Production of donor-derived sperm after spermatogonial stem cell transplantation in the dog.
Production of donor-derived sperm after spermatogonial stem cell transplantation in the dog.
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DOI：
10.1530/rep-08-0226
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发表时间：
2008-12
期刊：
影响因子：
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通讯作者：
Travis AJ
中科院分区：
文献类型：
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作者：
Kim Y;Turner D;Nelson J;Dobrinski I;McEntee M;Travis AJ
精原干细胞移植提供了研究精原干细胞（SSC）和操纵雄性生殖系的独特方法。我们在这里报告了这项技术在狗身上的首次成功表现，狗是人类疾病的重要模型。首先，我们研究了一种照射方案，以消耗受体睾丸中的内源性雄性生殖细胞。组织学检查证实内源性精子发生消耗>95%，但保留了正常睾丸结构。然后，在移植混合生精小管细胞之前，对五个月大的受体狗（n = 5）的睾丸进行局部照射[新鲜（n = 2）或培养两周后（n = 3）]。接受培养细胞的狗立即表现出过敏反应，并通过姑息治疗迅速消退。在接受新鲜细胞的狗中没有观察到这种反应，但使用了不同的注射介质。注射后 12 个月，受者被阉割并从附睾中收集精子。我们进行了微卫星分析，将附睾精子的 DNA 与受者和供者的基因组 DNA 进行了比较。我们使用 6 个标记来证明来自一名新鲜混合小管细胞受体的精子中存在供体等位基因。在其他受者的精子中没有检测到供体等位基因的证据。使用基于单核苷酸多态性的定量 PCR，显示约 19.5% 的精子来自受者的供体。我们的结果表明，首次在狗身上成功完成了 SSCT，这是在这一有价值的生物医学模型中通过雄性种系进行转基因的重要一步。
Spermatogonial stem cell transplantation offers unique approaches to investigate spermatogonial stem cells (SSC) and to manipulate the male germline. We report here the first successful performance of this technique in the dog, which is an important model of human diseases. First, we investigated an irradiation protocol to deplete endogenous male germ cells in recipient testes. Histologic examination confirmed >95% depletion of endogenous spermatogenesis, but retention of normal testis architecture. Then, five month-old recipient dogs (n=5) were focally irradiated on their testes prior to transplantation with mixed seminiferous tubule cells [fresh (n=2) or after two weeks of culture (n=3)]. The dogs receiving cultured cells showed an immediate allergic response, which subsided quickly with palliative treatment. No such response was seen in the dogs receiving fresh cells, for which a different injection medium was used. 12 months post injection, recipients were castrated and sperm was collected from epididymides. We performed microsatellite analysis comparing DNA from the epididymal sperm with genomic DNA from both the recipients and the donors. We used 6 markers to demonstrate the presence of donor alleles in the sperm from one recipient of fresh mixed tubule cells. No evidence of donor alleles was detected in sperm from the other recipients. Using quantitative PCR based on single nucleotide polymorphisms, about 19.5% of sperm were shown to be donor-derived in the recipient. Our results demonstrate the first successful completion of SSCT in the dog, an important step toward transgenesis through the male germline in this valuable biomedical model.