Systematic screening and dynamic profiling of germline regulatory pathways and spermatogonial surface markers in a...

Spermatogonial cells are capable of transmitting genetic information to the next generation and have the potential for self-renewal. However, research on spermatogonial cells is generally limited by the paucity of cell surface markers, which are not universal among species. In this study, we developed a systematic screening strategy for germline regulatory pathways and spermatogonial surface markers in non-model organisms. This was achieved by combining weighted gene co-expression network analysis (WGCNA), differential expressed gene (DEG) overrepresentation analysis, and functional annotation. This strategy was employed to identify a spermatogonia-related module, which was found to be enriched with stem cell-related pathways in the scallop Patinopecten yessoensis. This module contained a transmembrane protein, fibroblast growth factor receptor (FGFR). A single copy of Fgfr (PyFgfr) was confirmed in the P. yessoensis genome, which exhibited the canonical functional domains of FGFRs. PyFgfr was universally expressed in multiple tissues, including the testis. The highest expression was observed at the resting stage of the testis, with exclusive localization in spermatogonia. To obtain antibodies that recognize the cell surface region, the extracellular domains of PyFGFR were used as an antigen to prepare antiserum. Western blotting, immunohistochemical, and immunofluorescent analyses demonstrated that the antiserum specifically detected PyFGFR in the spermatogonia. This study demonstrates the feasibility of this strategy for screening spermatogonial surface markers in the scallop. This approach will facilitate the culture and manipulation of spermatogonia in non-model organisms, which may contribute to genetic improvement in aquaculture.