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

Unlocking the potential of koala spermatogonial stem cells for conservation: optimising isolation, characterisation, and culture approaches (127942)

Katerina B Damyanova 1 2 , Brett Nixon 1 2 , Joshua Fisher 3 4 , Stephen Johnston 5 6 , Andres Gambini 6 7 , Patricio P Benitez 7 , Yolande Campbell 5 , Tessa Lord 1 2
  1. Infertility and Reproduction Research Program, Hunter Medical Research Institute, Newcastle, NSW, Australia
  2. Centre for Reproductive Science, School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, Newcastle, NSW, Australia
  3. Mothers and Babies Research Program, Hunter Medical Research Institute, Newcastle, NSW, Australia
  4. School of Medicine and Public Health, College of Health, Medicine and Wellbeing, University of Newcastle, Newcastle, NSW, Australia
  5. School of Environment, The University of Queensland, Gatton, Queensland, Australia
  6. School of Veterinary Science, The University of Queensland, Gatton, Queensland, Australia
  7. School of Agriculture and Food Science, The University of Queensland, Gatton, Queensland, Australia

The koala (Phascolarctos cinereus), an iconic Australian marsupial, faces escalating threats from habitat loss, disease, and climate-related disasters, leading to its endangered status in several states. Preserving male germline potential through spermatogonial stem cell (SSC) biobanking offers a promising avenue for long-term fertility preservation and assisted breeding programs. However, the limited understanding of SSC biology in marsupials hinders the development of such technologies.

This study aimed to characterise koala SSCs and develop protocols for their isolation and in vitro maintenance. Using immunohistochemistry on paraffin-embedded testis sections, we identified 8 stages of the seminiferous epithelium based on acrosome development (PNA-lectin labelling). Upon identifying the conserved expression of 4 spermatogonia markers in the koala testis we defined the distribution of undifferentiated (PLZF+) and differentiating (STRA8+) spermatogonia across the seminiferous cycle as well as their proliferation via co-staining with PCNA.

In addition, transmission electron microscopy has been used to investigate the ultrastructure of koala germ cells within the seminiferous tubules. This revealed defining morphological features of different germ cell types and the presence of cytoplasmic bridges between dividing spermatogonia, providing novel insights into spermatogonial morphology and behaviour in situ.

To support downstream applications, several SSC enrichment methods have been tested, including differential plating, Percoll gradient separation, and magnetic-activated cell sorting (THY1+), with assessments of enrichment efficiency underway via quantification of PLZF+ cells. Trials of short-term in vitro culture of koala spermatogonia are in progress, using both feeder-free (laminin) and feeder-dependent (mitotically inactivated embryonic mouse-derived fibroblasts) systems in serum-free and FBS supplemented medium, with and without growth factors (GDNF, FGF2). Cultures are being monitored for viability, morphology, and marker expression (PLZF, STRA8, DDX4, UCHL1, PCNA, EPAS1).

Together, these approaches bring us closer to establishing SSC-based conservation tools for koalas, laying a critical foundation for SSC biobanking and assisted reproduction in endangered marsupials.