Alterations induced by E-cadherin and beta-catenin antibodies during the development of Bufo arenarum (Anura-Bufonidae)

E(epithelial)-cadherin is a member of a calcium-dependent family of cell surface glycoproteins involved in cell-cell adhesion and morphogenesis. Catenins are a large family of proteins that connect the cadherins to the cytoskeleton. They are important for cadherin function and for transducing signals involved in specification of cell fate during embryogenesis. The best characterized catenins include alpha-, beta-, gamma-, and p120-catenin. Using specific antibodies, we studied the expression and distribution of E-cadherin, and alpha- and beta-catenin in developmental stages of Bufo arenarum toad. The three proteins were found co-localized in stages 19 to 41 of development. Surprisingly, E-cadherin was the only of these three proteins found earlier than stage 19. To test whether E-cadherin and beta-catenin have a functional role in Bufo arenarum embryogenesis, stage 17 whole embryos were incubated with anti-E-cadherin and beta-catenin antibodies. Both anti-E-cadherin and anti-beta-catenin antibodies induced severe morphological alterations. However, while alterations produced by the anti-beta-catenin antibody, showed some variability from the most severe (neural tube and notochord duplication) to a simple delay in development, the alterations with anti-E-cadherin were homogeneous. These observations suggest a critical role for E-cadherin and beta-catenin in the early embryonic development of the Bufo arenarum toad. Our results are consistent with the developmental role of these proteins in other species. One of the most surprising findings was the blockage with the anti-beta-catenin antibodies on later embryo stages, and we hypothesize that the partial axes duplication could be mediated by the notochord induction.     

Characterization of Bufo arenarum oocyte plasma membrane proteins that interact with sperm

Sperm-oocyte plasma membrane interaction is an essential step in fertilization. In amphibians, the molecules involved have not been identified. Our aim was to detect and characterize oocyte molecules with binding affinity for sperm. We isolated plasma membranes free from vitelline envelope and yolk proteins from surface-biotinylated Bufo arenarum oocytes. Using binding assays we detected a biotinylated 100 kDa plasma membrane protein that consistently bound to sperm. Chromatographic studies confirmed the 100 kDa protein and detected two additional oocyte molecules of 30 and 70 kDa with affinity for sperm. Competition studies with an integrin-interacting peptide and cross-reaction with an anti-HSP70 antibody suggested that the 100 and 70 kDa proteins are members of the integrin family and HSP70, respectively. MS/MS analysis suggested extra candidates for a role in this step of fertilization. In conclusion, we provide evidence for the involvement of several proteins, including integrins and HSP70, in B. arenarum sperm-oocyte plasma membrane interactions.     

Serum antigens detected in pars recta luminal fluid and coelomic envelope surrounding Bufo arenarum eggs

In anurans, protease activity from the pars recta portion of the oviduct (under regulation by 17β-estradiol), is known to cause ultrastructural alterations on the oocyte surface rendering fertilizability. In mammals, the presence of serum proteins in oviductal fluid via transudation is also well known. In the present study we determined the plasma proteins of the anuran Bufo arenarum that are present in pars recta fluid and oocyte extracellular matrix and characterized the 17β-estradiol-induced proteins synthesized de novo and secreted into the pars recta lumen. Rabbit polyclonal antibodies against the soluble proteins in pars recta fluid cross-reacted with anuran plasma proteins and with the extracellular matrix of coelomic eggs based on immunoelectrophoresis and immunohistochemistry, respectively. Using radiolabeled leucine in the absence and presence of 17β-estradiol, we show that a polypeptide of 66 kDa molecular mass is the principal protein synthesized and secreted into the pars recta lumen.     

Participation of the 39-kDa glycoprotein (gp39) of the vitelline envelope of Bufo arenarum eggs in sperm-egg interaction

The acquisition of egg fertilizability in Bufo arenarum takes place during the oviductal transit and during this process the extracellular coelomic envelope (CE) of the eggs is converted into the vitelline envelope (VE). It has been stated that one of the necessary events leading to a fertilizable state is the proteolytic cleavage of CE glycoproteins in the oviductal pars recta by oviductin, a serine protease. Consequently, there is a marked increase in the relative quantity of glycoproteins with 39 (gp39) and 42 kDa (gp42) in the VE. In the present study, sperm-VE binding assays using heat-solubilized biotin-conjugated VE glycoproteins revealed that both gp39 and gp42 have sperm binding capacity. According to this result, our study was focused on gp39, a glycoprotein that we have previously reported as a homologue of mammalian ZPC. For this purpose, rabbit polyclonal antibodies against gp39 were generated at our laboratory. The specificity of the antibodies was confirmed with western blot of VE glycoproteins separated on SDS-PAGE. Immunohistochemical and immunoelectron studies showed gp39 distributed throughout the width of the VE. In addition, immunofluorescence assays probed that gp39 bound to the sperm head. Finally, as an approach to elucidate the possible involvement of gp39 in fertilization, inhibition assays showed that pretreatment of eggs with antibodies against gp39 generated a significant decrease in the fertilization rate. Therefore, our findings suggest that gp39, which is modified by oviductal action, participates as a VE glycoprotein ligand for sperm in Bufo arenarum fertilization.     

Role of oviductal pars recta extract on interspecific fertilization between anurans

During their journey through the oviductal pars recta, the vitelline envelope (VE) of Bufo arenarum oocytes encounter structural alterations that make them sensitive to attack by sperm lysin and thus to penetration by sperm cells. The role of pars recta (PR) on the specificity of fertilization between amphibians was analyzed by conditioning Bufo arenarum oocytes with either homologous PR extract (PRE) or Leptodactylus chaquencis PRE. The oocytes were thereafter exposed to sperm lysin preparations from both species. Lysis of the VE only took place when the oocytes were exposed to the homologous PRE. The pattern of protein composition of PRE of these species was strikingly different as shown by Coomassie blue staining of SDS-PAGE. Moreover, antibodies against PR fluid (PRF) of Bufo arenarum produced seven bands of immunoprecipitation in electrophoresed homologous PRE and only one faint band in Leptodactylus chaquencis PRE. Here we show that: (i) the biological activity of PR from Bufo arenarum and Leptodactylus chaquencis over the VE of Bufo arenarum oocytes is species-specific; (ii) this specificity seems to be based in differences in protein structure, which was indicated by the fact that proteins from PRE of Leptodactylus chaquencis and Bufo arenarum were antigenically distinct; (iii) the specificity was solely related to PR activity and not to sperm lysin activity since sperm lysin preparations from both species showed comparable activity.     

Protein synthesis in enuleated fertilized and unfertilized mouse eggs

Summary Fertilized and unfertilized C57BL/6J eggs were microsurgically enucleated and then analyzed for their capacity to synthesize proteins using 2-dimensional polyacrylamide gel electrophoresis. In both types of enucleated eggs (cytoplasts), protein synthesis continued and was still detected up to three days in culture. Shortly after enucleation, the pattern of polypeptides remained similar to the respective non-operated control eggs but it later became gradually reduced in intensity and complexity. After two days of culture the appearance of some new proteins typical for 2-cell embryos was observed in enucleated fertilized eggs only. Our findings suggest that maternal mRNA stored during oogenesis is utilized during the preimplantation period.     

A Protein That Binds an Exogastrula-Inducing Peptide, EGIP-D, in the Hyaline Layer of Sea Urchin Embryos

Exogastrula-inducing peptides are present in eggs and embryos of the sea urchin Anthocidaris crassispina . They induce exogastrulation when added exogenously to the embryos. In the present study, we investigated an EGIP-D-binding protein in the embryos. EGIP-D was incubated with homogenates of embryos. EGIP-D was then cross-linked to the binding protein by use of disuccinimidyl suberate (DSS) and the complex was analyzed by western blotting with an EGIP-D-specific antibody. A 30-kDa protein was detected in both eggs and embryos. To examine the localization of this protein, EGIP-D was added to intact embryos, cross-linked to proteins by use of DSS, and the complexes were again analyzed by western blotting. The EGIP-D-binding protein was detected in intact embryos but not in embryos treated with Ca²⁺- and Mg²⁺-free seawater (CMF-SW) that removes the hyaline layer (HL). It appeared, therefore, that this protein was present on the outer surface of the embryo, being a constituent of the HL. The CMF-SW extract that contained EGIP-D-binding protein, inhibited the induction of exogastrulation by EGIP-D. Furthermore, the treatment of embryos with CMF-SW prevented EGIP-D from inducing exogastrulation. Our observations indicate that the interaction between EGIP-D and the binding protein is a prerequisite for induction of exogastrulation by EGIP-D.     

Studies of oogenesis in Bufo arenarum (author's transl)

Based on a series of macroscopic and histological observations, during an annual cycle, the main stages of oogenesis in Bufo arenarum (Hensel) have been recognized, pointing out the most significant features. The analysis has established five characteristic stages which permit the individualization of the maturation stage of the oocyte in the ovary. All the information obtained has provided the possibility of drawing up a synthetic table so that the oogenetic stages of this amphibian species, very much used in Argentine experimentation, could be easily recognized.     

Characterization of urea transport in Bufo arenarum oocytes

Xenopus laevis oocytes have been extensively used for expression cloning, structure/function relationships, and regulation analysis of transporter proteins. Urea transporters have been expressed in Xenopus oocytes and their properties have been described. In order to establish an alternative system in which urea transporters could be efficiently expressed and studied, we determined the urea transport properties of ovarian oocytes from Bufo arenarum, a toad species common in Argentina. Bufo oocytes presented a high urea permeability of 22.3 x 10(-6) cm/s, which was significantly inhibited by the incubation with phloretin. The urea uptake in these oocytes was also inhibited by mercurial reagents, and high-affinity urea analogues. The urea uptake was not sodium dependent. The activation energy was 3.2 Kcal/mol, suggesting that urea movement across membrane oocytes may be through a facilitated urea transporter. In contrast, Bufo oocytes showed a low permeability for mannitol and glycerol. From these results, we propose that one or several specific urea transporters are present in ovarian oocytes from Bufo arenarum. Therefore, these oocytes cannot be used in expression studies of foreign urea transporters. The importance of Bufo urea transporter is not known but could be implicated in osmotic regulation during the laying of eggs in water.     

Tyrosination-detyrosination of the COOH-terminus of alpha-tubulin in oocytes and embryos of Bufo arenarum

Soluble tubulin from Bufo arenarum oocytes and early embryos was shown to be composed mainly of the non-tyrosinable species. The low proportion of tyrosinable tubulin was almost exclusively constituted by the tyrosinated form. Compared with oocytes and embryos, toad brain contained a higher proportion of tyrosinable tubulin constituted mainly by the non-tyrosinated form. Tubulin carboxypeptidase was detected in toad brain but not in oocytes and embryos.     

Oviductal protease and trypsin treatment enhance sperm-envelope interaction in Bufo arenarum coelomic eggs

We describe the morphological and biochemical changes in Bufo arenarum coelomic egg envelopes (CE) following passage through the oviduct. In this species, the transformation of the CE into the vitelline envelope (VE) leads to the acquisition of fertilizability and involves the cleavage of a glycoprotein component. Electrophoretic patterns indicate that a pars recta oviductal protease selectively hydrolyzes in vitro the 84 and the 55 kDa glycoproteins of the CE. During the CE to VE transformation, the relative concentrations of gp48, 42 and 39 kDa also change. In in vitro tests, sperm binding to envelope glycoprotein occurs when they are exposed to VE but not when treated with CE, and VE labeled glycoproteins bind to the head and mid piece of the sperm. The gp39 VE component has 100% identity with internal domains of the sequence deduced from ovarian cDNA for the homologous zona pellucida glycoprotein type C (ZPC) protein precursor in B. arenarum. The effects of trypsin as a substitute for oviductal protease were also examined. Trypsin selectively attacks the 84 and the 55 kDa glycoproteins without hydrolyzing other components and renders coelomic eggs fertilizable in a jelly water preparation. Therefore, trypsin can mimic in vitro the biological action of the oviductal protease. However, it does not wholly mimic the biological action of the oviduct which, in B. arenarum at least, exceeds a mere proteolytic effect. This fact was verified by the lower fertility rates and the abnormal embryo development found when trypsin-treated coelomic eggs were fertilized in vitro.     

A cadherin-like protein in eggs and cleaving embryos of Xenopus laevis is expressed in oocytes in response to progesterone

A new cadherin-like protein (CLP) was identified in oocytes, eggs, and cleavage stage embryos of Xenopus laevis. As a probe for detecting new cadherin proteins, an antiserum was raised to a 17 amino acid peptide derived from a highly conserved region in the cytoplasmic domain of all cadherins which have been sequenced to date. This antipeptide antibody recognized Xenopus E-cadherin and a polypeptide in Xenopus brain extracts similar to N-cadherin, which were independently identified by specific mAbs. In extracts of eggs and midblastula stage embryos the antipeptide antibody recognized specifically a 120-kD glycoprotein that migrated faster on SDS gels than the 140-kD E- and N-cadherin polypeptides. This 120-kD polypeptide was not recognized by the mAbs specific to E- and N-cadherin. In fact, E- and N-cadherin were not detectable in eggs or midblastula stage embryos. The possible relationship of CLP to P-cadherin, which has been identified in mouse tissues, has not yet been determined. CLP was synthesized by large, late stage oocytes. When oocytes were induced to mature in vitro with progesterone it accumulated to the same level found in normally laid eggs. It did not accumulate further to any significant extent during the early cleavage stages. CLP was detected on the surface of stage 8 blastomeres by cell surface biotinylation, but only after the tight junctions of the blastula epithelium were opened by removal of Ca2+. We conclude that CLP is a maternally encoded protein that is the major, if not only, cadherin-related protein present in the earliest stages of Xenopus development, and we propose that it may play a role in the Ca2(+)-dependent adhesion and junction formation between cleavage stage blastomeres.     

Characterization of somatic embryogenesis in sandalwood (Santalum album L.)

In the synchronous embryogenesis system of sandalwood developed in our laboratory, we observed that the early events of differentiation from freshly induced callus (stage 0) are accomplished in three distinct stages viz., preglobular masses (stage 1), globular embryos (stage 2), and bipolar embryos (stage 3). Transition from stage 0 to 1 was accomplished using 2,4-D and involves a stage specific appearance of two polypeptides of 15 and 30 kDa molecular weight. A 24 kDa polypeptide that was detected as a marked band in extracts of primary callus was not detected in stages 1, 2, and 3. Further, the tissue level of a 50 kDa glycoprotein decreased during transition from stage 2 to stage 3. However, the levels of glycoproteins in the medium were markedly higher in stage 0 cultures compared to those in stage 1. The activities of a protein kinase, glycosidase, and xylanase increased markedly with progressing embryogenesis. Our observations suggest that in addition to being controlled at the level of stage-specific gene expression, somatic embryogenesis in sandalwood is also regulated at the level of controls on cell wall flexibility and posttranslational changes in the pool of preexisting proteins.     

Polyphosphoinositide synthesis and protein phosphorylation in the plasma membrane from full-grown Bufo arenarum oocytes.

1. Polyphosphoinositide content and phosphorylation of lipids and proteins were analyzed in oocytes of the toad Bufo arenarum Hensel. 2. Plasma membrane-enriched fractions obtained from full-grown, prophase-arrested oocytes incorporated 32P into both phospholipids and proteins after incubation with [gamma-32P]ATP in an Mg(2+)-containing medium. Phosphatidylinositol 4-phosphate (PIP), phosphatidate (PA) and phosphatidylinositol-4,5-bisphosphate (PIP2) were the only labelled lipids. The 32P incorporation depended on incubation time, the amount of protein, and the ATP concentration. 3. Autoradiography of polyacrylamide gel electropherograms and scintillation counting showed that the radioactivity was mainly associated with a group of membrane proteins having an M(r) of 87,000. 4. This paper provides evidence for the capacity of prophase-arrested oocytes from Bufo arenarum to synthesize polyphosphoinositides and to phosphorylate distinct membrane proteins.     

Functional role of a high mol mass protein complex in the sea urchin yolk granule

We have investigated the biochemical and functional characteristics of the major protein constituents of the yolk granule organelle present in sea urchin eggs and embryos. Compositional analysis, using sodium dodecyl sulfate polyacrylamide gel electrophoresis, revealed distinctly different polypeptide patterns under reducing and non-reducing conditions. In the presence of reducing agent, a 240 kDa species dissociated into polypeptides of apparent mol mass 160, 120 and 90 k. The relatedness of these polypeptides to the 240 kDa species was demonstrated in protein gel blot and peptide mapping analyses. The profile of yolk granule polypeptides was dynamic during embryonic development with the disappearance of the 160 kDa species and the coincidental appearance of lower mol mass polypeptides. However, the 240 kDa complex was detected even after the disappearance of the 160 kDa polypeptide. The 240 kDa complex was released from yolk granules in the absence of calcium and the purified species was shown to bind liposomes in a calcium-dependent manner. In addition, the 240 kDa complex possessed a calcium-dependent, liposome aggregating activity. The 240 kDa species could also induce the aggregation of yolk granules, previously denuded of the complex following treatment with either ethylenediaminetetraacetic acid or trypsin. Collectively, these results demonstrate the dynamic characteristics of the yolk granule 240 kDa protein complex and offer insights into a possible functional role.     

Mesodermal and axial determinants contribute to mesoderm regionalization in <Emphasis Type="Italic">Bufo arenarum</Emphasis> embryos

The existence of mesodermal determinants in the equator of Bufo arenarum embryos has been previously demonstrated. In this work, their role in dorso-ventral regionalization of mesoderm was studied by transferring the determinants to animal blastomeres. The transfer was performed by cleavage reorientation and cytoplasmic microinjection. Forced inclination during early cleavage caused deviation of the third cleavage plane and annexation of equatorial cytoplasm into animal quartets. Animal blastomeres from embryos oriented with the dorsal side up, incorporated ventro-equatorial cytoplasm and formed blood cells, mesenchyme, and coelomic epithelium. In contrast, animal blastomeres from embryos oriented with the ventral side up, acquired dorso-equatorial cytoplasm and developed notochord, somites, mesenchyme, coelomic epithelium and nervous tissue. In order to investigate if this dorso-ventral differentiation pattern responds to an interaction of mesodermal and axial factors, isolated 8-cell-stage animal quartets were microinjected with subcortical cytoplasm from: (a) the ventro-equatorial region of synchronous embryos; (b) the vegetal pole of uncleaved eggs; (c) a combination of both cytoplasms. As expected, the implanted ventro-equatorial cytoplasm promoted ventral mesoderm differentiation. Conversely, the joint transfer of ventro-equatorial cytoplasm and vegetal pole cytoplasm behaved as the dorso-equatorial cytoplasm, promoting dorso-lateral mesoderm and neural formation. Thus, mesoderm regionalization in B. arenarum embryos seems to be caused by a concurrent action of both mesodermal and axial determinants.