Microvascularization and histomorphology of lateral line organs in adult Xenopus laevis

The microvasculariaztion of the lateral line organs (LLOs) of the adult pipid frog, Xenopus laevis was studied by scanning electron microscopy of vascular corrosion casts (VCCs) and correlative light microscopy of paraplast embedded tissues sections. Scanning electron micrographs of VCCs revealed that each neuromast within the LLO rests on a distinct bowl‐like capillary network (vascular bowl). One to three vascular bowls were supplied by an ascending arteriole and drained by a descending venule towards the skin deep dermal vascular network. Blood flow regulation mechanisms in form of intimal cushions were present at the origin of ascending arterioles supplying LLOs, microvenous valves were present at the confluence of deep dermal venules and veins. This together with sprouting and nonsprouting angiogenesis (intussusceptive microvascular growth) found in vascular bowls demonstrate that in adult Xenopus the capillary bed of LLO's still can be adjusted to changing energetic needs. J. Morphol. 275:497–503, 2014. © 2013 Wiley Periodicals, Inc.     

Incorporation of 35S-sulfate into yolk platelets of Xenopus laevis embryos. A study using electron microscope autoradiography.

Inorganic 35S-sulfate was injected into Xenopus laevis embryos before first cleavage to study incorporation of the label into the yolk platelets in order to localize glycosaminoglycan synthesis. Electron microscope autoradiography of embryonic thin sections from blastulae and gastrulae revealed that the primary site of label incorporation is at the edge of the yolk platelets, and, to a lesser extent, in their interiors. Autoradiography of isolated yolk platelets, lacking unit membranes, indicated the absence of label. Thus, edge associated label comes from the yolk platelets membrane, and interior label is solubilized in the glycerol-water gradient during yolk platelets isolation. Ruthenium red staining of yolk platelet in situ shows haavy deposits of the dye on the yolk platelet membrane surface facing the cytoplasmic surface. The crystalline main body of isolated yolk platelets does not take up the dye. It appears that continuous synthesis or sulfation of glycosaminoglycan occurs primarily at the outer surface yolk platelet membranes during early development, providing a novel site for this process.     

Sperm incorporation in Xenopus laevis: characterisation of morphological events and the role of microfilaments

Scanning and transmission electron microscopy were used to determine the morphological changes in the egg plasma membrane associated with sperm binding, fusion and incorporation in Xenopus laevis. Sperm incorporation in Xenopus is rapid, occurring within 3-5 min following addition of sperm. Images have been obtained of both early sperm-egg interactions and fertilisation bodies. Additionally, two drugs that specifically alter F-actin dynamics, latrunculin and jasplakinolide, were used to determine whether sperm incorporation is a microfilament-dependent process. Jasplakinolide did not prevent sperm incorporation, cortical granule exocytosis or cortical contraction, suggesting these events can occur without depolymerisation of existing, stabilised filaments. Latrunculin A, which competes with thymosin beta4 in ooplasm for binding actin monomer, did not inhibit cortical granule exocytosis, but blocked cortical contraction in 100% of eggs at a concentration of 5 microM. Although a single penetrating sperm was found on an egg pretreated in latrunculin, fertilisation bodies were never observed. At < 5 microM latrunculin, many eggs did undergo cortical contraction with some exhibiting severe distortions of the plasma membrane and abnormal accumulations of pigment granules. Preincubation of eggs in jasplakinolide before latrunculin mitigated both these effects to some degree. However, eggs incubated in latrunculin either prior to or after insemination never progressed through first cleavage.     

Inositol incorporation into phosphoinositides in retinal horizontal cells of Xenopus laevis: enhancement by acetylcholine, inhibition by glycine

The absorption of light by photoreceptor cells leads to an increased incorporation of [2-3H]inositol into phosphoinositides of horizontal cells in the retina of Xenopus laevis in vitro. We have identified several retinal neurotransmitters that are involved in regulating this response. Incubation with glycine, the neurotransmitter of an interplexiform cell that has direct synaptic input onto horizontal cells, abolishes the light effect. This inhibition is reversed by preincubation with strychnine. Acetylcholine added to the culture medium enhances the incorporation of [2-3H]inositol into phosphoinositides in horizontal cells when retinas are incubated in the dark. This effect is inhibited by preincubation with atropine. However, atropine alone does not inhibit the light-enhanced incorporation of [2-3H]inositol into phosphoinositides in the retina. gamma-Aminobutyric acid, the neurotransmitter of retinal horizontal cells in X. laevis, as well as dopamine and norepinephrine, have no effect on the incorporation of [2-3H]inositol into phosphoinositides. These studies demonstrate that the light-enhanced incorporation of [2-3H]inositol into phosphoinositides of retinal horizontal cells is regulated by specific neurotransmitters, and that there are probably several synaptic inputs into horizontal cells which control this process.     

Permeability of Xenopus laevis embryos: Specific incorporation of precursors into eukaryotic proteins and nucleic acids

All amino acids and several nucleic acid precursors are taken up by Xenopus laevis embryos. The embryos are completely intact and not modified in any way. These precursors are directly incorporated into the macromolecules of Xenopus embryos and not prokaryotic contaminants as has been previously claimed. Radioactive leucine is incorporated into Xenopus laevis ribosomal proteins as characterized by sucrose gradient centrifugation. The uptake of the amino acids is cycloheximide sensitive and unaffected by chloramphenicol. Radioactive adenosine and orotic acid are taken up and incorporated into tRNA and rRNA at high levels as characterized by sucrose gradients and electrophoresis. These characterizations of labeled macromolecules unequivocally show that normal Xenopus laevis embryos will take up and incorporate labeled precursors to levels which are sufficient to study cellular biochemical events at such early stages of development.     

Comparison by electrophoresis of proteins characteristic of the lateral line and skin of Xenopus laevis

In experiments aimed at determining acousticolateralis marker proteins, fractions of lateral-line organs and skin of Xenopus laevis were analyzed by one- and two-dimensional polyacrylamide-gel electrophoresis. A protein fraction of approximately 44K mol. wt (K = 1000 daltons) and isoelectric pH 6.3, consisting of at least two components, was enhanced in lateral-line neuromast tissue (containing hair cells) and was decreased in tactile organs and skin (lacking hair cells). This "neuromast-marker-protein" fraction had a mol. wt close to that of actin but was shown to be different from actin. Two other major proteins, at mol. wts 16 and 28K, were present in gels of skin and absent in gels of lateral-line tissue. These proteins were shown to be due to secretion of the amphibian granular glands and were designated "negative marker proteins".     

Comparative actions of GABA and acetylcholine on the Xenopus laevis lateral line

The effects of GABA, acetylcholine and carbachol on the spontaneous activity of afferent nerve fibers in the lateral line of Xenopus laevis are characterized. Atropine and bicuculline were also tested on drug- and water motion-evoked activity. GABA (0.019-1.25 mM) suppressed and both acetylcholine (1.25-80 microM) and carbachol (1.25-40 microM) increased spontaneous activity. These actions were blocked by bicuculline (100 microM) and atropine (4 microM) respectively. Atropine (20 microM) and bicuculline (100 microM) had no effect on water motion-evoked activity. The results characterize actions of GABA and acetylcholine not previously described and provide evidence that does not support the hypothesis that GABA or acetylcholine are the afferent transmitter.     

Acrosome reaction in sperm of the frog, Xenopus laevis: its detection and induction by oviductal pars recta secretion

Previous electron microscopic observations have shown that the acrosome of the sperm of the frog, Xenopus laevis, comprises a membrane-bounded vesicle covering the anterior-most position of the head. We obtained a sperm suspension from the testes and stained it with LysoSensor Green for observation under a confocal laser scanning microscope and found a bright fluorescence reflecting the presence of the acrosomes at the top of the sperm head in about 64% of the sperm, with no deterioration of their capacity to fertilize. About 40% of the sperm with an acrosome underwent an acrosome reaction in response to Ca(2+) ionophore A23187, as evidenced by a loss of LysoSensor Green stainability, accompanied by breakdown of the acrosomal vesicle. About 53% of the sperm bound to isolated vitelline envelopes underwent an acrosome reaction, whereas both jelly water and solubilized vitelline envelopes weakly induced an acrosome reaction. When the sperm were treated with an oviductal extract obtained from the pars recta, but not the pars convoluta region, about 40% of the sperm with acrosomes underwent an acrosome reaction. The substance containing acrosome reaction-inducing activity in the pars recta extract seemed to be a heat-unstable substance with a molecular weight of greater than 10 kDa. The activity was not inhibited by protease inhibitors but required extracellular Ca(2+) ions. These results indicate that the acrosome reaction occurs on the vitelline envelopes in response to the substance deposited from the pars recta during the passage of the oocytes through the oviduct.     

Functional expression of mammalian glucose transporters in Xenopus laevis oocytes: evidence for cell-dependent insulin sensitivity.

We report the functional expression of two different mammalian facilitative glucose transporters in Xenopus oocytes. The RNAs encoding the rat brain and liver glucose transporters were transcribed in vitro and microinjected into Xenopus oocytes. Microinjected cells showed a marked increase in 2-deoxy-D-glucose uptake as compared with controls injected with water. 2-Deoxy-D-glucose uptake increased during the 5 days after microinjection of the RNAs, and the microinjected RNAs were stable for at least 3 days. The expression of functional glucose transporters was dependent on the amount of RNA injected. The oocyte-expressed transporters could be immunoprecipitated with anti-brain and anti-liver glucose transporter-specific antibodies. Uninjected oocytes expressed an endogenous transporter that appeared to be stereospecific and inhibitable by cytochalasin B. This transporter was kinetically and immunologically distinguishable from both rat brain and liver glucose transporters. The uniqueness of this transporter was confirmed by Northern (RNA) blot analysis. The endogenous oocyte transporter was responsive to insulin and to insulinlike growth factor I. Most interestingly, both the rat brain and liver glucose transporters, which were not insulin sensitive in the tissues from which they were cloned, responded to insulin in the oocyte similarly to the endogenous oocyte transporter. These data suggest that the insulin responsiveness of a given glucose transporter depends on the type of cell in which the protein is expressed. The expression of hexose transporters in the microinjected oocytes may help to identify tissue-specific molecules involved in hormonal alterations in hexose transport activity.     

Purification and partial characterization of Xenopus laevis tenascin from the XTC cell line.

We report here the purification of tenascin, an extracellular matrix molecule involved in the control of morphogenesis, from the conditioned medium of the Xenopus XTC cell line. Tenascin was purified by affinity chromatography on a column of the monoclonal antibody mAb TnM1; the molecule eluted from this column has a relative molecular mass of 210 kDa after reduction. Electrophoretic analysis under non-reducing conditions shows that the purified components are oligomeric disulfide-linked complexes which barely enter a 4% polyacrylamide gel. Upon rotary shadowing these molecules appear to possess a central globular domain to which pairs or triplets of arms are attached. Polyclonal antibodies have been raised against purified Xenopus tenascin. They recognise specifically the antigen on Western blots of XTC conditioned medium and adult brain, by immunofluorescence, these antibodies reveal large amounts of tenascin in the secretory vesicles as well as in the extracellular matrix of XTC cells. In the Xenopus tadpole, they stain the developing cartilage, the basal lamina of skin epidermis, myotendinous ligaments and restricted regions of the central nervous system.     

Immunolocalization of a mammalian aquaporin 3 homolog in water-transporting epithelial cells in several organs of the clawed toad Xenopus laevis

Nucleotide sequences of cDNA were used to construct antibodies against an aquaporin (AQP) expressed in the clawed toad, Xenopus laevis, viz., Xenopus AQP3, a homolog of mammalian AQP3. Xenopus AQP3 was immunolocalized in the basolateral membrane of the principal cells of the ventral skin, the urinary bladder, the collecting duct and late distal tubule of the kidney, the absorptive epithelial cells of the large intestine, and the ciliated epithelial cells of the oviducts. Therefore, we designated this AQP as basolateral Xenopus AQP3 (AQP-x3BL). The intensity of labeling for AQP-x3BL differed between the ventral and dorsal skin, with the basolateral membrane of the principal cells in the ventral skin showing intense labeling, whereas that in the dorsal skin was lightly labeled. AQP-x3BL was also immunolocalized in the basolateral membrane of secretory cells in the small granular and mucous glands of the skin. As AQP-x5, a homolog of mammalian AQP5, is localized in the apical membrane of these same cells, this provides a pathway for fluid secretion by the glands. Although Hyla AQP-h2 is translocated from the cytoplasm to the apical membrane of the Hyla urinary bladder in response to arginine vasotocin (AVT), AQP-h2 immunoreactivity in Xenopus bladder remains in the cytoplasm and barely moves to the apical membrane, regardless of AVT stimulation. AQP-x3 is localized in the basolateral membrane, even though the AVT-stimulated AQP-h2 does not translocate to the apical membrane. These findings provide new insights into AQP function in aquatic anurans.     

Evidence for basolateral Cl- channels as modulators of apical Cl- secretion in pulmonary epithelia of Xenopus laevis

Pulmonary epithelia of air-breathing vertebrates are covered by a thin, fluid layer that is essential for immune defense and gas diffusion. The composition of this layer is maintained by ion transport mechanisms, including Cl(-) transport. The present study focuses on the function of basolateral Cl(-) channels in Xenopus pulmonary epithelia, since knowledge concerning this issue is limited. Therefore, Ussing chamber measurements were performed, and transepithelial short-circuit currents (I(SC)) were monitored. Basolateral application of the Cl(-) channel inhibitor N-phenylanthranilic acid (DPC) resulted in an increase of the I(SC), indicating a DPC-sensitive Cl(-) conductance. This observation was confirmed in experiments using an apical-to-basolateral Cl(-) gradient, with and without nystatin (apical side) to permeabilize the epithelia as well as by establishing an iodide gradient. The DPC-sensitive Cl(-) conductance was influenced by procedures interfering with apical Cl(-) secretion. For example, the effect of forskolin was increased when basolateral Cl(-) channels were blocked by the simultaneous application of DPC. Activation of apical Cl(-) secretion by forskolin/IBMX and subsequent DPC application resulted in a significantly reduced DPC effect. Accordingly, DPC led to an increased apical Cl(-) secretion estimated by an increased 5-nitro-2-(3-phenylpropylamino)benzoic acid-sensitive I(SC). Furthermore, inhibition of basolateral anion exchangers responsible for Cl(-) uptake resulted in a decreased DPC-sensitive current. Taken together, we have evidence concerning the function of basolateral Cl(-) channels in Xenopus pulmonary epithelium and that these channels play a significant role in mediating apical Cl(-) secretion involving a novel Cl(-) recycling mechanism across the basolateral membrane.