The effects of growth factors on proliferation, colonization and gene expression of Caspian trout spermatogonial stem cells in vitro

The present study aimed to examine the effect of different growth factors on Caspian trout spermatogonial cell proliferation, colony formation and germ cell specific gene expression. Spermatogonial cells were isolated from testes of juvenile male Caspian trout by two enzymatic digestion methods and purified by differential plating technique. The suspended cells containing spermatogonial stem cells were cultured with different growth factors (bFGF, GDNF, IGF-I, EGF and LIF) for 14 days. The area of colony was measured in all experimental groups at day 14. Also, Immunofluorescence staining was used for qualitative and quantitative evaluations of DDX4/VASA-positive cells. The expression levels of Vasa and Gfrα1, as germ cell markers, was assessed in the experimental groups. Spermatogonial cells treated with a combination of growth factors bFGF, GDNF, IGF-1 and EGF had a positive effect on the area of colonies (P< 0.001). However, in the presence of LIF, no considerable effect detected on the spermatogonial cell proliferation. Immunofluorescence staining showed spermatogonial colonies were positive for germ cell-specific marker. With the exception of LIF-treated group, no significant difference was observed in the number of DDX4/VASA-positive spermatogonial cells among the experimental groups (P>0.05). A combination of growth factors did not have detectable effects on the expression levels of Vasa and Gfrα1 (P>0.05). The findings indicated that recombinant human growth factors has no significant effects on the in vitro proliferation of Caspian trout spermatogonia.