Morphological characterization of spermatogenesis and spermatogonial stem cells in Larimichthys crocea, a seasonal breeding teleost

Abstract Seasonal spermatogenesis in fish is a complex and highly regulated process in which spermatogonial stem cells (SSCs) undergo a series of cellular changes to differentiate into mature sperm. In this study, we systematically described testicular development and identified thirteen different germ cell types throughout the reproductive cycle in large yellow croaker (Larimichthys crocea), a commercially important marine cultured fish in East Asia. Using a set of specific antibodies (VASA, PCNA, DMC1, NANOS2 and GSDF), we developed a high-throughput immunohistochemistry method to identify different types of spermatogenic cells, with a particular focus on distinguishing spermatogonial subtypes. VASA was strongly expressed in all four types of spermatogonia (As, Apr, Adiff, and B) and decreased progressively during spermatogenesis. DMC1 exhibited distinct expression patterns in different spermatocytes subtypes, and GSDF was highly expressed in somatic cells surrounding type A spermatogonia. Particularly, NANOS2 was highly specific to As and Apr spermatogonia, supporting their role as SSC candidates. By morphological observation and co-staining of VASA and PCNA, we found that As spermatogonia exhibited dynamic development characteristics during the annual reproductive cycle. These findings provide a valuable tool for reproductive studies and potential applications in surrogate reproduction through SSCs transplantation in teleost fish.