Human amnion mesenchymal stem cells restore spermatogenesis in mice with busulfan-induced testis toxicity by inhibiting apoptosis and oxidative stress

BACKGROUND: Before starting gonadotoxic therapies, cryopreservation of mature sperm has been proposed worldwide as a method for male fertility preservation and for enabling the conception of a healthy baby with assisted reproductive technology (ART); however, these technologies are not feasible for prepubertal boys and men with spermatogenic failure. Transplantation of mesenchymal stem cells has exhibited successful therapeutic benefits in restoring spermatogenesis via gonadal graft angiogenesis, transplanted cell clonogenesis, and disordered somatic compartment recovery. This study aimed to elucidate the fertility protective effects and the underlying mechanisms of human amnion mesenchymal stem cells (hAMSCs) against busulfan-induced testis toxicity. METHODS: An in vivo busulfan-induced testis toxicity mouse model and an in vitro busulfan-administered mouse Sertoli cell line were employed to evaluate the efficacy and mechanisms of hAMSC transplantation on male fertility preservation. The process of spermatogenesis was evaluated histologically, and the percentage of seminiferous tubules with vacuoles was evaluated by HE staining. Semen parameters were calculated by computer-assisted semen analysis. ELISA was employed to test the testosterone concentration and the levels of oxidative- and antioxidative-associated substances LDH, MDA, GR, SOD, GPx, and CAT. The rates of proliferation (Ki67), apoptosis (Annexin V), and ROS were measured by FACS. The fluorescence intensity of a marker of apoptosis (TUNEL) and a meiosis gene in spermatogenesis (SCP3) were detected by immunofluorescence assay. The expression of mRNA in germ cell-specific (GCS) genes (Dazl, Ddx4, and Miwi) and meiosis genes (Scp3, Cyclin A1, and Stra8) was tested by qPCR. The expression of antiapoptotic proteins (SURVIVIN and BCL2), apoptotic proteins (CASPASE3 and CASPASE9), GCS proteins (Dazl, Ddx4, and Miwi), and meiosis proteins (Scp3, Cyclin A1, and Stra8) was tested by western blotting. RESULTS: hAMSC transplantation following disruption by busulfan-induced testis toxicity restored spermatogenesis, elevating testosterone levels and enhancing testicular weight, size, and semen parameters in vivo. In addition, hAMSCs clearly ameliorated cell apoptosis, enhanced cell proliferation, repressed oxidative damage, and augmented oxidative defense in vivo and in vitro. Moreover, hAMSCs distinctly increased the expression of the GCS genes Dazl, Ddx4, and Miwi and the meiosis genes Scp3, Cyclin A1, and Stra8 in vivo. CONCLUSIONS: hAMSCs might represent a promising tool for the use in regenerative medicine, as these cells can restore spermatogenesis in a busulfan-induced testis toxicity mouse model and facilitate activity in a busulfan-administered mouse Sertoli cell line by resisting apoptosis and oxidative stress.