The biology of spermatogonial development in sheep

精原干细胞（stem Sg）是精子的成体前体；它们的健康对物种的繁殖至关重要。精原体移植是一种从供体睾丸中分离出精原体干细胞并将其注射到受体动物的精小管中，使受体动物产生供体精子的技术。小鼠精原体移植已被证明是研究精子发生的一种有价值的新工具，并为利用干细胞保存和传播遗传开辟了可能性。特别是，绵羊产业将受益于无需运输动物的额外遗传方式，以及将基因直接引入种系的成本较低的方式。公羊是研究哺乳动物精原细胞出生后发育的良好选择。它们相对较小，价格便宜，而且公羊羔羊没有被阉割，所以有现成的来自屠宰场的各种年龄的睾丸供研究。研究干细胞Sg必须克服的一个主要问题是，它们是睾丸中的少数群体。因此，为了寻找对其健康重要的分子，我们将用激光从绵羊睾丸的冷冻切片中收集单个干细胞Sg，然后从细胞中提取RNA，通过PCR定量重要基因转录物。RNA也将用于通过微阵列对这些细胞表达的基因进行广泛筛选。这些信息将用于提高公羊精原体移植的效率。我们还将开发一种使用逆转录病毒将基因永久植入供体干细胞的方法。最后，我们将使用精原移植和转导的供体干细胞Sg来测试在筛选中鉴定的基因在公羊精原发育中的功能。在这个过程中，我们将为农场动物产业和寻找大型动物疾病模型的研究人员展示精子移植的效率。

Spermatogonial stem cells (stem Sg) are the adult precursors of sperm; their health is critical to propagation of species.  Spermatogonial transplantation is a technique that isolates stem Sg from donor testes and injects them into the seminiferous tubules of a recipient for production of donor sperm by the recipient animal.  Spermatogonial transplantation in mice has proven to be a valuable new tool for studying spermatogenesis, and has opened the possibility of using stem Sg for preservation and dissemination of genetics.  In particular the sheep industry would benefit from additional ways to transport genetics without shipping animals, and from less costly ways to introduce genes directly into the germ line. Rams are a good alternative to rodents for studying post-natal development of spermatogonia in mammals.  They are comparatively small and cheap, and ram lambs are not castrated, so there is a ready supply of testes of all ages from the abattoir for study.  A major problem that must be overcome in studying stem Sg is that they are a minor population in the testis.  Therefore in order to look for molecules important to their health, we will collect individual stem Sg from frozen sections of ram lamb testes with a laser and then extract RNA from the cells to quantify important gene transcripts by PCR.  The RNA also will be used for a broad screening of genes expressed by these cells using microarrays.  The information will be used to increase the efficiency of spermatogonial transplantation in rams.  We also will develop a way to permanently incorporate genes into donor stem Sg using a retrovirus.  Finally we will use spermatogonial transplantation and transduced donor stem Sg to test the function of genes identified in the screens in development of ram spermatogonia.  In the process, we will demonstrate efficiency of spermatogonial transplantation for the farm animal industries and for researchers seeking large animal models of disease.