Protein synthesis and morphogenesis are not tightly linked during embryogenesis in Fucus

Fertilized eggs of the brown alga Fucus have long been used as model organisms for investigating the early events in the establishment of polarity and subsequent embryogenesis since large numbers of zygotes can easily be obtained. We have analyzed protein synthesis in eggs and embryos during the first day of development using two-dimensional gels and found that synthesis of 12 of the 60 most prominent proteins changed either qualitatively or quantitatively. Actin and beta-tubulin were identified by immunoblotting; synthesis of these cytoskeletal proteins was initiated at different times during the first 12 hr of development. Unique, reproducible patterns of protein synthesis observed during development in the light permitted accurate staging of developing embryos. Inhibitors such as cytochalasin and sucrose, however, blocked morphogenesis without affecting protein synthesis, and, conversely, growth in the dark delayed protein synthesis but had very little effect on the timing of morphogenesis. The data are consistent with morphogenesis and protein synthesis being relatively independent during early embryogenesis. Actinomycin D added soon after fertilization had no effect on protein synthesis 1 day later, indicating that the proteins analyzed were encoded by maternal mRNA stored in the egg.     

Program of early development in the mammal: Changes in absolute rates of synthesis of ribosomal proteins during oogenesis and early embryogenesis in the mouse

The absolute rates of synthesis of specific ribosomal proteins have been determined during growth and meiotic maturation of mouse oocytes, as well as during early embryogenesis in the mouse. These measurements were made possible by the development of a high-resolution twodimensional gel electrophoresis procedure capable of resolving basic proteins with isoelectric points between 9.1 and 10.2. Mouse ribosomal proteins were separated on such gels and observed rates of incorporation of [³⁵S]methionine into each of 12 representative ribosomal proteins were converted into absolute rates of synthesis (femtograms or moles synthesized/hour/oocyte or embryo) by using previously determined values for the absolute rates of total protein synthesis in mouse oocytes and embryos (R. M. Schultz, M. J. LaMarca, and P. M. Wassarman, 1978,Proc. Nat. Acad. Sci. USA,75, 4160;R. M. Schultz, G. E. Letourneau, and P. M. Wassarman, 1979,Develop. Biol.,68, 341–359). Ribosomal proteins were synthesized at all stages of oogenesis and early embryogenesis examined and, while equimolar amounts of ribosomal proteins were found in ribosomes, they were always synthesized in nonequimolar amounts during development. Rates of synthesis of individual ribosomal proteins differed from each other by more than an order of magnitude in some cases. Synthesis of ribosomal proteins accounted for 1.5, 1.5, and 1.1% of total protein synthesis during growth of the oocyte, in the fully grown oocyte, and in the unfertilized egg, respectively. During meiotic maturation of mouse oocytes the absolute rate of synthesis of ribosomal proteins decreased about 40%, from 620 to 370 fg/hr/cell, as compared to a 23% decrease in the rate of total protein synthesis during the same period. On the other hand, during early embryogenesis the absolute rates of synthesis of each of the 12 ribosomal proteins examined increased substantially as compared with those of the unfertilized egg, such that at the eight-cell stage of embryogenesis synthesis of ribosomal proteins (4.17 pg/hr/embryo) accounted for about 8.1% of the total protein synthesis in the embryo. Consequently, while the absolute rate of total protein synthesis increased about 1.5-fold during development from an unfertilized mouse egg to an eight-cell compacted embryo, the absolute rate of ribosomal protein synthesis increased more than 11-fold during the same period. These results seem to reflect the differences reported for the patterns of ribosomal RNA synthesis during early development of mammalian, as compared to nonmammalian, animal species. The results are compared with those obtained using oocytes and embryos fromXenopus laevis.     

Proteomic analysis of egg white proteins during the early phase of embryonic development

Avian egg albumen participates in embryonic development by providing essential nutrients as well as antimicrobial protection. Although various biological functions of egg white proteins were suggested during embryogenesis, global changes of these proteins under incubation conditions remained uninvestigated. This study presents a proteomic analysis on the change of egg white proteins during the first week of embryonic development. By using 2-DE, together with MALDI-TOF MS/MS, thirty protein spots representing eight proteins were identified showing significant changes in abundance during incubation. An accelerating degradation of ovalbumin was observed in a wide range of molecular weight. In addition, four protein complexes were predicted according to the detected molecular weight increase. Among these speculated protein complexes, an ovalbumin spot coupled with RNA-binding protein was detected. The absence of these protein complexes before incubation, followed by the constant increase in abundance during incubation indicates conceivable pivotal roles in embryonic development. To better understand the function of the proteins identified in this study, discrepancies of egg white protein changes between fertilized and unfertilized chicken eggs were additionally demonstrated. These findings will provide insight into the embryogenesis process to improve our knowledge of egg white proteins in regulating and supporting early embryonic development.     

Dynamic distribution of region-specific maternal protein during oogenesis and early embryogenesis of Xenopus laevis

Summary For analysing spatial distribution of maternal proteins in an amphibian egg, monoclonal antibodies specific to certain regions were raised. One monoclonal antibody was found (MoAB Xa5B6) which reacted specifically with the animal hemisphere of the mature Xenopus laevis egg. The maternal protein that reacted with the MoAb Xa5B6 was shown to be distributed asymmetrically along the dorso-ventral axis in the upper region of the equatorial zone of the fertilized egg. At late blastula stage, the antigen protein could be observed clearly in both the marginal zone and animal cap. It was localized predominantly in mesodermal and ectodermal cells of late neurula embryos. The Xa5B6 antigen accumulated during oogenesis. The distribution pattern of maternal protein was remarkably different in the developmental stages of the oocyte. The pattern in the mature oocyte was completely different from that of the immature egg in which the antigen was located in the radial striations of the oocyte cytoplasm. After maturation, the distribution pattern changed drastically to an animal-vegetal polarization and the striation labellings were no longer observed. By Western blot examination, it was confirmed that the amounts of antigen protein were constant during early embryogenesis and the mesoectoderm contained a greater amount of antigens than the endoderm at late blastula. The antibody detected two bands of approximately 70 × 10³ and 30 × 10³ Mr by Western blot analysis. The latter molecule may possibly be a degrading moiety of the former. The results were discussed in relation to establishment of animal-vegetal (A/V) and dorso-ventral (D/V) polarization at the molecular level. Offprint requests to: A.S. Suzuki     

Histomorphological study on embryogenesis of the honeybee Apis cerana

As important pollination species, honeybees play substantial impacts on the balance of global ecosystem, including two best-known honeybees Apis mellifera and Apis cerana. Embryogenesis is a fundamental stage of honeybee development and plays important roles in supporting the whole-life developmental process. However, few studies were reported on honeybee embryonic morphology using egg section, possibly due to the fragility of honeybee eggs and the difficulty of making embryonic sections. In this study, we reported a simply equipped method of frozen sectioning and PI (propidium iodide) staining to show the inner structure and cell distribution of A. cerena embryos at the different embryonic developmental stages. We found that the stages of A. cerena embryogenesis could also be typically classified into ten developmental stages, which are similar with the sister honeybee species, A. mellifera. To be noted, besides the cell distribution in the whole egg, we clearly observed the migration route of embryonic cells during the early embryonic development in A. cerena. This study provides a new insight into the whole process of honeybee embryogenesis from the perspective of egg sectioning, a histological basis for genetic manipulation using A. cerena eggs, and a reference method for egg sectioning for other insect species.     

Spatial distribution of abundant proteins in oocytes and fertilized eggs of the Mexican axolotl (Ambystoma mexicanum)

Two-dimensional protein patterns were compared from sections along the longitudinal axis of oocytes and fertilized eggs of the Mexican axolotl (Ambystoma mexicanum). Only a few differences were observed between four different sections through both oocyte and fertilized eggs. A set of proteins (14 out of 120 proteins) were found that reside only in the germinal vesicles (GV) of the fully grown oocyte. Two of these were observed exclusively in the vegetal half, and one in the animal half after GV breakdown, while other proteins were randomly distributed within the fertilized egg. One cytoplasmic protein was present only in the vegetal half of the mature oocyte and became present also in the animal half of the fertilized egg. Additional proteins were observed in all transverse sections of both mature oocyte and fertilized eggs. It is proposed that these proteins are modified rather than newly synthesized proteins.     

Silkworm egg proteins at the germ-band formation stage and a functional analysis of BmEP80 protein

The patterning of embryos in early stages is a critical process for embryo development. In order to understand the molecular mechanism of early embryogenesis in silkworm, 2-DE combined with MALDI-TOF-MS technologies were used to analyze the proteins from diapause-destined eggs at the germ-band formation stage. From over 1000 spots, 93 were selected for analysis and data were obtained from 59 revealing 42 proteins. Gene Ontology annotation showed these proteins were involved in several biological processes at the germ-band formation stage, including cell stress response and protein folding, cell growth and migration, termination of diapause, and nutrition storage. Prominent among them was a new 80 kDa protein, named Bombyx mori egg protein 80 (BmEP80). BmEP80 was a component of the eggshell which was secreted by follicle cells during the late vitellogenesis stage to early choriogenesis stage (FCs −5 to +10). It disappears during early embryogenesis and RNAi against it resulted in the collapse of eggs, thus it is likely that BmEP80 is a new component of the silkworm vitelline membrane.     

Lie Group Analysis of the Photo-Induced Fluorescence of Drosophila Oogenesis with the Asymmetrically Localized Gurken Protein

Lie group analysis of the photo-induced fluorescence of Drosophila oogenesis with the asymmetrically localized Gurken protein has been performed systematically to assess the roles of ligand-receptor complexes in follicle cells. The (2×2) matrix representations resulting from the polarized tissue spectra were employed to characterize the asymmetrical Gurken distributions. It was found that the fluorescence of the wild-type egg shows the Lie point symmetry X ₂₃ at early stages of oogenesis. However, due to the morphogen regulation by intracellular proteins and extracellular proteins, the fluorescence of the embryogenesis with asymmetrically localized Gurken expansions exhibits specific symmetry features: Lie point symmetry Z ₁ and Lie point symmetry X ₁. The novel approach developed herein was successfully used to validate that the invariant-theoretical characterizations are consonant with the observed asymmetric fluctuations during early embryological development.     

Stage-specific protein regulation during somatic embryo development of Carica papaya L. ‘Golden’

Somatic embryogenesis is an important biotechnological technique for large-scale propagation of elite genotypes. Identifying stage-specific compounds associated with somatic embryo development can help elucidate the ontogenesis of Carica papaya L. somatic embryos and improve tissue culture protocols. To identify the stage-specific proteins that are present during the differentiation of C. papaya somatic embryos, proteomic analyses of embryos at the globular, heart, torpedo and cotyledonary developmental stages were performed. Mass spectrometry data have been deposited in the ProteomeXchange with the dataset identifier PXD021107. Comparative proteomic analyses revealed a total of 801 proteins, with 392 classified as differentially accumulated proteins in at least one of the developmental stages. The globular-staged presented a higher number of unique proteins (16), and 7 were isoforms of 60S ribosomal proteins, suggesting high translational activity at the beginning of somatic embryogenesis. Proteins related to mitochondrial metabolism accumulated to a high degree at the early developmental stages and then decreased with increasing development, and they contributed to cell homeostasis in early somatic embryos. A progressive increase in the accumulation of vicilin, late embryogenesis abundant proteins and chloroplastic proteins that lead to somatic embryo maturation was also observed. The differential accumulation of acetylornithine deacetylase and S-adenosylmethionine synthase 2 proteins was correlated with increases in putrescine and spermidine contents, which suggests that both polyamines should be tested to determine whether they increase the conversion rates of globular- to cotyledonary-staged somatic embryos. Taken together, the results showed that somatic embryo development in C. papaya is regulated by the differential accumulation of proteins, with ribosomal and mitochondrial proteins more abundant during the early somatic embryo stages and seed maturation proteins more abundant during the late stages.     

Histone phosphorylation during sea urchin development

Studies on histone phosphorylation during transitions in chromatin structure occurringin vivoduring spermatogenesis and early embryogenesis in sea urchins are reviewed and evaluated in the light of recent studies on histone phosphorylation occurring during chromatin synthesis in frog egg extractsin vitroand evidence that protein kinases and phosphatases play direct roles in the regulation of cellular structure. Sperm-specific histone variants Sp H1 and Sp H2B are maintained as phosphorylated derivatives N and O/P throughout spermatogenesis and early embryogenesis and egg specific histone variants CS H1 and CS H2A are phosphorylated during early embryogenesis. These developmental correlations provide clues about the roles of histone phosphorylation in control of chromatin structurein vivoand provide a basis for the interpretation of data obtained from in-vitro sperm chromatin remodeling in egg extracts and from biochemical studies on the effects of histone phosphorylation on DNA binding. The potential consequences for chromatin structure of the various histone phosphorylation events observed in sea urchins and frog egg extracts are discussed.     

Repair of a genetically-caused defect in oogenesis in Drosophila melanogaster by transplantation of cytoplasm from wild-type eggs and by injection of pyrimidine nucleosides

Eggs produced by homozygous mutant rudimentary (r⁹, 154.5) females of Drosophila melanogaster die during embryogenesis, apparently because the mutant female fails to incorporate certain substances into the egg during oogenesis. These eggs can be rescued by injecting them at the preblastoderm stage with wild-type nuclei and cytoplasm or wild-type cytoplasm alone from unfertilized eggs. Some preblastoderm eggs injected with 1.5% of egg volume of cytoplasm from unfertilized wild-type eggs were able to complete both embryonic and postembryonic development and emerged as adults, whereas not a single uninjected control egg was able to complete embryonic development. The eggs of rudimentary mothers can also be rescued by injecting each egg at the blastoderm stage with 0.01 μg of pyrimidine nucleosides. The results demonstrate that a pyrimidine deficiency is the cause of abortive embryogenesis, and confirm the previous finding that the rudimentary mutation leads to pyrimidine auxotrophy.     

Growth and protein profile changes in Lepidium sativum L. plantlets exposed to cadmium

Plant metabolic response to heavy metal stress is still to be clarified. The present investigation was undertaken to examine the influence of different concentrations of cadmium on the Lepidium sativum L. plantlets. Exposure of seedlings of L. sativum L. to increasing concentrations of cadmium results in the growth inhibition and in the accumulation of proteins in the 10–25 kDa range in cotyledons and hypocotyls of the plantlets. Most of these proteins are also found in extracts of L. sativum seeds. Analysis by ESI-MS after two-dimensional electrophoresis showed that these proteins exhibit sequences similar to those of storage proteins from various Cruciferae species. The response to metal exposure during germination and initial plantlet elongation thus involves inhibition of both storage protein catabolism and plant protein anabolism. In addition, two of the proteins present in higher amounts in plantlets exposed to cadmium heat-shock, in agreement with literature data, and jasmonate-like inducible protein are related to cellular stress and another two (LEAs or late embryogenesis abundant) are involved in embryogenesis. Changes in protein expression can be detected by two-dimensional electrophoresis after exposure to heavy metal concentrations lower than those at which morphometric changes become evident. Proteomics of germinating L. sativum thus constitutes a very sensitive tool for evaluating environmental pollution.     

Site and timing of synthesis of tubulin and other proteins during oogenesis in Drosophila melanogaster

Protein synthetic patterns during oogenesis in Drosophila melanogaster were examined; in particular the site, time, and rate of tubulin synthesis and accumulation during oogenesis were determined. Ovarian proteins were labeled with [³⁵S]methionine in vivo or in organ culure in vitro, and the proteins synthesized in egg chambers of specific developmental stages displayed by two-dimensional gel electrophoresis. A dissection technique was devised to examine proteins synthesized in each of the three cell types present in stage 10B egg chambers. The majority of proteins which were resolved by two-dimensional gel electrophoresis, including tubulin and actin, were synthesized throughout oogenesis and, at least to some extent, in each of the stage 10B cell types. Protein synthesis specific to developmental stage and/or cell type was also observed; for example, two nonchorion proteins were synthesized only in follicle cells and primarily at stage 10. A sensitive and specific radioimmune assay was developed in order to quantitate tubulin accumulation. Synthesis of several α-tubulin subunits and one β-tubulin subunit was observed. The tubulin content per egg chamber increased from 3 ng in stage 9 to 17 ng in stage 14, a period of about 13 hr. An accumulation rate of 1 ng/hr suggests that tubulin mRNA can account for about 4% of the total, nonmitochondrial, poly(A)⁺ RNA of the egg. Analysis of separated cell types at stage 10B revealed that both the follicle and nurse cells synthesize and accumulate appreciable amounts of tubulin. The stage 10B oocyte contains relatively little tubulin but actively synthesizes it. These two complementary analyses demonstrate that the tubulin present in the egg is synthesized within the oocyte-nurse cell syncytium, first in the nurse cells and later in the oocyte.     

A Chemical and Physical Characterization of Echinoid RNA during Early Embryogenesis

The properties of the major RNA components prepared from Arbacia punctulata have been characterized by sedimentation in sucrose gradients and analysis of base composition. Comparison of the components found in the unfertilized egg with those observed subsequent to fertilization revealed no differences in any of the embryonic stages examined. The base composition and sedimentation profiles of RNA from unfertilized nuclear egg fragments and from 105,000 g pellet were similar to those of the intact egg. It is concluded that the early stages of embryogenesis are not accompanied by detectable alteration of the physical or chemical characteristics of the major RNA components found in the unfertilized egg.     

Effects of jasmonic Acid on embryo-specific processes in brassica and linum oilseeds

A number of effects on embryogenesis of the putative phytohormone jasmonic acid (JA), and its methyl ester (MeJA), were investigated in two oilseed plants, repeseed (Brassica napus) and flax (Linum usitatissimum). Results from treatments with JA and MeJA were compared with those of a known effector of several aspects of embryogenesis, abscisic acid (ABA). Jasmonic acid was identified by gas chromatography-mass spectrometry as a naturally occurring substance in both plant species during embryo development. Both JA and MeJA can prevent precocious germination of B. napus microspore embryos and of cultured zygotic embryos of both species at an exogenous concentration of >1 micromolar. This dose-response was comparable with results obtained with ABA. Inhibitory effects were also observed on seed germination with all three growth regulators in rapeseed and flax. A number of molecular aspects of embryogenesis were also investigated. Expression of the B. napus storage protein genes (napin and cruciferin) was induced in both microspore embryos and zygotic embryos by the addition of 10 micromolar JA. The level of napin and cruciferin mRNA detected was similar to that observed when 10 micromolar ABA was applied to these embryos. For MeJA only slight increases in napin or cruciferin mRNA were observed at concentrations of 30 micromolar. Several oilbody-associated proteins were found to accumulate when the embryos were incubated with either JA or ABA in both species. The MeJA had little effect on oilbody protein synthesis. The implications of JA acting as a natural regulator of gene expression in zygotic embryogenesis are discussed.     

Immunolocalization of Non-Symbiotic Hemoglobins During Somatic Embryogenesis in Chicory

Hemoglobins are ancient O₂-binding proteins, ubiquitously found in eukaryotes. They have been categorized as symbiotic, nonsymbiotic and truncated hemoglobins. We have investigated the cellular localization of nonsymbiotic hemoglobin proteins during somatic embryogenesis in Cichorium hybrid leaves (Cichorium intybus L. var. sativum × C. endivia var. latifolia) using immunolocalization technique. These proteins were detected during the two steps of culture: induction and expression. In leaves, hemoglobins colocalised with plastids, which were dispersed in the parietal cytoplasm as well as in the two guard cells of a stomata, but not in epidermis cells. Upon induction of embryogenesis, in the dark, this pattern disappeared. During the induction phase, where competent cells reinitiate the cell cycle and prepare for mitosis, hemoglobins appeared initially near chloroplasts, and then in the vicinity of vascular vessels especially in the phloem and in cells surrounding the xylem vessels. When leaf fragments were transferred to another medium for the expression phase, hemoglobins were observed in the majority of the leaf blade cells and in small young embryos but not in the older ones. Hemoglobins were also detected in other leaves cells or tissues all along the process. The role of these nonsymbiotic hemoglobins during somatic embryogenesis is discussed.Key Words: chicory, immunolocalization, nonsymbiotic hemoglobin, somatic embryogenesis     

Proteomic analysis of egg white proteins during storage

Egg storage causes egg white to lose its viscous nature to form a thin liquid, commonly referred to as egg white thinning. To understand the mechanisms underlying egg white thinning, white-shell eggs were used in the present study to determine the proteome-level changes of egg white proteins occurred during storage. Egg white thinning was observed visually after 20 days of storage at ambient temperature (22 ± 2 °C) when the maximum number of proteome-level changes occurred. The proteins that showed significant changes in abundance during storage included ovalbumin, clusterin, ovoinhibitor, ovotransferrin, and prostaglandin D2 synthase. Among these, only the abundance of clusterin was observed to change continuously during the storage period. Hence, it is expected that the increase in the concentrations of clusterin and ovoinhibitor along with the change of ovalbumin content during storage might contribute to egg white thinning. Degradation of ovalbumin/clusterin during egg storage may be due to the combined effect of proteolysis and increase in pH; this may also be partly responsible for egg white thinning phenomenon.