Nephroblastoma in the clawed frog Xenopus laevis.

One 2.5-3 year old female clawed frog (Xenopus laevis), out of a consignment of 4,000 frogs, was found to have an abnormal abdominal growth, weighing 7.9 g. The growth was examined histologically and on the basis of the abundant stroma and serially-arranged tubules nephroblastoma was diagnosed. The growth is not considered to be transmissible.     

Xanthine dehydrogenase activity in the clawed frog, Xenopus laevis

Xanthine dehydrogenase (XDH; EC 1.2.1.37) activity in the clawed frog, Xenopus laevis, was detected in kidney tissue homogenates, but not in skin, liver, ovaries or gut tissues. The enzyme migrated as a single band of activity on both polyacrylamide and starch gel electropherograms, exhibited substrate inhibition, and did not appear developmentally until feeding larval stages. The tissue specificity, post-fertilization stage of appearance and single isozymic form make this a useful enzyme marker for further study concerning its developmental appearance and maintenance as a kidney-specific protein.     

Biophysics of underwater hearing in the clawed frog,Xenopus laevis

Anesthetized clawed frogs (Xenopus laevis) were stimulated with underwater sound and the tympanic disk vibrations were studied using laser vibrometry. The tympanic disk velocities ranged from 0.01 to 0.5 mm/s (at a sound pressure of 2 Pa) in the frequency range of 0.4–4 kHz and were 20–40 dB higher than those of the surrounding tissue. The frequency response of the disk had two peaks, in the range of 0.6–1.1 kHz and 1.6–2.2 kHz, respectively. The first peak corresponded to the peak vibrations of the body wall overlying the lung. The second peak matched model predictions of the pulsations of the air bubble in the middle ear cavity. Filling the middle ear cavity with water lowered the disk vibrations by 10–30 dB in the frequency range of 0.5–3 kHz.Inflating the lungs shifted the low-frequency peak downwards, but did not change the high-frequency peak. Thus, the disk vibrations in the frequency range of the mating call (main energy at 1.7–1.9 kHz) were mainly caused by pulsations of the air in the middle ear cavity; sound transmission via the lungs was more important at low frequencies (below 1 kHz). Furthermore, the low-frequency peak could be reversibly reduced in amplitude by loading the larynx with metal or tissue glue. This shows that the sound-induced vibrations of the lungs are probably coupled to the middle ear cavities via the larynx. Also, anatomical observations show that the two middle ear cavities and the larynx are connected in an air-filled recess in submerged animals.This arrangement is unique to pipid frogs and may be a structural adaptation to connect all the air spaces of the frog and improve low-frequency underwater hearing. Another function of the recess may be to allow cross-talk between the two middle ear cavities. Thus, the ear might be directional. Our pilot experiments show up to 10 dB difference between ipsi- and contralateral stimulus directions in a narrow frequency range around 2 kHz.     

Outgrowth dependency of forelimb regeneration on nerves in adult African clawed frog,Xenopus laevis

Summary The relationship between the size and shape of regenerative outgrowth and the quantity of innervation was studied in adult Xenopus laevis. The forelimbs, of which the nerve supply was artificially altered, were amputated midway through the stylopodium and were kept for 1 year. The regenerative outgrowths that formed in normal limbs with an intact nerve supply were mainly spike-shaped and occasionally rod-shaped. However, when the nerve supply to the distal part of the forelimb was augmented by surgically diverting ipsilateral sciatic nerve bundles, the quantity of innervation was increased to about two and a half times that of the normal limb. These hyperinnervated outgrowths were somewhat larger than those of the normally innervated outgrowths and the majority of them were oar-shaped, a type hardly ever encountered in normal regeneration. In contrast, when partial denervation was performed concomitantly with limb amputation, by ablation of the N. radialis at the shoulder joint, the quantity of innervation decreased to about one half that of the normal limb. The outgrowths obtained were spike-shaped in all cases, with their size being about half that of the normally innervated outgrowths. Furthermore, when both the N. radialis and N. ulnaris were ablated in the same way, the amputated limbs were mostly non-regenerative, but some of them regenerated small conical outgrowths. Based on these results, a discussion is presented concerning the relationship between a regenerative outgrowth and the innervation of the forelimb in Xenopus.     

Artificial fertilization of gametes from the South African clawed frog,Xenopus laevis

A reproducible and effective method for fertilization eggs of Xenopus laevis was developed based of systematic manipulation of environmental factors. The effects of varying concentrations of individual components of a fertilization medium were tested by measuring jelly swelling, sperm motility, and sperm longevity. Results were used to develop an improved medium for fertilization, consisting of 41.25 mM NaCl, 1.25 mM KCl, 0.25 mM CaCl₂, 0.0625 mM MgCl₂, 0.5 mM Na₂HPO₄, 2.5 mM HEPES, 1.9 mM NaOH, final pH(2°) 7.8.     

Factors affecting oogenesis in the South African clawed frog (Xenopus laevis)

Xenopus laevis, commonly known as the South African Clawed frog, is a hardy adaptable species that is relatively easy to maintain as a laboratory animal. Gametogenesis in wild Xenopus laevis is continuous and under ideal conditions, reproduction can occur year round. This unique aspect of amphibian reproduction offers an advantage over mammalian model systems: the eggs and oocytes collected from laboratory maintained Xenopus laevis provide an abundant and readily obtainable supply of material for cellular and biological research. However, many investigators report that laboratory Xenopus laevis go through periods of unexplained inefficient or complete failure of oocyte production or the production of poor quality oocytes. This results in experimental delays, inability to reproduce data, and ultimately the use of more animals. There is a lack of evidenced based information regarding the housing conditions that are necessary to optimize the health and fecundity of this species in captivity, but studies of wild Xenopus laevis have shown that temperature, age of the female, and nutrition are of key importance. The objective of this report is to review oogenesis with a special emphasis on these factors as they pertain to laboratory Xenopus laevis maintained for the purpose of providing a steady supply of eggs and oocytes. Harvesting methods and other experimental techniques that affect the quality of eggs and oocytes are also discussed.     

Wave frequency recognition and absolute pitch for water waves in the clawed frog,Xenopus laevis

Summary The ability of adultXenopus laevis to identify water wave frequencies was demonstrated by go/no-go conditioning. The acuity of frequency recognition is of absolute-pitch quality.Abbreviations S+ stimulus with response reward - S- stimulus with response punishment - u discrimination index     

Ontogeny of excitatory and inhibitory control of gastrointestinal motility in the African clawed frog, Xenopus laevis

The transparent body wall of Xenopus laevis larvae during the first developmental stages allows in vivo studies of gastrointestinal tract activity. The purpose of this study was to chart the ontogeny of gut motility in Xenopus larvae and to identify the most important control systems during the first developmental stages. Coordinated descending contraction waves first occurred in the gut at Nieuwkoop and Faber stage 43 [0.8 +/- 0.1 contractions/min (cpm)] and increased to 4.9 +/- 0.1 cpm at stage 47. The cholinergic receptor agonist carbachol (5-10 microM) increased contraction frequency already at stage 43, as did neurokinin A (NKA, 0.3-1 microM). The muscarinic antagonist atropine (100 microM) first affected contraction frequency at stage 45, which coincides with the onset of feeding. The tachykinin antagonist MEN-10,376 (6 microM) blocked NKA-induced contractions but not spontaneous motility. Both sodium nitroprusside [nitric oxide (NO) donor, 1-10 microM] and vasoactive intestinal peptide (VIP, 0.1-1 microM) inhibited contractions from the earliest stage onward. Blocking NO synthesis using NG-nitro-L-arginine methyl ester (100 microM) had no effect per se, but antagonized VIP evoked inhibition at stage 47. We conclude that gastrointestinal motility is well developed in the Xenopus laevis larvae before the onset of feeding. Functional muscarinic and tachykinin receptors are present already at the onset of motility, whereas a cholinergic tone develops around the onset of feeding. No endogenous tachykinin tone was found. Functional VIP receptors mediate inhibition at the onset of motility. NO seems to mediate the VIP effect at later stages.     

Female sex behaviors in the South African clawed frog, Xenopus laevis: gonadotropin-releasing, gonadotropic, and steroid hormones

The goals of this study were to characterize sex behaviors of female South African clawed frogs, Xenopus laevis, and to explore the behavioral effects of endocrine manipulation. The responses of females to clasp assaults by sexually active males were observed. Two patterns of female responses predominated. In one, females exhibited extreme leg extension and ticking vocalizations when clasped (unreceptive behaviors). In the other, females responded to being clasped by adduction of the thighs and increased flexion at the knee; ticking vocalizations were absent (receptive behaviors). When the female was unreceptive, clasps by males generally lasted less than 1 min. With a receptive female, on the other hand, amplexus could last up to 2 days. In intact females, injection of human chorionic gonadotropin (HCG) or of luteinizing hormone-releasing hormone (LHRH) into the dorsal lymph sac results in significant increases in receptivity. These hormones do not promote receptivity in ovariectomized females. Neither estradiol (E) nor progesterone (P) when administered alone was effective in restoring receptivity to ovariectomized females. In combination, E + P increased sexual receptivity. The releasing hormone, LHRH, when given to ovariectomized, E + P-treated females, further increased receptivity and led to the prolonged amplexus otherwise observed with an HCG-injected intact female. The behavioral effects of LHRH may be independent of action on the pituitary since they are not mimicked by gonadotropin.     

Differential antigen expression during metamorphosis in the tripartite olfactory system of the African clawed frog, Xenopus laevis

In the adult African clawed frog, Xenopus laevis, olfactory epithelium is housed in three separate nasal cavities: the principal cavity, the middle cavity, and the vomeronasal organ. The sensory epithelium in each of these cavities has distinct cellular features, and presumed physiological and behavioral functions, which arise during metamorphosis. Most notably, the middle cavity is formed de novo, and the principal cavity is transformed from a larval sensory epithelium with water exposure to an adult olfactory epithelium with air exposure. To understand the cellular nature of this plasticity more clearly, we characterized the staining patterns generated in the olfactory system of X. laevis with a new monoclonal antibody, anti-E7. The olfactory epithelium is first stained with anti-E7 during late embryonic development. Transection of the olfactory nerves during metamorphosis eliminates all staining and indicates that the staining is associated with mature or nearly mature olfactory receptor neurons. The antibody diffusely stains the vomeronasal organ throughout development and in adults. In the larval principal cavity, the olfactory receptor neurons are brightly stained, but this cellular staining is lost after metamorphosis. The mucus from Bowman's glands in the principal cavity, however, is intensely stained in adults. The middle cavity, throughout development and in adulthood, has the same staining characteristics as the larval principal cavity. Thus, the E7 antibody can distinguish the three areas of the olfactory epithelium, allowing measurement of sensory epithelium volume, and serves as an excellent marker for the changes in the sensory epithelium that occur during metamorphosis.     

Isolation and characterization of the third component of complement in the serum of the clawed frog, Xenopus laevis

The hemolytic activity against SRBC in the serum of normal Xenopus is dependent on specific antibody and both Ca++ and Mg++, whereas the activity against RRBC is dependent on Mg++ alone. Both of these hemolytic activities disappeared after treatment of the serum with zymosan or with the specific rabbit antiserum against one of the zymosan-binding proteins in Xenopus serum. By using this antiserum as a probe, a complement component (XC) was purified as a single entity from the Xenopus plasma after polyethylene glycol precipitation, DEAE-Sepharose CL-6B, Sepharose CL-6B, and Sephadex G-200 column chromatographies. The XC, contained at 2.3 mg/ml in normal serum, showed an electrophoretic mobility of beta-globulin, with a m.w. of 204,000 (204K) comprising two distinct subunits of 125K and 85K, which are linked with each other by disulfide bonds. The 204K protein exhibited a strong hemolytic activity in association with other components in Xenopus serum. Digestion of 204K protein by trypsin resulted in a specific cleavage of the 125K subunit and a conversion of its immunoelectrophoretic mobility to the anodal side, leaving the 85K subunits intact. The treatment of XC with SDS and urea resulted in the splitting of 125K subunits into 78K and 40K, but this splitting was inhibited upon pretreatment with methylamine, suggesting the presence of a thiol ester bond in the XC. The amino acid composition of the XC revealed a striking resemblance to that of mammalian C3. In all aspects, the 204K protein (XC) is regarded as representing the C3 of Xenopus laevis, which plays a key role in both the classical and alternative hemolytic pathways.     

Development of adrenergic and cholinergic cardiac control in larvae of the African clawed frog Xenopus laevis

Cardiac responses (heart rate, stroke volume, and cardiac output) to cholinergic and adrenergic receptor stimulation were investigated in developing larvae of Xenopus laevis from Nieuwkoop and Faber (NF) stage 33/34 (newly hatched) to NF stage 53 (22 d after hatching). Effects on heart rate (fH), stroke volume (SV), and cardiac output (CO) were analyzed using in situ preparations and video-microscopic techniques to record the continually beating heart. The results show that administration of acetylcholine to the heart decreases heart rate as early as NF stage 40. A significant reduction in SV and CO following acetylcholine administration to the heart was found at NF stages 45-53. Epinephrine had no significant effect on fH, SV, or CO at any of the stages investigated. However, an adrenergic tonus on the heart is present already at NF stage 40 (11%). This tonus increases up to a maximum (44%) at NF stages 45-47, when the maximal heart rate is found during development of X. laevis. We conclude that acetylcholine has a negative chronotropic and possibly also inotropic effect on the heart very early in development of X. laevis. We also hypothesize that the high adrenergic tonus found at NF stages 45-47 is responsible, at least in part, for the peak in heart rate seen at these stages.     

Developmental changes of purinergic control of intestinal motor activity during metamorphosis in the African clawed frog, Xenopus laevis

Little is known about the purinergic regulation of intestinal motor activity in amphibians. Purinergic control of intestinal motility is subject to changes during development in mammals. The aim of this study was to investigate purinergic control of intestinal smooth muscle in the amphibian Xenopus laevis and explore possible changes in this system during the developmental phase of metamorphosis. Effects of purinergic compounds on mean force and contraction frequency in intestinal circular muscle strips from prometamorphic, metamorphic, and juvenile animals were investigated. Before metamorphosis, low concentrations of ATP reduced motor activity, whereas the effects were reversed at higher concentrations. ATP-induced relaxation was not inhibited by the P2-receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) but was blocked by the ecto-nucleotidase inhibitor 6-N,N-diethyl-d-beta,gamma-dibromomethylene ATP (ARL67256), indicating that an ATP-derived metabolite mediated the relaxation response at this stage. Adenosine induced relaxation before, during, and after metamorphosis, which was blocked by the A(1)-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). The stable ATP-analog adenosine 5'-[gamma-thio]-triphosphate (ATPgammaS) and 2-methylthioATP (2-MeSATP) elicited contractions in the circular muscle strips in prometamorphic tadpoles. However, in juvenile froglets, 2-MeSATP caused relaxation, as did ATPgammaS at low concentrations. The P2Y(11)/P2X(1)-receptor antagonist NF157 antagonized the ATPgammaS-induced relaxation. The P2X-preferring agonist alpha-beta-methyleneadenosine 5'-triphosphate (alpha-beta-MeATP) evoked PPADS-sensitive increases in mean force at all stages investigated. This study demonstrates the existence of an adenosine A(1)-like receptor mediating relaxation and a P2X-like receptor mediating contraction in the X. laevis gut before, during, and after metamorphosis. Furthermore, the development of a P2Y(11)-like receptor-mediated relaxation during metamorphosis is shown.     

The primary structures and myotropic activities of two tachykinins isolated from the African clawed frog,Xenopus laevis

Two peptides with limited structural similarity to mammalian substance P (SP) and neurokinin A (NKA) have been isolated from extracts of the intestine of the African clawed frog (Xenopus laevis). The primary structure of an SP-like peptide was established as: Lys-Pro-Arg-Pro-Asp-Gln-Phe-Tyr-Gly-Leu-Met.NH(2), which is identical to the previously characterized peptide, bufokinin isolated from the toad Bufo marinus. The primary structure of an NKA-related peptide was established as Thr-Leu-Thr-Thr-Gly-Lys-Asp-Phe-Val-Gly-Leu-Met.NH(2). Only the five amino acids at the C-terminal region of the peptide are identical to mammalian NKA whereas the N-terminal region shows no structural similarity to previously characterized tachykinins. Immunohistochemical investigations of the gut wall revealed a dense network of nerve fibres and nerve cell bodies containing SP/NKA-like substances. The myotropic effects of the Xenopus tachykinins were compared with the contractile effect of mammalian SP and NKA on isolated strips of circular smooth muscle from Xenopus stomach. No significant differences in potencies (-log EC(50)) or in intrinsic activities were observed between the Xenopus and mammalian peptides. The potencies for the Xenopus SP-like (8.49+/-0.15) and the NKA-like peptide (8.12+/-0.06) were similar suggesting that the amino acid sequence at the N-terminal region of the tachykinins is not important in activating the tachykinin receptors in Xenopus gastric smooth muscle. The maximum response to Xenopus SP (alpha=0.59+/-0.06) was significantly lower than to the NKA-like peptide (alpha=1.0) suggesting a more effective interaction of the NKA-like peptide with the tachykinin receptor(s) in Xenopus stomach.     

Patterns of spatial and temporal cranial muscle development in the African clawed frog, Xenopus laevis (Anura: Pipidae)

The African Clawed Toad, Xenopus laevis, has been a major vertebrate model organism for developmental studies for half a century. Because most studies have focused on the early stages of development, this has had the effect that many aspects of organogenesis and later development remain relatively poorly known in this species. In particular, little is known about cranial muscle development even at the level of morphology and histological differentiation of muscle anlagen and muscle fibers. In this study, we document the morphogenesis and histological differentiation of cranial muscles in X. laevis. We provide a detailed account of the timing of development for each of the cranial muscles, and also describe a new muscle, the m. transversus anterior. The cranial musculature of X. laevis larvae generally develops in a rostrocaudal sequence. The first muscles to differentiate are the extrinsic eye muscles. Muscles of the mandibular and hyoid arches develop almost simultaneously, and are followed by the muscles of the branchial arches and the larynx, and by the mm. geniohyoideus and rectus cervicis. Despite the fact that differentiation starts at different stages in the different muscles, most are fully developed at Stage 14. These baseline data on the timing of muscle differentiation in the X. laevis can serve as a foundation for comparative studies of heterochronic changes in cranial muscle development in frogs and other lissamphibians.