Fate of Spermatogonial Stem Cells

描述(申请人提供)：我们的长期目标是更好地了解精子发生的初始步骤，即精原细胞的增殖和分化在当前授权期的调控，我们证明了A型精原细胞的增殖是由干细胞因子(SCF)刺激的。其他研究表明，支持细胞-精原细胞共培养允许精原细胞分化，即分离的A型精原细胞(可能是干细胞)多次分裂，产生相互关联的分化A型精原细胞。我们还证实PI-3K/AKT/p70S6信号通路参与了SCF诱导的A型精原细胞的增殖。此外，我们还证明了A型向精子的分化与端粒长度和端粒酶活性的变化有关。在这个竞争性的更新应用中，在目标1中，我们将进一步研究A型精原细胞的生物学。我们推测，除了SCF，白血病抑制因子(LIF)和胶质细胞源性神经营养因子(GDNF)等其他配体对精原细胞的发育可能也很重要。我们的初步结果表明，这两种配体的受体(GFRalpha1和LIFR)表达于A型精原细胞表面。我们将使用全套免疫细胞化学方法检测是否所有A型精原细胞都具有GFRalpha1、c-kit和LIF受体。使用免疫磁珠程序，我们将分离GFRalpha1阳性细胞组以及c-kit和LIFR阳性细胞组，并用各自的配体刺激每组细胞。为了确定这些A型精原细胞亚群是否保持其分化能力，我们将它们移植到不育的受体小鼠中，并在体内跟踪精子发生。在目标2中，我们将继续研究与精原细胞增殖有关的分子信号。我们已经产生了令人兴奋的初步数据，表明SCF和LIF可能启动了不同的细胞内信号通路。通过这些信号通路，特定的候选基因被配体诱导，导致精原细胞的增殖。在第三个目标中，我们将使用基因表达序列分析(SAGE)和DNA阵列程序来检测分离的A型精原细胞和培养的生精小管中的基因表达。这项建议中概述的实验应该提供关于精原细胞基本生物学的新数据。此外，这些研究将有助于开发体外精子发生技术，并有助于使用健康的生殖细胞进行辅助生殖。

DESCRIPTION (provided by applicant): Our long-term objective is to understand better the regulation of the initial steps of spermatogenesis, namely spermatogonial proliferation and differentiation In the current granting period, we demonstrated that proliferation of type A spermatogonia is stimulated by stem cell factor (SCF) Other studies demonstrated that Sertoli cell-spermatogonial co-cultures allow for spermatogonial differentiation, i.e., the isolated type A (presumably stem cells) divided a number of times to yield rows of interconnected differentiated type A. We also established that the PI-3 kinase/AKT/p70 S6 kinase signaling pathway is involved in SCF-induced proliferation of type A spermatogonia. In addition, we demonstrated that differentiation of type A into sperm is associated with changes in telomere length and telomerase activity. In this competing renewal application, in aim 1, we will further examine the biology of the type A spermatogonia. We hypothesize that in addition to SCF, other ligands such as leukemia inhibitory factor (LIF) and glial cell line-derived neurotrophic factor (GDNF) may be important for spermatogonial development. Our preliminary results indicate that receptors (GFRalpha1 and LIFr) for these two ligands are expressed on the surface of type A spermatogonia. We will use whole mount immunocytochemistry to examine whether all type A spermatogonia possess the GFRalpha1, c-kit, and LIF receptors. Using the immunomagnetic bead procedure, we will separate a GFRalpha1 positive group of cells as well as a c-kit and LIFr positive group of cells and stimulate each group with the respective ligands. To determine whether these subsets of type A spermatogonia retain their ability to differentiate, we will transplant them into sterile recipient mice and follow spermatogenesis in vivo. In aim 2, we will continue to investigate the molecular signals responsible for spermatogonial proliferation. We have generated exciting preliminary data to suggest that SCF and LIF may initiate distinct intracellular signaling pathways. Through these signaling pathways, specific candidate genes are induced by ligands leading to spermatogonial proliferation. In the third aim, we will examine gene expression in isolated type A spermatogonia and in seminiferous tubules in culture using serial analysis of gene expression (SAGE) and DNA array procedures in response to ligands. The experiments outlined in this proposal should provide new data on the basic biology of the spermatogonia. Furthermore, these studies will help in developing techniques of in-vitro spermatogenesis and contribute to the use of healthy germ cells in assisted reproduction.