Developmental Changes in the Free Amino Acid Pool of Rice(Oryza sativa L. subsp,japonica) Bmbryos

The level of/various amino acids in rice embryos rose sharply during embryo differentiation (7–9 days after anthesis). Then it increased steadily or tended to become stable at mid-maturation stage (13–18 days after anthesis), thereafter continuing to increase, except that the contents of aspartate and glutamate decreased significantly. The total free amino acid pool expanded rapidly during the differential stage. There after the pool capacity showed only a slightly increase until the end of embryogenesis. Both on embryo cell and dry weight bases, the capacity reached the maximum at the 9th day after anthesis, then decreased at the 13th day, and later remained stable. We deemed that the establishment of the free amino acid pool is one of the events which occur in the process of rice embryo differentiation. By the fulfillment of the differentiation (the 13th dray after anthesis), the pool capacity within the embryo cells remained stable on the whole. The free amino acid pool was dominated by serine, alanine, aspartate and glutamate during the differentiation stage. In the maturation stage, serine, alanine, arginine and lysine were the main components. These predominant; amino acids may play an important role in regulating the availability of the whole amino acid pool.     

向日葵幼胚培养中体细胞胚胎发生的观察

1.向日葵不同品种体细胞胚胎发生的情况不同。2.较高浓度的蔗糖有利于向日葵幼胚的体细胞胚胎发生。3.在同样条件下,2mm长的幼胚较其它时期的幼胚体细胞胚胎发生的频率高。4.在蔗糖浓度为17.5％并分别加入0.5—10.0ppm玉米素的Nitsch培养基中,向日葵幼胚产生体细胞胚胎发生的频率随着玉米素浓度的增高而增加。5. 2,4-D能使体细胞胚胎发生,但不能分化器官。6.切片观察表明:在含玉米素的培养基上,幼胚产生了胚性细胞团和胚状体。并多数发生于子叶与下胚轴的深层。胚性细胞团周围细胞退化,使其与周围组织之间形成间隙。     

水稻胚胎发育时期的特异性蛋白质

对不同发育时期的水稻胚蛋白质,进行了单向SDS聚丙烯酰胺凝胶电泳和双向凝胶电泳分析。单向电泳图中存在20kD左右、胚分化完成期以后特有的蛋白质。根据双向电泳图谱,稻胚总蛋白按其在胚胎发育过程中的动态可分为7类,其中组成性的蛋白质约占85％,在不同发育时期基本不变;具有发育时期特异性的蛋白质约占15％。以分化期胚蛋白质的抗体,对各时期的胚蛋白质进行交叉免疫电泳,所得图谱总体上是相似的,但不同时期的图谱之间出现沉淀峰的峰形、位置和数目的差异。交叉免疫电泳图谱的这种变化可能反映了稻胚发育过程中某些分化期蛋白质发生的变化。     

Somatomedins/insulin-like growth factors, their receptors and binding proteins are present during mouse embryogenesis

Somatomedins/insulin-like growth factors (Sm/IGFs) are considered to have important roles in regulating fetal growth; however, because of limited quantities of tissue, few studies have been performed on their effects on embryonic growth. To assess a potential role for these factors, we evaluated mouse embryonic tissues for the presence of Sm/IGF and insulin receptors and Sm/IGF-binding proteins by chemical affinity labelling. In addition, we measured extractable Sm-C/IGF-I radioimmunoactivity in mouse embryonic tissues. Finally, we compared these data with those from the embryonal carcinoma cell line, PC13. All embryos from day 9 (3-4 somites) to day 12 (45 somites) possessed both Sm-C/IGF-I and IGF-II receptors in apparent greater abundance than insulin receptors. The visceral yolk sac appeared to have proportionally more insulin receptors than the corresponding embryonic tissue. Extracts from the embryos contained immunoreactive Sm-C/IGF-I and binding proteins of 30-45 X 10(3) Mr. PC13 cells possessed all three receptors and the apparent abundance of the insulin and IGF-II receptors was reduced after differentiation was induced with retinoic acid. PC13 cells released both immunoreactive Sm-C/IGF-I- and Sm-C/IGF-I-binding proteins into their medium. When differentiated, the binding proteins resembled the native ones extracted from the intact embryos. The presence of Sm/IGF activity, receptors and binding proteins in early embryogenesis suggests a role for these factors in embryonic growth. The PC13 cell line appears to only partially reflect normal development.     

Protein synthesis in embryonic tissues during mouse postimplantation development

Mouse embryos of the NMRI strain between the 7th and 9th day of gestation were isolated from the uterus and dissected into the various tissue derivatives in order to investigate newly synthesized proteins during morphogenesis. The day 7 embryo was fragmented into trophoblast and ectoplacental cone, distal and proximal endoderm, extraembryonic and embryonic ectoderm. The day 8 and day 9 embryos were divided into trophoblast and placental anlage, yolk sac, amnion, and allantois, as well as cranial, central, and caudal embryonic tissue. The intact embryos were incubated in Dulbecco's minimum essential medium in the presence of 35S-methionine for 4 h, then dissected into the various fragments, and further processed for two-dimensional gel electrophoresis. Protein synthesis of the isolated tissue derivatives was analyzed and compared for the three developmental stages. Concerning the proteins with isoelectric points in the range of 4.5 to 8.0 and molecular weight ratio (M(r)) values between 20,000 and 200,000, we found several significant quantitative and qualitative differences in the various tissue fragments. In addition, we observed further quantitative and qualitative differences in protein synthesis during the postimplantation period investigated. We propose that the differences reflect some of the cell lineage- and developmental stage-specific changes in gene expression during early mammalian differentiation.     

Differential expression of c-Jun proteins during Müllerian duct growth and apoptosis: caspase-related tissue death blocked by diethylstilbestrol

To elucidate whether the differentiation of the Müllerian duct (MD) is mediated by c-Jun proteins, Western immunoblot with c-Jun/sc-45 antibody was used to investigate these proteins in female chick left and right MDs (LMD and RMD, respectively). The content of these proteins (e.g., the 66-kDa, 45-kDa, and 39-kDa forms) in the LMD or RMD of various stages of embryos was detected by measuring their density in autoradiograms by a Spot-denso-program with Alpha Ease software. In the LMD, the growing embryonic sex tract, the content of the 66-kDa and 39-kDa proteins increased to their highest level in 9th to 12th day embryos and then declined thereafter. In the RMD, the apoptotic embryonic sex tract, the content of these proteins also showed a linear increase from the 9th to 10th day and then declined at the 13th day. When the RMD entered the apoptosis stages (14th to 18th day of incubation), these proteins were persistently overexpressed. Another protein (45 kDa) was detected in both ducts only at the 9th to 13th days, and its content was higher in RMD than in the LMD. In parallel to this finding, high caspase-3 activity (determined by the measurement of the fragmented 85-kDa poly ADP-ribose polymerase) was found in the RMD during apoptosis. The apoptotic death of RMD was prevented by in vivo diethylstilbestrol treatment, which inhibited the overexpression of the 66-kDa and 45-kDa proteins, the fragmentation of DNA, and the activity of caspase-3. No inhibitory effect was found for the 39-kDa protein.     

水稻胚与胚乳分化发育中的内源多胺

稻胚发育过程中,其内源多胺以腐胺、亚精胺为主。在幼胚分化期,腐胺和亚精胺的含量很高;幼胚分化完成时,其含量急剧下降;直至分化后期才趋稳定。在胚及胚乳发育时期,还出现一种未知多胺X_(22),其含量除在胚分化完成时较少外,在胚发育的其他各期中,含量则一直很高。DNA和蛋白质含量的变化,从分化期开始递增直至物质积累成熟期,其趋势均相同。多胺可能参与胚与胚乳中核酸和蛋白质合成的调节。     

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.     

Characteristics of embryos from repeat breeder and virgin heifers

The characteristics of 7-day-old embryos non-surgically collected from 35 repeat breeder heifers (RBH) and 24 virgin heifers (VH) were compared by repeated observations within each animal. A higher incidence of the embryos collected from the VH was classified as normal and had reached a more advanced developmental stage than embryos from the RBH. Nearly all VH yielded normal (N) embryos, but morphologically deviated (MD) or degenerated embryos (D) appeared occasionally in many VH. The RBH group contained three subgroups of animals. One group of RBH yielded a high percent of N embryos. A second group yielded mostly MD or D embryos with an occasional N embryo, and the third group only D embryos or no embryos. Heifers from which no embryos were recovered on day 7 yielded uncleaved ova, apparently retarded embryos or no embryos when slaughtered three days after insemination. It is concluded that retarded embryonic development may be a common factor for most RBH. The embryo morphology and the degree of retardation differ among animals and between oestrous periods in the same animal.     

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.     

Novel clues on abiotic stress tolerance emerge from embryo proteome analyses of rice varieties with contrasting stress adaptation

Cereal embryos sustain severe water deficit at the final stage of seed maturation. The molecular mechanisms underlying the acquisition of desiccation tolerance in seed embryos are similar to those displayed during water deficit in vegetative tissues. The genetic variation among six rice genotypes adapted to diverse environmental conditions was analysed at the proteome level to get further clues on the mechanisms leading to water-stress tolerance. MS analysis allowed the identification of 28 proteins involved in stress tolerance (late embryogenesis abundant proteins), nutrient reservoir activity, among other proteins implicated in diverse cellular processes potentially related to the stress response (e.g., mitochondrial import translocase). Hierarchical clustering and multidimensional scaling analyses revealed a close relationship between the stress-sensitive genotypes, whereas the stress-tolerant varieties were more distantly related. Besides qualitative and significant quantitative changes in embryo proteins across the distinct varieties, we also found differences at post-translational level. The results indicated that late embryogenesis abundant Rab21 was more strongly phosphorylated in the embryos of the sensitive varieties than in the embryos of the tolerant ones. We propose that the differences found in the phosphorylation status of Rab21 are related to stress tolerance.     

The role of arabinogalactan proteins binding to Yariv reagents in the initiation, cell developmental fate, and maintenance of microspore embryogenesis in Brassica napus L. cv. Topas

Arabinogalactan proteins (AGPs) are extracellular proteoglycans involved in plant growth and development. The addition of beta-D-glucosyl Yariv reagent (betaGlcY), a synthetic phenylglycoside that specifically reacts with AGPs, to the culture medium notably disturbed microspore embryogenesis in a concentration-dependent manner. The initiation of microspore embryogenesis was clearly inhibited by 30 microM betaGlcY and completely inhibited by 50 microM betaGlcY. The transfer of microspore-derived embryos at different developmental stages into NLN6 medium containing 50 microM betaGlcY prohibited their normal development, as approximately 21.24, 43.99, and 59.73%, respectively, of the treated globular-, heart-, and torpedo-stage embryos exhibited numerous root hair-like structures. Both heart-stage and torpedo-stage embryos showed a rapid growth of roots with a large number of clustered root hairs. Some root hair-like structures were also observed on the apical portions of embryos. Microscopy of the treated embryos revealed that the basic patterns of cells at both the radial and apical-basal axes were greatly altered, such that the cells lost their ability to carry out programmed embryogenesis. These results show that the betaGlcY-AGP interaction modulates the developmental fate of embryonic cells, especially epidermal cells, and thereby strongly affects root generation and development. Immunofluorescence microscopy revealed that both JIM8 and JIM13 binding to AGP co-localize with betaGlcY-binding sites. Thus, AGPs binding to betaGlcY, co-localized with Jim8- and Jim13-binding protein, appear to play a crucial role in the initiation of Brassica microspore embryogenesis and the maintenance of cell differentiation during embryonic development. In addition, these proteins may also be involved in the regulation of root generation.     

Embryogenic and callus-specific proteins in somatic embryogenesis of the grass,Dactylis glomerata L.

Somatic embryogenesis occurs spontaneously in some monocotyledoneous callus and cell suspension cultures maintained in suitable culture conditions. Nevertherless, the processes involved in somatic embryo development, and factors inducing this differentiation, are poorly understood. In order to study the changes in protein composition accompanying embryogenesis in cell suspension cultures of Dactylis glomerata L., embryos of various sizes and “undifferentiated” callus cells were separated and their total cellular protein extracts analyzed by two-dimensional polyacrylamide gel electrophoresis. Several proteins could be identified that are specific for embryos or callus under various culture conditions. Three independent detection methods were employed: silver-staining of proteins, in vivo labeling of proteins with [³⁵S]methionine, and in vitro translation of poly(A)⁺ RNA. All culture conditions tested, including those that induce embryonic proteins in carrot, fail to induce embryonic proteins in D. glomerata callus cells.     

Cell differentiation of the head ectoderm in mouse embryos in vitro

Cell differentiation has been studied in the explants of head ectoderm of 8, 9 and 10 day old mouse (CBA) embryos and of head epidermis of 13 day old embryos. Pieces of ectoderm were taken from the temporal region. It was established by indirect immunofluorescence that within 10, 15 and 20 days of cultivation spheroids with keratins or crystallins in some groups of fibres formed in the head ectoderm explants from 9 and 10 day old embryos. When cultivating the regions of head epidermis from 13 day old embryos, spheroids formed with keratin only in their cells. The data obtained suggest that there appear to be two clones of cells determined to the synthesis of keratins or crystallins in the head ectoderm of early mouse embryos. During embryogenesis, the number of cells determined to the synthesis of keratins appears to increase in the regions not related to the eye area. At the same time, the clone of cells determined to the synthesis of crystallins appears to be eliminated.     

Comparative analysis of early embryonic sunflower cDNA libraries

To gain information concerning cell functions and activities during sunflower embryogenesis, an expressed sequence tag (EST) approach was used to analyse gene expression in the early stages of sunflower embryos development. Confocal microscopy observations of whole-mounted embryos allowed us to identify precisely the major steps of the zygotic embryonic development. A time-course analysis was then employed to collect the embryonic material. Three cDNA libraries were constructed from microdissected embryos, and three other cDNA libraries were created using a classical day after pollination schedule. A total of 7106 ESTs were produced and assembled. The total number of putative different genes represents about 43.1 (3064 tentative contigs and singlets) of the analysed sequences. The unigenes that showed similarity to proteins with known or predicted functions (50.3) were classified into 15 different functional categories. The functional profiles were found to be quite similar for all studied embryo stages but statistical analysis revealed that successive and coordinate sets of genes are expressed at each embryonic stage. The analysis allowed us to identify abundant and differentially expressed genes at the early stages of embryos development as well as some putatively interesting genes, showing strong similarities with genes playing key roles in plant and animal embryogenesis. The data presented in this study not only provide a first global overview of the genes expression profile during sunflower embryogenesis but also represent an original and valuable tool for developmental genomics studies on exalbuminous dicots.     

Effect of acute hypoxia on embryonal death and the sex ratio of the progeny

The effect of acute hypoxia on mouse embryogenesis and its influence on the sex ratio in the progeny was studied. On the 7th--10th day of pregnancy mice were subjected to hypoxia at 230 mm HG for 3 hours. They were sacrificed on the 18th day of pregnancy, and gonads of their embryos were taken for investigation. The sex ratio was determined as the ratio of the number of males to that of females. It has been shown that acute hypoxia on the 7th--10th days of pregnancy, resulting in high mortality of adult animals, had no substantial effect on the embryonic development. No selective death of the embryos of either sex was observed both in experimental and control groups.