Qualitative patterns of protein synthesis in the preimplantation mouse embryo: I. Normal pregnancy

Qualitative patterns of protein synthesis in preimplantation mouse embryos were examined by SDS-polyacrylamide-gel electrophoresis followed by autoradiography. The results demonstrate that the qualitative pattern of protein synthesis in newly fertilized eggs (day 1) is very similar to the protein pattern obtained from ovulated, unfertilized eggs. By late day 1 or early day 2, most of these “maternal” proteins are no longer being synthesized by the embryo, and many new autoradiographic bands are apparent. The most intriguing aspect of this study is the observation that all major changes in the qualitative pattern of protein synthesis take place between fertilization and the four- to eight-cell stage (day 3). From early day 3 onward, the qualitative pattern of protein synthesis remains essentially unchanged.Many of the major autoradiographic bands observed in mouse embryos from the four- to eight-cell stage and onward are also observed in protein patterns obtained from blastocyst-stage rabbit embryos. The changing patterns of protein synthesis revealed in this study occur before any gross differentiation of the embryos is evident (delineation of the inner cell mass and trophoblast) and before a marked increase in the relative rate of incorporation of l-[³⁵S]methionine takes place. However, the qualitative changes in the pattern of protein synthesis do coincide with a period of extensive fine structural differentiation.     

Regulation of stage-specific gene expression during early mouse development: effect of cytochalasin B and aphidicolin on stage-specific protein synthesis in mouse eggs

Mechanisms regulating stage-specific translation in mouse embryos were studied by inhibitor experiments. When fertilized eggs were continuously treated with cytochalasin B, cleavage was prevented, whereas karyokinesis proceeded, resulting in protein synthesis patterns changing stage-specifically as in control embryos through preimplantation development. When fertilized eggs were continuously exposed to aphidicolin, cleavage and DNA synthesis were inhibited, thus keeping their protein synthesis at the level of fertilized eggs with few new polypeptides appearing after one day. The next day these eggs stopped translation almost completely. Stage-specific translation therefore might be controlled by nuclear replications rather than by cytoplasmic clock.     

Situs inversus in the developing mouse: proteins affected by the iv mutation (genocopy) and the teratogen retinoic acid (phenocopy)

To decipher genes that are important in the determination of laterality, we compared two-dimensional protein gels from wild-type C57BL/6J mice and C57BL/6J mice that carried the iv mutation, which confers random determination of visceral situs. To span the time period(s) during which laterality determination occurs, we compared computer-analyzed two-dimensional protein gels from wild-type mouse embryos and iv/iv mouse embryos at 7.5, 8.0, and 8.5 days post-coitum. One polypeptide that was expressed only on day 8.0 of development and only in wild-type embryos represents a particular candidate for determination of laterality. Day 8.5 postcoitum represents the earliest time in murine development that laterality is manifest. Two-dimensional gels were compared from 8.5 day embryos that were C57BL/6J wild-type, C57BL/6J iv/iv, or C57BL/6J wild-type and exposed to the teratogen retinoic acid late on day 7. Reproducible alterations of protein synthesis were observed in both the iv genocopy and retinoic acid phenocopy, yielding abnormal laterality determination. The intersection of these peptide changes identifies a protein likely to play a role in the determination of laterality.     

Analysis of proteins synthesized by 9.5 day mouse embryos: determination of cardiac and noncardiac proteins.

To catalog polypeptides that were specific to developing hearts, we separated 35S-methionine-labeled 9.5 day mouse embryos into cardiac and noncardiac (carcass) components. Two-dimensional gels were then used to analyze the polypeptides synthesized in these two fractions. As a result, we were able to distinguish polypeptides that were specific to or increased in the heart as well as those polypeptides that were specific to or increased in the embryo minus the dissected heart. Using this analysis, there were two polypeptides that were cardiac-specific and 17 that were expressed at increased levels by at least twofold in the heart. The cardiac-specific polypeptides may be used in further studies to identify early cardiac tissue. Conversely, there were 26 polypeptides unique to noncardiac structures and an additional 15 that were increased in the carcass more than twofold relative to the heart. The noncardiac-specific polypeptides may be used to define contamination of putative cardiac tissue with noncardiac material. Two of the polypeptides expressed more abundantly in the carcass appeared to correspond to known proteins in the mouse fibroblast database, cyclin and tropomyosin 4. Thus the heart at 9.5 days of murine development can be distinguished readily from the remainder of the embryonic mouse both macroscopically and on two-dimensional gels.     

Control of protein synthesis during blastocyst formation in the mouse.

In order to evaluate the dependence of the embryo on new mRNA synthesis during the period leading to blastulation, quantitative and qualitative aspects of protein synthesis in developing mouse morulae were investigated using α-amanitin, an inhibitor of RNA polymerase II. Only 1 of 423 early morulae cultured for 27 hr in the presence of 11 μg/ml α-amanitin cavitated, although most progressed as far as fully compacted morulae. About two-thirds of the untreated embryos cavitated during the same period. Incorporation of [³⁵S]methionine into protein was measured at 3- or 4-hr intervals over a 24-hr period and showed a two- to fivefold increase in control embryos. This increase was blocked in the α-amanitin-treated group although initial levels of incorporation were maintained. Total uptake of the amino acid appeared to be unaffected by the inhibitor. RNA synthesis, as measured by [³H]uridine incorporation over the same period, was reduced by between 5 and 52%, and the preblastulation surge in RNA synthesis was also blocked by α-amanitin. Two-dimensional polyacrylamide gel electrophoresis of labeled polypeptides synthesized by the embryos after 24-hr incubation in the presence or absence of the inhibitor revealed three distinct classes of polypeptide. The majority of polypeptides continued to be synthesized in the presence of α-amanitin whereas a small number of polypeptides, the synthesis of which would normally have increased during the development of the morula to the blastocyst, were prevented from doing so. A few polypeptides which normally cease to be synthesized over this period continued to be synthesized in the presence of α-amanitin. It is concluded that, while most of the proteins detectable at the morula stage are synthesized on mRNA templates of relatively long translational life, the general surge in protein synthesis, including the increased synthesis of a few species of polypeptide, are dependent on continuous translational activity.     

Differential timing of nuclear lamin A/C expression in the various organs of the mouse embryo and the young animal: a developmental study

In mouse embryos, acquisition of the nuclear lamin polypeptides A/C varies according to developmental stage and tissue type. In order to determine the precise time points and cell types in which lamin A/C are first observed, we have used two monoclonal antibodies in immunofluorescence studies of different tissues of developing mouse embryos and of young mice. One antibody (mAB346) is specific for lamins A and C, while the other (PKB8) detects lamins A, B and C. Dividing uterine development into three phases--germ layer formation, organogenesis and tissue differentiation--our results show that lamin A/C expression in the embryo proper is not observed until the third phase of development. Lamin A/C first appears at embryonic day 12 in muscle cells of the trunk, head and the appendages. Three days later it is also seen in cells of the epidermis where its appearance coincides with the time of stratification. In the simple epithelial of lung, liver, kidney and intestine, as well as in heart and brain, lamins A/C do not appear until well after birth. Embryonal carcinoma (EC) cells express lamin B but not lamin A/C. Lamin A/C expression is noted in some EC cells after they are induced to differentiate and in several differentiated teratocarcinoma cell lines. Our results suggest that commitment of a cell to a particular pathway of differentiation (assayed by cell-type-specific expression of intermediate filament proteins) usually occurs prior to the time that lamin A/C can be detected. Thus lamin A/C expression may serve as a limit on the plasticity of cells for further developmental events.     

Global gene expression during the human organogenesis: from transcription profiles to function predictions

Human embryogenesis includes an integrated set of complex yet coordinated development of different organs and tissues, which is regulated by the spatiotemporal expression of many genes. Deciphering the gene regulation profile is essential for understanding the molecular basis of human embryo development. While molecular and genetic studies in mouse have served as a valuable tool to understand mammalian development, significant differences exists in human and mouse development at morphological and genomic levels. Thus it is important to carry out research directly on human embryonic development. Here we will review some recent studies on gene regulation during human embryogenesis with particular focus on the period of organogenesis, which had not been well studied previously. We will highlight a gene expression database of human embryos from the 4(th) to the 9(th) week. The analysis of gene regulation during this period reveals that genes functioning in a given developmental process tend to be coordinately regulated during human embryogenesis. This feature allows us to use this database to identify new genes important for a particular developmental process/pathway and deduce the potential function of a novel gene during organogenesis. Such a gene expression atlas should serve as an important resource for molecular study of human development and pathogenesis.     

Comparative proteome analysis of the embryo proper and yolk sac membrane of day 11.5 cultured rat embryos

BACKGROUND: Proteomic analysis of cultured postimplantation rat embryos is expected to be useful for investigation into embryonic development. Here we analyzed protein expression in cultured postimplantation rat embryos by two-dimensional electrophoresis (2-DE) and mass-spectrometric protein identification. METHODS: Rat embryos were cultured from day 9.5 for 48 h or from day 10.5 for 24 h. Proteins of the embryo proper and yolk sac membrane were isolated by 2-DE and differentially analyzed with a 2-D analysis software. Selected protein spots in the 2-DE gels were identified by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometric analysis and protein database search. RESULTS: About 800 and 1,000 protein spots were matched through the replicate 2-DE gels each from one embryo in the embryo proper and yolk sac membrane, respectively, and virtually the same protein spots were observed irrespective to the length of culture period. From protein spots specific to the embryo proper (126 spots) and yolk sac membrane (304 spots), proteins involved in tissue-characteristic functions, such as morphogenesis and nutritional transfer, were identified: calponin, cellular retinoic acid binding protein, cofilin, myosin, and stathmin in the embryo proper, and Ash-m, dimerization cofactor of hepatocyte nuclear factor, ERM-binding phosphoprotein, cathepsin, and legumain in the yolk sac membrane. CONCLUSION: Proteomic analysis of cultured postimplantation rat embryos will be a new approach in developmental biology and toxicology at the protein level.     

Polar lobe specific regulation of translation in embryos of Ilyanassa obsoleta.

Comparisons of the patterns of synthesis of ¹⁴C-labeled proteins of normal embryos of Ilyanassa with the patterns of synthesis of ³H-labeled proteins of embryos from which the polar lobes had been removed at the trefoil stage were made by coelectrophoresis on polyacrylamide disc gels. Controls utilizing biochemical and morphological markers were performed to assure that normal and delobed embryos were developing at equivalent rates. The expression of significant differences in the patterns of protein synthesis were found between normal and delobed embryos, and these differences were not dependent upon concomitant RNA synthesis. These differences were observable as early as after only 24 hr of development, although organogenesis does not begin until much later in development. Therefore, the observed differences probably reflect determininative events. The results support the hypothesis that the developmental determinants of the polar lobe may include specific, preformed mRNA sequences, or specific regulators of translation.     

African swine fever virus-induced polypeptides in porcine alveolar macrophages and in Vero cells: two-dimensional gel analysis

High-resolution two-dimensional electrophoresis followed by computer analysis has been used to study quantitatively the patterns of protein synthesis produced in porcine alveolar macrophages and in Vero cells infected with African swine fever virus (ASFV). Initially, a protein database for each cell type was constructed. The porcine alveolar macrophage database includes 995 polypeptides (818 acidic, isoelectric focusing (IEF) and 177 basic, nonequilibrium pH gradient electrophoresis (NEPHGE)) whereas the Vero database contains 1,398 polypeptides (1,127 acidic, IEF and 271 basic, NEPHGE). Taking these databases as reference, ASFV highly virulent strain E70 induces 57 acid and 43 basic polypeptides in porcine alveolar macrophages, which account for most of the information content of the virus DNA. The kinetics of synthesis of the virus-induced polypeptides showed the existence of three classes of proteins: one whose synthesis starts early after infection, continues for a period and then switches off; another whose synthesis also starts early but continues for prolonged periods; and a third which requires DNA replication. The attenuated, cell adapted, strain BA71V induces 92 acidic and 37 basic proteins in Vero cells. Significant differences were observed when comparing the patterns of polypeptides induced by the two viral strains. In both cell systems studied, ASFV infection produces a general shutoff of protein synthesis that affects up to 65% of the cellular proteins. Interestingly, 28 proteins of porcine alveolar macrophages and 48 proteins of Vero cells are stimulated at least two times by ASFV infection.     

A set of stage-dependent embryonic antigens expressed in cell cultures of BALB/c mouse embryos and in transformed cell lines

A rabbit antiserum (A2) directed against the detergent-solubilized fraction of the simian virus 40-transformed mouse embryo fibroblast cell line VLM detects common antigens in primary cell cultures from BALB/c mouse embryos and in transformed cell lines from various species. Positively reacting cell cultures show a set of polypeptides with molecular weight species p86, p74, p68, p46, p42, p40, and p35. As tested by Western blotting procedures, all immunoprecipitated proteins carry immunologically reactive determinants. By analysis with two-dimensional gel electrophoresis, all precipitated polypeptides show charge heterogeneities. Concerning the two major members of the protein set, p40 consists of at least four subspecies with isoelectric points in the range of pH 6.2-6.8, whereas p35 is composed of two subspecies focusing between pH 6.4 and pH 7.2. By comparison of the two-dimensional patterns of p35 of various transformed cell lines, a basic (pH 6.6-7.2) and an acidic (6.4-6.6) charge type of p35 could be observed. Comparative analyses of primary cell cultures from 12-16-day mouse embryos show the immunoprecipitated set of polypeptides only in the 16-day embryo cell cultures. After six further propagations, these cells express the immunoreactive proteins as strongly as the primary cell cultures. In embryonic cell cultures of day 14 of gestation the expression of this set of antigens is induced only when cells are propagated at least six times. Under identical conditions these proteins could not be induced in cell cultures of 18-day-old mouse embryos. None of the polypeptides could be immunoprecipitated from primary mouse kidney cell cultures of 12-day-old mice even when the cultures were propagated at least 15 times. This set of polypeptides is also present in simian virus 40-transformed cells of hamster, rat, monkey, and human origin. These findings suggest that in simian virus 40-transformed mouse cells, in addition to p53, the synthesis of other embryonic antigens is reactivated. The presence of the described set of polypeptides in polyoma virus-transformed cells of rat and mouse origin and in cell lines derived from malignant human tumors might indicate common functions in metabolic patterns of transformed cells.     

Pattern of expression of HtrA1 during mouse development.

The human HtrA family of proteases consists of four members: HtrA1, HtrA2, HtrA3, and HtrA4. In humans the four HtrA homologues appear to be involved in several important functions such as cell growth, apoptosis, and inflammatory reactions, and they control cell fate via regulated protein metabolism. In previous studies it was shown that the expression of HtrA1 was ubiquitous in normal adult human tissues. Here we examined the expression of HtrA1 protein and its corresponding mRNA during mouse embryogenesis using Northern blotting hybridization, RT-PCR, and immunohistochemical staining analyses. Our results indicate that HtrA1 is expressed in a variety of tissues in mouse embryos. Furthermore, this expression is regulated in a spatial and temporal manner. Relatively low levels of HtrA1 mRNA are detected in embryos at the beginning of organogenesis (E8), and the levels of expression increase during late organogenesis (E14-E19). Our results show that HtrA1 was expressed during embryonic development in specific areas where signaling by TGFbeta family proteins plays important regulatory roles. The expression of HtrA1, documented both at mRNA and protein levels by RT-PCR and immunohistochemistry in the developing nervous system, is consistent with a possible role of this protein both in dividing and postmitotic neurons, possibly via its documented inhibitory effects on TGFbeta proteins. An exhaustive knowledge of the different cell- and tissue-specific patterns of expression of HtrA1 in normal mouse embryos is essential for a critical evaluation of the exact role played by this protein during development.     

Effect of Abscisic Acid, Osmoticum, and Desiccation on Synthesis of Storage Proteins during the Development of White Spruce Somatic Embryos

The effect of abscisic acid (ABA), non-permeating osmoticumand desiccation treatment on storage protein synthesis duringmaturation of somatic embryos of Picea glauca (Moench) Voss.was examined. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot analysis demonstrated that someof the major crystalloid and matrix polypeptides were absentfrom somatic embryos maturing on medium containing ABA and lowosmoticum. However, treatment with polyethylene glycol-4000(PEG) in combination with ABA resulted in the synthesis of aspectrum of storage polypeptides resembling that of mature zygoticembryos. These storage proteins accumulated throughout an 8-weekculture period, resulting in a threefold higher protein contentthan somatic embryos maturing for the same time in the absenceof PEG. The structure and distribution of protein bodies incells of these osmotically treated somatic embryos was similarto that in cells of mature zygotic embryos. Treatment with 5·0-7·5%PEG prevented catabolism of the accumulated storage polypeptidesduring desiccation. The optimal culture conditions for somaticembryo maturation and storage protein deposition was 16 µMABA and 7·5% PEG for 8 weeks followed by desiccation.Analysis of mRNAs by in vitro translation and immunoprecipitationof translated products showed that the crystalloid protein mRNAprofiles of zygotic and those of somatic embryos maturing on16 µM ABA in the absence of PEG were similar. The differencesobserved in the pattern of accumulated polypeptides in thesesomatic embryos and those of mature zygotic embryos, therefore,indicates that storage-protein synthesis in response to osmoticumis in part regulated at the translational level. During regenerationof somatic embryos to plantlets the storage polypeptides wererapidly utilized in a manner similar to that in zygotic seedlings.Copyright1993, 1999 Academic Press Desiccation, osmotic stress, storage proteins, Picea, embryogenesis—somatic, mRNA (crystalloid protein)     

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.     

Regulation of Protein Synthesis in Embryos of Ilyanassa obsoleta

Much of the protein synthesis during early development in Ilyanassaresults from the translation of oogenetic mRNA. We show thatmicrotubule proteins are products of this translation, and thattheir synthesis is subject to translation level regulation.We also show that translation level regulation is involved inthe function of the polar lobe by making comparisons of theelectrophoretic patterns of synthesis of ¹⁴C labeled proteinsof normal embryos with the patterns of synthesis of ³H labeledproteins of embryos from which the polar lobes had been removedat the trefoil stage. Controls utilizing biochemical and morphologicalmarkers were performed to assure that normal and delobed embryoswere developing at equivalent rates. The expression of significantdifferences in the patterns of protein synthesis were foundbetween normal and delobed embryos, and these differences werenot dependent upon concomitant RNA synthesis. These differenceswere observable as early as after only 24 hours of development,although organogenesis does not begin until much later in development.Therefore, the observed differences probably reflect determinativeevents. The results support the hypothesis that the developmentaldeterminants of the polar lobe may include specific, preformedmRNA sequences, or specific regulators of translation.     

Temporal synthesis of mitochondrial proteins from mouse epididymal spermatozoa

Mitochondria from ejaculated bovine spermatozoa contain a group of polypeptides ranging in molecular weights from 13,000 to 35,000 not found in other bovine or murine testicular mitochondria [Hecht and Bradley, 1981]. These proteins are present in the mitochondria isolated from both epididymal and ejaculated spermatozoa. To establish when during epididymal transport, spermiogenesis, and/or meiosis these proteins are synthesized, the synthesis intervals for the mitochondrial proteins from cauda epididymal spermatozoa were established following intratesticular injection of (³⁵S)methionine. Mice were killed every third day over a 33-day period and cauda epididymal spermatozoa were fractionated into mitochondrial and head components. Radioactivity in each fraction was monitored by liquid scintillation counting. Maximal incorporation was observed during spermiogenesis, although substantial amounts of protein were synthesized during meiosis. Analysis of the mitochondrial polypeptides by gel electrophoresis revealed that many polypeptides such as the cysteine-rich structural protein of the mitochondrial capsule were synthesized over prolonged intervals of spermiogenesis and meiosis rather than in a brief specific time period. These results suggest that spermatozoal mitochondria are produced by a sequential substitution of new proteins into the differentiating mitochondria rather than the abrupt appearance of a new class of mitochondria during spermatogenesis.     

Responses of mitochondrial biogenesis and function to maternal diabetes in rat embryo during the placentation period

Mitochondria are cellular organelles that have been reported to be altered in diabetes, being closely related to its associated complications. Moreover, mitochondrial biogenesis and function are essential for proper embryo development throughout the placentation period, occurring during organogenesis, when a great rate of congenital malformations have been associated with diabetic pregnancy. Thus, the aim of the current work was to investigate the effect of the diabetic environment on mitochondrial function and biogenesis during the placentation period. For this purpose, we studied the oxidative phosphorylation system (OXPHOS) enzymatic activities as well as the expression of genes involved in the coordinated regulation of both mitochondrial and nuclear genome (PGC-1alpha, NRF-1, NRF-2alpha, mtSSB, and TFAM) and mitochondrial function (COX-IV, COX-I, and beta-ATPase) in rat embryos from control and streptozotocin-induced diabetic mothers. Our results reflected that diabetic pregnancy retarded and altered embryo growth. The embryos from diabetic mothers showing normal morphology presented a reduced content of proteins regulated through the PGC-1alpha mitochondriogenic pathway on gestational day 12. This fact was accompanied by several responses that entailed the activation of OXPHOS activities on the same day and the recovery of the content of the studied proteins to control levels on day 13. As a result, the mitochondria of these embryos would reach a situation close to control on day 13 that could allow them to follow the normal mitochondriogenic schedule throughout a gestational period in which the mitochondrial differentiation process is critical. Nevertheless, malformed embryos from diabetic mothers seemed to show a lower adaptation capability, which could exacerbate their maldevelopment.     

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.