Isolation, characterization, in vitro expansion and transplantation of Caspian trout (Salmo caspius) type a spermatogonia

Abstract

Sprmatogonial stem cells (SSCs) are valuable for preservation of endangered fish species, biological experimentation, as well as biotechnological applications. However, the rarity of SSCs in the testes has been a great obstacle in their application. Thus, establishment of an efficient in-vitro culture system to support continuous proliferation of SSCs is essential. The present study aimed to establish an efficient and simple method for in vitro culture of Caspian trout undifferentiated spermatogonial cells. Using a two-step enzymatic digestion, testicular cells were isolated from immature testes composed of mainly undifferentiated spermatogonial cells with gonadosomatic indices of <0.05%. The spermatogonial cells were purified by differential plating through serial passaging. The purified cells indicated high expression of type A spermatogonia-related genes (Ly75, Gfrα1, Nanos2, Plzf and Vasa). Proliferation of purified cells was confirmed by BrdU incorporation. Co-culture of purified cells with testicular somatic cells as a feeder layer, resulted in continuous proliferation of type A spermatogonia. The cultured cells continued to express type A spermatogonia-specific markers after one month culture. The cultured spermatogonia were successfully incorporated into the germline after being intraperitoneally transplanted into sterile triploid rainbow trout hatchlings. These results, for the first time, demonstrated that the somatic microenvironment of the rainbow trout gonad can support the colonization and survival of intraperitoneally transplanted cells derived from a fish species belonging to a different genus. Therefore, the combination of in vitro culture system and xenotransplantation can be considered as a promising strategy for conservation of Caspian trout genetic resources.