Oatley Jon M, Avarbock Mary R, Telaranta Aino I, Fearon Douglas T, Brinster Ralph L
Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, 3850 Baltimore Avenue, Philadelphia, PA 19104, USA.
Proc Natl Acad Sci U S A. 2006 Jun 20;103(25):9524-9. doi: 10.1073/pnas.0603332103. Epub 2006 Jun 1.
Spermatogonial stem cells (SSCs) are the foundation for spermatogenesis and, thus, preservation of a species. Because of stem cell rarity, studying their self-renewal is greatly facilitated by in vitro culture of enriched biologically active cell populations. A recently developed culture method identified glial cell line-derived neurotrophic factor (GDNF) as the essential growth factor that supports in vitro self-renewal of SSCs and results in an increase in their number. This system is a good model to study mechanisms of stem cell self-renewal because of the well defined culture conditions, enriched cell population, and available transplantation assay. By withdrawing and replacing GDNF in culture medium, we identified regulated expression of many genes by using microarray analysis. The expression levels of six of these genes were dramatically decreased by GDNF withdrawal and increased by GDNF replacement. To demonstrate the biological significance of the identified GDNF-regulated genes, we examined the importance of the most responsive of the six, bcl6b, a transcriptional repressor. By using siRNA to reduce transcript levels, Bcl6b was shown to be crucial for SSC maintenance in vitro. Moreover, evaluation of Bcl6b-null male testes revealed degeneration and/or absence of active spermatogenesis in 24 +/- 7% of seminiferous tubules. These data suggest that Bcl6b is an important molecule in SSC self-renewal and validate the biological relevance of the GDNF-regulated genes identified through microarray analysis. In addition, comparison of data generated in this study to other stem cell types suggests that self-renewal in SSCs is regulated by distinctly different molecular mechanisms.
精原干细胞（SSCs）是精子发生的基础，因而也是物种得以延续的基础。由于干细胞数量稀少，通过体外培养富集的具有生物活性的细胞群体极大地促进了对其自我更新的研究。最近开发的一种培养方法确定了胶质细胞系衍生神经营养因子（GDNF）是支持SSCs体外自我更新并使其数量增加的关键生长因子。由于培养条件明确、细胞群体富集且有可用的移植检测方法，该系统是研究干细胞自我更新机制的良好模型。通过在培养基中去除和替换GDNF，我们利用微阵列分析确定了许多基因的调控表达。其中六个基因的表达水平在去除GDNF后显著降低，在替换GDNF后升高。为了证明所确定的GDNF调控基因的生物学意义，我们研究了六个基因中反应最强烈的转录抑制因子bcl6b的重要性。通过使用小干扰RNA（siRNA）降低转录水平，结果表明Bcl6b对体外SSCs的维持至关重要。此外，对Bcl6b基因敲除雄性小鼠睾丸的评估显示，24 +/- 7%的生精小管出现退化和/或缺乏活跃的精子发生。这些数据表明Bcl6b是SSCs自我更新中的一个重要分子，并验证了通过微阵列分析确定的GDNF调控基因的生物学相关性。此外，将本研究中生成的数据与其他干细胞类型的数据进行比较表明，SSCs的自我更新受截然不同的分子机制调控。
