Enrichment of spermatogonial stem cells and staging of the testis cycle in a dasyurid marsupial, the fat-tailed dunnart.

There is increasing interest in the use of marsupial models in research, for use in next-generation conservation by improving fitness through genetic modification, and in de-extinction efforts. Specifically, this includes dasyurid marsupials such as the Thylacine, Tasmanian devil, quolls, and the small rodent-like dunnarts. Technologies for generating genetically modified Australian marsupials remain to be established. Given the need to advance research in this space, the fat-tailed dunnart (Sminthopsis crassicaudata) is being established as a model for marsupial spermatogonial stem cell isolation, modification, and testicular transplantation. This species is small (60-90 mm body size), polyovulatory (8-12 pups per birth), and can breed in standard rodent facilities when housed in a 12:12 light cycle. To develop the fat-tailed dunnart as a model for next-generation marsupial conservation, this study aimed to enrich dunnart spermatogonial stem cells from whole testis digestions using a fluorescent dye technology and fluorescence-activated cell sorting. This approach is not dependent on antibodies or genetic reporter animals that are limiting factors when performing cell sorting on species separated from humans and mice by large evolutionary timescales. This study also assessed the development of spermatogonia and spermatogenesis in the fat-tailed dunnart, by making the first definition of the cycle of the seminiferous epithelium in any dasyurid. Overall, this is the first detailed study to assess the cycle of dasyurid spermatogenesis and provides a valuable method to enrich marsupial spermatogonial stem cells for cellular, functional, and molecular analysis.