African Clawed Frog Xenopus Laevis Research Articles

Toward the understanding of biology of oocyte life cycle in Xenopus Laevis: No oocytes left behind.

BackgroundFor the past more than 25 years, we have been focusing on the developmental and reproductive biology of the female gametes, oocytes, and eggs, of the African clawed frog Xenopus laevis.MethodsThe events associated with the life cycle of these cells can be classified into the four main categories: first, oogenesis and cell growth in the ovary during the first meiotic arrest; second, maturation and ovulation that occur simultaneously and result in the acquisition of fertilization competence and the second meiotic arrest; third, fertilization, that is sperm‐induced transition from egg to zygote; and fourth, egg death after spontaneous activation in the absence of fertilizing sperm.Main findingsOur studies have demonstrated that signal transduction system involving tyrosine kinase Src and other oocyte/egg membrane‐associated molecules such as uroplakin III and some other cytoplasmic proteins such as mitogen‐activated protein kinase (MAPK) play important roles for successful ovulation, maturation, fertilization, and initiation of embryonic development.ConclusionWe summarize recent advances in understanding cellular and molecular mechanisms underlying life cycle events of the oocytes and eggs. Our further intention is to discuss and predict potentially promising impact of the recent findings on the challenges facing reproductive biology and medicine, as well as societal contexts.

Reciprocal Matched Filtering in the Inner Ear of the African Clawed Frog (Xenopus laevis).

Anurans (frogs and toads) are the most vocal amphibians. In most species, only males produce advertisement calls for defending territories and attracting mates. Female vocalizations are the exceptions among frogs, however in the African clawed frog (Xenopus laevis) both males and females produce distinct vocalizations. The matched filter hypothesis predicts a correspondence between peripheral auditory tuning of receivers and properties of species-specific acoustic signals, but few studies have assessed this relationship between the sexes. Measuring hearing sensitivity with a binaural recording of distortion product otoacoustic emissions, we have found that the ears of the males of this species are tuned to the dominant frequency of the female's calls, whereas the ears of the females are tuned close to the dominant frequency of the male's calls. Our findings provide support for the matched filter hypothesis extended to include male-female calling. This unique example of reciprocal matched filtering ensures that males and females communicate effectively in high levels of background noise, each sex being most sensitive to the frequencies of the other sex's calls.

Xenopus laevis as UberXL for nematodes

The effect of invasive species on local parasite dynamics is often overlooked. The African Clawed Frog Xenopus laevis (Daudin, 1802) (Anura: Pipidae) is a global invader, with established populations on four continents and is a domestic exotic in southern Africa. Despite a century of parasitological surveys, the current study reports seven previously unrecorded nematode species parasitising X. laevis across South Africa. These are adult Capillaria sp. and Falcaustra sp. from the intestine, third stage larvae of Contracaecum sp. encysted in the body cavity, third stage larvae of Paraquimperia sp. and Tanqua sp. from the intestine and two different species of second stage nematode larvae from the lungs and kidneys, respectively. Morphological descriptions, photomicrographs and molecular data of the 18S and 28S rRNA and COI genes are provided to aid future investigations. We propose that these nematodes could well be using X. laevis as a definitive, paratenic and intermediate host, probably involving native fish, piscivorous birds, semi-aquatic reptiles and invertebrates in their life cycles. All recovered nematodes are recorded for the first time herein in association with X. laevis, except for the genus Contracaecum, members of which have previously been recorded from invasive X. laevis from California and Chile. The current study illustrates that X. laevis is an important parasite reservoir in its native range, with implications for its role in the invasive range. The fact that none of these nematodes could be identified to species level underscores the importance of provid ing morphological descriptions and molecular data when reporting on parasitological surveys, especially those of known invasive species.

PACT/PRKRA and p53 regulate transcriptional activity of DMRT1.

The transcription factor DMRT1 (doublesex and mab-3 related transcription factor) has two distinct functions, somatic-cell masculinization and germ-cell development in some vertebrate species, including mouse and the African clawed frog Xenopus laevis. However, its transcriptional regulation remains unclear. We tried to identify DMRT1-interacting proteins from X. laevis testes by immunoprecipitation with an anti-DMRT1 antibody and MS/MS analysis, and selected three proteins, including PACT/PRKRA (Interferon-inducible double-stranded RNA dependent protein kinase activator A) derived from testes. Next, we examined the effects of PACT/PRKRA and/or p53 on the transcriptional activity of DMRT1. In transfected 293T cells, PACT/PRKRA and p53 significantly enhanced and repressed DMRT1-driven luciferase activity, respectively. We also observed that the enhanced activity by PACT/PRKRA was strongly attenuated by p53. Moreover, in situ hybridization analysis of Pact/Prkra mRNA in tadpole gonads indicated high expression in female and male germline stem cells. Taken together, these findings suggest that PACT/PRKRA and p53 might positively and negatively regulate the activity of DMRT1, respectively, for germline stem cell fate.

Gelsolin and Related Proteins in Vertebrate Model Organisms

Gelsolin, encoded by the Gsn gene, is a highly conserved actin-binding protein detected in all studied eukaryotic organisms. Gelsolin is the founding member of the gelsolin superfamily of proteins, whose main functions are binding, severing, and capping actin filaments. Moreover, gelsolin is engaged in apoptosis regulation, signal transduction, and embryonic development. Gelsolin is also implicated in some human diseases such as Alzheimer disease and familial amyloidosis of the Finnish type (FAF). Because of its multifunctionality, gelsolin is a potential candidate for diverse therapeutic applications. Most of our knowledge about gelsolin superfamily proteins comes from investigations conducted on vertebrate model organisms such as zebrafish (Danio rerio), African clawed frogs (Xenopus laevis), chickens (Gallus gallus), and mice (Mus musculus). Here, we present fundamental findings as well as new knowledge to indicate main research directions and future possibilities. Different model organisms provide specific possibilities and present particular advantages. However, all of them give useful data, which allow us to gain a better understanding of the function of gelsolin and related proteins.

Assessing the effect of landscape features on pond colonisation by an elusive amphibian invader using environmental DNA

Abstract Environmental DNA (eDNA) is becoming an essential tool for detecting aquatic invasive species and investigating their spread. Surprisingly, this technique has been very rarely used to investigate habitat selection, site occupancy, and colonisation despite its higher capacity to detect many species. The African clawed frog (Xenopus laevis) is a principally aquatic amphibian introduced in several continents from South Africa. In western France, no recent systematic survey of the invasion range has been attempted, mainly because of the elusive nature of the species. Furthermore, the influence of landscape features on invasion has never been investigated, even if adults and juveniles are known to disperse overland and along river networks. Using presence–absence data generated by an eDNA survey conducted across the known invasion front of X. laevis in western France, we aimed to determine whether and how the landscape features surrounding a pond influence the probability that a pond is colonised. Xenopus laevis was detected well beyond the formerly known invasive distribution and at the outward end of some transects, suggesting that we did not reach the actual invasion front in these parts of the range. The landscape variables that best predicted the presence of X. laevis in a pond were topographic wetness index and grass cover within a buffer of 250 m. Higher values of both topographic wetness index and grass cover were negatively related to the occurrence probability. The effects of these two variables more likely to reflect dispersal behaviour than habitat preferences at the pond scale. By combining the high detection probability of eDNA survey techniques and a landscape ecology approach, we may gain valuable insight into the colonisation process of water bodies by elusive invasive species. Such information is crucial to prevent access to specific sites and locate invasion front areas where connectivity can be disrupted, thus increasing the effectiveness of management countermeasures.

Adrenal Adenoma in an Adult Female Xenopus laevis

Abstract This report describes a neoplasm involving the adrenal cortex of an aged female African clawed frog (Xenopus laevis). The animal was found to be in poor body condition and was subsequently...

Simple Method To Characterize the Ciliary Proteome of Multiciliated Cells.

Motile cilia of multiciliated epithelial cells have important roles in animal development and cell homeostasis. Although several studies have identified and reported proteins localized in this complex organelle and the related immotile primary cilia from various cell types, it is still challenging to isolate high quantities of ciliary proteins for proteomic analysis. In this study, African clawed frog (Xenopus laevis) embryos, which have many multiciliated cells in the epidermis, were treated with a simple ionic buffer to identify 1009 proteins conserved across vertebrates; these proteins were putatively localized in motile cilia. Using two ciliary proteome databases, we confirmed that previously validated cilia-associated proteins are highly enriched in our ciliary proteome. Proteins localized at the transition zone and Ellis-van Creveld zone, which are distinct regions at the base of cilia, near the junction with the apical cell surface, were isolated using our method. Among the newly identified ciliary proteins, we report that KRT17 may have an unrecognized function in motile cilia. Hence, the method developed in this study would be useful for understanding the ciliary proteome.

The role of ambient temperature and body mass on body temperature, standard metabolic rate and evaporative water loss in southern African anurans of different habitat specialisation.

Temperature and water availability are two of the most important variables affecting all aspects of an anuran’s key physiological processes such as body temperature (Tb), evaporative water loss (EWL) and standard metabolic rate (SMR). Since anurans display pronounced sexual dimorphism, evidence suggests that these processes are further influenced by other factors such as vapour pressure deficit (VPD), sex and body mass (Mb). However, a limited number of studies have tested the generality of these results across a wide range of ecologically relevant ambient temperatures (Ta), while taking habitat use into account. Thus, the aim of this study was to investigate the role of Ta on Tb, whole-animal EWL and whole-animal SMR in three wild caught African anuran species with different ecological specialisations: the principally aquatic African clawed frog (Xenopus laevis), stream-breeding common river frog (Amietia delalandii), and the largely terrestrial raucous toad (Sclerophrys capensis). Experiments were conducted at a range of test temperatures (5–35 °C, at 5 °C increments). We found that VPD better predicted rates of EWL than Ta in two of the three species considered. Moreover, we found that Tb, whole-animal EWL and whole-animal SMR increased with increasing Ta, while Tb increased with increasing Mb in A. delalandii and S. capensis but not in X. laevis. Whole-animal SMR increased with increasing Mb in S. capensis only. We did not find any significant effect of VPD, Mb or sex on whole-animal EWL within species. Lastly, Mb did not influence Tb, whole-animal SMR and EWL in the principally aquatic X. laevis. These results suggest that Mb may not have the same effect on key physiological variables, and that the influence of Mb may also depend on the species ecological specialisation. Thus, the generality of Mb as an important factor should be taken in the context of both physiology and species habitat specialisation.

Spatiotemporal expression profile of embryonic and adult ankyrin repeat and EF-hand domain containing protein 1-encoding genes ankef1a and ankef1b in zebrafish

Recent human next-generation sequencing (NGS) studies indicate a correlation between ANKEF1 (ankyrin repeat and EF-hand domain containing protein 1) expression and cilia formation or function. Additionally, a single study conducted in the African clawed frog (Xenopus laevis) showed ankef1 is down-regulated after pharmacological fibroblast growth factor (FGF) inhibition and plays a role in protocadherin-mediated cell protrusion and adhesion. That study also revealed a critical role for ankef1 in the embryonic development of the frog, with morphants exhibiting phenotypes including spina bifida and a shortened body axis. Interestingly, while little is known about ANKEF1 function in other vertebrate systems, recent proteomic data has shown ANKEF1 enriched in ciliated cells. Likewise, publicly available EST profile databases imply ANKEF1 expression in multiple human tissues, including high levels in the testes. Together, these previous studies suggest an important role for ANKEF1 in ciliated tissues and during embryonic development. Here, we report cloning of zebrafish (Danio rerio) ankef1a, as well as its paralog, ankef1b, and expression analyses by whole-mount in situ hybridization (WISH) and quantitative polymerase chain reaction (qPCR) during embryonic development and in adult tissues. WISH shows both forms are ubiquitously expressed early in development, with more discrete expression of both transcripts in embryonic tissues known to precede or possess motile cilia, including dorsal forerunner cells (DFC) and the otic vesicles, respectively. Additionally, both transcripts are enriched in the developing pharynx and swim bladder. Our qPCR results indicate enhanced expression in the testes, along with increased expression in brain. Certainly, our experiments in the zebrafish model system with ankef1a and ankef1b provide a solid foundation for future studies to uncover the molecular pathways through which Ankef1 acts in both healthy and disease states.

Evaluating the role of fish as surrogates for amphibians in pesticide ecological risk assessment

Ecological risk of chemicals to aquatic-phase amphibians has historically been evaluated by comparing estimated environmental concentrations in surface water to surrogate toxicity data from fish species. Despite their obvious similarities, there are biological disparities among fish and amphibians that could affect their exposure and response to chemicals. Given the alarming decline in amphibians, in which anthropogenic pollutants play at least some role, investigating the risk of chemicals to amphibians is becoming increasingly important. Here, we evaluate relative sensitivity of fish and larval aquatic-phase amphibians to 45 different pesticides using existing data from three standardized toxicity test designs: (1) amphibian metamorphosis assay (AMA) with the African clawed frog (Xenopus laevis); (2) fish short-term reproduction assay (FSTRA) with the fathead minnow (Pimephales promelas); (3) fish early life stage test (ELS) with fathead minnows or rainbow trout (Oncorhynchus mykiss). The advantage of this dataset over previous work is that the underlying studies are consistent in exposure method, study duration, test species, endpoints measured, and number of concentrations tested. We found very strong positive relationships between fish and frog lowest adverse effect concentrations (LOAEC) for survival [Spearman's rank correlation (rs) = 0.88], body weight (rs = 0.86), and length (rs = 0.89) with only one out of 45 chemicals (propiconazole) exhibiting 100-folder greater sensitivity in frogs relative to fish. While our results suggest comparable toxicity for pesticides between fish and aquatic-phase amphibians under these test conditions, further research with a greater diversity of amphibians and exposure scenarios will help determine the relevance of these results across species and life stages.

Cardiotoxicity in African clawed frog (Xenopus laevis) sub-chronically exposed to environmentally relevant atrazine concentrations: Implications for species survival

The toxic effects of different atrazine concentrations on tadpoles and adult male African clawed frogs (Xenopus laevis) were assessed in a controlled laboratory environment following 90 days’ exposure. The aim was to elucidate the danger of atrazine exposure on the cardiac tissue relative to its critical function of rhythmic contractility, fundamental for optimal blood circulation and homeostasis.Tadpoles and adult frogs were exposed to 0 μg/L (control), 0.01 μg L−1, 200 μg L−1 and 500 μg L−1 concentrations of atrazine for 90 days. Mortality was concenration-dependent and significantly increased in juvenile group (77%, 43%, 23% and 0 respectively for 500 μg L−1, 200 μg L−1, 0.01 μg L−1, and control group). While the mean juvenile heart area decreased concentration-dependently, adult frog mean heart area significantly increased in the 200 μg L−1 group only and mean heart weight change was variable across all exposure levels.Light microscopy of hematoxylin and eosin (H&E) and Mallory-Heidenhain rapid one-step staining techniques on cardiac tissue sections of the juvenile and adult frogs revealed shrinkage of cardiac muscle cells into thin wavy myocytes. Additionally, disorganized branching of muscle fibres with reduced striations were observed in 0.01 μg L−1 and 200 μg L−1 but hypertrophied myocytes, thickened intensely staining myofibrils in the 500 μg L−1 group in juvenile and adult frogs. Significant increase in the mean percentage area of connective tissue in all the treated groups (p < 0.036) were also recorded. Immunohistochemistry analysis showed decreased eNOS localization in cardiac tissue in 200 μg L−1 and 500 μg L−1 of both juvenile and adult group, suggestive of decreased cardiac contractility due to atrazine exposure.The results indicate that environmentally relevant atrazine concentrations cause significant mortality in tadpoles while concentrations ≥200 μg L−1 adversely affect cardiac muscle morphology and may induce functional perturbations in cardiac tissue contractility and consequent dysfunction which generally may have an adverse impact on their survival and longevity.

The plus maze and scototaxis test are not valid behavioral assays for anxiety assessment in the South African clawed frog.

There are no behavioral models for testing anxiety in amphibians, a group of animals widely used for developmental, ecotoxicological, and genetic research. We aimed to validate two common rodent paradigms, the plus maze and the scototaxis test, for use in the aquatic African clawed frog (Xenopus laevis). We predicted: (a) that frogs would prefer the dark, vs. light, portions of the testing arenas (face validity), (b) that this behavior could be altered with acute administration of anxio-selective drugs (construct validity), and (c) that time spent in the dark portions of the arenas would be positively correlated (predictive validity). Prior to testing, frogs were treated with fluoxetine (selective serotonin reuptake inhibitor [SSRI]), desipramine (serotonin- and norepinephrine-reuptake inhibitor), caffeine (methylxanthine, adenosine receptor antagonist, phosphodiesterase inhibitor), saline, or were left unmanipulated. Each drug was administered acutely (1h prior to testing; caffeine) or subacutely (24, 3, and 1h prior to testing; fluoxetine, desipramine) at one of three doses. Plus maze and scototaxis testing were separated by 1week; each frog completed both behavioral tasks and was treated with the same drug regimen prior to testing. Overall, both tests showed face validity, however, data suggest these paradigms lack both construct and predictive validity.

Is normalized hindlimb length measurement in assessment of thyroid disruption in the amphibian metamorphosis assay relevant?

The amphibian metamorphosis assay represents an OECD Level 3 and EDSP Tier 1 ecotoxicity test assessing thyroid activity of chemicals in African clawed frog (Xenopus laevis). To evaluate the effectiveness of snout-vent length (SVL) normalization of hindlimb length (HLL), correlation between the HLL and SVL or body weight was evaluated in the control groups of 10 individual studies from three laboratories. Two studies required separate analysis of the Nieuwkoop-Faber (NF) stage ≤60 and >60 animals creating a total of 12 data sets. On study day 7, significant positive correlation between HLL and SVL or body weight was observed in eight and seven of the 10 data sets, respectively (r=0.608-0.843 and 0.583-0.876). On study day 21, significant positive correlation between HLL and SVL or body weight was found in three and four of the 12 data sets, respectively (r=0.452, 0.480 and 0.553 and r=0.621, 0.546, 0.564 and 0.378). Significant positive correlation between HLL and SVL was found in three of five studies, including ≤NF stage 60 data (r=0.564, 0.546 and 0.621). In one of eight studies, including >NF stage 60 data, the positive correlation between HLL and body weight was determined (r=0.378). Negative or no correlation between HLL and SVL or body weight was found in the other late stage data sets. Therefore, use of SVL-normalized HLL to assess thyroid-mediated effects in X. laevis tadpoles is not warranted. HL stage relative to body stage should be considered.

Trace, Machine Learning of Signal Images for Trace-Sensitive Mass Spectrometry: A Case Study from Single-Cell Metabolomics.

Recent developments in high-resolution mass spectrometry (HRMS) technology enabled ultrasensitive detection of proteins, peptides, and metabolites in limited amounts of samples, even single cells. However, extraction of trace-abundance signals from complex data sets ( m/ z value, separation time, signal abundance) that result from ultrasensitive studies requires improved data processing algorithms. To bridge this gap, we here developed "Trace", a software framework that incorporates machine learning (ML) to automate feature selection and optimization for the extraction of trace-level signals from HRMS data. The method was validated using primary (raw) and manually curated data sets from single-cell metabolomic studies of the South African clawed frog ( Xenopus laevis) embryo using capillary electrophoresis electrospray ionization HRMS. We demonstrated that Trace combines sensitivity, accuracy, and robustness with high data processing throughput to recognize signals, including those previously identified as metabolites in single-cell capillary electrophoresis HRMS measurements that we conducted over several months. These performance metrics combined with a compatibility with MS data in open-source (mzML) format make Trace an attractive software resource to facilitate data analysis for studies employing ultrasensitive high-resolution MS.

Activity and intracellular localization of senescence-associated β-galactosidase in aging Xenopus oocytes and eggs.

Senescence-associated β-galactosidase (SA-β-gal) serves as a marker of senescence in aging somatic cells. However, little is known about SA-β-gal dynamics in aging gamete cells. To address this issue, here we investigated activity and intracellular localization of SA-β-gal in freshly obtained and aging oocytes and eggs of the African clawed frog Xenopus laevis. Data base mining revealed the presence of several homologous β-galactosidase sequences in the annotated Xenopus genome. Some of them were predicted to contain an N-terminal signal peptide sequence, suggesting enzyme translocation to cellular organelles. Biochemical and microscopic analyses confirmed SA-β-gal localization in the particulate and cytosolic fractions of oocytes and eggs. SA-β-gal activity was found to reside predominantly within a fraction of dense cytoplasmic granules that were extensively stained with the lysosome-specific dye LysoTracker Green DND-26 and had an average size of 8.9 ± 5.6 μm. These features identify the SA-β-gal-containing granules as a subpopulation of yolk platelets, specialized late endosomes or lysosomes that accumulate and store processed protein in frog oocytes. Further analysis revealed an increase of SA-β-gal activity in Xenopus eggs, but not in oocytes, aged in vitro over 48 h. Our data suggest that endosomal acidification during egg aging may be responsible for this increase.

Using Xenopus laevis Oocytes to Functionally Characterize Plant Transporters.

Functionally characterizing plant membrane transport proteins is challenging. Typically, heterologous systems are used to study them. Immature eggs (oocytes) of the South African clawed frog Xenopus laevis are considered an ideal expression system for such studies. These large oocytes have a low number of endogenous transport systems in their plasma membranes and highly express foreign mRNA; the oocyte plasma membrane is the default destination of integral membrane proteins that lack recognized organellar sorting signals. These features facilitate almost background-free characterization of putative plant membrane transporters. Here we describe how to isolate Xenopus laevis oocytes, prepare capped sense RNA (cRNA) of the maize boron importer TASSEL-LESS1 (TLS1) as an example, microinject the cRNA into the isolated oocytes, and functionally assess the boron import capabilities of TLS1 in an oocyte swelling assay. These protocols can be easily adapted to study other plant and non-plant transporters with putative import function. © 2019 by John Wiley & Sons, Inc.

Membrane Microdomains as Platform to Study Membrane-Associated Events During Oogenesis, Meiotic Maturation, and Fertilization in Xenopus laevis.

Studies on the egg plasma membrane-associated tyrosine kinase Src have shed light on the identity of the molecular machinery that is responsible for gamete interaction and possibly fusion in African clawed frog Xenopus laevis. Here we describe our protocol for identifying and analyzing molecular and cellular machinery that contributes to a variety of biological processes in the course of oogenesis, oocyte maturation, egg fertilization, and early embryogenesis in Xenopus. Our current special interest is to evaluate the hypothesis that the oocyte/egg membrane microdomain (MD)-associated uroplakin III-Src system is responsible for mediating sperm-egg membrane interaction/fusion signal to the oocyte/egg cytoplasm to initiate embryonic and zygotic development in this species. Therefore, this chapter contains a brief introduction to biology of oocytes and eggs in Xenopus and addresses the following questions: (1) What is oocyte/egg MD? (2) Why do we study oocyte/egg MD? (3) How to manipulate oocyte/egg MD? (4) What has been achieved by oocyte/egg MD studies? (5) What are the next steps in oocyte/egg MD studies?

High abundance of invasive African clawed frog Xenopus laevis in Chile: challenges for their control and updated invasive distribution

High abundance of invasive African clawed frog Xenopus laevis in Chile: challenges for their control and updated invasive distribution

Exploring the mysteries of megakaryocytopoiesis and thrombopoiesis through comparative hematology

In modern hematology, research on hematopoiesis and blood cells in vertebrates, such as birds, reptiles, amphibians, and fish, is lagging. This is because there are many experimental constraints when selecting subjects other than humans and mice as research subjects. Currently, the availability of flow cytometry to count classified nucleated blood cells and utilization of whole genome information have led to novel findings. For example, in case of amphibian hematopoiesis studies, megakaryocytes have been found to be present in African clawed frogs (Xenopus laevis), which do not have platelets but have circulating nucleated thrombocytes. Moreover, we shed light on several mysteries, such as the C-terminal region in human TPO molecules not being found in birds, amphibians, and fish TPO molecules and the functional universalities of mutant CALR-MPL binding and EPO-EphB4 binding, in conjunction with comparative hematology.