Xenopus is one of the premier model systems to study cell and developmental biology in vivo in vertebrates. Here we briefly review how this South African frog came to be favored by a large community of scientists after the explosive growth of molecular biology and examine some of the original discoveries arising from this sturdy frog. Experimental embryology started in Rana but developed in newt embryos for historical reasons. A long lineage of mentorship, starting with Theodor Boveri, Hans Spemann, Fritz Baltzer, Ernst Hadorn, and Michail Fischberg, used newt embryos. In Oxford, Fischberg made the transition to Xenopus laevis because it was widely available for human pregnancy tests and laid eggs year-round, and he fortuitously isolated a one-nucleolus mutant. This mutant allowed nuclear transfer experiments showing that genetic information is not lost during cell differentiation and the demonstration that the nucleolus is the locus of transcription of the large ribosomal RNAs. With the advent of DNA cloning, the great equalizer among all fields of biology, microinjected Xenopus oocytes became an indispensable tool, providing the first living-cell mRNA translation, polymerase II and III transcription, and coupled transcription-translation systems in eukaryotes. Xenopus embryos provide abundant material to study the earliest signaling events during vertebrate development and have been subjected to saturating molecular screens in the genomic era. Many novel principles of development and cell biology owe their origins to this remarkably resilient frog.

Lucia Arregui, Phd
Postdoctoral Researcher
Project Summary
The clawed frogs (Xenopus laevis and X. tropicalis) are valuable biomedical models. Production of transgenic and mutant lines in these species has led to a rapidly expanding need for a cost-effective and efficient way to maintain the increasing number of lines at the National stock center for Xenopus (National Xenopus Resource, NXR, Woods Hole, Massachusetts). Storage of cryopreserved germplasm in repositories can provide a way to protect such lines and reduce the number of live animals held at NXR. In collaboration with NXR we are developing a sperm cryopreservation user-centered pathway (combination of a protocol and its practical application) for Xenopus sp. for the establishment of a germplasm repository.

SPERM QUALITY EVALUATION
Cryopreservation is a multi-step process that can produce a significant loss on sperm quality. The first step for the development of a sperm cryopreservation protocol is to establish methods for quality evaluation of the sperm along the process of cryopreservation. For sperm, several parameters have been used for the evaluation of quality but the most common ones are motility and cell membrane integrity. We are standardizing for Xenopus a method for evaluation of motility using computer-assisted sperm analysis (CASA) as it allows performing a rapid, repeatable and accurate analysis. Membrane integrity is being quantified and compared using fluorescent (SYBR-14/PI) and non-fluorescent dyes (eosin Y) to open the opportunity to be performed using fluorescence or bright-field microscopy.


SPERM CRYOPRESERVATION PATHWAY
Once sperm quality could be assessed, we proceed to develop a cryopreservation protocol. We perform cryotoxicity test to select the less detrimental cryoprotectants and theirs concentration. Next, we evaluate each step of the cryopreservation process in relation to others. We assess the effect of sperm concentration, composition of cryodiluent and rate of freezing and thawing. Finally, we test fertilization capacity of sperm samples cryopreserved using the most suitable protocols. We are considering the application of this protocol together with the protocol itself. Therefore, we integrate many factors besides cryopreservation itself for the development of this pathway such as cost, labelling, simplicity, scalability or database requirements.
OPTIMIZING SPERM CRYOPRESERVATION PATHWAY
Multiple factors before, during and after freezing and thawing will be analyzed to consider its effect during the cryopreservation process. The role of temperature of animal housing and animal age on gamete quality will be determined. Also, sperm samples are collected through maceration of the testes and different methods of testes preparation (tearing with forceps, grinding with pestle) will be analyzed to assess the effect on sperm quality and freezability.

References
De Robertis ED, and Gurdon JB. 2021. A Brief History of Xenopus in Biology. Cold Spring Harb Protoc 12:pdb.top107615.
Horb M, Wlizla M, Abu-Daya A, McNamara S, Gajdasik D, Igawa T, Suzuki A, Ogino H, Noble A, Robert J, James-Zorn C, and Guille M. 2019. Xenopus Resources: Transgenic, Inbred and Mutant Animals, Training Opportunities, and Web-Based Support. Front Physiol 387.
Two species of the clawed frog family, Xenopus laevis and X. tropicalis, are widely used as tools to investigate both normal and disease-state biochemistry, genetics, cell biology, and developmental biology. To support both frog specialist and non-specialist scientists needing access to these models for their research, a number of centralized resources exist around the world. These include centers that hold live and frozen stocks of transgenic, inbred and mutant animals and centers that hold molecular resources. This infrastructure is supported by a model organism database. Here, we describe much of this infrastructure and encourage the community to make the best use of it and to guide the resource centers in developing new lines and libraries.
Pearl E, Morrow S, Noble A, Lerebours A, Horb M, and Guille M. 2017. An optimized method for cryogenic storage of Xenopus sperm to maximise the effectiveness of research using genetically altered frogs. Theriogenology 149-155.
Cryogenic storage of sperm from genetically altered Xenopus improves cost effectiveness and animal welfare associated with their use in research; currently it is routine for X. tropicalis but not reliable for X. laevis. Here we compare directly the three published protocols for Xenopus sperm freeze-thaw and determine whether sperm storage temperature, method of testes maceration and delays in the freezing protocols affect successful fertilisation and embryo development in X. laevis. We conclude that the protocol is robust and that the variability observed in fertilisation rates is due to differences between individuals. We show that the embryos made from the frozen-thawed sperm are normal and that the adults they develop into are reproductively indistinguishable from others in the colony. This opens the way for using cryopreserved sperm to distribute dominant genetically altered (GA) lines, potentially saving travel-induced stress to the male frogs, reducing their numbers used and making Xenopus experiments more cost effective.
Team Members

Rose Upton - Postdoctoral Researcher

Luke Turner - Undergraduate Student Worker

Lily Carter - Undergraduate Student Worker
Collaborators

Marko Horb - Director, The National Xenopus Resource

Nikko-Ideen Shaidani - Manager, The National Xenopus Resource

James Parente - Assistant manager, The National Xenopus Resource

Carl Anderson - Research Assistant, The National Xenopus Resource
Funding Organizations
