Marsupial conservation and research are limited, partly by the absence of assisted reproductive technologies (ART) and genomic modification techniques. Given their established roles in genetic modification and in vitro gametogenesis, spermatogonial stem cells (SSCs) – the undifferentiated precursors of sperm – present a promising and logical approach for the advancement of these technologies in marsupials. However, SSCs of Australian marsupials are uncharacterised and conditions for their long-term culture remain undefined, preventing the development of these fundamental techniques. Therefore, this research aimed to understand the effects of growth factors in established eutherian SSC culture conditions on marsupial SSCs.
Fat-tailed dunnart (Sminthopsis crassicaudata) adult testis was enzymatically digested and subjected to different culture conditions, including serum-free medium with combinations of essential growth factors such as GDNF, bFGF, EGF, and LIF. Cells were fractioned based on differential adherence, and SSC markers was assessed via qPCR and RNAseq to identify conditions that promote propagation and self-renewal of marsupial SSCs.
Morphology of dunnart germ cell colonies in serum-free conditions was similar to mitotic spermatogonia in vivo, which is supported by the increased expression of spermatogonial markers (GFRA1, KIT) compared to serum exposed colonies. After 40 days in vitro, RNAseq analysis demonstrated increased expression of key SSC regulatory genes (POU5F1, ID4, ZBTB16) in adherent fractions under serum-free conditions. These findings suggest synergistic effects of GDNF and bFGF with EGF and LIF in enhancing SSC marker expression, with no change in markers of differentiation.
These findings demonstrate sustained culture of dunnart SSCs and provides insights into the effects of various culture conditions and growth factors on dunnart SSC maintenance and differentiation. These initial advances in dunnart SSC culture provide a critical foundation to further SSC isolation and growth. This will facilitate the development of downstream SSC applications such as ART and genetic modification to enhance marsupial conservation.