The dynamic transcriptome profiles in turbot prepubertal testes and the important role of GDNF in maintaining spermatogonial stem cell self-renewal

Germ cell transplantation is an effective technique for producing surrogate offspring in fish by transplanting the donor-derived cells into allogeneic or xenogeneic recipients. Spermatogonia are the most commonly used donor stem cells for transplantation due to their easy large-scale acquisition. Despite significant advancements in germ cell transplantation, a comprehensive understanding of the complex regulation of spermatogonia differentiation and development remains a bottleneck in its application to aquaculture. Our study first unveiled the spermatogonia renewal and differentiation trajectory at a single-cell transcriptomic level in prepubertal male turbot ( Scophthalmus maximus ). Through scRNA-seq, Monocle analysis, and in situ hybridization, we found that both gdnfa and gdnfb were expressed in spermatogonial stem cells (SSCs) and somatic cells, suggesting the involvement of both autocrine and paracrine pathways of glial cell line-derived neurotrophic factor. Notably, gdnfb showed relatively high expression levels predominantly in SSC-1. Additionally, within the SSC-1 and SSC-2 clusters, we identified gfra1b , nanos2 , zbtb16a , and lin28a , which are essential for spermatogonial maintenance in mammals. We also identified grb7 and dnmt3bb.1 , two genes that were highly and exclusively expressed in SSC clusters. In the validation experiments, we found that the addition of GDNF to the SSC culture medium significantly promoted SSC self-renewal and inhibited differentiation. Co-injection of GDNF with turbot SSCs also improved the colonization frequency of donor SSCs in Paralichthys olivaceus triploid larvae. These results provide insights into the proliferation and differentiation trajectory of SSCs, highlighting key marker genes and signaling pathways that regulate SSC self-renewal and differentiation in commercial fish. • We developed a transcriptional single-cell atlas of the developing turbot testes during prepuberty. • We found that distinctive phases of spermatogonial stem cell (SSC) self-renewal and differentiation occur during prepuberty. • We examined the dynamics of gene expression along the pseudotime of SSC differentiation. • We found that glial cell line-derived neurotrophic factor enhances SSC self-renewal and transplantation efficiency. • We identified key marker genes regulating SSC self-renewal and differentiation in turbot.