph, where it is controlled by a physiological inhibitor. In R. microplus female gut, VTDCE is localized in areas of protein synthesis and trafficking with the underlying haemolymph. VTDCE is also localized in the ovary basal region, in vesicle membranes of ovary pedicel cells and in oocyte cytosol. These results suggest that VTDCE plays a role in vitellin digestion during tick development.
... Upon internalization by clathrin-coated vesicles, Vg is conveyed to the yolk granules and post-endocytically processed to yield a stable form of vitellin (Vt) (Raikhel and Dhadialla, 1992;Giorgi et al., 1997a). With the onset of embryonic development, Vt undergoes a process of proteolysis triggered by activation of latent pro-proteases (Fagotto, 1990b;Nordin et al., 1990). These pro-enzymes have been shown to be maternally derived gene products that are transferred endocytically to the oocyte during vitellogenesis (Fagotto, 1990a;Cho et al., 1991a;Giorgi et al., 1997b;Liu and Nordin, 1998;Cho et al., 1999). ...
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.
... In most non-mammalian oviparous organisms, one of the early events during embryo development is the initiation of proteolytic processing of VT. The regulation of this process is still unclear but a growing body of evidence (Liu and Nordin, 1998;Nordin et al., 1990;Fagotto, 1990;Mallya et al., 1992;Fagotto and Maxfield, 1994) indicates that pH may be one of the regulators of yolk degradation. Furthermore, granule restructuring is also linked with VT utilization. ...
Acid phosphatase activity, previously identified in Rhodnius prolixus oocytes, was studied during egg development. Fertilized eggs exhibited a five fold increase of total acid phosphatase activity during the first days of development. In contrast non-fertilized oviposited eggs showed no activation of this enzyme. An optimum pH of 4.0 for pNPP hydrolysis in a saturable linear reaction and a strong inhibition by lysosomal acid phosphatase inhibitors such as NaF (10 mM) and Na(+)/K(+) tartrate (0.5 mM) are the major biochemical properties of this enzyme. Fractionation of egg homogenates through gel filtration chromatography revealed a single peak of activity with a molecular mass of 94 kDa. The role of this enzyme in VT dephosphorylation was next evaluated. Western blots probed with anti-phosphoserine polyclonal antibody demonstrated that VT phosphoaminoacid content decreases during egg development. In vivo dephosphorylation during egg development was confirmed by following the removal of (32)P from (32)P-VT in metabolically labeled eggs. Vitellin was the only phosphorylated molecule able to inhibit pNPPase activity of partially purified acid phosphatase. These data indicate that acid phosphatase activation follows oocyte fertilization and this enzyme seems to be involved in VT dephosphorylation.
... To this end, they are partitioned in endosome-like organelles, the yolk granules (Opresko et al., 1980;Giorgi et al., 1999), by the ability of the growing oocyte to undergo receptor mediated endocytosis (Raikhel and Dhadialla, 1992;Sappington et al., 1995;Snigirevskaya et al., 1997). Due to their endocytic origin via membrane-bound organelles, vitellin polypeptides have long assumed to be degraded proteolytically along the lysosomal pathway Nordin et al., 1990). In line with this assumption, several lysosomal, maternally encoded, pro-proteases were found in developing embryos Cho et al., 1991a,b;Takahashi et al., 1993;Cho et al., 1999) and shown to be associated with yolk granules together with vitellin polypeptides (Takahashi et al., 1996;Giorgi et al., 1997). ...
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.
... This finding implied that removal of covalently bound phosphate groups is a part of a programmed pathway for appropriate embryo utilization of yolk reserves. Such biochemical pathway appears to be unique to R. prolixus, once B. germanica developing eggs, where the same phenomenon was investigated, do not present such alterations (Nordin et al. 1990). ...
In triatomines, as well as in other insects, accumulation of yolk is a process in which an extra-ovarian tissue, the fat body, produces yolk proteins that are packed in the egg. The main protein, synthesized by the fat body, which is accumulated inside the oocyte, is vitellogenin. This process is also known as vitellogenesis. There are growing evidences in triatomines that besides fat body the ovary also produces yolk proteins. The way these yolk proteins enter the oocyte will be discussed. Yolk is a complex material composed of proteins, lipids, carbohydrates and other minor components which are packed inside the oocyte in an organized manner. Fertilization triggers embryogenesis, a process where an embryo will develop. During embryogenesis the yolk will be used for the construction of a new individual, the first instar nymph. The challenge for the next decade is to understand how and where these egg proteins are used up together with their non-protein components, in pace with the genetic program of the embryo, which enables cell differentiation (early phase of embryogenesis) and embryo differentiation (late phase) inside the egg.
... The primary constituents of crustacean yolk are lipovitellins and lipid droplets. During embryogenesis, proteases are activated which are important in the degradation of lipovitellin to amino acids and peptides (Chaffoy de Courcelles and Kondo, 1980;Ezquieta and Vallejo, 1985;Warner and Shridar, 1985;Medina et al., 1988;Nordin et al., 1990;Yamamoto et al., 1994). Also active are lipases that hydrolyze the lipids derived from lipovitellin and lipid droplets (Subramoniam, 1991). ...
Embryos of the blue crab Callinectes sapidus develop in egg sacs carried on the abdomen of the female. They develop over a period of 10-13 days at 28 degrees C and are nutritionally dependent on yolk until they emerge from the egg sacs as free-swimming zoeae. The principal component of blue crab yolk is lipovitellin (LpII), a water-soluble lipoprotein composed of approximately equal amounts of lipid and protein. We followed changes in the concentration of apoproteins of LpII during embryogenesis by ELISA and Western blots, using monoclonal antibodies against two LpII apoprotein associated peptides identified as Protein A (107 kDa) and Protein B (75 kDa). During embryogenesis there was a decrease in Protein B but an increase in two smaller peptides (52 and 35 kDa) that reacted with the Protein B antibody. Utilization of LpII during embryogenesis was also followed morphologically by immunohistochemistry. Utilization of LpII was slow in early embryonic stages, followed by rapid utilization in late embryonic stages, such that only traces of LpII were present at the end of embryogenesis. The cells of the developing hepatopancreas appear to play an important role in the utilization of LpII.
LDL isolated from eggs of the sand crayfish Ibacus ciliatus had proteins of 8 to 15 kDa with proteinase activity, which was detected by casein SDS-PAGE, and digested native vitellogenin of sand crayfish. Ca²⁺ was essential for the proteinase activity of LDL and its activity was inhibited by EDTA. Addition of the digested vigellogenin to the native vitellogenin stimulated the proteolysis of the vitellogenin. It was revealed by casein SDS-PAGE that the vitellogenin digested by LDL had a protein of 100 kDa with proteinase activity, but native vitellogenin itself had no proteinase activity. These results indicate that the native vitellogenin has proteinase activity as a latent form and the proteinase activity appears after digestion of the vitellogenin by LDL. The pattern of apoliporoteins of the proteolyzed vitellogenin on a gel after SDS-PAGE was very similar to that of lipovitellin of eggs of sand crayfish. These results suggest that the LDL and the proteolyzed vitellogenin take part in the proteolytic processing of the vitellogenin into yolk proteins.
Oocytes and embryos of the cockroach Blattella germanica were examined by optical and electron microscopy to study yolk granule degradation during embryo development. During vitellogenesis, progressively larger yolk granules are formed in the ooplasm and by chorionogenesis, the mature granules are packed so tightly that their shape is highly distorted. Throughout ovarian development, endosymbiotic bacteria lie at the follicle cell/oocyte interface. Just prior to chorionogenesis the endosymbionts transit the oocyte plasma membrane and cluster at the periphery. Bacteria become more numerous over the ventral region of the egg by day 1 postovulation and begin to invade the interior of the yolk mass from the ventral periphery. At that time, lysis of the nearby yolk granules occurs while those in the central ooplasm remain intact and free of bacteria up to day 4. Vitellophages become evident by day 2 postovulation. These cells are also distributed over the egg's periphery but are most numerous in the ventral region. Vitellophages, in association with the endosymbionts, protrude toward the yolk granules and extend filo- and lamellipodia over the granule surface. Portions of the yolk granules are then engulfed and sequestered as large vacuoles in the vitellophage's cytoplasm. The vacuoles then become vesiculated. As embryo development proceeds, the vesiculated portions partition into smaller multivesicular bodies. This study describes the dynamics of yolk granule-vitellophage interaction in embryos of B. germanica and suggests that yolk utilization entails the cooperative efforts of both vitellophages and endosymbiont bacteria.
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.
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.
Yolk proteins (YP1, YP2, and YP3) of the fall webworm, Hyphantria cunea, are of relatively low molecular weight. Yolk protein-2 (YP2) was purified from gel slices and by KBr density gradient ultracentrifugation followed by ion exchange chromatography. YP2 is composed of one subunit with a molecular weight of 35.5 kDa. YP2 contains neutral lipids (diacylglycerol and triacylglycerol) and phospholipids (phosphatidylcholine and phosphatidylethanolamine). The neutral lipids are largely composed of lauric acid and palmitoleic acid. YP2 contains relatively large amounts of glutamic acid and aspartic acid but small amounts of tyrosine, phenylalanine, and methionine. YP2 is a vitellin (Vn) synthesized by the fat body. Vitellogenin-2 (Vg2), the precursor of YP2, is present in very small amounts in the hemolymph. Lipophorin and storage protein also are found in the ovary of H. cunea, and these proteins do not immunologically cross-react with YP2. YP2 is detected in first instar larvae but completely disappears during the second instar, indicating that YP2 is intensively utilized during postembryonic development. Anti-YP2 antibodies cross-react with ovarial extracts of Bombyx mori but not with those of insects from other orders such as Cletus schmidti (Hemiptera), Lucilia illustris (Diptera), Anechura japonica (Dermaptera), Periplaneta americana (Dictyoptera), and Ducetia japonica (Orthoptera). © 1995 Wiley-Liss, Inc.
Newly laid eggs of the stick insect Carausius morosus contain two native vitellins (Vit A and Vit B). Under denaturing conditions, these vitellins resolved into 3 (A1, A2, and A3) and 2 (B1 and B2) polypeptides. All of these polypeptides had counterparts in the female hemolymph from which they were shown to be derived by in vivo labelling. During ovarian development, the 2 vitellins changed both in charge and polypeptide composition. In EV and LV follicles, Vit A resolved into 4 distinct vitellin polypeptides (A0, A1, A2 and A3). Using a panel of monoclonal antibodies, polypeptide A0 proved to be immunologically related to polypeptide A2. In follicles about to begin choriongenesis, polypeptide A3 was gradually replaced by a lower Mr polypeptide. Over the same time period, polypeptide B1 changed in charge, but not in Mr. To confirm the existence of a polypeptide processing in C. morosus, ovarian follicles of different developmental stages were exposed in vivo to [35S]-methionine from 6 to 72 h. Data showed that A0 and B1 were the polypeptides most heavily labelled after short time exposures to the radioisotope. Polypeptides B2 and A3 were also labelled to some extent. With progressively longer exposures, polypeptides A1 and A2 also became labelled. In vivo exposure to [3H]-GlcNAc caused all vitellin polypeptides to become heavily labelled. Autoradiographic analysis of ovarian follicles labelled this way showed that, during development, radioactivity was gradually transferred from newly formed yolk spheres in the cortical ooplasm to the central ooplasm. Data were interpreted as suggesting a causal relationship between polypeptide processing and progressive yolk sphere fusion to yield the central ooplasm. © 1993 Wiley-Liss, Inc.
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.
Oöplasmic pH and the electrical potential difference across the egg coverings (PDegg) were measured in developing locust (Locusta migratoria) eggs with double-barrelled pH microelectrodes. Successful microelectrode impalements were obtained up to the ninth day of development, the day before hatching at 37°C. PDegg for eggs bathed in control saline changed substantially during development, from a minimum of −23 mV in day-1 eggs to a maximum of −78 mV in day-3 eggs, whereas oöplasmic pH remained near 7.2–7.3 before fertilization and up to day 5. Dechorionation reduced PDegg in day-3 eggs by only 5 mV. Input resistance decreased from 1.2 MΩ in chorionated eggs before fertilization to less than 0.2 MΩ in day-1 to day-5 eggs. PDegg changed by up to 19 mV for a 10-fold change in bathing saline Cl− concentration, but less than 5 mV for a 10-fold change in the concentration of Na+ or K+. Experiments with internally perfused eggs indicated that the Cl−-dependent component of PDegg is developed primarily across the serosal cuticle, and that up to 28% of PDegg consists of a second potential developed across the serosal epithelium. Contribution of a metabolic pump to the latter component was suggested by the effects of chilling or anoxia on PDegg in intact eggs. Acidification of egg oöplasm by 0.2 pH units in hypercapnic bathing saline (7% carbon dioxide) was associated with a 66% hyperpolarization of PDegg. The results suggest that oöplasmic pH increases as ambient pCO2 declines during oviposition, and that an electrogenic proton pump contributes to both PDegg and oöplasmic pH regulation.
1.1. Two proteinases have been identified in yolk granules of Nereis diversicolor mature oocytes, an aminopeptidase and an acid cysteine proteinase.2.2. The aminopeptidase was identified as a metallo-enzyme having a molecular weight of about 260 kDa.3.3. Except that the acid cysteine proteinase is a high molecular weight protein (200 kDa) and has a very low pH optimum (3.0), the enzyme possesses properties resembling those of mammalian cathepsin L.4.4. The cathepsin L-like proteinase was found to be liable to the in vitro proteolysis of the yolk granule proteins and is therefore suggested to be involved in yolk protein processing.
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.
Low-density lipoprotein (LDL) with large amounts of phospholipid but not triacylglycerol was isolated from the egg yolk of crustacea sand crayfish Ibacus ciliatus as well as lipovitellin. LDL possessed vitellogenin-degrading proteinase activity. Hemolymph vitellogenin was degraded by incubating with LDL at pH 4.5 for 72 hr at 35 degrees C and apolipoprotein profiles of vitellogenin degraded by LDL were very similar to those of lipovitellin in the egg.
Two vitellins (Vn-1 and Vn-2) were identified in cockroach ovary. Vn-2 was purified by ion-exchange chromatography using DEAE cellulose and Sepharose CL-6B, and gel filtration using Sephadex G-200 and Sepharose 4B. Vn-2 comprises three subunits with molecular weights of 102, 72 and 40 kDa. Vn-2 contains diacylglycerol and triacylglycerol; their fatty acids are mostly lauric acid and palmitoleic acid. Vn-2 also has phosphatidylcholine and phosphatidylethanolamine but no sphingomyelin. Vn-2 contains large amounts of glutamic acid and aspartic acid but small amounts of tyrosine, phenylalanine, and methionine. Vn-2 gradually decreases during embryonic development; none remains in first instar larvae. It was found that Vn-2 partially reacted immunologically with ovarian extracts of Periplaneta fulginosa but not with those of Blattella germanica. Vn-2 also showed no immunological reaction with extracts from other orders; including Cletus schmidti (Hemiptera), Lucilia illustris (Diptera), Anechura japonica (Dermaptera), Bombyx mori (Lepidoptera), and Ducetia japonica (Orthoptera).
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.
Changes in polypeptides pattern of haemolymph, midgut, ovary and salivary glands of female mosquito A. stephensi were studied when fed upon anti-mosquito haemolymph antibodies. The expression of almost all polypeptides was reduced in haemolymph and ovary of the immune fed mosquitoes as compared to control. However, there was no significant difference in case of midgut and salivary glands. Seven polypeptides 100, 90, 84, 80, 62, 19 and 12.5 kDa were absent in haemolymph and five 92, 90, 80, 60 and 55 kDa were absent in ovaries. Changes in the polypeptide pattern have been correlated with the fecundity reduction due to immunized blood feeding.
Multiple vitellogenins (VG) are found in species from all major families of cockroaches. Proof that observed multiple VGs are actually distinct proteins and not artifacts of differential processing requires careful examination. In Periplaneta americana two immunologically and electrophoretically distinct vitellins (VTs) of similar native size exist in the egg. VT1 is composed of four major peptides of molecular weights 170, 105, 92 and 78 K. VT2 is composed of three major peptides of molecular weights 105, 101 and 60 K. A peptide with molecular weight of about 105 K is found in both VG1 and VG2 and a similar sized peptide is also conserved in the VGs of all other Blatinae subfamily members examined despite the distant immunological relation of these proteins. The 170 K peptide of VT1 is likely to be a processing intermediate of the 92 K and the 78 K peptides. Each VT from a freshly ovulated egg is immunologically identical to and has essentially the same peptide substructure as its serum precursor. The cumulative of the constituent peptides of each VG is approx. 260 K, consistent with 17S native dimeric molecules of approx. 520 K . A specific and limited cleavage of VT major peptides occurs during early embryogenesis (at 30°C, 9 days after ovulation) resulting in a partial loss of immunological determinants. During subsequent yolk utilization the VTs undergo gradual degradation.
To determine whether a low pH intracellular "sorting" step is required to route peptides into secretory granules, the effects of pH altering drugs on the biosynthesis and secretion of peptides by AtT-20 mouse corticotrope tumor cells and rat intermediate pituitary cells were examined. Doses of each drug maintaining normal protein synthesis and cell morphology, while obliterating the intracellular pH gradients detected by acridine orange fluorescence, were experimentally determined. Regions of the cell rich in secretory granules were localized by immunocytochemistry and were found to coincide with organelles with a low internal pH. Biosynthetic labeling experiments were coupled with immunoprecipitation and sodium dodecyl sulfate polyacrylamide gel analyses to examine the biosynthesis and secretion of corticotropin (ACTH(1-39], alpha-melanotropin, ACTH(18-39), beta-endorphin, gamma-melanotropin, alpha-amidated joining peptide, and the NH2-terminal region of pro-ACTH/endorphin. Chloroquine (20-40 microM) and a mixture of NH4Cl and methylamine (2-5 mM each) dissipated pH gradients but had no effect on the synthetic rate of pro-ACTH/endorphin, the extent and rate of precursor processing to smaller peptides, the rate of basal secretion of the various peptides, or the extent to which secretion of each of the peptides could be stimulated by secretagogues. Monensin (0.1-1 microM) had no discernible effect on intracellular pH gradients yet totally blocked proteolytic processing of pro-ACTH/endorphin. Thus, a monensin-blockable step occurs in peptide processing, presumably in the trans Golgi region; however, a low pH chloroquine-sensitive sorting step is not required for processing or for routing peptides to a stable storage form which can be released in response to secretagogues.
That the yolk proteins (YPs), or vitellins, stored in the oocytes of insects are a nutritional store for subsequent embryogenesis has long been assumed. Exhaustive data base searching programs revealed highly significant sequence similarity between the three YPs of Drosophila melanogaster and part of the triacylglycerol lipase of the domestic pig. Based upon time of degradation of YPs during embryogenesis, existence of maternally stored ecdysteroid conjugates in embryos, location of these conjugates in locust embryos, and the fact that free active ecdysteroid hormones are released at a specific time in embryogenesis to trigger cuticle deposition, we postulate that the similarity reflects a common property of Drosophila YPs--the ability to bind the fatty acid ecdysteroid conjugates. Our finding of conjugated ecdysteroids tightly bound to purified Drosophila YP supports this prediction.
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.
Cell adhesion in the sea urchin blastula is mediated by a 22S genus-specific glycoprotein complex consisting initially of six 160-kDa subunits that are processed proteolytically as development proceeds. Noncytolytic removal of the 22S particle from the surface with either 2.5% butanol or trypsin renders dissociated cells reaggregation incompetent, and addition restores reaggregation and development. Polyclonal antibodies against the 22S complex prevent reaggregation in a genus-specific manner while monoclonal antibodies stain cell surface structures in a pattern consistent with a code that specifies the position of a cell in the embryo by a unique combination of subunits in its cell adhesion particles. The existence of similar particles in Drosophila and amphibian embryos suggests that these glycoprotein complexes are a general class of organelles, the toposomes, that in the embryo mediate cell adhesion and express positional information.
This volume was first published in 1980. Coated vesicles are distinct organelles whose existence has been recognized for many years. Since the original observations of the invaginations of erythroblast and oocyte membranes, coated vesicles have been found associated with a wide variety of membranes and they have been seen to be of importance in the uptake and transport of peptides, proteins and lipoproteins as well as in some cases as mediators of immune function. This book is a comprehensive treatment of coated vesicles and their involvement in the process of endocytosis (the engulfment of material in membranous enclosures).
Various aspects of the processing of Blattella germanica vitellin (Vt) in the oocyte and egg have been investigated. Employing subunit specific antibodies, the precursor product relationships among the subunits of this Vt have been determined. After endocytosis of Vt by the oocyte, the Mr 160,000 subunit Vt is cleaved to products of Mr 95,000 and Mr 50,000. In association with an unprocessed Mr 102,000 peptide, these form the subunits of the Vt of freshly ovulated eggs. Between 4 and 5 days post ovulation (at 30°C), all three subunits of Vt are again processed proteolytically before use by the embryo. Although Vt's high mannose-type oligosaccharides are trimmed during embryogenesis, their modification occurs subsequent to the day 4–5 proteolysis, precluding the possibility that changes in oligosaccharide content or structure contribute to regulating this second proteolytic event. Although the predominant oligosaccharide of Vt is Man9GlCNAc2, the Mr 50,000 subunit of egg-borne Vt contains a much higher proportion of Man6GlCNAc2 than the other two subunits; therefore, this portion of the precursor vitellogenin must be more accessible to the processing mannosidases of the endoplasmic reticulum during its biosynthesis. A microtechnique for aspirating the yolk from individual eggs in an oothecapermits its isolation free of contamination by embryonic tissue. With this procedure, the specific activity profiles of exo-α-mannosidase, exp-β-N-acetylglucosaminidase, α-glucosidase and acid phosphatase were monitored during the first 6 days after ovulation, and some of their properties were also determined. Expression of the acid phosphatase and exo-β-N-acetyl-glucosaminidase activities coincide with the day 4–5 proteolysis, while α-mannosidase remains relatively constant throughout the first 6 days. Functions for these enzymes and the oligosaccharides of Vt during Vt storage and utilization are proposed.
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.
Vitellin was isolated from öocytes of Leucophaea maderae and fractionated into 14S (subunit structure ABC2D2) and 28S (subunit structure AB3C2) complexes. Chromatography on DEAE-cellulose in 8 M urea partially separated the subunits from each other and provided evidence for two kinds of B subunit in 28S Vt. Amino acid analysis of each subunit purified by SDS gel electrophoresis showed significant differences in the contents of a few amino acids. The leucine, lysine and valine content of A14 and A28 differed from the other subunits and the alanine content of C14 and C28 was less than half of the others. B14, D14 and B28 had similar amino acid compositions. Using antisera prepared against each subunit, a strong immunologic identity among the B14, D14 and B28 subunits was found, with weak cross-reaction to A14 or A28. A14 appeared identical to A28 and the C14 and C28 were identical to each other, but the C subunits did not cross react with any other subunit. Trypsin treatment of native 14S and 28S complexes showed a high susceptibility of subunit A to this enzyme. In contrast, all subunits were digested at similar rates by chymotrypsin, papain and subtilisin.
A serine proteinase (molecular mass, about 25,000 Da) has been found in Drosophila mature oocytes. (1) The detected activity increases exponentially during embryogenesis. (2) The subcellular localization changes from the yolk granules, in oocytes, to the soluble fraction, in late embryos. (3) The proteinase cleaves by basic residues, but arginine is preferred (about 7-fold) over lysine.
Publisher Summary This chapter describes two types of purification methods for Cathepsin B, Cathepsin H, and Cathepsin L. Method I is applicable to large amounts of frozen tissues, whereas method II is used with flesh tissue and takes advantage of a 50-fold purification factor attainable by isolation of lysosomes: it has the further advantage of separating the enzymes from inhibitors that are present in the cytosol and plasma. In first purification method, cathepsins B and H are purified from human liver. Method II involves purification of Cathepsins B, H, and L from rat liver. Method I include: extraction, autolysis, and acetone fractionation and DEAE-cellulose chromatography. The pool of cathepsin B from DEAE-cellulose is further purified by covalent chromatography on a column of aminophenylmercuric acetate coupled to Sepharose. Method II include: homogenization and cell fractionation gel; chromatography on Sephadex G-75; CM-Sephadex chromatography; chromatography of cathepsin L on concanavalin A-Sepharose. Cathepsin B can be with BZ-DL-Arg-NPhNO 2 or Bz-Arg-2-NNap as substrate, wheras, Cathepsin H can be assayed selectively by use of an unblocked substrate such as Leu-NNap, Arg-NNap, or Arg-NMec. Three synthetic substrates have been used for cathepsin L assay: Bz-Arg-NH 2 , Z-Lys-OPhNO 2 , and Z-Phe-Arg-NMec.
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.
The fate of vitellin (VT), the major yolk protein in Rhodnius prolixus, was studied during embryogenesis by electrophoretic and immunological techniques.Analysis of the protein content of the eggs by polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions shows a continuous alteration in the VT molecule following oviposition. Two distinct phases of VT processing can be observed: (1) when limited proteolysis is dominant; and (2) when an extensive degradation of VT occurs inside the digestive system of the embryo.In the first phase (days 0–10 after oviposition), as embryogenesis proceeds, the two principal bands of VT give rise to several bands that migrate progressively further into the gel. Analysis of the same samples by SDS-PAGE reveals a continuous proteolysis of the subunits of the VT molecule. When the VT is analysed by rocket immunoelectrophoresis very little alteration in the total VT content per egg is observed from day 0 to day 10, suggesting that the antibodies used also recognize polypeptides produced by partial proteolysis. Western blot analysis demonstrated that some of the new polypeptides (112, 103, 64 and 56 kDa) generated during embryonic development are in fact derived from VT by limited proteolysis. After cleavage, the polypeptides remain in the same lipoprotein macromolecular complex.In the second phase of VT degradation, an extensive degradation of VT takes place, such that by day 14 after oviposition (hatching), VT content has declined to about half of the initial value. The VT remaining in the first instar is located entirely in the crop and disappears within 5 days after hatching.
1.
The amino acid composition and subunit structure of locust vitellogenin have been investigated. Glycine was the predominant amino acid of the vitellogenin
2.
By combining previous data on the free amino acids of the developing egg and on the degradation of the newly characterized vitellogenin, the daily metabolism of amino acids could be calculated.
3.
Most amino acids were metabolized extensively at the beginning of embryogenesis. During blastokinesis metabolism slowed then speeded up with the approach of hatching.
4.
Serine, glycine and tyrosine demonstrated metabolic activity at variance with this general trend. Serine was the most abundant free amino acid. Presumably much of the glycine released from the vitellogenin contributed to the serine pool. Tyrosine metabolism appeared to be correlated to cuticle sclerotization.
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.
Vitellogenin uptake in vitro was measured in artificial and in haemolymph media. Survival of follicles was much longer in the latter. Vitellogenin was taken up specifically and uptake was linear for 8–15 h in both media. The composition and concentration of ions in the artificial media strongly influenced vitellogenin uptake and distinct pH-optima at 7.3 and between 7.7 and 8.0 were observed. In haemolymph media, vitellogenin uptake was dependent on the haemolymph donor: uptake was highest in haemolymph containing high vitellogenin concentrations. When artificial media were supplemented with increasing vitellogenin concentrations, vitellogenin uptake increased. The uptake process was saturable. Adding unlabelled vitellogenin at concentrations below 3 mg/ml stimulated uptake of radio-labelled vitellogenin. The effect of juvenile hormone III on in vitro vitellogenin uptake was studied in haemolymph from pregnant females, which contains no endogenous juvenile hormone, and a medium vitellogenin concentration. In the presence of juvenile hormone III, vitellogenin uptake increased in follicles of 28% of the ovaries tested, was unaffected in 59% and decreased in the remainder. These data indicate that juvenile hormone III can directly influence vitellogenin uptake and suggest that the responsiveness of vitellogenic follicles to juvenile hormone is variable.
The vitellophags perform 2 essential functions during embryogenesis of the migratory locust, Locusta migratoria (Orthoptera : Acrididae). They contribute to yolk digestion and probably to translocation of the released nutrients toward the embryo. When the germ band, the amnion, and the serosa are segregated, the vitellophags become far more efficient than the various cells bordering the yolk. Their activity increases as the yolk cleavage progresses. The 2 kinds of yolk inclusions are modified differently. (i) The lipoglycoprotein bodies become altered very early through erosion, followed by fragmentation. The vitellophagic lysosomes increase in number, while the matrix of certain adjacent yolk bodies reacts to a test for acid phosphatases. (ii) The lipid droplets are modified in such a manner that more and more vitellophags become laden with tiny lipid globules. Moreover, several vitellophags phagocytize cells that die during normal embryogenesis. (i) Some of them ingest prematurely dead cells that are pushed aside from the germ anlage, then from the inner layer of germ band, toward the adjacent yolk. (ii) Before katatrepsis, a few vitellophags phagocytize some of the amniotic cells that degenerate ventrally to head and thorax. (iii) During and after the completion of dorsal closure, numerous vitellophags engulf and digest most of the extraembryonic cells that have been carried into the yolk mass.
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.
Food is an important extrinsic factor in the control of moulting as well as in the control of reproduction in colonies of Blattella germanica. Starvation following a moult or a parturition delays the initiation of another moulting cycle or female reproductive cycle. The initiation of a moulting cycle after a period of starvation requires only a short period of food availability (12 hr). To be able to postpone development until adequate food is available is advantageous to intermittent feeders and scavengers, such as cockroaches, which must forage for food. Synchronization of the development of colonies of cockroaches by controlling the availability of the food is a valuable research tool.
1.1. The electron microscopic analysis of the intermediates of yolk degradation in Artemia has revealed that the process is not homogeneous. Three different mechanisms of degradation (1, 2 and 3) with different complexity appear to occur during development.2.2. We have described previously that yolk degradation in Artemia occurs through five cycles.3.3. The three mechanisms have not been found in every cycle.4.4. Mechanism 2 has been observed in cycle one, two and five, and both mechanisms 1 and 3, in the third and fourth cycles.5.5. Mechanism 3 releases mitochondria thus confirming previous results on the storage of mitochondria in the yolk granules of Artemia.
Eggs from reciprocal crosses of T(3;12)/3;12 in Blattella germanica , which were arrested early in development, all showed gross defects. They were grouped into three types corresponding in frequency to that expected from fertilization by three of the four df/dp gametes resulting from adjacent chromosome disjunction in T (3;12)/3;12 males. A later egg lethal (stage VII) corresponded in frequency to the fourth gamete. An earlier lethality was associated with paternally derived genetic unbalance, especially in one egg type. It is suggested that the genetic complement of the male pronucleus affects some early process(es), and also that the zygotic genome of B. germanica may act earlier in the control of development than is the case in holometabolous insects.
Fluorescent amines, 9-aminoacridine, acridine orange and quinacrine, were used as probes for a pH gradient (deltapH) across gastric microsomal vesicles. Analysis of probe uptake data indicates that 9-aminoacridine distributes across the membrane as a weak base in accordance with the deltapH. On the other hand, acridine orange and quinacrine show characteristics of binding to membrane sites in addition to the accumulation in response to deltapH. A discussion of the advantages and limitations of the probes is presented. Application of these probes to pig gastric microsomal vesicles indicates that that K+-stimulated ATPase is responsible for the transport of H+ into the vesicles and thus develops a deltapH across the membrane. The deltapH generated by the K+-ATPase has a definite requirement for internal K+. The proton gradient can be discharged slowly after ATP depletion or rapidly either by detergent disruption of the vesicles or by increasing their leakiness using both H+ and K+ ionophores. On the other hand, the sole use of the K+ ionophore, valinomycin, stimulates the ATP-induced formation of deltapH by increasing the availability of K+ to internal sites. This stimulation by valinomycin requires the presence of permeable anions like Cl-. Analysis of the Cl- requirement indicates that in the presence of valinomycin the net effect is the accumulation of HCl inside the gastric vesicles. With an external pH of 7.0, the ATP-generated deltapH was calculated to be from 4 to 4.5 pH units. The results are consistent with the hypothesis that the K+-stimulated ATPase drives a K+/H+ exchange across the gastric vesicles. Since other lines of evidence suggest that these gastric microsomes are derived from the tubulovesicular system of the oxyntic cell, the participation of the ATP-driven transport processes in gastric HCl secretion is of interest.
Crosslinking with dimethyl suberimidate reveals a chain of histone octamers in chromatin. The octamer can be isolated free in solution at high ionic strength and pH. The identification of dimers formed by crosslinking reveals two or more contacts of each histone with others within the octamer. The molecular weight (110,000) and pattern of dissociation of the octamer are compatible with the composition (F2A1)2(F3)2(F2A2)2(F2B)2.
In contrast to previous findings, three major yolk proteins have been identified in the oocytes and eggs of Drosophila melanogaster. They are also present as major proteins in the haemolymph of mature females and in trace amounts in the haemolymph of young females; the male haemolymph lacked all three proteins. Female fat body contained the three proteins and, surprisingly, trace amounts were also present in the male fat body. The accumulation and degradation of the three yolk proteins by oocytes and embryos was asynchronous suggesting that independent controls may exist.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Two new reciprocal translocations in the German cockroach have been analyzed. They were identified cytologically to be T(3;12) and T(7;12). Linkage studies showed that groups XI, IX, and IV are on chromosomes 12, 3, and 7, respectively, and clearly demonstrated sex differences in recombination. Each of these chromosomes have distinctive morphological features that facilitate their identification, and permit breakpoint and centromere localization. A sex difference in fecundity is associated with T(7;12), but not T(3;12). About 40 percent mortality occurred when T(3;12) males or females and T(7;12) females were outcrossed. Outcrossing T(7;12) males produced the expected 50 percent mortality. Cell counts at metaphase I revealed that T(3;12) males exhibit directed segregation, while T(7;12) males do not. Tests for homozygosity indicated that the T(7;12) homozygote is viable. A map of chromosome 12 is presented showing the tentative placement of linkage group XI with respect to interchange breakpoints and chromosome morphology. The results are discussed in relation to possible sex differences in chiasma localization.
A cysteine, cathepsin B-like proteinase activity has been found in Drosophila embryos. It appears associated with yolk granules and its activity during embryogenesis correlates well with the degradation of these organelles. In mature oocytes, the enzyme is found in an inactive form which may be activated by limited proteolysis by a serine proteinase also present in oocytes. In early embryos, when solubilized in vitro, the cathepsin B-like proteinase is found in a form of high molecular mass (approx 1000 kDa). This decreases with development down to about 39 kDa, likely the mass of the free proteinase. The heavy form apparently results from the tight association with a yolk protein complex. The proteinase has been found in vitro to degrade readily the yolk polypeptides. The proteinase activity increases during early embryogenesis in parallel with the decrease in molecular weight of the heavy form, and decreases to low values in late embryos. We have also found that ammonium chloride can inhibit in vivo the degradation of yolk and, in parallel, the developmental inactivation of the proteinase. The results altogether suggest that the cathepsin B-like proteinase is implicated in yolk degradation in Drosophila.
Several discrete events were resolved in the processing of vitellogenin in Blattella germanica. Using tunicamycin to inhibit the synthesis of high-mannose oligosaccharide, a high molecular weight pro-vitellogenin peptide (apo-proVG, Mr 215,000) was identified in fat body. Dosages of tunicamycin which inhibited glycosylation of vitellogenin by 98% inhibited its synthesis by as much as 59%, yet led to an intracellular accumulation of apo-proVG. Reversibility and dose dependency of these effects on vitellogenin synthesis, glycosylation, proteolytic processing, and secretion were demonstrated. In control insects, glycosylation of apo-proVG yielded a Mr 240,000 pro-vitellogenin peptide (proVG). FITC-Concanavalin A bound to purified proVG but not to apo-proVG, thus confirming an absence of high-mannose oligosaccharide in the apo-protein. Following its glycosylation, proVG was processed rapidly in fat body to Mr 160,000 (VG160) and Mr 102,000 (VG102) peptides which subsequently were secreted into hemolymph. After uptake into developing oocytes, the VG160 peptide was processed further prior to chorionation, yielding subunits of Mr 95,000 and 50,000. Uniqueness of the peptides of mature vitellin (Mr 102,000, 95,000, and 50,000) was indicated by comparison of the CNBr fragments of each purified subunit. Staining of CNBr fragments with FITC-Concanavalin A also indicated that high-mannose oligosaccharides are attached at one or more sites within each vitellin subunit. Resolution of the substructure of this insect vitellin and identification of events involved in the processing and secretion of its fat body apo-protein provide a basis for further study of the assembly and transport of vitellogenin, its packaging in eggs, and utilization during embryogenesis.
Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.
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.
Differential embryonic development, as seen within egg cases of the German cockroach, serves to distinguish matings of interchange heterozygotes from those of wild type. In T (3;12), one group of fertilized eggs ceases development during stage I of embryonic development; a second group, during stage VII. The frequency of the two groups correlates closely with that of adjacent-1 vs adjacent-2 disjunction. It also does not differ significantly from the expected frequency if zygotes fertilized by one of the two types of adjacent-1 gametes reach a more advanced stage of development than those of the other three types of duplication-deficiency gametes. The absence of a sex difference in the stages of embryonic death indicates that it makes little difference whether aneuploid gametes are of maternal or paternal origin.
Insects can be divided into three groups based on the sizes of the polypeptide constituents of their vitellogenins and vitellins. In order to determine the relationships between these groups, antisera to the vitellins of seven insects from six taxonomic orders were used to assess immunological cross-reactivity. Antigenic relatedness was observed only between vitellins from species within the same family. Amino acid compositional data for vitellins from nine species were used to assess homology by difference matrices. The S delta Q values were similar for both intra-order and inter-order comparisons and strongly suggested relatedness. The S delta n comparisons supported the immunological data that indicated that the vitellins were evolving rapidly. For most insect vitellins there are two distinct size classes of polypeptides that seem to be derived from a single asymmetric proteolytic cleavage of a precursor. We propose a model that suggests that the different size polypeptides represent distinct domains and that in the evolution of the vitellogenin genes of the Diptera and Hymenoptera there has been domain elimination.
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Evidence that yolk granules of Bltzftellu germmica become acidified during early embryo development
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Biochemical properties of the two forms of Leucophea maderae vitellin and their subunits
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