Defining the molecular identity of marsupial spermatogonia for germline conservation technologies — ASN Events
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  3. Defining the molecular identity of marsupial spermatogonia for germline conservation technologies

Defining the molecular identity of marsupial spermatogonia for germline conservation technologies (#469)

Patrick R Tatt 1 , Gerard A Tarulli 1 , Benjamin R Carone 2 , Savannah Hoyt 3 , Rachel O'Neill 3 , 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

Background

Ongoing declines in marsupial populations highlight the need for more advanced conservation technologies. Spermatogonial stem cells (SSCs), which sustain lifelong male fertility, are a promising cell type for developing germline conservation technologies, including SSC transplantation and in vitro spermatogenesis. Molecular markers used to identify and isolate SSCs are largely characterised in eutherians, but not in marsupials. Marsupials and eutherians diverged approximately 160 million years ago, and it is unknown whether these markers are conserved. Our aim was to define markers for marsupial spermatogonia to enable their identification and isolation for future spermatogonial-based conservation technologies.

Methods

Single nuclei RNA sequencing was performed on two adult fat-tailed dunnart (Sminthopsis crassicaudata) testes. Somatic and germ cell types were annotated and the localisation of canonical spermatogonial markers was assessed. Differential gene expression and immunofluorescne analysis was used to identify candidate dunnart spermatogonial genes to compare with eutherian marker genes, and for use in enrichment through live cell sorting.

Results

Differential expression analysis identified approximately 70 genes enriched in the spermatogonial population. Three genes with known expression in eutherian spermatogonia (PIWIL4, ELAVL2, TCF7) were validated in dunnart by immunofluorescence. Interestingly, some known eutherian spermatogonial markers were not localised exclusively to spermatogonia in the adult dunnart testis, with GFRA1 expressed in spermatids and ZBTB16 in Leydig cells, while other established markers were not detected. In addition, we identified CD9, GPR125, ABCG2 and CXCR4 as potential targets for spermatogonial enrichment, with marsupial and eutherian spermatogonia sharing similar epitope patterns. Comparative analysis to identify unique marsupial spermatogonial markers remains ongoing.

Conclusion

This study provides the first validated spermatogonial marker panel for an Australian marsupial, demonstrates distinct expression patterns of canonical eutherian spermatogonial markers within the marsupial testis, and identifies candidate targets for spermatogonial enrichment to advance future germline-based conservation strategies.