Oral Presentation ESA-SRB-ANZOS 2025 in conjunction with ENSA

Enrichment and long-term culture of marsupial spermatogonia: A foundation of next generation conservation technologies. (128446)

Patrick R. Tatt 1 , Gerard A Tarulli 1 , Benjamin R Carone 2 , Savannah Hoyt 3 , Rachel O'Neill 3 , Stephen R Frankenberg 1 , Andrew J Pask 1
  1. School of Biosciences, University of Melbourne, Melbourne, VIC, Australia
  2. Rowan University, Glassboro, New Jersey, USA
  3. University of Connecticut, Mansfield, Connecticut, USA

Ongoing declines in Australian marsupial populations highlight the limitations of conventional conservation strategies and the need for innovative approaches. Spermatogonial stem cells (SSCs), the source of the male germline, have been used in advanced techniques that may prove valuable for marsupial conservation efforts, including SSC transplants, in vitro spermatogenesis, and derivation into induced pluripotent stem cells. However, marsupial SSCs, and the techniques required to utilise them, are not defined. This research aimed to establish reliable methods to culture and enrich marsupial spermatogonia to advance marsupial SSC-based research and conservation technologies.

Fat-tailed dunnart (Sminthopsis crassicaudata) testis cells were cultured under various common eutherian SSC conditions. Antibody-based cell sorting and differential adhesion were assessed for spermatogonial enrichment and subsequently cultured for spermatogonia colony formation. Spermatogonial enrichment, colony formation and maintenance were evaluated using gene expression analysis (RT-PCR, RNAseq) and immunofluorescent staining for germ cell (UCHL1, DDX4) and somatic cell (GATA4) markers.

ITGA6-based cell sorting and differential adhesion resulted in a 13-fold and 8-fold increase in spermatogonial gene expression (ID4, UCHL1, GFRA1), respectively, with the assessment of colony formation in enriched fractions currently ongoing. Dunnart spermatogonial culture was most robust under serum-free DMEM/F12 when compared to StemPro-34-SFM and MEMĪ±. These conditions supported spermatogonia for ~50 days and through passage, suggesting continual support of the stem cell population. Differences between conditions were more pronounced after passage, with DMEM/F12 show up to 10-fold higher expression (p<0.0001) of key spermatogonial regulatory genes (ID4, ETV5, GFRA1) relative to MEMĪ±.

This study significantly advances our knowledge of the enrichment methods and culture conditions needed for isolation and extended in vitro expansion of marsupial spermatogonia, enabling developments towards dunnart SSC transplants and investigations into marsupial SSC regulation. This also provides essential tools for developing advanced SSC-based applications that will be invaluable for marsupial research and conservation.