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

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.

Male germ cell proliferation and apoptosis in sexually immature meagre Argyrosomus regius (Asso, 1801) treated with recombinant follicle stimulating hormone

“…On the contrary, we did not find any evidence that rFsh administration was effective in stimulating stem spermatogonia self-renewal. In fact, after 6 weeks of rFsh treatment, the density of anti-PCNA positive SpA und decreased significantly compared to control fish and this reduction was so marked that only sparse stem spermatogonia self-renewal was observed in the peripheral region of the testis, which is usually the main spermatogonial proliferative region in teleosts, including meagre 41 . Therefore, the 6-week rFsh treatment appeared to stimulate differentiation of SpA und and their progress towards spermatogenesis.…”

Cryopreservation and transplantation of spermatogonia stem cells in piracanjuba Brycon orbignyanus (Characiformes: Characidae), an endangered fish species

“…Studies involving the histological characterization and isolation of SSCs are essential to identify the moment the testes produce higher quantities of SSCs. Identifying this spermatogenesis stage is essential to obtain a higher concentration and better puri cation of SSCs, which can be used later in cell transplantation, cryopreservation, or in vitro cultivation (Poursaeid et al 2020;Zupa et al 2020).…”

Effects of Cryoprotective Medium Composition, Dilution Ratio, and Freezing Rates on Spotted Halibut (Verasper variegatus) Sperm Cryopreservation

“…A variety of factors, such as formation of ice crystal within the cell, changes of pH, sudden immersion in cold water, osmotic shock, and CPA toxicity, affect cryopreserved sperm in a cumulative manner. Furthermore, DNA damage during cryopreservation is caused by the formation of reactive oxygen species that change DNA by oxidizing bases and creating breaks in the helix [ 51 , 52 , 53 ].…”