精原干细胞冷冻保存后再移植的生精功能

Spermatogenic function of transplanted spermatogonial stem cell following cryopreservation

背景:精原干细胞的冷冻保存在男性不育治疗方面具有潜在的临床应用价值,但冷冻保存过的精原干细胞移植后,其精子发生过程及生精能力目前尚不完全清楚。目的:观测精原干细胞冷冻保存后移植的生精功能。方法:采用复合酶消化、差速贴壁结合非连续性Percoll密度梯度离心的方法获取雄性SD大鼠及雄性C57BL/6小鼠精原干细胞;以雄性BALB/c裸小鼠为受体,出生后6周予腹腔注射白消安破坏其内源性生精功能。将供体精原干细胞分为冷冻保存组与非冷冻保存组,分别以曲细精管微注射的方法异种移植精原干细胞,采用形态学方法观察生精过程;采集附睾精液,观察精子形态并进行体外受精实验。移植后1,2,3个月取受体睾丸组织,采用免疫组化SABC法检测α6-Integrin蛋白表达情况;移植后1周、1个月、3个月取受体睾丸组织,应用扫描电镜观察生精过程。结果与结论:冷冻保存前的精原干细胞活性率高于冷冻保存后的细胞活性率(P<0.01)。冷冻保存组与未冷冻保存组受体睾丸组织精原细胞膜和细胞质中均有α6-Integrin的阳性表达,随时间增加而增加,两组间α6-Integrin蛋白的表达无统计学差异;SD大鼠精原干细胞移植后能在BALB/c裸小鼠生精上皮中克隆增殖,并能分化形成大鼠形态特征的精子,在小鼠精子发生巢内既有大鼠来源的精子发生,又有小鼠内源性精子发生,其供体来源的精子数量较小鼠自身来源的精子数量多。通过体外受精实验提示移植后的精原干细胞在受体内增殖分化形成的精子功能正常,具有受精的能力。结果说明采用常规方法冷冻保存的精原干细胞,移植后能在受体生精上皮中克隆增殖,并能分化形成成熟的精子。

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.