Cellular Architecture and Stage-Specific Germ Cell Dynamics of the Koala Seminiferous Epithelium

The koala (Phascolarctos cinereus), recently listed as endangered, faces significant reproductive challenges compounded by the high prevalence of Chlamydia pecorum infection, which may cause severe urogenital pathology and infertility. Spermatogonial stem cell (SSC) biobanking represents a promising conservation strategy, but its implementation requires detailed knowledge of spermatogenic organisation and SSC identity in this species. Here, we provide the first integrated histological, immunofluorescent, and ultrastructural characterisation of the koala testis, with a focus on spermatogonia and their somatic support cells. Quantitative morphometric analysis of H&E-stained sections revealed that seminiferous tubules occupy a lower proportion of the testicular parenchyma in the koala (∼62%) than reported for other mammalian species, and that urogenital disease status is associated with graded impairment of spermatogenic epithelial integrity. Transmission electron microscopy confirmed the ultrastructural features of koala Sertoli, Leydig, and peritubular myoid cells, and enabled identification of Type A, intermediate, and Type B spermatogonia based on nuclear morphology and chromatin organisation as well as intercellular cytoplasmic bridges consistent with clonal spermatogonial development. Using PNA-lectin acrosomal labelling, an eight-stage classification system for the koala seminiferous epithelial cycle was adapted and refined, with stage frequencies quantified. Immunofluorescent screening of seventeen antibody targets identified PLZF and STRA8 as conserved markers of undifferentiated and differentiating spermatogonial populations, respectively, enabling their abundance, proliferative activity, and distribution across the seminiferous epithelial cycle to be mapped. Together, these findings establish the first molecular and morphological framework for the koala spermatogonial compartment, providing a foundation for future SSC identification, isolation, and biobanking.