Spermatogonial Transplantation

描述（由申请人提供）：此更新申请将集中在该补助金的前4.5年中所做的观察。发现在移植实验中，从出生前或刚出生后直到出生后约3天的小鼠中分离的生殖细胞不能有效地发挥干细胞的功能。在这些相同的实验中，从出生后4天以上的动物中分离的细胞有效地发挥了干细胞的作用。移植感受态细胞出现的时间大致与生殖细胞有丝分裂的恢复和生殖细胞从小管内部迁移到基底膜形成A精原细胞的时间一致。初步研究表明，一些3日龄或3日龄以下动物的生殖母细胞可迁移至基膜，甚至可进行有丝分裂，但它们总是不能启动精子发生。即使在移植后数周，仍有少量生殖母细胞留在基底膜上，但没有生精细胞集落形成，这些未成熟的生殖母细胞不能作为生精干细胞发挥功能。我们的假设是，在生殖细胞中发生的基因表达的变化是至关重要的功能性（即移植能力）生殖系干细胞的形成称为“成熟”的生殖细胞。本申请中提出的实验将研究这一假设，并试图确定在此期间在生殖细胞和睾丸体细胞中基因表达发生了什么变化。我们的方法的主要优点是使用干细胞移植作为功能性干细胞的测定。本实验主要有三个目的：1）确定细胞培养条件是否能将生殖细胞转化为具有移植活性的干细胞。2)确定在生殖细胞和体细胞中发生的基因表达的变化，以允许转化为可移植干细胞。3)使用RNAi系统抑制特定mRNA，测试候选基因在成熟过程中的作用。拟议的研究具有重要意义，在获得一个基本的理解是什么需要形成一个生精干细胞，并在获得更实际的洞察力，什么操作可能会进行对原始生殖细胞和生殖细胞，以创建一个来源的细胞移植。这项研究可能对解决人类不育问题有意义，因为原始生殖细胞或生殖细胞成熟为功能性干细胞是异常的。

DESCRIPTION (provided by applicant): This renewal application will focus on an observation made in the first 4.5 years of this grant. It was found that gonocytes isolated from mice before or right after birth up until about 3 days after birth could not function efficiently as stem cells in transplant experiments. Cells isolated from animals 4 days plus after birth functioned efficiently as stem cells in these same experiments. The time period for this appearance of transplantation competent cells coincides roughly with the resumption of gonocyte mitosis and the migration of the gonocytes from the interior of the tubule to the basement membrane to form A spermatogonia. Preliminary studies indicate that some of the gonocytes from 3 day old or younger animals migrate to the basement membrane and may even undergo some mitosis but they always fail to initiate spermatogenesis. Even weeks after transplantation a few gonocytes remain on the basement membrane but no colonies of spermatogenic cells are formed and these immature gonocytes fail to function as spermatogenic stem cells. It is our hypothesis that changes in gene expression that are occurring in gonocytes are crucial to the formation of functional (i.e. transplantation competent) germ line stem cells referred to as the "maturation" of gonocytes. The experiments proposed in this application will investigate this hypothesis and attempt to define what changes in gene expression take place during this time in both gonocytes and in testicular somatic cells. The major advantage of our approach is the use of stem cell transplantation as an assay for functional stem cells. Experiments are proposed in 3 Specific aims: 1) Determine if cell culture conditions can transform gonocytes into transplantation competent stem cells. 2) Determine what changes in gene expression occur in the gonocytes and the somatic cells to allow the transformation into transplantation competent stem cells. 3) Test the role of candidate genes in the maturation process using the RNAi system to inhibit specific mRNAs. The proposed studies have significance in obtaining a basic understanding of what is required to form a spermatogenic stem cell and in gaining more practical insight into what manipulations could possibly be performed on primordial germ cells and gonocytes to create a source of cells for transplantation. This research could have implications in approaching human infertility problems where the maturation of primordial germ cells or gonocytes into functional stem cells is aberrant.