Spermatogonial Transplantation

描述（由申请人提供）：此续期申请将重点关注本资助的前4.5年的观察结果。研究发现，从小鼠出生前或出生后3天左右分离的性腺细胞在移植实验中不能作为干细胞有效地发挥作用。在同样的实验中，从出生后4天以上的动物身上分离的细胞有效地发挥了干细胞的功能。移植能态细胞出现的时间大致与性腺细胞有丝分裂的恢复和性腺细胞从小管内部向基底膜迁移形成精原细胞的时间一致。初步研究表明，来自3日龄或更小的动物的一些性腺细胞迁移到基底膜，甚至可能进行一些有丝分裂，但它们总是不能启动精子发生。即使移植几周后，基底膜上仍有少量的性腺细胞，但没有形成生精细胞的集落，这些未成熟的性腺细胞不能作为生精干细胞发挥作用。我们的假设是，在性腺细胞中发生的基因表达变化对功能性（即移植能力）生殖系干细胞的形成至关重要，即性腺细胞的“成熟”。本应用程序中提出的实验将研究这一假设，并试图确定在这段时间内性腺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。实验提出了三个具体目标：1)确定细胞培养条件是否可以将淋细胞转化为移植能力干细胞。2)确定在性腺细胞和体细胞中发生的基因表达变化，以使其转化为移植能力强的干细胞。3)利用RNAi系统抑制特异性mrna，测试候选基因在成熟过程中的作用。提出的研究对于获得对形成生精干细胞所需条件的基本理解，以及对可能对原始生殖细胞和性腺细胞进行哪些操作以创建用于移植的细胞来源获得更实际的见解具有重要意义。这项研究可能会对解决人类不育问题产生影响，其中原始生殖细胞或性腺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.