Establishment of mud crab (Scylla paramamosain) spermatogonial stem cell line: A potential tool for immunological...

Spermatogonial stem cells (SSCs) can differentiate into sperm and are important for studying on genetic information transmission of animals. However, the establishment of the SSC line in crustaceans is still in its infancy. This study aimed to establish a method for the isolation, culture, and identification of SSCs derived from the gonad of a marine crustacean (mud crab, Scylla paramamosain), and evaluate their differentiation ability and potential application in immunological research, in vitro. SSCs showed robust growth, proliferation, and passaging ability (up to 35 passages) in germ cell culture medium. Proteomic analysis showed that the protein expression profile of SSC was closely related to the gonadal tissue. SSCs were found to be able to express male-specific and pluripotent markers, such as CD9, PIWI, DDX4, DAZL, NANOG, SOX2, and EPHA1. Furthermore, SSCs were differentiated into osteoblasts and adipocytes under in vitro induction. Green fluorescent protein (GFP), packaged by lentivirus, was able to be overexpressed in SSCs after infection. In addition, the infection of white spot syndrome virus (WSSV) simulated the expression of inflammation-associated factors, including TRAF6, TNF-α, MyD88, Dorsal, and Relish, and apoptosis-related genes (BAX and Bcl2) in SSCs. Thus, SSCs were initially isolated and characterized from mud crabs for the first time. Our results proved that SSCs can be used in reproduction technology, germplasm conservation, and immunological studies in crustaceans.