Serum protein synthesis in the early chick embryo

Isolated yolk-sacs of chick embryos secreted serum proteins when incubated in buffered chick Ringer's solution. The presence of serum transferrin, two embryo-specific alpha-globulins, and a prealbumin were demonstrated by acrylamide gel analysis. Yolk-sacs from embryos explanted at 11-13 somites (40 hr preincubation) and cultured for 48 hr secreted in addition a protein with the mobility of serum albumin. Incubation of yolk-sacs in the presence of radioactive valine indicated that serum proteins were synthesized as early as the primitive streak stage. By incubating isolated yolk-sacs and embryos from 48-hr explants in the presence of radioactive valine, the synthesis of serum proteins was found to be restricted to the yolk-sac at this stage of development. Culturing explants on various nutrient proteins as well as protein starvation medium altered the relative synthesis of several serum proteins. We have proposed that morphological and biochemical changes in embryos resulting from altered nutrition may be mediated by the proteins of the serum.     

Analysis of proteins secreted by mouse embryos developing in vivo and in vitro

Proteins secreted by mouse blastocysts developing in vitro were compared to these from blastocysts developing in utero to determine if a simple medium supporting blastocyst development also supports secreted protein expression. In-vivo embryos were collected on days 3, 4, or 5 of pregnancy and incubated in 35S-methionine to produce conditioned medium containing released, labeled proteins. Embryos for culture were collected on day 3 and after 48 or 72 h labeled conditioned medium was produced. Labeled proteins were separated by two-dimensional electrophoresis and compared using a digital image analysis system. Day 3 embryos did not release proteins in detectable amounts, although synthesis of intracellular proteins was substantial. Day-4 and -5 blastocysts released proteins in increasing amount and complexity, consistent with previous results. When day-3 embryos were cultured in medium containing 4 mg/ml BSA for 48 h, secreted protein patterns were similar but not identical to those of day-5 uterine blastocysts. Although most of the proteins produced by uterine blastocysts were secreted by cultured embryos, differences were found in the relative quantities of certain proteins. Neither crystallized BSA nor polyvinyl alcohol at 4 mg/ml supported development of protein secretion as well as the crude fraction-V BSA. Blastocysts restricted to the oviduct also exhibited quantitative differences in protein secretion patterns compared to uterine blastocysts. Thus, although blastocyst development and the expression of many secreted proteins are supported outside the uterus, the full pattern of secretion characteristic of the peri-implantation embryo may be dependent on specific uterine influences.     

Identification of proteins secreted from axons of embryonic dorsal-root-ganglia neurons

Secretion of proteins from the growth cone has been implicated in axon growth and synapse formation and might be involved in the transmission of a variety of axon-derived regulatory signals during neurogenesis. In order to identify axonally secreted proteins, dorsal-root-ganglia neurons from chicken embryos were cultured in a compartmentalized cell culture system that allows separate access to neuronal cell somas and axons. The proteins synthesized by the neurons were metabolically labeled by addition of [35S]methionine to the compartment containing the cell somas; the proteins released from the axons were harvested from the culture medium of the axonal compartment. Two-dimensional gel electrophoresis revealed two axonally secreted proteins with apparent molecular mass of 132-140 kDa and 54-60 kDa; they were termed axonin-1 and axonin-2, respectively. Both axonins were found to be secreted from a variety of neuronal cell cultures, but not from any of the nonneuronal cultures investigated, and hence might be neuron-specific. Virtual absence of these proteins from the axonal protein pattern suggests constitutive secretion. The information acquired on coordinates and spot morphology of these proteins in two-dimensional gel electrophoresis provides a useful assay for their purification.     

Uterine and oviducal protein secretion during early pregnancy in the mouse

Changes in the protein composition of the embryo's environment during early development were studied by analysis of proteins synthesized and secreted by oviducal and uterine explants on Days 1-6 of pregnancy. Although secretions from ampullar and isthmic oviduct and uterus contained many proteins in common, each area also produced its own characteristic proteins. In the uterus, changes in the secretion pattern were found during the peri-implantation period, including both increases and decreases in particular proteins which appear to be dependent on the presence of embryos. Embryo-induced effects on uterine secretion began between 09:00 h of Day 4 and 09:00 h of Day 5. Oviducal secretions exhibited many of the embryo-dependent proteins found in the uterus, but the expression of these proteins did not appear to be influenced by the presence of embryos on Day 1 or Day 3. The characteristic pattern of secreted protein expression by each portion of the reproductive tract may reflect the specialization of each area for certain developmental events.     

Cotesia kariyai teratocytes: growth and development

The growth and development of teratocytes was examined in the Cotesia kariyai-Pseudaletia separata system. Cotesia kariyai embryos released an average of 163 teratocytes at the time of hatching, 3.5 days after oviposition. The cells increased in diameter from 30 to 77 μm until 7 days post-parasitization, after which there was no significant increase in average diameter. However, there was significant variation in diameter within the population of teratocytes during the later developmental stages of the parasitoid larvae. The DNA contents increased up to day 7. The ploidy level of teratocytes increased 4-fold (2C to 8C) from days 4 to 7 and thereafter remained the same. Scanning electron microscopy (SEM) revealed that the surface of the teratocytes was covered with microvilli during all developmental stages, although on days 9 to 10 post-parasitization, bleb structures were also observed on a few. In vitro analysis of the proteins secreted from teratocytes following labeling with (35)S-methionine showed that many proteins were synthesized de novo and secreted by the cells until 9 to 10 days post-parasitization. These results indicate that teratocytes in later stages of development maintain their activity and regulate the physiological state of the host.     

A comparison of protein synthetic patterns in normal and animalized sea urchin embryos

Newly synthesized proteins from normal and animalized sea urchin embryos were compared by the technique of double labeling. Total embryonic protein was solubilized in SDS, urea, and 2-mercaptoethanol. The proteins were examined by coelectrophoresis on an SDS-polyacrylamide gel. The gels were sliced and the radioactivity determined. A standardized ratio of the isotopes served as the basis of comparison. A comparison of newly synthesized proteins from normal embryos 24 and 48 h old showed a shift from larger to smaller molecular weight proteins. Animalized embryos showed a similar shift. When normal and animalized embryos of the same ages were compared, differences were found. The differences were distributed over the entire range of molecular weights. These results show that although differences in protein synthesis between animalized and normal embryos are evident by 24 h, most of the changes in protein synthesis that occur in normal embryos are unaffected by animalization.     

The metencephalic floor plate of chick embryos expresses two secretory glycoproteins homologous with the two glycoproteins secreted by the subcommissural organ

The nature and the function of the compounds secreted by the floor plate (FP) of the metencephalon are little known. The FP cells of the hindbrain react with antibodies (AFRU) against the glycoproteins secreted by the subcommissural organ (SCO). One of the these proteins, RF-Gly I, is a 540-kDa core glycosylated protein. The aims of the present investigation were to identify by immunoblot the AFRU-immunoreactive compound secreted by the FP of chick embryos, to establish temporal and regional patterns of this secretory activity, and to obtain information about the fate of these compounds. It was established that the SCO and FP of chick embryos secrete two AFRU-immunoreactive compounds of 540 and 230 kDa. The two compounds secreted by the FP have been designated as FP-Gly I and FP-Gly II. The expression of these proteins was circumscribed to the metencephalic FP, and occurred from HH 29 to HH 36. Within the FP cells, FP-Gly I and FP-Gly II were confined to the supranuclear and apical regions, which under the electron microscope displayed numerous cisternae of the rough endoplasmic reticulum and granules. Aggregates of AFRU-immunoreactive material appeared on the free surface of the FP. The possibility that FP-Gly I and FP-Gly II are released into the ventricle to reach distant targets is discussed.     

Extracellular proteins in embryogenic suspension cultures of Norway spruce (Picea abies)

Embryogenic cell lines of Norway spruce ( Picea abies ) varying in growth habit and morphology were compared as regards profiles of extracellular proteins. Similar proteins were detected in the culture medium by SDS PAGE and in vivo labeling experiments, indicating that the proteins were secreted. Approximately 20 protein bands could be detected in the medium of each cell line. Three of the bands represented glycosylated proteins, as revealed by Concanavalin A staining. Some of the secreted proteins were similar for all tested embryogenic lines of Norway spruce, others were either specific for a group of cell lines or for individual cell lines. A correlation was observed between the morphology of the somatic embryos in a cell line and the presence of secreted proteins. The embryogenic cell lines of Norway spruce can be divided into two main groups. A and B, where A is characterized by somatic embryos with dense embryoheads and B by somatic embryos with loosely aggregated cells in their embryoheads. When proteins secreted from a cell line belonging to group A were added to cell lines belonging to group B, the somatic embryos of the B type developed further and became more similar in morphology to A-type embryos. These observations indicate that cell lines belonging to group A secrete certain proteins to the culture medium that are essential for the development of somatic embryos of Norway spruce.     

Synthesis of heat shock proteins in Drosophila melanogaster embryos

The pattern of polypeptides synthesized in a cell-free protein synthesizing system containing polysomes isolated from heat-shocked (37 C) Drosophila embryos showed significant differences when compared with the pattern obtained when polysomes from normal embryos were used. The synthesis of normal embryonal proteins was reduced and the heat shock proteins were the major products of elongation. After short, 10 min, heat treatment mainly quantitative changes were observed suggesting that normal mRNAs were still present on polysomes, and their products could be completed in vitro in the heterologous cell-free system. The mRNAs coding for normal embryonal proteins were present in almost unchanged amounts in heat-shocked embryos as could be judged from the pattern of proteins synthesized in heterologous cell-free system supplemented with cytoplasmic RNA from normal and heat-shocked embryos. Thus the change in protein synthesis in heat-shocked embryos is not associated with degradation of normal embryonal mRNAs but with their inaccessibility for translation.     

Characterization of a diverse secretome generated by the mouse preimplantation embryo in vitro

This study investigates the suitability of surface-enhanced laser desorption and ionization time-of-flight (SELDI-TOF) and electrospray ionization (ESI) mass spectrometry for analysis of the proteins released by the mouse preimplantation embryo in vitro. SELDI-TOF analysis with CM10 or IMAC30 (but not Q10) protein chips detected a protein peak at m/z ~8570 released by both C57BL6 and hybrid embryos. No other peaks unique to the presence of the embryo were identified with this method. ESI mass spectrometry of tryptic digests of embryo-conditioned media identified a total of 20 proteins released during development from the zygote to blastocyst stage. Four proteins were expressed in at least 7 out of 8 cultures tested, one of these (lactate dehydrogenase B) was in all cultures. A further five proteins were in at least half of the cultures and 11 more proteins were in at least one culture. The expression of two of these proteins is essential for preimplantation embryo development (NLR family, pyrin domain containing 5 and peptidyl arginine deiminase, type VI). A further four proteins detected have roles in redox regulation of cells, and three others are capable of inducing post-translational modifications of proteins. This study shows the feasibility of ESI mass spectrometry for identifying the proteins secreted by the preimplantation embryo in vitro. This analysis identifies a range of targets that now require detailed functional analysis to assess whether their release by the embryo is an important property of early embryo development.     

Patterns of protein synthesis and metabolism during sea urchin embryogenesis

We have analyzed the patterns of protein synthesis in developing embryos of the sea urchin Strongylocentrotus purpuratus by two-dimensional gel electrophoresis. There was an increase in the number of proteins detectably synthesized during development, as well as significant changes in relative rates of synthesis involving approximately 20% of the nearly 900 newly synthesized polypeptides. The majority of these changes were increases rather than decreases in synthesis; about half were of at least 10-fold, while a few were of more than 100-fold. Very few changes were detected upon fertilization and during the first several hours of development, while about 60% of the changes detected occurred between the hatching and the beginning of invagination. An analysis of proteins detected by silver staining indicated that most remained nearly constant in mass during embryonic development, but several increased or declined substantially. Many proteins present in eggs were not detectably synthesized in either eggs or embryos.     

Mouse Wnt receptor gene Fzd5 is essential for yolk sac and placental angiogenesis

Wnts are secreted signaling molecules implicated in various developmental processes and frizzled proteins are the receptors for these Wnt ligands. To investigate the physiological roles of frizzled proteins, we isolated and characterized a novel mouse frizzled gene Fzd5. Fzd5 mRNA was expressed in the yolk sac, eye and lung bud at 9.5 days post coitum. Fzd5 specifically synergized with Wnt2, Wnt5a and Wnt10b in ectopic axis induction assays in Xenopus embryos. Using homologous recombination in embryonic stem cells, we have generated Fzd5 knockout mice. While the heterozygotes were viable, fertile and appeared normal, the homozygous embryos died in utero around 10.75 days post coitum, owing to defects in yolk sac angiogenesis. At 10.25 days post coitum, prior to any morphological changes, endothelial cell proliferation was markedly reduced in homozygous mutant yolk sacs, as measured by BrdU labeling. By 10.75 days post coitum, large vitelline vessels were poorly developed, and the capillary plexus was disorganized. At this stage, vasculogenesis in the placenta was also defective, although that in the embryo proper was normal. Because Wnt5a and Wnt10b co-localized with Fzd5 in the developing yolk sac, these two Wnts are likely physiological ligands for the Fzd5-dependent signaling for endothelial growth in the yolk sac.     

Stage-specific inhibition of Xenopus embryogenesis by aprotinin, a serine protease inhibitor.

We examined the effects of various protease inhibitors on Xenopus laevis embryogenesis. Aprotinin, a serine protease inhibitor, was found to inhibit embryogenesis markedly, but other protease inhibitors had virtually no effect. The inhibitory effect of aprotinin was specific for embryos at the blastula or gastrula stage. These results suggest that an aprotinin-sensitive protease involved in embryonic development is secreted from the embryos or appears on the surface of embryonic cells at these stages. We found that various serine proteases are in fact secreted from the embryos with their development and that some of them are sensitive to aprotinin.     

Estradiol secretion by the ovary of 19-day-old hypophysectomized and sham-operated chick embryos

The amounts of estradiol released into culture media by ovaries from 19-day-old hypophysectomized (decapitated) and sham-operated chick embryos were determined by RIA. Per single ovary, ovaries from decapitated embryos secreted slightly less estradiol than ovaries from sham-operated ones during a 4-h culture period (837 +/- 413 vs 935 +/- 378 pg), but this difference was not significant. On an ovarian weight basis, ovaries from decapitated embryos secreted slightly more estradiol than ovaries from sham-operated ones (1.15 vs 0.91 ng), but this difference was not significant either. It is concluded from these results that the hypophysis does not control estradiol secretion by the ovary in the 19-day-old chick embryo.     

Retinoic acid induction of stress proteins in fetal mouse limb buds

Retinoic acid (RA) is teratogenic in rodent embryos. Several teratogens have been shown to induce the synthesis of a subset of heat shock proteins (stress proteins) in Drosophila. To determine if RA induces the synthesis of these proteins in rodent embryos, pregnant ICR mice were dosed with 100 mg/kg RA on Day 11 of gestation. Forelimb buds were removed from embryos 2.5 hr post-RA-treatment and nuclei were isolated, stained, and sorted from stages of the cell cycle. Nuclear proteins were extracted and analyzed by two-dimensional polyacrylamide gel electrophoresis. Nuclear proteins with molecular weights of approximately 84 and 25 kDa were synthesized in embryos in the G0 + G1 phase after pregnant dams were treated with RA. Isoelectric points, molecular weights, immunochemical blotting, and polypeptide mapping demonstrated that these proteins are indistinguishable from stress proteins isolated under a variety of conditions from rat submaxillary glands and mouse lymphoma cells. These results suggest that treatment with RA induces the synthesis of a subset of stress proteins; the role of these proteins in the teratogenic effects of RA is not known.     

Developmental changes in teratocytes of the braconid wasp Cotesia congregata in larvae of the tobacco hornworm,Manduca sexta

The endoparasitic wasp Cotesia congregata develops in the hemocoel of larval stages of the tobacco hornworm, Manduca sexta. Teratocytes were released from the serosal membrane during hatching of the first instar wasp larva at 2-3days after oviposition; about 160 cells were released per embryo. The cells increased in diameter from about 10 to >200&mgr;m prior to wasp emergence. Nascent microvilli, visible on the cell surface before hatching of the first instar larva, rapidly increased in length and number following release of the cells. Irrespective of when the wasps were due to emerge, or how many parasitoids were present in the host, dramatic cytological changes occurred in the cells during the last instar of the host's development. Many of these morphological and ultrastructural changes were symptomatic of the cytological features of degenerating or apoptotic cells, and large numbers of vesicles appeared interspersed amongst the microvilli. The nucleus developed extensive dentritic ramifications, and the chromatin condensed in large clumps on the inner nuclear membrane. At the final stages of the wasps' development, the nucleus occupied the bulk of the interior of the cell. The cytoplasm gradually grew dramatically more electronluscent and less granular, as did the nucleoplasm, which is also indicative of impending cell death. Following the parasites' emergence, many of the cells underwent extensive blebbing of the cell surface. Teratocytes within a host appeared heterogeneous with respect to their morphological appearance. Analysis of the proteins secreted by teratocytes in vitro following labelling with (35)S-methionine showed that many (>30) polypeptides were synthesized de novo and secreted by the cells; some proteins were clearly targeted for secretion. We presume that the cells likely secrete a large number of proteins in vivo as well as in vitro.     

HMGB1, an architectural chromatin protein and extracellular signalling factor,has a spatially and temporally restricted expression pattern in mouse brain

HMGB1 is an abundant chromatin component, so far considered ubiquitous. HMGB1 also has an extracellular signalling role: when passively released by necrotic cells, it triggers inflammation; moreover, it can be actively secreted by myeloid cells, neurons and neuronal cancer cells. We show here that HMGB1 protein is undetectable in most cells in adult mouse brain, and is present in a subset of brain cells during development, with a very complex temporal, spatial and subcellular expression pattern. HMGB1 is expressed in the cortical plate of E14.5 embryos, predominantly in the nucleus, although roughly 1% of cells show a cytoplasmic localization as well. In E16 embryos, HMGB1 is nuclearly expressed in scattered cells apparently moving from the ventricular zone to the cortical plate. HMGB1 expression is strongly down-regulated at later developmental stages; in adult mice significant expression is maintained only in areas of continuing neurogenesis. Finally, HMGB1 subcellular localization changes during retinoic acid induced differentiation of P19 neuroblastoma cells.     

Protein secretion by the mouse trophoblast during attachment and outgrowth in vitro

Protein secretion from mouse blastocysts undergoing attachment and trophoblast outgrowth in vitro was assessed. When Day 5 blastocysts were cultured in serum-containing medium, secretion of several 'attachment-associated' proteins (PAS) was initiated within 24 h, coincident with attachment and outgrowth. Those proteins characteristic of the pre-attachment blastocyst disappeared or made-up only a small portion of the secretions once attachment began. The major secreted protein from attached embryos, PA1, is a 35,000-45,000 Mr acidic glycoprotein with multiple isoelectric forms. PA2, a group of basic 40,000 Mr proteins and PA3 a group of 72,000 Mr proteins were also produced during outgrowth. PAS were secreted during outgrowth on fibronectin-coated plastic in serum-free medium, but not by blastocysts held in a non-attachment state during culture in serum-free medium on uncoated plastic. In pre-attachment blastocysts, secreted proteins were produced by trophoblast vesicles, but not by isolated inner cell masses. Both trophoblast vesicles growing out in vitro and surgically isolated trophoblast from spreading blastocysts had secreted protein patterns qualitatively similar to those of intact blastocyst outgrowths. The results indicate that development of trophoblast protein secretion continues through the period of outgrowth and giant cell transformation. These changes are apparently dependent on attachment of the blastocyst to a suitable substrate, but not dependent on any other serum influence.     

Streptococcus pneumoniae proteins released into medium upon inhibition of cell wall biosynthesis

Inhibition of murein biosynthesis in Streptococcus pneumoniae by either penicillin or bacitracin leads to an increase in the amount of protein secreted into the medium. This process was studied in wild-type cells grown under lysis-permissive conditions as well as in an autolysin-deficient mutant. The time course of secretion did not follow cellular lysis but commenced immediately after the addition of the cell wall inhibitor in a manner similar to that described recently for cell wall and membrane components in various tolerant streptococci. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that this increase was not due to the stimulation of release of three protein components which are secreted under normal growth conditions; rather, a complex set of cellular proteins escaped from the antibiotic-treated pneumococci. The proteins released during bacitracin treatment was slightly different from those observed when penicillin was used. Analysis on sucrose gradients indicated that the secreted proteins were membrane bound rather than soluble. Membrane vesicles could indeed be detected by electron microscopy of negative-stained secreted material.