Spermatogonial Transplantation

描述(由申请人提供)：本续期申请将侧重于在这笔拨款的头4.5年中所作的观察。在移植实验中，从出生前或出生后即刻直到出生后大约3天的小鼠身上分离的性腺细胞被发现不能有效地作为干细胞发挥作用。在同样的实验中，从出生后4天以上的动物身上分离出来的细胞可以有效地作为干细胞发挥作用。具有移植能力的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迁移到基底膜以形成A精原细胞的时间相吻合。初步研究表明，3日龄或更小的动物的性细胞部分迁移到基底膜，甚至可能经历一些有丝分裂，但它们总是不能启动精子发生。即使在移植后几周，基底膜上仍有少量的性腺细胞，但没有形成生精细胞集落，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，发生在性细胞中的基因表达的变化对于功能性(即具有移植能力的)生殖系干细胞的形成至关重要，这种干细胞被称为性细胞的“成熟”。本申请中提出的实验将研究这一假说，并试图确定在这段时间内性细胞和睾丸体细胞中基因表达发生了什么变化。我们方法的主要优势是使用干细胞移植作为一种功能干细胞的检测方法。实验有三个具体目的：1)确定细胞培养条件是否能将性腺细胞转化为具有移植能力的干细胞。2)确定性腺细胞和体细胞中的基因表达发生了什么变化，以允许转化为具有移植能力的干细胞。3)利用RNAi系统抑制特定的mRNAs，测试候选基因在成熟过程中的作用。拟议的研究对于基本了解形成生精干细胞所需的条件，以及更实际地了解可能对原始生殖细胞和性腺细胞进行哪些操作以创造移植细胞来源具有重要意义。这项研究可能对解决人类不孕不育问题有意义，在人类不孕不育问题上，原始生殖细胞或性细胞成熟为功能干细胞是异常的。

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.