Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses
Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses
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DOI：
10.3791/61800
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发表时间：
2020-10-01
影响因子：
1.2
通讯作者：
Kotaja, Noora
中科院分区：
文献类型：
--
作者：
Makela, Juho-Antti;Cisneros-Montalvo, Sheyla;Kotaja, Noora
精子发生是一个独特的分化过程，最终产生身体最独特的细胞类型之一，精子。生殖细胞的分化发生在体细胞支持细胞的细胞质袋中，支持细胞同时宿主4至5代生殖细胞，并协调和同步它们的发育。因此，在横截面内生殖细胞类型的组成是恒定的，并且这些细胞缔合也被称为生精上皮周期的阶段（I-XII）。重要的是，还可以基于通过透照揭示的它们的差异光吸收/散射特性以及阶段以数字顺序沿着小管彼此沿着的事实，从完整的生精小管中识别阶段。本文介绍了一种透射辅助显微切割方法，用于分离代表小鼠生精上皮细胞周期特定阶段的曲细精管节段。首先在解剖显微镜下检查生精小管的光吸收模式，然后切割代表特定阶段的小管片段并用于下游应用。在这里，我们描述了免疫染色协议的阶段特定的南瓜制剂和完整的小管节段。这种方法使研究人员能够专注于在精子发生的特定阶段发生的生物学事件，从而为精子发生的发育，毒理学和细胞学研究及其分子机制提供了独特的工具。
Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. Differentiation of germ cells takes place in the cytoplasmic pockets of somatic Sertoli cells that host 4 to 5 generations of germ cells simultaneously and coordinate and synchronize their development. Therefore, the composition of germ cell types within a cross-section is constant, and these cell associations are also known as stages (I-XII) of the seminiferous epithelial cycle. Importantly, stages can also be identified from intact seminiferous tubules based on their differential light absorption/scatter characteristics revealed by transillumination, and the fact that the stages follow each other along the tubule in a numerical order. This article describes a transillumination-assisted microdissection method for the isolation of seminiferous tubule segments representing specific stages of mouse seminiferous epithelial cycle. The light absorption pattern of seminiferous tubules is first inspected under a dissection microscope, and then tubule segments representing specific stages are cut and used for downstream applications. Here we describe immunostaining protocols for stage-specific squash preparations and for intact tubule segments. This method allows a researcher to focus on biological events taking place at specific phases of spermatogenesis, thus providing a unique tool for developmental, toxicological, and cytological studies of spermatogenesis and underlying molecular mechanisms.