Molecular Analysis of Cell Type-Specific Gene Expression Profile During Mouse Spermatogenesis by Laser Microdissection and qRT-PCR
Molecular Analysis of Cell Type-Specific Gene Expression Profile During Mouse Spermatogenesis by Laser Microdissection and qRT-PCR
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DOI：
10.1177/1933719112452939
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发表时间：
2013-03-01
影响因子：
2.9
通讯作者：
Moley, Kelle H.
中科院分区：
文献类型：
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作者：
Esakky, Prabagaran;Hansen, Deborah A.;Moley, Kelle H.
激光显微切割 (LMD) 是一种选择性细胞分离技术，能够在直接显微镜观察下从复杂组织（例如睾丸）中分离出所需的同质细胞亚群。 LMD 结合定量逆转录酶聚合酶链式反应 (qRT-PCR) 是量化特定细胞群中信使 RNA (mRNA) 表达的不可或缺的工具。基因表达在精子发生的有丝分裂、减数分裂和减数分裂后三个连续阶段在时间和空间上受到调节。本研究展示了基于苏木精和伊红 (H&E) 染色的简短修改 LMD 方案。通过使用“标记基因”的 mRNA 分析来验证阶段特异性 LMD 的成功，这些基因在物种间保守，并且已知在精子发生过程中存在差异表达。 Magea4、Hspa2、Cox6b2、Tnp1、Prm1 和 Prm2 用于区分显微解剖的细胞群，分别是精原细胞（I 组）、精母细胞（II 组）、圆形和浓缩精子细胞（III 组）以及细长和浓缩精子细胞（IV 组）。 LMD 与 qRT-PCR 结合进一步扩展，可评估选定应激反应基因（如 Hsp90aa1、Gpx4、Ucp2、Sod1 和 Sod2）的细胞阶段特异性分布。生殖细胞特异性 mRNA 谱通过 LMD 样品的蛋白质印迹、免疫组织化学和相应蛋白质的共聚焦定位进行适当补充。目前的研究表明，LMD 可以成功地从睾丸的复杂组织中分离出细胞亚群；并建立所选应激反应基因和蛋白质的细胞阶段特异性基础表达模式。我们的假设是，应激反应基因的基线表达会因细胞阶段而异，从而产生针对生殖毒物的不同阶段特定的脆弱性。
Laser microdissection (LMD) is a selective cell isolation technique that enables the separation of desired homogenous cell subpopulations from complex tissues such as the testes under direct microscopic visualization. The LMD accompanied by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) represents an indispensable tool in quantifying messenger RNA (mRNA) expression among defined cell populations. Gene expression is temporally and spatially regulated at 3 sequential phases of mitotic, meiotic, and postmeiotic stages of spermatogenesis. The present study demonstrates a short modified LMD protocol based upon hematoxylin and eosin (H&E) staining. Stage-specific LMD success was validated by the use of mRNA profiling of "marker genes" which are conserved across species and are known to be differentially expressed during spermatogenesis. Magea4, Hspa2, Cox6b2, Tnp1, Prm1, and Prm2 are used to differentiate among the microdissected cell populations, namely spermatogonia (group I), spermatocytes (group II), round and condensing spermatids (group III), and elongated and condensed spermatids (group IV), respectively. The LMD combined with qRT-PCR is further extended to assess the cell stage-specific distribution of selected stress response genes such as Hsp90aa1, Gpx4, Ucp2, Sod1, and Sod2. The germ cell-specific mRNA profiles are suitably complemented by Western blot of the LMD samples, immunohistochemistry, and confocal localization of the corresponding proteins. The current study suggests that LMD can successfully isolate cell subpopulations from the complex tissues of the testes; and establish cell stage-specific basal expression patterns of selected stress response genes and proteins. It is our hypothesis that the baseline expression of stress response genes will differ by cell stage to create discrete stage-specific vulnerabilities to reproductive toxicants.