A simple and effective approach for the isolation and identification of germline stem cells in mature large yellow croaker (Larimichthys crocea)

Spermatogonial stem cells (SSCs) and oogonial stem cells (OSCs) are important germline stem cells located in the gonads, responsible for maintaining fertility throughout the reproductive lifespan. However, at present, there is still a lack of optimized protocols specifically for the isolation and identification of SSCs and OSCs in large yellow croaker (Larimichthys crocea), a marine economic fish. Therefore, this study aimed to explore a simple, inexpensive, efficient, and practical method for isolating and identifying germline stem cells in this species. SSCs were enriched using a three-layer discontinuous Percoll density gradient (15%, 25%, and 50%), while OSCs were obtained by sequential filtration through 300, 200, and 40 μm nylon mesh membranes following enzymatic digestion. Quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH) were employed to evaluate the expression patterns of candidate stem cell markers in the isolated cell fractions and gonadal tissues. The results demonstrated that oct4 served as a specific marker for undifferentiated type A spermatogonia (Aund), ly75 was preferentially expressed in differentiated type A spermatogonia (Adiff) and type B spermatogonia (SpgB), and nanos2 functioned as a broader spermatogonial marker. Notably, klf4 was identified as a novel and specific marker for fish oogonia, alongside nanos2 and vasa, expanding the molecular toolkit for OSCs research. The meiotic marker sycp3 further assisted in distinguishing OSCs from differentiated oogonia. The current research contributes to cell culture, cryopreservation, and transplantation in L. crocea and provides a reference for the isolation and identification of germline stem cells from other mature economic fish species.