Chinese Journal of Tissue Engineering Research ›› 2010, Vol. 14 ›› Issue (31): 5767-5772.doi: 10.3969/j.issn.1673-8225.2010.31.017

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Spermatogenic function of transplanted spermatogonial stem cell following cryopreservation

Ma Liang-hong1, Ding Qiang2, Feng Li-xin3, Li Pei-jun1, Chen Fu-bao1   

  1. 1 Department of Urology, Affiliated Hospital of Ningxia Medical University, Yinchuan  750004, Ningxia Hui Autonomous Region, China; 2 Institute of Urology, Huashan Hospital, Fudan University, Shanghai  200040, China; 3 Laboratory for Germline Stem Cell Research, Shanghai Jiao Tong University School of Medicine, Shanghai  200025, China
  • Online:2010-07-30 Published:2010-07-30
  • About author:Ma Liang-hong☆, Doctor, Associate chief physician, Department of Urology, Affiliated Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China mmm1770@163.com
  • Supported by:

     the National Natural Science Foundation of China, No. 30860282*; the Natural Science Foundation of Ningxia, No. NZ08107*

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Abstract:

BACKGROUND: Cryopreservation of spermatogonial stem cell has potential clinical value for the treatment of male infertility. However, the process and ability of spermatogenesis of stem cell after cryopreservation is not yet entirely clear.
OBJECTIVE: To observe the spermatogenic function of spermatogonial stem cell cryopreserved 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. The recipients were male BALB/c nude mice, in which endogenous spermatogenesis were destroyed by intraperitoneal injection of busulfan at 6 weeks of age. The donor stem cells were divided into cryopreservation and non-cryopreservation groups, and xenotransplanted into the seminiferous tubules. Morphological methods were used to observe the process of spermatogenesis, and the epididymal semen was collected to observe sperm shape and perform in vitro fertilization (IVF). The expression level of α6-Integrin protein was analyzed by immunohistochemical SABC staining at months 1, 2 and 3 after transplantation. The process of spermatogenesis was observed by scanning electron microscope at week 1, months 1 and 3 after transplantation.
RESULTS AND CONCLUSION: The cell viability rate was higher before cryopreservation than after cryopreservation (P < 0.01). SABC staining showed that spermatogonia expressed α6-Integrin positively on cytomembrane and cytoplasm in both non-cryopreservation group and cryopreservation group, and the expression level increased with time prolonged, but there were no significant difference between two groups. When germ cells of SD rats were transplanted into testes of BALB/c mice, spermatogonial stem cell could colonize and proliferate in the recipient seminiferous epithelium, and produce plenty of spermatozoa with rat-sperm shape, both the rat-derived spermatogenesis and the endogenous spermatogenesis came from the mouse spermatogenesis nest in which the number of sperm originated from the donor were more than those from the recipient themselves. IVF experiment indicated that spermatozoa generated from donor spermatogonial stem cells had normal function and capability to fertilize. These results show that spermatogonial stem cell can colonize and proliferate in the recipient seminiferous epithelium, and differentiate to form mature sperm after conventional cryopreservation and transplantation.

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