Fig. 2

a Canine SSCs supplemented with FSH in vitro and the control group were evaluated at 0, 72 and 120 h. a–c cSSCs (asterisk) supplemented with FSH in culture showed an increase in the number of cells and formed germ cell clumps after 72 h (arrow). These cells did not show a difference in morphological features compared with the control group up to at least 120 h. ai–ci These cells formed clumps after 72 h in vitro (scale bar = 100 μm). b Illustration demonstrating the influence of FSH on the cSSCs. Specific receptors in the testes, namely, follicle-stimulating hormone receptor (FSHR), are bound in Sertoli cells and Leydig cells. Under the influence of FSH, Sertoli cells release GDNF, and this paracrine factor then binds to the GFRA1/Ret protein localized in the membrane of SSCs and initiates the self-renewal process in these cells. c Flow cytometric analysis of the percentage of GFRA1positive cSSCs after 72 and 120 h of treatment with FSH supplementation and the control (p ≤ 0.05). d Graph showing the phenotypes of the cSSCs for germ cell markers (GFRA1, c-kit, DAZL, CD49f, PLZF, OCT4, STRA8) before xenotransplantation of the cSSCs (p < 0.05). e Gene expression of mGFRA1 evaluated at 72 and 120 h after FSH supplementation and in the control (p < 0.05)