Changes in lipid droplet localization during embryogenesis of the silkworm, Bombyx mori

Lipid droplets are considered one of the most important energy sources in lepidopteran eggs during late embryogenesis, but the process of their incorporation into the embryo is as yet unknown. The present study focused on the process of transition of lipid droplets from the extraembryonic yolk to the embryo of the silkworm Bombyx mori, using morphological and biochemical approaches. The morphological study revealed that the incorporation of lipid droplets from the extraembryonic yolk into the embryo occurs at three points and in three different ways during the development of the embryo. Some lipid droplets were translocated directly from the extraembryonic yolk to the embryo before the blastokinesis stage. However, the majority of lipid droplets together with the other components of the extraembryonic yolk were incorporated in the embryo via both morphogenetic inclusion during dorsal closure and ingestion of the extraembyonic yolk by the developing caterpillar prior to hatching. Similar results were obtained from the biochemical study. Thus, we propose that there are three steps in the incorporation of lipid droplets from the extraembryonic yolk into the embryo. In addition, morphological and biochemical data concerning the total amount of lipid droplets in the egg suggested that lipid droplets were mainly consumed during late embryogenesis, seeming to synchronize with tracheal development.     

Multiple vitellogenin-derived yolk proteins in gray mullet (Mugil cephalus): disparate proteolytic patterns associated with ovarian follicle maturation

Disparate proteolytic patterns of yolk proteins, derived from three types of vitellogenin (VgA, VgB, and VgC), were observed in gray mullet. Immuno-biochemical analyses of extracts obtained from vitellogenic ovaries (VO) and ovulated eggs (OE) confirmed that a large proportion of VgA-derived lipovitellin (LvA) was degraded into free amino acids (FAAs) during ovarian follicle maturation. The maturation-associated alteration of VgB-derived Lv (LvB) involved only limited proteolysis; the heavy and light LvB chains were dissociated into at least three and one polypeptide fragments, respectively. The native mass of VgC-derived Lv (LvC) exhibited little difference between VO and OE, although it was apparent that the LvC was 'nicked' during maturation, resulting in the appearance of several bands in OE. Similar analyses confirmed that VgA-derived beta'-component (beta'-cA) and VgB-derived beta'-c (beta'-cB) decreased during maturation in both quantity and native mass, while phosvitin derived from either VgA (PvA) or VgB (PvB) appeared to be degraded into FAAs. The pattern of maturation-associated proteolysis of mullet yolk proteins is similar to that reported for other marine teleosts spawning pelagic eggs. However, the depository ratio of the three distinct types of Lv in the mullet VO appeared to be different from that estimated for another marine pelagophil, the barfin flounder. These results support a recent paradigm regarding the significance of Vg multiplicity upon successive physiological events in this group of fishes including the hydration of maturing oocytes, the acquisition of proper egg buoyancy, and the generation of requisite nutrient stocks for each stage of embryogenesis and larval development.     

The embryo pancreas is a source of increased yolk amylase in the fertilized eggs of domestic fowls.

The amylases were studied in the yolk of fertilized eggs and in the pancreases of the embryos of domestic fowls. The amylase activity in the yolk increased markedly from 13 days of incubation until hatching, but the activity decreased when the embryos were taken out of the eggs. The isoamylases in the yolk and in the pancreas of the embryo were identical electrophoretically. The amylase occurs mainly in the pancreas of the embryo. We think that the increase in amylase activity in the yolk of fertilized eggs during incubation depends upon the accumulation of pancreatic amylase synthesized by the developing embryo in the egg.     

Purification and characterization of vitellogenin and lipovitellins of the sand crab Emerita asiatica: molecular aspects of crab yolk proteins.

In the mole crab Emerita asiatica, the main yolk proteins consist of two slow moving lipovitellins (Lv I and Lv II) of glycolipoprotein nature. Lv I cleaves into subunits (MW: 109,000 and 105,000) and Lv II gives rise to six subunits (MW: 65,000, 54,000, 50,000, 47,000, 44,000, and 42,000) in SDS-PAGE (with beta-mercaptoethanol). In order to observe the stability of Lv II as well as to achieve better resolution of the proteins, two different buffer systems (Phosphate buffered saline and tris-buffered saline), 40% sucrose, and glass distilled water were used as homogenizing media. Among them, better resolution was achieved with tris-buffered saline and 40% sucrose, and tris-buffered saline seems to be the ideal medium for elution of Lv II. The analysis of biochemical constituents of the major Lv II reveals a percentage composition of 69.325, 27.927, and 2.753 respectively for protein, lipid, and bound sugars. In the I stage embryo, protein comprises about 67.276%, lipid 29.65%, and bound sugars 3.015%. Vitellogenin (Vg) electrophoretically corresponding to the Lv I and Lv II was present in the female haemolymph during the entire period of embryogenesis. The number of subunits (8) of Vg in all stages remained unaltered and their approximate molecular weights were Vg1, 91,000; Vg2, 87,000; Vg3, 83,000; Vg4, 61,000; Vg5, 58,000; Vg6, 45,000; Vg7, 42,000; and Vg8, 38,000. Different proteins present in the embryos (I and IV stage) and the serum obtained from the animal carrying the I stage embryo were separated by gel-filtration in high performance liquid chromatography (HPLC). Sephadex (G-200) gel filtration chromatography was used to purify the Lv II in large quantity. Total lipid extracted from Lv II as well as the embryos belonging to different stages of development were separated into their constituent neutral, glycolipids, and phospholipids, using silicic acid column chromatography. Thin layer chromatography (TLC) was used to isolate the different phospholipids purified from various stages of embryos and Lv II. As many as seven different phospholipids were separated from Lv II and I and IX stage embryos; and whereas thin layer chromatogram of V and VI stage embryos showed six different phospholipids, embryos of VII and VIII stage contained four phospholipid species. Cholesterol, glycolipids, and individual phospholipids isolated from the Lv II and I stage embryo were quantified spectrophotometrically and the results were discussed.     

Lipid and fatty acid composition during embryo and larval development of puye Galaxias maculatus Jenyns, 1842, obtained from estuarine, freshwater and cultured populations

Galaxias maculatus eggs and larvae obtained from broodfish captured either in an estuarine or a freshwater environment, as well as from cultured broodstock were analysed to compare their lipid and fatty acid profiles. Results showed a lower lipid content in embryos and larvae from estuarine populations than those from fresh water, denoting the influence of environmental conditions. The n-3:n-6 ratio was higher in eggs from estuarine and cultured populations, being in the range of marine fishes, whereas for eggs from freshwater fish was lower and typical of freshwater fishes. The polyunsaturated fatty acids (PUFA), particularly docosahexaenoic acid (22:6n-3) and eicosapentaenoic acid (20:5n-3), were higher in eggs and larvae of broodstock coming from culture or estuarine environments than in those from fresh water. Moreover, these fatty acids markedly increased after hatching in larvae coming from estuarine populations, suggesting the effect of the environment on fatty acid profiles to physiologically prepare the larvae to adapt to higher salinity conditions. Linoleic acid (18:2n-6) content was higher in fresh water fish and its reduction during embryo and larval development was accompanied by a significant increase of arachidonic acid (20:4n-6), which was not observed in embryos or larvae from broodstock fish from estuary or aquaculture origin. Both environment and diet of broodstock fish affected lipid and fatty acid composition of G. maculatus embryo and larvae as well as their changes during development.     

Embryo lipoproteins and yolk lipovitellin consumption during embryogenesis in Macrobrachium borellii (Crustacea: Palaemonidae)

The prawn Macrobrachium borellii has lecithotrophic eggs with highly-abbreviated development. The major yolk component is lipovitellin (LV), a lipoprotein with 30% lipids (by weight). LV consumption during embryogenesis was followed by ELISA and Western blot analysis using an anti-LV polyclonal antibody. No cross-reacting proteins were observed and LV-like lipoproteins were strongly recognized by the antibody in hemolymph (vitellogenin), yolk (LV) and embryos (LVe), as determined by Western Blot analysis. LV decreased significantly along development from 9.4 to 1.1 microg/mg egg. Consumption rate of LV was slow in early embryogenesis, followed by a rapid utilization in late embryonic stages. Significant LVe amounts were still present at hatching. LV apolipoproteins were selectively degraded during embryo development, being the highest molecular weight subunit the most affected. Comparison among in vitro, in vivo and theoretical proteolysis suggested that trypsin may be involved in LV degradation during late embryogenesis. Embryo lipoprotein (HDLe) synthesis was first detected at stage 6. HDLe shared the same density, MW and subunit composition as adult hemolymph HDL(1) and did not cross-react with LV-like lipoproteins. Though expressed at low concentration, it fulfilled embryo needs for lipid transport among organs.     

Anopheles gambiae lipophorin: characterization and role in lipid transport to developing oocyte

Lipid transport in arthropods is achieved by highly specialized lipoproteins, which resemble those described in vertebrate blood. Here, we describe purification and characterization of the lipid-apolipoprotein complex, lipophorin (Lp), in the malaria vector mosquito Anopheles gambiae. We also describe the Lp-mediated lipid transfer to developing eggs and the distribution of the imported lipid in developing embryos. The density of the Lp complex was 1.135 g/ml with an apparent molecular weight of 630 kDa. It is composed of two major polypeptides, apoLp I (260 kDa) and apoLp II (74 kDa) and composed of 50% protein, 48% lipid and 2% carbohydrate (w/w). Hydrocarbon, cholesterol, phosphatidyl choline, phosphatidyl ethanolamine, cholesteryl ester and diacylglyceride were the major Lp-associated lipids. Using fluorescently tagged lipids, we observed patterns that suggest that in live developing oocytes, the Lp was taken up by a receptor-mediated endocytic process. Such process was blocked at low temperature and in the presence of excess unlabeled Lp, but not by bovine serum albumin. Imported Lp was segregated in the spherical yolk bodies (mean size 1.8 microm) and distributed evenly in the cortex of the oocyte. In embryonic larvae, before hatching, a portion of the fatty acid in vesicles was found evenly distributed along the body, whereas portion of phospholipids was accumulated in the intestine.     

Proteolytic activity in the yolk sac membrane of quail eggs.

Extraembryonal degradation of yolk protein is necessary to provide the avian embryo with required free amino acids during early embryogenesis. Screening of proteolytic activity in different compartments of quail eggs revealed an increasing activity in the yolk sac membrane during the first week of embryogenesis. In this tissue, the occurrence of cathepsin B, a lysosomal cysteine proteinase, and cathepsin D, a lysosomal aspartic proteinase, has been described recently (Gerhartz et al., Comp Biochem Physiol, 118B:159-166, 1997). Determination of cathepsin B-like and cathepsin D-like proteolytic activity in the yolk sac membrane indicated a significant correlation between growth of the yolk sac membrane and proteolytic activity, shown by an almost constant specific activity. Both proteinases could be localized in the endodermal cells, which are in direct contact to the yolk. The concentration of proteinases in the endodermal cells appears to be almost unaltered in the investigated early stage of quail development, whereas the amount of endodermal cells increases rapidly, seen by a complicated folding of the yolk sac membrane. In the same cells quail cystatin, a potent inhibitor of quail cathepsin B (Ki 0.6 nM), has been localized at day 8 of embryonic development. Approximately at this stage of development, the quail embryo stops metabolizing yolk. In conclusion, it is strongly indicated that the amount of available free amino acids, produced by proteolytic degradation and supporting embryonic growth, is regulated by the growth of the yolk sac membrane.     

Amino acid and lipid metabolism in embryos of flesh fowl with different yolk mass

It has been proposed that variations in relative yolk mass in a population of flesh fowl be used as a model of development of nidicolous and nidifugous birds. During development of the eggs with a high proportion of yolk, an excess of lipids is cleaved at a higher rate and oxidized until day 17 of incubation, while in the embryos developing from the eggs with a low relative yolk mass, amino acids are intensely cleaved during the period preceding the hatching. Significant differences in the body content of cystine were found in 17-day embryos and upon hatching, thus suggesting a delayed activity of the genes encoding keratins in the group corresponding to the seminidicolous type according to the egg content of lipids. These biochemical differences question the widespread concept on the occurrence of dichotomy by the end of embryogenesis and beginning of neonatal growth of nidifugous and nidicolous birds.     

Vitellogenin cleavage products as indicators for toxic stress in zebra fish embryos: a proteomic approach

Vitellogenins (Vtgs) are the major yolk proteins in all oviparous animals. Systematic and regulated processing of these during embryogenesis is crucial for embryonic development. In the present study, toxicant-induced disturbance of Vtg degradation processes during Danio rerio (DR) embryogenesis was analysed to establish a sensitive tool for monitoring toxic stress at the molecular level. A 2-DE-based proteomic approach for whole DR embryos was established to study Vtg cleavage products (lipovitellin (Lv) derivatives). Ethanol was chosen as a positive control for a toxicity related change in the proteome of whole zebra fish embryos. Protein extracts from embryos treated with two ethanol concentrations, 0.5 and 2% v/v, showing either no or very strong visible effects, like absent heartbeat and blood circulation, were examined. Significant changes in the Lv pattern were detected for both conditions. The results are interpreted as scope for the use of the high abundant Lv derivatives as sensitive stress indicators in zebra fish embryos reflecting the overall fitness of the intact organisms.     

Dynamics of Ubiquitin Pools in Developing Sea Urchin Embryos

The sea urchin embryo is a closed metabolic system in which embryogenesis is accompanied by significant protein degradation. We report results which are consistent with a function for the ubiquitinmediated proteolytic pathway in selective protein degradation during embryogenesis in this system. Quantitative solid- and solution-phase immunochemical assays, employing anti-ubiquitin antibodies, showed that unfertilized eggs of Strongylocentrotus purpuratus have a high content of unconjugated ubiquitin ( ca . 8 × 10⁸ molecules), and also contain abundant conjugates involving ubiquitin and maternal proteins. The absolute content of ubiquitin in the conjugated form increases about 13-fold between fertilization and the pluteus larva stage; 90% or more of embryonic ubiquitin molecules are conjugated to embryonic proteins in hatched blastulae and later-stage embryos. Qualitatively similar results were obtained with embryos of Lytechinus variegatus . The results of pulse-labeling and immunoprecipitation experiments indicate that synthesis of ubiquitin in S. purpuratus is developmentally regulated, with an overall increase in synthetic rate of 12-fold between fertilization and hatching. Regulation is likely to occur at the level of translation, since others have shown that levels of ubiquitin-encoding mRNA remain virtually constant in echinoid embryos during this developmental interval. The sea urchin embryo should be a useful system for characterizing the role of ubiquitination in embryogenesis.     

非洲爪蟾孵化酶分子对卵黄膜作用方式的研究

非洲爪蟾的孵化液对卵黄膜和二甲基酷蛋白具有降妥活性。用非洲爪蟾孵化酶的特异性抗ＧＳＴ－ＵＶ．２抗体进行Ｗｅｓｔｅｒｎ杂交的结果表明,孵化液中出现一种分子量为６０ｋＤ的大组分,有时也会出现一种分子量为４０ｋＤ的小组分。     

Ecdysteroid titres and location in developing eggs of Schistocerca gregaria

Ecdysteroid levels in the separated embryo and yolk fractions of Schistocerca gregaria eggs have been determined at each of the developmental stages. The major hormones present both in the free and conjugated state are ecdysone, 20-hydroxyecdysone and 2-deoxyecdysone. At the beginning of embryonic development the ecdysteroids occur only in the yolk whereas, after blastokinesis, they are found in the embryo. The levels of conjugates fall during embryonic development, whereas a decrease of free hormone titres in early embryogenesis is followed by a marked increase in late embryos (stage 26 and 28). The possible role of ecdysteroids in relation to the morphogenetic processes of egg development and the site of origin of the free ecdysteroid peaks are discussed.     

Accumulation of calcium and phosphorus in pigeon (Columba livia) embryos

Summary Calcium and phosphorus were measured in the yolk and albumen of fertile pigeon (Columba livia) eggs incubated for 0–17 days, and in embryos and hatchlings. Shell provided most of the calcium for skeletal mineralization of the embryos, whereas phosphorus was derived from the yolk and albumen. Mobilization of calcium from the shell to the embryo commenced at approximately day 11 of incubation, accumulating both in the embryo and the yolk sac. There was 1.4 times more calcium in squab yolk sacs than that contained in newly laid egg yolks. The results suggest that whereas general patterns of calcium and phosphorus accumulation during embryogenesis in altricial birds closely resemble those of precocial birds, calcium mobilization from the shell begins later, proceeds at a slower rate and results in a less mineralized hatchling.CIDA/NSERC Visiting Research Associate Permanent address: Department of Animal Science, University of Peradeniya, Peradeniya, Sri Lanka     

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.     

Metabolic fates of yolk lipid and individual fatty acids during embryonic development of the coot and moorhen

There is currently little information regarding the metabolic fates of yolk lipid and individual fatty acids during embryonic development of free-living avian species. Here we report the pattern of lipid utilization during embryonic development of the coot (Fulica atra) and the moorhen (Gallinula chloropus), two related species producing precocial offspring from eggs with a distinctive fatty acid composition and with an incubation period similar to that of the chicken. By the time of hatching, the proportions of the initial yolk lipid that had been transferred to the embryo were 88.2% and 79.8% for the coot and moorhen respectively. During the whole incubation period, 42.9% and 40.0% of the initial yolk lipid of the coot and moorhen respectively were lost from the system due to oxidation for energy, equating to 47.8% and 50.0% respectively of the actual amount of lipid transferred over this time. Thus, the lipid received by the embryos of both species is partitioned almost equally between the alternative fates of energy metabolism and incorporation into tissue lipids. In the coot, this 50:50 split between oxidation and tissue formation was maintained during the hatching process. The proportions of arachidonic (20:4n-6) and docosahexaenoic (22:6n-3) in the yolk lipids of these species were 2.5-3.5 times higher than in eggs of domestic poultry. In contrast to the situation in the chicken, there was no preferential uptake of 22:6n-3 from the yolk during coot and moorhen development. The fatty acid compositions of the whole body lipids of the coot and moorhen hatchlings were almost identical to those of the initial yolks indicating that, unlike the chicken, these species display relatively little overall biomagnification of 20:4n-6 and 22:6n-6 during development. It is suggested that the yolk fatty acid profiles of the coot and moorhen are particularly well matched to the requirements of the embryo, reducing the need for selective uptake of 22:6n-3 and for the overall biomagnification of 22:6n-3 and 20:4n-6.     

Resolution of the subunit composition of a cytokinin-binding protein from wheat embryos

When the wheat germ cytokinin-binding protein (CBF-1) is isolated from excised wheat embryos only one group of polypeptides near 54 000 is observed as opposed to three polypeptide groups reported for CBF-1 from commercially milled wheat germ. The other two lower molecular mass polypeptide groups previously reported are probably proteolytic degradation products of the 54 000 species resulting from excessive heat or physical damage during milling. The CBF-1 polypeptide accumulates rapidly in the embryo after 20 days post-anthesis. A larger set of polypeptides near 66 000 – 68 000 appears during embryo development and also reacts with anti-CBF-1 serum. These polypeptides are also observed upon immunoprecipitation of 20-day embryo polyadenylated RNA translation products although the data are not definitive enough to prove a precursor relationship to the CBF-1 54 000 polypeptide. The new findings are discussed in regard to previous characterizations of CBF-1.     

The metabolism of neutral and polar lipids in eleuthero-embryos and starving larvae of the African catfish Clarias gariepinw

Lipid class analysis was carried out on developing eggs, eleuthero-embryos (yolk sac larvae) and starving larvae of the freshwater species Clarias gariepinus , using thin layer chromatography. Samples were taken at fixed intervals from a large pool of fertilized eggs obtained through induced reproduction of several parent fish. The total lipid content of fertilized eggs fluctuated around 22% of the dry weight and decreased from 21% at hatching to about 12–5% at yolk absorption. In starving larvae, the amount of total lipid per individual remained relatively constant. Polar lipids [phosphatidylcholine (PC) and phosphatidylethanolamine (PE)] together accounted for 73·6 to 80% of total lipid. PC was by far the most abundant lipid class during the entire experimental period (70–75% of total lipid). PC was catabolized proportionally to total lipid, demonstrating its role as the main energy supplier. All yolk PE was converted to body tissue. The neutral Hpids consisted of triglycerides (TAG), cholesterol and cholesteryl esters (respectively 12·5, 10 and 3% of total lipid in newly fertilized eggs). All TAG were depleted before complete yolk absorption.