In vivo activation of pro-form Bombyx cysteine protease (BCP) in silkmoth eggs: localization of yolk proteins and BCP,and acidification of yolk granules

The present study was designed to investigate the process of acidification of yolk granules during embryogenesis. In oocytes of mature Bombyx mori silkmoth, yolk proteins and a cysteine protease (pro-form BCP) were found in yolk granules. BCP was localized in small sized yolk granules (SYG, 3-6 microm in diameter) and yolk proteins in large sized granules (LYG, 6-11 microm in diameter), which might result in a spatial separation of protease and its substrates to avoid unnecessary hydrolysis. The granules were isolated on Percoll density gradient centrifugation. Although separation of LYG and SYG was incomplete, the granules sedimented in different fractions when using unfertilized egg extract, in which LYG was recovered from heavier fractions and BCP from lighter fractions. Acid phosphatase, as well as other lysosomal marker enzymes tested, was recovered from LYG-containing fractions. When extracts were prepared from developing eggs (day 3), some BCP-containing granules co-sedimented with LYG. The inactive pro-form BCP was activated in vivo, in parallel with yolk protein degradation, and as demonstrated previously in vitro under acidic conditions (). These results suggest that acidification occurs in yolk granules during embryogenesis. This was also confirmed using acridine orange fluorescent dye. In early development, most yolk granules were neutral, but became acidic during embryonic development. SYG were progressively recovered in heavier density fractions, displaying acidic interior. In this fraction, BCP-containing granules seem to be associated with larger granules (6-11 microm in size). In addition, SYG (BCP containing granules) were likely to be acidified earlier than LYG. Our results suggest that acidification initiates yolk degradation through activation of pro-form BCP.     

Cytochemical characterization of the endomembranous system during oocyte secondary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Characidae)

The endomembranous system of Serrasalmus spilopleura oocyte secondary growth was analysed using structural and ultrastructural cytochemical techniques. In vitellogenic oocytes, the endoplasmic reticulum components, the nuclear envelope intermembranous space, some Golgi dictiossomes, lysosomes, yolk granules, regions of the egg envelope and sites of the follicle cells react to acid phosphatase detection (AcPase). The cortical alveoli, some heterogeneous cytoplasmic structures, regions of the egg envelope, and sites of the follicle cells are strongly contrasted by osmium tetroxide and zinc iodide impregnation (ZIO). The endoplasmic reticulum components, some vesicles, and sites of the follicle cells also react to osmium tetroxide and potassium iodide impregnation (KI). The biosynthetic pathway of lysosomal proteins, such as acid phosphatase, required for vitellogenesis, involves the endoplasmic reticulum, Golgi complex, vesicles with inactive hydrolytic enzymes, and, finally, lysosomes. In S. spilopleura oocytes at secondary growth, the endomembranous system takes part in the production of the enzymes needed for vitellogenesis, and in the metabolism of yolk exogenous components (AcPase detection). The endomembranous system compartments also show reduction capacity (KI reaction) and are involved in the metabolism of proteins rich in SH‐groups (ZIO reaction).     

Degradation of yolk platelets in the early amphibian embryo is regulated by fusion with late endosomes

The eggs of many animal species contain a large store of yolk platelets, lipid droplets and glycogen granules; these are consumed during early embryogenesis. However, the mechanisms by which degradation of these stored materials occurs during early embryogenesis are not clearly understood. The mechanisms underlying yolk degradation in amphibian (newt) embryos were investigated. Electron microscopy using an anion marker, cationic ferritin, revealed that yolk platelets were degraded after fusion with late endosomes containing primary lysosomes. Electron microscopy and the results of experiments using a number of reagents with selective effects on intracellular transport suggested that yolk degradation activity in early amphibian embryos may be regulated at the point of fusion between late endosomes and yolk platelets.     

Yolk Degradation in Tick Eggs: III. Developmentally Regulated Acidification of the Yolk Spheres

Yolk spheres in tick eggs contain a latent procathepsin L, which is activated in vivo , in parallel with yolk degradation, and in vitro by acid treatment (Fagotto, F., Arch. Insect Biochem. Physiol., 1990b in press). Mature cathepsin L hydrolyzes vitellin at acidic pH (Fagotto, F., Arch. Insect Biochem. Physiol., 1990a in press). Here, yolk spheres' pH has been estimated using acridine orange. In the early development, all yolk spheres are neutral, then an increasing number acidify, until hatching, where general acidification seems to occur. This fits well with vitellin utilized slowly during embryogenesis, more intensely at hatching (Chinzei, Y. and I. Yano, Experientia 41 , 948, 1985), and can be related to sequential degradation of individual spheres observed during embryonic development, then extensive yolk liquefaction in the larva. Different yolk spheres populations have been separaded on Percoll density gradients. In freshly laid eggs, yolk spheres are dense, neutral, undegrated and contain exclusively the precursor of cathepsin L. In later stages, yolk spheres are progressively recovered in lower density fractions, displaying acidic interior and cytological signs of degradation. They cosediment with mature cathepsin L. It is concluded that acidification initiates yolk degradation through procathepsin L activation.     

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

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

Identification and comparisons of the yolk polypeptides and the genes which code for them in D. melanogaster sibling species

Molecular Genetics and Genomics - The yolk proteins stored in Drosophila, oocytes for utilisation during embryogenesis are an ideal system for studying the regulation of gene expression during...     

Inorganic polyphosphate inhibits an aspartic protease‐like activity in the eggs of Rhodnius prolixus (Stahl) and impairs yolk mobilization in vitro

Inorganic polyphosphate (poly P) is a polymer of phosphate residues that has been shown to act as modulator of some vertebrate cathepsins. In the egg yolk granules of Rhodnius prolixus, a cathepsin D is the main protease involved in yolk mobilization and is dependent on an activation by acid phosphatases. In this study, we showed a possible role of poly P stored inside yolk granules on the inhibition of cathepsin D and arrest of yolk mobilization during early embryogenesis of these insects. Enzymatic assays detected poly P stores inside the eggs of R. prolixus. We observed that micromolar poly P concentrations inhibited cathepsin D proteolytic activity using both synthetic peptides and homogenates of egg yolk as substrates. Poly P was a substrate for Rhodnius acid phosphatase and also a strong competitive inhibitor of a pNPPase activity. Fusion events have been suggested as important steps towards acid phosphatase transport to yolk granules. We observed that poly P levels in those compartments were reduced after in vitro fusion assays and that the remaining poly P did not have the same cathepsin D inhibition activity after fusion. Our results are consistent with the hypothesis that poly P is a cathepsin D inhibitor and a substrate for acid phosphatase inside yolk granules. It is possible that, once activated, acid phosphatase might degrade poly P, allowing cathepsin D to initiate yolk proteolysis. We, therefore, suggest that degradation of poly P might represent a new step toward yolk mobilization during embryogenesis of R. prolixus. J. Cell. Physiol. 222: 606–611, 2010. © 2009 Wiley‐Liss, Inc.     

家蝇卵黄蛋白基因的克隆与结构序列分析

家蝇卵黄蛋白基因编码的卵黄蛋白是家蝇胚胎发育的重要营养来源 .根据 3种家蝇卵黄蛋白cDNA保守序列设计引物 ,用PCR技术从家蝇基因组DNA中扩增到大小为 76 8bp的mdYP1基因的部分DNA片段 .经地高辛标记成特异性探针 ,从构建的家蝇基因组文库中筛选出一个阳性克隆 ,并从该克隆中分离到大小为 3991bp的mdYP1基因组基因 .序列分析显示 ,该基因组序列含有约1 6kb的 5′ 上游区和 1 0kb的 3′ 下游区 ,编码区由一个 6 1bp的内含子和大小分别为 2 2 2bp和10 2 8bp的 2个外显子组成 .5′ 上游区含有典型的CAAT TATA盒 .     

Drosophila cathepsin B-like proteinase: a suggested role in yolk degradation

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.     

Biochemical characterization of lysosomes in unfertilized eggs of Xenopus laevis.

Relations between lysosomes and yolk platelets of amphibian eggs have been suggested. This work demonstrates the presence of acid hydrolases in oocytes induced to ovulate in vitro. About 40% of the acid hydrolases are found in a sedimentable fraction, and, in accordance with the lysosomal concept, they display structural latency. Biochemical data did not indicate any association between lysosomal enzymes and yolk platelets. The mechanism of yolk resorption is discussed and it is suggested that the fusion of lysosomes and yolk platelets might be one of the mechanisms involved in yolk digestion.     

Biochemical characterization of lysosomes in unfertilized eggs of xenopus laevis

Relations between lysosomes and yolk platelets of amphibian eggs have been suggested. This work demonstrates the presence of acid hydrolases in oocytes induced to ovulate in vitro. About 40% of the acid hydrolases are found in a sedimentable fraction, and, in accordance with the lysosomal concept, they display structural latency. Biochemical data did not indicate any association between lysosomal enzymes and yolk platelets. The mechanism of yolk resorption is discussed and it is suggested that the fusion of lysosomes and yolk platelets might be one of the mechanisms involved in yolk digestion.     

THE ROUTE OF ENTRY AND LOCALIZATION OF BLOOD PROTEINS IN THE OOCYTES OF SATURNIID MOTHS

The oocytes of saturniid moths take up proteins selectively from the blood. The distribution of blood proteins in the ovary during protein uptake was investigated by staining 2 µ sections of freeze-dried ovaries with fluorescein-labeled antibodies. The results indicate that blood proteins occur primarily in the intercellular spaces of the follicle cell layer, in association with a brush border at the surface of the oocyte, and within the oocyte in the yolk spheres. That proteins derived from the blood are associated with the yolk spheres was confirmed by isolating these bodies and showing that lysis, which can be induced by any of a number of mechanical means, causes them to release immunologically defined proteins known to be derived from the blood. That the level of blood proteins in the cytoplasm is low relatively to that in the yolk spheres was confirmed by the observation that the yellow pigments associated with several blood proteins, although conspicuous in the yolk spheres, are not visible in the translucent layer of centrifuged oocytes. From these and previous physiological observations, it is proposed that blood proteins reach the surface of the oocyte by an intercellular route, that they combine with some component of the brush border, and that they are transformed into yolk spheres by a process akin to pinocytosis.     

Lysosomes and yolk formation in the oocytes of Asterina gibbosa (Echinodermata,Asteroidea)

Summary

Primary lysosomes appear in the oocytes of A. gibbosa at the end of previtellogenesis. The lysosomes fuse with the cisternae of the endoplasmic reticulum and give rise to yolk globules containing acid phosphatase. The yolk globules then grow by fusion.     

Salt concentration-dependency of vitellogenin processing by cathepsin D in Xenopus laevis

The mechanism by which cathepsin D produces only limited proteolysis of vitellogenins (VTG) was studied in Xenopus oocytes. We first examined mature oocytes for the existence of cathepsin D; immunoblot and biochemical analyses revealed the existence of a 43kDa enzyme protein and its proteolytic activity in oocytes during and after the vitellogenesis. By determining the proteolytic activity of the fractions after subcellular fractionation of oocytes, we confirmed that cathepsin D is preserved in the yolk plasma of mature yolk platelets. The reaction of VTG with cathepsin D was examined in vitro at pH 5.6 as a function of NaCl concentrations. Lipovitellins generated from the VTG were preserved for several days at 37°C in the presence of the enzyme if the NaCl concentration was 0.15 mol/L or lower. The amount of lipovitellins decreased with increased molarity of the salt and at 0.5 mol/L NaCl they were rapidly degraded. The precipitates, growing in the reaction tube with 0.15 mol/L NaCl, included all constituents of yolk proteins and were ultrastructurally shown to have crystal structures perforated by empty cavities. No precipitates appeared at 0.5 mol/L NaCl. The results indicate that the limitation on proteolysis of the VTG by cathepsin D is due to the insolubility of yolk proteins at physiological salt concentrations, which explains why yolk can be stored stably in the presence of acid hydrolases over a long period.     

Bombyx acid cysteine proteinase

Summary

Three kinds of yolk proteins (vitellin, egg-specific protein and 30 k-proteins) are found in silkmoth eggs and have been well characterized. Essentially these proteins are considered to be amino acid reserves for developing embryos. Since at an early stage of egg development the cysteine proteinase accounts for the majority of the total proteinase activity, it may be involved in the degradation of yolk proteins. The enzyme is stored in the eggs as an inactive pro-form, indicating that the activation of the enzyme might be one of the key steps in yolk protein degradation. To investigate at the molecular level how yolk proteins degradation takes place, we have studied Bombyx acid cysteine proteinase (BCP) during an early period of embryonic development. We summarize how proteinases are regulated and are involved in the degradation of Bombyx yolk proteins during embryogenesis. These will be discussed mainly in light of recent results obtained from eggs of the silkmoth, Bombyx mori.     

Proton-pyrophosphatase and polyphosphate in acidocalcisome-like vesicles from oocytes and eggs of Periplaneta americana

Acidocalcisomes are acidic organelles containing large amounts of polyphosphate (poly P), a number of cations, and a variety of cation pumps in their limiting membrane. The vacuolar proton-pyrophosphatase (V-H⁺-PPase), a unique electrogenic proton-pump that couples pyrophosphate (PPi) hydrolysis to the active transport of protons across membranes, is commonly present in membranes of acidocalcisomes. In the course of insect oogenesis, a large amount of yolk protein is incorporated by the oocytes and stored in organelles called yolk granules (YGs). During embryogenesis, the content of these granules is degraded by acid hydrolases. These enzymes are activated by the acidification of the YG by a mechanism that is mediated by proton-pumps present in their membranes. In this work, we describe an H⁺-PPase activity in membrane fractions of oocytes and eggs of the domestic cockroach Periplaneta americana. The enzyme activity was optimum at pH around 7.0, and was dependent on Mg²⁺ and inhibited by NaF, as well as by IDP and Ca²⁺. Immunolocalization of the yolk preparation using antibodies against a conserved sequence of V-H⁺-PPases showed labeling of small vesicles, which also showed the presence of high concentrations of phosphorus, calcium and other elements, as revealed by electron probe X-ray microanalysis. In addition, poly P content was detected in ovaries and eggs and localized inside the yolk granules and the small vesicles. Altogether, our results provide evidence that numerous small vesicles of the eggs of P. americana present acidocalcisome-like characteristics. In addition, the possible role of these organelles during embryogenesis of this insect is discussed.     

Verteilungsmuster und Ultrastruktur des Glykogens in der Oocyte vonMusca domestica

Summary Fully grown oocytes ofMusca domestica contain large amounts of glycogen distributed in a characteristic pattern. Three cytoplasmic layers can be distinguished: 1. The periplasm which is free of carbohydrates and merely contains some lipid and protein yolk. 2. A zone of large glycogen clods. 3. Adjacent to this the central ooplasm where numerous lipid droplets and protein yolk spheres are found beside medium size glycogen clods. The glycogen areas are not surrounded by membranes, in contrast to the other yolk inclusions. Some possible interpretations of this ooplasmic pattern, which is already established during oogenesis, are discussed.     

Histochemical observation of the cortical zone of oocyte of Indian wall lizard, Hemidactylus flaviviridis (Rüppel).

The cortical zone of oocytes, which lies just below the follicular epithelium appears in the early stages of development, but reaches its fullest growth in vitellogenic oocytes. In the present studies it was found that the cortical zone of Hemidactylus flaviviridis consists of proteins, lipoproteins, carbohydrates, fatty yolk, RNA and little amount of DNA in mature oocytes along with mitochondria and Golgi bodies. In the early oocyte, this zone is fine granular in nature, but during the yolky stages of oocyte, it becomes filled by the vacuolar structure, which shows in it's the presence of fatty and compound yolk. The L1 and L2 types of lipid globules are also observed in the cortical zone during vitellogenic oocyte.