Preliminary Experimental Study On Spermatogonial Stem Cell Transplantation To Cure Infertility Owing To High-dose Chemotherapy

With the development of modern medicine, the clinical curative rate of prepubertal oncological patients has been increased remarkably, the survival rate of these patients has been improved significantly. However, many male patients have to face permanent sterility caused by high-dose chemotherapy, there is no definite method to treat this iatrogenic infertility at present. Spermatogonial stem cells are immortal and pluripotent as precursor cells of spermatozoa formation, which can proliferate and differentiate in testicular microenvironment. It has opened a new door for clinical application in therapy of male infertility. In this study, we will explore the surface markers and the approach of isolation and purification of spermatogonial stem cells, observe the process of transference, proliferation and differentiation in vivo and evaluate the capability to form mature spermatozoa after spermatogonial stem cell transplantation. Our study can provide experimental evidence for applying the method of spermatogonial stem cell transplantation to cure infertility owing to high-dose chemotherapy.Part I Investigation of immunochemical characteristics of spermatogonial stem cellsin testicular tissue of ratObjective To explore the surface markers of spermatogonial stem cells in rat. Methods Histological sections of testes from Sprague-Dawley (SD) rats aged 10-day were examined by hematoxylin and eosin (HE) stain. The expression and distribution of α₆-Integrin and c-kit on testicular tissue were examined by means of immunohistochemistry and immune electron microscopy.Results HE stain showed that the seminiferous epithelium was mainly composed of spermatogonia and sertoli cells in 10-day SD rat. Spermatogonia were relatively large cells with round or orbicular-ovate nucleus, the sharper nuclear membrane, the higher nucleus:cytoplasm ratio, and many cytoplasmic inclusions mostly concentrated at one side of the cell. Sertoli cells were irregular shape with weak-stained nucleus. Using α₆-Integrin as cell marker to perform SABC stain, the expression of spermatogonia at outer compartment of seminiferous tubules werepositive on cytomembrane and cytoplasm. Sertoli cells expressed negatively α₆-Integrin on seminiferous tubules. Observed by electron microscopy after immunochemical stain of α₆-Integrin, electron dense deposition were in cytomembrane and cytoplasma of spermatogonia at outer compartment of seminiferous tubules, no electron dense deposition could be observed in cytomembrane and cytoplasma of sertoli cells. Using c-kit as cell marker to perform immunofluorescence stain, a large number of spermatogonia in seminiferous tubules adjacent to cavity expressed positively c-kit on cytomembrane and cytoplasm, while spermatogonia in seminiferous tubules adjacent to basal membrane expressed negatively c-kit. Sertoli cells also expressed negatively c-kit on seminiferous tubules.Conclusion In testicular tissue, all spermatogonia express uniquely α₆-Integrin, including undifferentiated spermatogonia and differentiating spermatogonia. Differentiating spermatogonia express positively c-kit. C-kit and α₆-Integrin can be used as the molecular markers to identify spermatogonia in special period. Spermatogonial stem cells express positively α₆-Integrin or c-kit on both cytomembrane and cytoplasm, thus α₆-Integrin and c-kit can be used as the surface markers of spermatogonial stem cells in proliferating and differentiating. Part II Isolation, purification and immunochemical identification of spermatogonialstem cells in ratObjective To evaluate the feasibility of the approach in isolating and purifying spermatogonial stem cells and its immunochemical characteristics. Methods Compound enzymatic digestions were used to prepare germ cell suspensions of SD rats at 10 days, velocity sedimentation and discontinuous percoll density gradient centrifugation were used to isolate and purify spermatogonia. Using c-kit and α₆-Integrin as markers respectively, we observed the immunochemical characteristics of spermatogonia in testicular tissues and purified cells, and detected the positive rates of purified cells by flow cytometry.Results C-kit and α₆-Integrin were expressed positively among the purified cells. The results showed consistence with those in testicular sections. Using c-kit as the cellmarker, the positive rate was( 1.59±0.04) % in the unpurified group and(68.33±2.45) % in the purified group (P <0.01) respectively. Using α₆-Integrin as the cell marker, the positive rate was (2.38±0.60) % in the unpurified group and (72.04±3.65) % in the purified group (P<0.01) respectively. Moreover ,both trypan blue stain and propidium iodide(PI) stain showed that the cell viability rates were more than 95 % in the purified cells. After the purified cells were cultured for 3 days, we observed the spermatogonial stem cells growing with typical chain or thyrsoid shape. Conclusion Spermatogonia with high purity and viability can be obtained via the following steps, including digestions with enzymes, velocity sedimentation and discontinuous percoll density gradient centrifugation. These methods can enrich the amount of spermatogonial stem cells relatively.Part III Research on transference, proliferation and differentiation of spermatogonialstem cells in vivo after transplantationObjective To explore a new method to cure infertility owing to high-dose chemotherapy by observing the transference, proliferation and differentiation of spermatogonial stem cells in vivo after transplantation.Methods Using C57BL/6 mice of postnatal 6-10 days as the germ cell donors, male germ cells were obtained by combination with compound enzymatic digestions, velocity sedimentation and discontinuous percoll density gradient centrifugation, cells were then labeled with the fluorescent PKH26 dye. Male C57BL/6 mice were used as the recipients which were homologous with the donor mice, they had been injected busulfan (40 mg/Kg, intraperitoneally) at 6 weeks of age, which could destroy endogenous spermatogenic cells. The busulfan-treated males were used as recipients beginning 4-6 weeks after injection. We traced the fluorescent-labeled cells in the recipient testes after transplantation and observed the process of cell transference in vivo. Normal mice without receiving busulfan treatment and cell transplantation were selected as the parallel positive control group (group 1 ) , mice which were microinjected cells into seminiferous tubules in unilateral testes were the experiment group ( group 3) , the same mice which were microinjected media into seminiferous tubules in contralateral testes were the parallel negative control group (group 2 ) . Inorder to observe the process of proliferation and differentiation in vivo after spermatogonial stem cell transplantation, we obtained the testes from three groups, performed HE stain and detected the expression of α₆-Integrin,c-kit and SCF in protein and mRNA levels in testicular tissues by Western Blot and real-time fluorescence quantitative-polymerase chain reaction assay (RFQ-PCR) respectively. Results Tracing the PKH26 fluorescent-labeled cells, partial transplanted cells immigrated into basal membrane of the seminiferous tubules in one week after transplantation. There were no transplanted cells in lumina of the seminiferous tubules in one month after transplantation, because these cells had immigrated into the basal membrane and had division growth. The transplanted spermatogonial stem cells could colonize the recipient testes and form plenty of spermatids in lumina of the seminiferous tubules in three months after transplantation.HE stain showed that nearly all endogenous spermatogonia disappeared and sertoli cells decreased after establishment of infertility model with busulfan. In group 1, spermatogonia in different stages were aligned on the seminiferous epithelium orderly and spermatozoa were formed in lumina of the seminiferous tubules during three months. With the extension of observing time, sertoli cells increased and few endogenous spermatogonia emerged in the seminiferous epithelium in group 2. In group 3, spermatogonia in different stages were aligned gradually in the seminiferous epithelium orderly, spermatozoa were formed in lumina of the seminiferous tubules, and sertoli cells increased. Abnormal nuclear division phases were not observed in the testicular cells and inflammatory cells did not infiltrate into the testicular tissues in group 3.Using Western Blot to analyze three sorts of proteins in testicular tissues during three-month observation, the expression level of a6-Integrin protein in group 3 was lower than group 1, higher than group 2 (both P <0.05) , but α₆-Integrin protein expression level increased gradually in each group, especially in group 1 and group 3 (all P <0.01) . C-kit protein expression level in group 3 was lower than group 1, higher than group 2 (both P <0.05) , but c-kit protein expression level increased gradually in each group, especially in group 1 and group 3 (all P <0.01) . SCF protein expression level was lower in group 3 than group 1 (all P <0.01) , there were no significant difference between group 3 and group 2. In group 1, SCF protein expression level was higher at the end of the second month than that at the end of the first month (P <0.01) after transplantation. In group 2 and group 3 , SCF proteinexpression levels increased gradually (all P <0.05) .RFQ-PCR was used to detect mRNA level in testicular tissues during three months. The expression level of α₆-Integrin mRNA in group 3 was lower than group 1 , higher than group 2 (both P <0.01) , but α₆-Integrin mRNA expression level increased gradually in each group, especially in group 1 and group 3 (all P <0.01) . In group 3, c-kit mRNA expression level was lower than group 1 , higher than group 2 (both P <0.01) , but c-kit mRNA expression level increased gradually in each group, especially in group 1 and group 3 (all P <0.05) . In group 1, the expression level of SCF mRNA was higher than either group 2 or group 3 (all P <0.05 ) .There were no significant difference between group 3 and group 2. SCF mRNA expression level increased gradually in each group (all P <0.05) .Conclusion Spermatogonial stem cells firstly transfer from the lumen of seminiferous tubule to the basal membrane after transplantation. In first month after transplantation, all of them have immigrated into the basal membrane and begun to proliferate and differentiate in the recipient testes. In third month after transplantation, the spermatogonial stem cells have formed spermatids. Busulfan has a less slight toxic effect on sertoli cells than on spermatogonia, but has detrimental influence on spermatogonia in the seminiferous tubules. Furthermore, the number of sertoli cells can restore when the toxic effect fade away. After high-dose chemotherapy, there are a certain number of sertoli cells in the seminiferous epithelium, this microenvironment of spermatogenesis is not been destroyed. Recipient sertoli cells can provide donor spermatogonial stem cells with proper microenvironment to proliferate and differentiate after donor spermatogonial stem cells are infused into the recipient seminiferous tubules. Exogenous spermatogonial stem cells can colonize the recipient seminiferous epithelium and then proliferate and differentiate after transplantation. Our research provide the compelling evidence for using spermatogonial stem cell transplantation to cure infertility owing to high-dose chemotherapy. Part IV Research on spermatogenesis function of spermatogonial stem cells aftertransplantation Objective To establish theoretical foundation for using spermatogonial stem celltransplantation to cure infertility by observing the spermatogenesis and the influence of cryopreservation on proliferation and differentiation of spermatogonial stem cells after transplantation.Methods Using C57BL/6 mice of postnatal 6-10 days and SD rat of postnatal 10 days as germ cell donors respectively, male germ cells were obtained by combination with compound enzymatic digestions, velocity sedimentation and discontinuous percoll density gradient centrifugation. Cell viability was detected by flow cytometry before and after cryopreservation. The recipients were male BALB/c nude mice, in which endogenous spermatogenesis were destroyed by intraperitoneal injection of busulfan at 6 weeks of age. 4-6 weeks after injection, the donor cells were transplanted into the seminiferous tubules of the busulfan-treated males. Whole experiment was divided into three stages. At the first stage, fresh germ cells of C57BL/6 mice were transplanted into the testes of BALB/c nude mice ( as non-cryopreservation group) , to observe the process of spermatogenesis by scanning electron microscope (SEM) and analyze α₆-Integrin protein expression at different time following transplantation, perform in vitro fertilization three months later. At the second stage, germ cells of SD rats were transplanted into the testes of BALB/c nude mice, to observe the spermatozoa morphology by SEM three months later. At the third stage, cryopreserved germ cells of C57BL/6 mice were transplanted into the testes of BALB/c nude mice (as cryopreservation group) , to analyze α₆-Integrin protein expression at different time following transplantation and compare it with the non-cryopreservation group.Results The cell viability rate was higher before cryopreservation than after cryopreservation[ ( 98.39±0.59 ) % vs. ( 59.90±3.87 ) % , P<0.01]. Immunohistochemical stain showed that spermatogonia expressed positively α₆-Integrin on cytomembrane and cytoplasm in both non-cryopreservation group and cryopreservation group. During three-month observation, there were no significant difference in the expression levels of α₆-Integrin protein between two groups, but α₆-Integrin expression level increased gradually in each group (all P <0.01) .SEM showed that the transplanted cells appeared in lumina of the seminiferous tubules in one week after transplantation. Lots of spermatogenic cells emerged on the seminiferous tubules in one month after transplantation. Spermatogonia increased dominantly and formed plenty of spermatozoa in three months after transplantation. When germ cells of SD rats were transplanted into testes of BALB/c mice, threemonths later, SEM showed that there were more spermatozoa with rat-sperm shape than those originated from the recipient themselves in the recipient testes, in addition, the rat spermatozoa also appeared in the recipient semen.When spermatogonial stem cells of C57BL/6 mice were transplanted into the testes of BALB/c nude mice, We collected semen in the recipient epididymis three months later, and performed in vitro fertilization with spermatozoa from BALB/c nude mouse and oocytes from female F1 white mice, then obtained 6 two-cell embryos, which were transplanted into oviduct of the pseudopregnant recipient mouse. Two young were born 20 days later, one was black, another was plum. By analyzing the hereditary character of the black young, its male gamete should come from the donor C57BL/6 mouse, we could assume that spermatozoa generated from donor spermatogonial stem cells had capability to fertilize. By analyzing the hereditary character of the plum young, its male gamete should come from the recipient BALB/c nude mouse, suggested that both the donor-derived spermatogenesis and the recipient-derived spermatogenesis came forth after the donor stem cells were transferred to the recipient testis.Conclusion Following transplantation, spermatogonial stem cells can colonize the recipient testes, initiate spermatogenesis and produce sperm with the capability of fertilization. Conventional cryopreservation of spermatogonial stem cells before transplantation do not interfere repopulation in the recipient testes and reinitiation of spermatogenesis, above data is important to apply spermatogonial stem cell transplantation in clinic. Before high-dose chemotherapy treatment on prepubertal oncological patients, we can extract spermatogonial stem cells from the testicular tissues and cryopreserve it, then transplant it to the patients having recovered from the cancer. These transplanted cells will regenerate spermatogenesis. These procedures can make the patients keep long time fertility. The combination of spermatogonial stem cell cryopreservation and transplantation is valuable for clinical application in curing infertility owing to high-dose chemotherapy.