Effects of cell heterogeneity on production of polypeptide growth factors and mesoderm-inducing activity by Xenopus laevis XTC cells

The Xenopus laevis XTC cell line has been analyzed for the production of polypeptide growth factors and mesoderm-inducing activity. By the use of specific biological assays, it is shown that XTC cells produce a growth factor functionally related to the platelet-derived growth factor (PDGF) and two transforming growth factor (TGF) beta-like activities. Mesoderm-inducing activity, as measured on X. laevis ectodermal explants from stage 10 embryos, was found to coelute on a Bio-Gel P-100 column with one of the TGF beta-like activities at an apparent molecular weight of 6-10 kDa. Analysis of the DNA content from XTC cells by flow cytometry demonstrated that the cell line is heterogeneous and consists of both tetraploid and diploid cells. Cloning of the XTC cells and selecting single-cell colonies on the basis of their ability to grow in soft agar resulted in the isolation of several homogeneous, morphologically different clonal derivatives. Analysis of conditioned medium from these clonal derivatives showed that only one of them, the only diploid line among six investigated, produced a strong heat- and acid-stable mesoderm-inducing activity that induced notochord and muscle formation in stage 10 X. laevis ectodermal explants. The relation between this activity and a recently described TGF beta-like mesoderm-inducing factor obtained from XTC-conditioned medium will be discussed. In conclusion, a clonal cell line derived from X. laevis XTC cells which provides a good source for further characterization of mesoderm-inducing factors has been established.     

The cell cycle of Xenopus laevis cells in monolayer culture

Analysis of the cell cycle of recently isolated Xenopus laevis cells in culture gave mean values for the duration of G1, S and G2 of 18.0, 8.2 and 5.3 h respectively. After 70 weekly subcultures, cells with 38 chromosomes had equivalent values of 4.7, 6.3 and 3.0 h, and for tetraploid cells of 7.0, 8.9 and 5.0 h. The duration of mitosis was 1.0 h.     

Synacrosomal formation after cell fusion of round spermatids of Xenopus laevis

Cell fusion was induced by hypotonic medium in pairs of spermatids which were derived from single secondary spermatocytes. In a pair of fused spermatids, a single acrosome (synacrosome) eventually formed whenever the cell fusion was induced during the course of acrosomal formation. Direct observation of the process of synacrosomal formation was made on pairs of fused spermatids which had completed acrosomal formation. Two patterns occurred, namely, fusion of two acrosomes or enlargement of one with diminution of the other. The total volume of the two acrosomes before synacrosomal formation almost equaled the volume of the coalesced synacrosomes in fused spermatids. Neither colchicine nor cytochalasin B prevented synacrosomal formation in spermatids which were fused after each had completed acrosomal formation. These results indicate that neither microtubules nor microfilaments seem to play a role in the formation of a synacrosome in pairs of fused spermatids. However, cycloheximide did inhibit acrosomal formation when present during the early stage of acrosome differentiation in pairs of spermatids which had been fused just after second meiotic division. This fact indicates that acrosomal formation is mediated by some protein(s) which are synthesized during the initial period of acrosomal formation.     

Effects of polyamines on the first division cycle of Xenopus laevis eggs

The involvement of polyamines in cytokinesis has been examined in eggs of the amphibian X. laevis. Microinjection of spermine or spermidine into unfertilized eggs induced a shortening of the first division cycle and early formation of cleavage-like constrictions. Eggs were activated by injection and developed furrows about 45 min later, whereas the first division normally occurred around 120 min after activation. In terms of concentration, spermine was slightly more effective than spermidine, but putrescine had no influence on the division cycle.     

Isolation of globin messenger RNA of Xenopus laevis

The polypeptide chains of Xenopus laevis hemoglobin have been analyzed by sodium dodecyl sulfate (SDS) and acid-urea gel electrophoresis. Four components can be distinguished, each having an approximate molecular weight of 13,000 daltons. Messenger RNA coding for the globin chains has been isolated and characterized. In a denaturing acrylamide gel the mRNA has an approximate molecular weight of 250,000 daltons. The complexity of the RNA is consistent with the presence of four different mRNA molecules, each of this molecular weight. When the mRNA is assayed in a wheat germ in vitro translation system, four polypeptides are synthesized corresponding to the four globin subunits. The relative proportion of the four synthesized polypeptides appears to vary according to the developmental stage of the red blood cells used for mRNA isolation. Hybridization of a complementary DNA (cDNA) copy of the globin mRNA to Xenopus laevis DNA in DNA excess indicates that each of the globin genes is present in one to three copies per haploid genome.     

Mitochondrial DNA synthesis in oocytes of Xenopus laevis

Mitochondria isolated from stage 3 (about half-grown) oocytes of Xenopus laevis exhibit a DNA synthetic rate in vitro of 2.35 ± 0.28 pg/oocyte/h. Similarly, stage 6 (full-grown) oocyte mitochondria synthesize DNA (mtDNA) at 0.28 ± 0.02 pg/oocyte/h. By comparison, the rate of mtDNA synthesis by intact stage 6 oocytes following microinjection of [³H]-dTTP was calculated to be 0.43 ± 0.08 pg/oocyte/h, indicating that the observed in vitro rates may represent minimum values. Measurements of DNA polymerase activity associated with mitochondria isolated from stage 3 oocytes are almost three times those recorded with stage 6 oocyte mitochondria. It appears that active replication of complete mtDNA molecules, which accompanies accumulation of mitochondria by the egg, is terminated midway through oogenesis.     

Exogeneous diacylglycerols downregulate the activity of Na-K pump in Xenopus laevis oocytes

In this study, cell permeable diacyglycerols, sn-1,2-dioctanoglycerol (DiC8), and sn-1-oleoyl-2-acetylglycerol (OAG) were found to downregulate the activity of Na⁺-K⁺ pump in Xenopus laevis oocytes. Both DiC8 and OAG decreased the binding of [³H]ouabain to intact oocytes while phorbol esters did not appreciably influence the same. These diacylglycerols inhibited the amiloride-sensitive ²²Na⁺ influx and ouabain-sensitive ⁸⁸Rb⁺ uptake in the oocytes. Furthermore, DiC8 prevented the ²²Na⁺ efflux from the oocytes preloaded with ²²Na⁺. Addition of H-7 to DiC8- and OAG-treated oocytes stimulated the pump activity curtailed by the two latters. The impairment of Na⁺-K⁺ pump activity by diacylglycerols suggests that protein kinase C activators may stimulate endocytosis of membrane-coupled Na⁺-K⁺ ATPase.     

Production of a functional full-length Xenopus laevis c-Myc protein in insect cells

C-Myc is a nuclear phosphoprotein whose normal cellular function has not yet been clearly defined. Studies with this protein have always been constrained by the difficulty of obtaining full-length c-Myc in an active form, whatever the expression system used. We report here experimental conditions optimized to increase the solubility and the purification of c-Myc in a baculovirus expression system. Such conditions allow the production of both soluble and active fulllength c-Myc. Interestingly, soluble c-Myc is found associated with a 500-kDa high-molecular-mass complex comparable to that found in human and Xenopus laevis embryos, and which may be required for its function in vivo.     

Production of WW super females by diploid gynogenesis in Xenopus laevis

Summary In Xenopus laevis, which does not show sex chromosomal dimorphism, the female is heterogametic (WZ) and the male is homogametic (ZZ). By activating eggs with UV-irradiated spermatozoa, and by inhibiting the formation of the second polar body gynogenetic diploids were obtained, including some WW females. These super-females are fertile and not sublethal; by gynogenetic reproduction they in turn generate only WW females, while after mating with a male they give rise to WZ females exclusively.From the sex ratio of the gynogenetic progeny of normal WZ females, the map distance between the centromere and the sex determining gene(s) has been calculated. By examining the sex ratio and the distribution of individuals exhibiting the phenotype of periodic albinism in the gynogenetic offspring of a^p/+females, it has been demonstrated that the a^p gene and the sex determining gene(s) are not linked.     

Argyrophilic nuclear and nucleolar proteins of Xenopus laevis oocytes identified by gel electrophoresis

In order to identify argyrophilic proteins of nuclei and nucleoli, in particular those responsible for the ‘nucleolar Ag staining’ widely used in cytology, we have utilized oocytes of Xenopus laevis because of the abundance of ‘pure’ extrachromosomal nucleoli. Examination of oocytes by light and electron microscopy shows that the large extrachromosomal nucleoli are heavily stained with the Ag technique and that the Ag deposits are largely enriched in, if not exclusive to, the internal, fibrillar region. The same pattern of Ag staining in internal regions of nucleoli is observed in isolated nucleoli from which soluble nuclear proteins were removed by extensive washing. Argyrophilic proteins of isolated oocyte nuclei and purified nucleoli have been identified by reaction with AgNO₃ and formaldehyde on gel-electrophoretically separated polypeptides. Among nuclear proteins, the most prominent argyrophilia is associated with nucleoplasmin, a soluble MW 30000 phosphoprotein of the nuclear sap. In addition, four minor Ag-staining nuclear proteins have been observed. By contrast, the only strongly argyrophilic protein observed on gel electrophoresis of proteins of purified nucleoli is a high molecular weight component (apparent MW 195000) which is often resolved in a characteristic ‘pair’ of closely spaced polypeptide bands. The enrichment of this high molecular weight argyrophilic protein in isolated nucleoli and the corresponding absence of argyrophilic proteins of the nuclear sap, including nucleoplasmin, indicates that this protein contributes to the nucleolus-specific Ag staining observed in histological sections. The possible nature of this polypeptide of MW 195000 is discussed.     

Cell-free translation of messenger RNA from oocytes of Xenopus laevis

The poly(A)⁺ RNA which accumulates during oogenesis in the amphibian Xenopus laevis is shown to be functional mRNA; the RNA was active in the mRNA-dependent “shift assay” for initiation sites in the rabbit reticulocyte lysate, and was an efficient template for protein synthesis in the wheat-germ cell-free system. Analysis of the in vitro protein products showed no differences between the coding properties of poly(A)⁺ RNA extracted from oocytes at all stages of development from previtellogenesis to maturity. In previtellogenic oocytes, the in vitro products of polysomal and of mRNP-associated poly(A)⁺ RNA were also identical. Neither was there any evidence for changes in the coding properties of the poly(A)⁺ mRNA of the oocyte. However, the patterns of oocyte in vivo protein synthesis changed markedly during early vitellogenesis. We conclude that the mRNP-associated poly(A)⁺ RNA present in mature oocytes constitutes the stored maternal mRNA, and that during oogenesis the coding composition of the poly(A)⁺ mRNA synthesised does not change markedly, while some form of translational control operates to direct the changing pattern of protein synthesis.     

In vitro growth of adult amphibian (Xenopus laevis) hepatocytes and characterization of hepatocyte-proliferating activity in homologous serum

Adult frog (Xenopus laevis) hepatocytes were found to proliferate in a culture medium containing adult homologous serum. Insulin and dexamethasone were required for a net proliferation of hepatocytes. Dose-response analysis showed that a low concentration of serum (greater than or equal to 0.5%) was enough to induce DNA synthesis and mitosis, but a higher concentration (5%) caused certain necrotic changes. Under optimal conditions, there was a two- to threefold increase in nuclei per culture 10 days after serum treatment. Heterologous sera (fetal bovine, calf and chick) showed less proliferative activity. Based on our results, hepatocyte-proliferating activity in adult frog serum is considered to be heat-unstable and acidic protein(s). Thus, adult frog serum may contain hepatopoietin possibly different from well-known growth factors.     

The restriction of the heart morphogenetic field in Xenopus laevis

We have examined the spatial restriction of heart-forming potency in Xenopus laevis embryos, using an assay system in which explants or explant recombinates are cultured in hanging drops and scored for the formation of a beating heart. At the end of neurulation at stage 20, the heart morphogenetic field, i.e., the area that is capable of heart formation when cultured in isolation, includes anterior ventral and ventrolateral mesoderm. This area of developmental potency does not extend into more posterior regions. Between postneurula stage 23 and the onset of heart morphogenesis at stage 28, the heart morphogenetic field becomes spatially restricted to the anterior ventral region. The restriction of the heart morphogenetic field during postneurula stages results from a loss of developmental potency in the lateral mesoderm, rather than from ventrally directed morphogenetic movements of the lateral mesoderm. This loss of potency is not due to the inhibition of heart formation by migrating neural crest cells. During postneurula stages, tissue interactions between the lateral mesoderm and the underlying anterior endoderm support the heart-forming potency in the lateral mesoderm. The lateral mesoderm loses the ability to respond to this tissue interaction by stages 27–28. We speculate that either formation of the third pharyngeal pouch during stages 23–27 or lateral inhibition by ventral mesoderm may contribute to the spatial restriction of the heart morphogenetic field.     

Calcium sequestering activities of reticulum vesicles from Xenopus laevis oocytes

Membrane vesicles isolated from Xenopus laevis full-grown stage VI and mature oocytes accumulate 45Ca in the presence of ATP and oxalate. The Ca2+-pumping activity measured in vitro does not appear to be modified during meiotic maturation; it is not affected by the complex Ca2+-calmodulin. Preliminary experiments have shown that the addition of Na+ (30 mM) rapidly discharges accumulated 45Ca into oocyte vesicles indicating that a Na+/Ca2+ exchange system occurs in this membrane fraction. During progesterone-induced maturation, the different intracellular membranes undergo morphological changes. We suggest that intracellular movement of membrane vesicles could be involved in the local regulation of Ca2+ levels.     

Distribution of Lectin Binding Sites in Xenopus laevis Egg Jelly

Eggs from the anuran Xenopus laevis are surrounded by a thick jelly coat that is required during fertilization. The jelly coat contains three morphologically distinct layers, designated J1, J2, and J3. We examined the lectin binding properties of the individual jelly coat layers as a step in identifying jelly glycoproteins that may be essential in fertilization. The reactivity of 31 lectins with isolated jelly coat layers was examined with enzyme-linked lectin-assays (ELLAs). Using ELLA we found that most of the lectins tested showed some reactivity to all three jelly layers; however, two lectins showed jelly layer selectivity. The lectin Maackia amurensis (MAA) reacted only with J1 and J2, while the lectin Trichosanthes kirilowii (TKA) reacted only with J2 and J3. Some lectins were localized in the jelly coat using confocal microscopy, which revealed substantial heterogeneity in lectin binding site distribution among and within jelly coat layers. Wheat germ agglutinin (WGA) bound only to the outermost region of J3 and produced a thin, but very intense, band of fluorescence at the J1/J2 interface while the remainder of J2 stained lightly. The lectin MAA produced an intense fluorescence-staining pattern only at the J1/J2 interface. Several lectins were also tested for the ability to inhibit fertilization. WGA, MAA, and concanavalin A significantly inhibited fertilization and WGA was found to block fertilization by preventing sperm from penetrating the jelly. Using Western blotting, we identified high-molecular-weight components in J1 and J2 that may be important in fertilization.     

Scaling effects on muscle function in fast and slow muscles of Xenopus laevis

Fibre bundles or whole muscles from Xenopus laevis, ranging in size from 0.5-60g, were studied. Maximum power output of predominantly fast (sartorius) and slow (adductor magnus) muscles was measured at cycle frequencies between 0.5 and 20Hz, using the work loop technique. Power output was highly dependent on cycle frequency, and in 50-60g adults was maximal at 6 Hz for fast fibres (65 Wkg^-1), and 2 Hz for slow fibres (14 Wkg^-1). The cycle frequency for maximum power output was dependent on body mass (M_b), and decreased as a function of M_b^-0.07 in fast fibres, and M_b^-0.23 in slow fibres. The functional significance of these differences is discussed.     

Silver positivity of the NORs during embryonic development of Xenopus laevis

Bovine corneal endothelial cells adhered equally well to a variety of collagens (types I, III, IV and V) consistent with a role for fibronectin in this process. They did not exhibit a preferential binding to collagen type IV--as might be anticipated if laminin were to play a significant role in their adhesion. Inhibition studies with anti-fibronectin antibodies demonstrated the importance of endogenous fibronectin in the mediation of attachment. Consistent with this, binding did not appear to require the presence of exogenous protein, since cells bound to collagens equally well in the presence or absence of added fibronectin and binding was not stimulated by pretreatment of collagens with this protein.