Cryopreservation of Xenopus Sperm and In Vitro Fertilization Using Frozen Sperm Samples.

The cryopreservation of Xenopus sperm allows for a significant reduction of the number of animals that must be kept, more efficient archiving of genetically altered (GA) lines, and easy exchange of lines with other laboratories, leading to improvements in animal welfare and cost efficiency. In this protocol, sperm from Xenopus laevis or Xenopus tropicalis are frozen using straightforward techniques and standard laboratory equipment. Testes are macerated in Leibovitz's L-15 medium, mixed with a simple cryoprotectant made from egg yolk and sucrose, and frozen slowly overnight in a polystyrene box at -80°C. Unlike mouse sperm, Xenopus sperm can be stored at -80°C rather than in liquid nitrogen, further reducing costs. The frozen sperm are then used for in vitro fertilization.

The skin microbiome of Xenopus laevis and the effects of husbandry conditions.

BACKGROUND: Historically the main source of laboratory Xenopus laevis was the environment. The increase in genetically altered animals and evolving governmental constraints around using wild-caught animals for research has led to the establishment of resource centres that supply animals and reagents worldwide, such as the European Xenopus Resource Centre. In the last decade, centres were encouraged to keep animals in a "low microbial load" or "clean" state, where embryos are surface sterilized before entering the housing system; instead of the conventional, "standard" conditions where frogs and embryos are kept without prior surface treatment. Despite Xenopus laevis having been kept in captivity for almost a century, surprisingly little is known about the frogs as a holobiont and how changing the microbiome may affect resistance to disease. This study examines how the different treatment conditions, "clean" and "standard" husbandry in recirculating housing, affects the skin microbiome of tadpoles and female adults. This is particularly important when considering the potential for poor welfare caused by a change in husbandry method as animals move from resource centres to smaller research colonies. RESULTS: We found strong evidence for developmental control of the surface microbiome on Xenopus laevis; adults had extremely similar microbial communities independent of their housing, while both tadpole and environmental microbiome communities were less resilient and showed greater diversity. CONCLUSIONS: Our findings suggest that the adult Xenopus laevis microbiome is controlled and selected by the host. This indicates that the surface microbiome of adult Xenopus laevis is stable and defined independently of the environment in which it is housed, suggesting that the use of clean husbandry conditions poses little risk to the skin microbiome when transferring adult frogs to research laboratories. This will have important implications for frog health applicable to Xenopus laevis research centres throughout the world.

A newly identified Rab-GDI paralogue has a role in neural development in amphibia.

Vesicle shuttling is critical for many cellular and organismal processes, including embryonic development. GDI proteins contribute to vesicle shuttling by regulating the activity of Rab GTPases, controlling their cycling between the inactive cytosol and active membrane bound states. While identifying genes controlled by A-form DNA sequences we discovered a previously unknown member of the GDI family, GDI3. The GDI3 gene is found only in amphibians and fish and is developmentally expressed in Xenopus from neurula stages onwards in the neural plate, and subsequently in both dorsal and anterior structures. Depletion or over-expression of the GDI3 protein in Xenopus embryos gives rise to very similar phenotypes, suggesting that strict control of GDI3 protein levels is required for correct embryonic development. Our analysis suggests the evolutionary origins of GDI3 and that it is functionally distinct from GDI1. Predicted structural analysis of GDI3 suggests that the key difference between GDI1 and GDI3 lies in their lipid binding pockets.

Inbreeding Ratio and Genetic Relationships among Strains of the Western Clawed Frog, Xenopus tropicalis.

The Western clawed frog, Xenopus tropicalis, is a highly promising model amphibian, especially in developmental and physiological research, and as a tool for understanding disease. It was originally found in the West African rainforest belt, and was introduced to the research community in the 1990s. The major strains thus far known include the Nigerian and Ivory Coast strains. However, due to its short history as an experimental animal, the genetic relationship among the various strains has not yet been clarified, and establishment of inbred strains has not yet been achieved. Since 2003 the Institute for Amphibian Biology (IAB), Hiroshima University has maintained stocks of multiple X. tropicalis strains and conducted consecutive breeding as part of the National BioResource Project. In the present study we investigated the inbreeding ratio and genetic relationship of four inbred strains at IAB, as well as stocks from other institutions, using highly polymorphic microsatellite markers and mitochondrial haplotypes. Our results show successive reduction of heterozygosity in the genome of the IAB inbred strains. The Ivory Coast strains clearly differed from the Nigerian strains genetically, and three subgroups were identified within both the Nigerian and Ivory Coast strains. It is noteworthy that the Ivory Coast strains have an evolutionary divergent genetic background. Our results serve as a guide for the most effective use of X. tropicalis strains, and the long-term maintenance of multiple strains will contribute to further research efforts.

APTE: identification of indirect read-out A-DNA promoter elements in genomes.

BACKGROUND: Transcriptional regulation is normally based on the recognition by a transcription factor of a defined base sequence in a process of direct read-out. However, the nucleic acid secondary and tertiary structure can also act as a recognition site for the transcription factor in a process known as indirect read-out, although this is much less understood. We have previously identified such a transcriptional control mechanism in early Xenopus development where the interaction of the transcription factor ilf3 and the gata2 promoter requires the presence of both an unusual A-form DNA structure and a CCAAT sequence. Rapid identification of such promoters elsewhere in the Xenopus and other genomes would provide insight into a less studied area of gene regulation, although currently there are few tools to analyse genomes in such ways. RESULTS: In this paper we report the implementation of a novel bioinformatics approach that has identified 86 such putative promoters in the Xenopus genome. We have shown that five of these sites are A-form in solution, bind to transcription factors and fully validated one of these newly identified promoters as interacting with the ilf3 containing complex CBTF. This interaction regulates the transcription of a previously uncharacterised downstream gene that is active in early development. CONCLUSIONS: A Perl program (APTE) has located a number of potential A-form DNA promotor elements in the Xenopus genome, five of these putative targets have been experimentally validated as A-form and as targets for specific DNA binding proteins; one has also been shown to interact with the A-form binding transcription factor ilf3. APTE is available from http://www.port.ac.uk/research/cmd/software/ under the terms of the GNU General Public License.