Oral Presentation ESA-SRB-NZSE-CaSR 2026 in conjunction with ENSA

Establishing the first spermatogonial enrichment and biobanking pipeline for the koala (Phascolarctos cinereus) (143519)

Katerina B Damyanova 1 2 , Bryana Childs 1 2 , Brett Nixon 1 2 , Joshua Fisher 2 3 , Stephen Johnston 4 5 , Andres Gambini 5 6 , Patricio D Palacios 6 , Yolande Campbell 4 , Tessa Lord 1 2
  1. Centre for Reproductive Science, School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, Newcastle, NSW, Australia
  2. Reproductive and Family Health Research Program, Hunter Medical Research Institute, Newcastle, New South Wales, Australia
  3. School of Medicine and Public Health, College of Health, Medicine and Wellbeing, University of Newcastle, Newcastle, NSW, Australia
  4. School of Environment, The University of Queensland, Gatton, Queensland, Australia
  5. School of Veterinary Science, The University of Queensland, Gatton, Queensland, Australia
  6. School of Agriculture and Food Science, The University of Queensland, Gatton, Queensland, Australia

Background: The endangered koala presents an urgent case for reproductive biobanking, yet spermatogonial stem cell (SSC)-based strategies, increasingly applied in human fertility preservation, remain unexplored as a conservation tool. Here, we present the first systematic evaluation of testicular cell isolation, SSC enrichment and short-term culture in the koala, directly testing whether SSC biobanking protocols built for eutherian mammals translate to a marsupial.

Methods: To encourage SSC enrichment, testicular cell suspensions from n=9 koalas were subjected to Percoll density-gradient centrifugation, differential plating, and magnetic-activated cell sorting (MACS) using ITGA6. Fractions were assessed by qPCR, immunocytochemistry using DDX4, PLZF, STRA8, SOX9 and Vimentin antibodies, and short-term culture recovery. Data were analysed using paired t-tests, Kruskal–Wallis/Dunn or ANOVA/Tukey tests, as appropriate.

Results: Unselected suspensions contained DDX4+ germ cells, including PLZF+ SSCs (<11% of total cell population) and STRA8+ differentiating spermatogonia, alongside SOX9+ and Vimentin+ somatic cells. In contrast to eutherian mammals, Percoll centrifugation did not enrich for koala spermatogonia, indicating species-specific buoyant density. Differential plating depleted STRA8+ differentiating spermatogonia (p=0.0249), however only modestly enriched PLZF+ SSCs (1.6-fold, p=0.0757). Unexpectedly, ITGA6-MACS failed to enrich PLZF+ SSCs in the selected population, but rather depleted Vimentin+ stromal cells (p=0.0372), creating a 2.4-fold enrichment in PLZF+ SSCs in the flow-through (p=0.0400). qPCR analysis mirrored these findings, with a 1.8-fold increase in Plzf transcript abundance in the flow-through relative to the selected fraction, with similar trends for additional SSC markers, Gfra1 and Uchl1. Promisingly, when placed in culture, flow-through cells displayed characteristic spermatogonial morphology and superior short-term recovery.

Conclusion: This study establishes the first platform for koala SSC isolation and culture, revealing that enrichment strategies routinely used in eutherian mammals do not translate to marsupials. By identifying the flow-through as an unexpectedly SSC-enriched fraction, this work lays essential groundwork for koala-specific biobanking and future genetic rescue efforts.Â