Meagre Argyrosomus regius (Asso, 1801) Stem Spermatogonia: Histological Characterization, Immunostaining, In Vitro Proliferation, and Cryopreservation

SIMPLE SUMMARY: The meagre, Argyrosomus regius, is a commercial fish species of which aquaculture production is limited due to technological bottlenecks such as inconstant reproduction in captivity. In addition, there are only a few broodstocks in European farms, which rises the problem of limited genetic variability of the cultured meagre population. The development of germ cell xenotransplantation technology might increase aquaculture production through the production of fertile meagre gametes in a host species for which farming technology is well established. Spermatogonial stem cells have the capacity to proliferate and to proceed through the spermatogenesis process, eventually giving rise to mature spermatozoa. In the present study, meagre spermatogonial stem germ cells were identified, isolated, cultured in vitro, and cryopreserved. Isolated spermatogonial stem cells proliferated, maintained their stem properties in culture, and were still viable after cryopreservation and thawing. This work represents a first step towards the development of a xenotransplantation technology that might facilitate the production of this valuable species in captivity. ABSTRACT: The meagre, Argyrosomus regius, is a valued fish species of which aquaculture production might be supported by the development of a stem germ cell xenotransplantation technology. Meagre males were sampled at a fish farm in the Ionian Sea (Italy) at the beginning and end of the reproductive season. Small and large Type A undifferentiated spermatogonia were histologically identified in the germinal epithelium. Among the tested stemness markers, anti-oct4 and anti-vasa antibodies labeled cells likely corresponding to the small single Type A spermatogonia; no labeling was obtained with anti-GFRA1 and anti-Nanos2 antibodies. Two types of single A spermatogonia were purified via density gradient centrifugation of enzymatically digested testes. Testes from fish in active spermatogenesis resulted in a more efficient spermatogonial stem cell (SSC) yield. After cell seeding, meagre SSCs showed active proliferation from Day 7 to Day 21 and were cultured up to Day 41. After cryopreservation in dimethyl-sulfoxide-based medium, cell viability was 28.5%. In conclusion, these results indicated that meagre SSCs could be isolated, characterized, cultured in vitro, successfully cryopreserved, and used after thawing. This is a first step towards the development of a xenotransplantation technology that might facilitate the reproduction of this valuable species in captivity.