Spermatogonial Transplantation

精原细胞移植

• 批准号: 7089966
• 负责人: MICHAEL D GRISWOLD
• 金额: $ 25.16万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7089966
• 关键词: RNA interference; cell cell interaction; cytogenetics; gene expression; genetic regulation; laboratory mouse; laboratory rat; messenger RNA; microarray technology; organ culture; polymerase chain reaction; sperm; spermatogenesis; stem cell transplantation; testis; tissue /cell culture; xenotransplantation

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.

描述（由申请人提供）：本次续展申请将重点关注本次拨款前 4.5 年的观察结果。研究发现，在移植实验中，从小鼠出生前或出生后立即分离到出生后约3天的生殖母细胞不能有效地发挥干细胞的功能。在这些相同的实验中，从动物出生后 4 天以上分离出的细胞可以有效地发挥干细胞的功能。移植感受态细胞出现的时间大致与生殖母细胞有丝分裂的恢复和生殖母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，来自三天龄或更小的动物的一些生殖母细胞迁移到基底膜，甚至可能经历一些有丝分裂，但它们总是无法启动精子发生。即使在移植后数周，一些生殖母细胞仍保留在基底膜上，但没有形成生精细胞集落，并且这些未成熟的生殖母细胞无法发挥生精干细胞的作用。我们的假设是，生殖母细胞中发生的基因表达的变化对于功能性（即具有移植能力）生殖系干细胞（称为生殖母细胞的“成熟”）的形成至关重要。本申请中提出的实验将研究这一假设，并尝试确定在此期间生殖母细胞和睾丸体细胞中基因表达发生哪些变化。我们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3个具体目标：1）确定细胞培养条件是否可以将生殖母细胞转化为具有移植能力的干细胞。 2) 确定生殖母细胞和体细胞中基因表达发生哪些变化，以允许转化为具有移植能力的干细胞。 3) 使用RNAi系统抑制特定mRNA来测试候选基因在成熟过程中的作用。拟议的研究对于获得对形成生精干细胞所需的条件的基本了解以及获得更实际的了解可以对原始生殖细胞和生殖母细胞进行哪些操作以创建用于移植的细胞来源具有重要意义。这项研究可能对解决人类不孕问题产生影响，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。