Mäkelä Juho-Antti, Cisneros-Montalvo Sheyla, Lehtiniemi Tiina, Olotu Opeyemi, La Hue M, Toppari Jorma, Hobbs Robin M, Parvinen Martti, Kotaja Noora
Institute of Biomedicine, Integrative Physiology and Pharmacology Unit, University of Turku, Turku, Finland;
Institute of Biomedicine, Integrative Physiology and Pharmacology Unit, University of Turku, Turku, Finland.
J Vis Exp. 2020 Oct 7(164). doi: 10.3791/61800.
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.
精子发生是一个独特的分化过程，最终产生身体中最独特的细胞类型之一——精子。生殖细胞的分化发生在支持细胞的细胞质凹陷处，支持细胞同时容纳4到5代生殖细胞，并协调和同步它们的发育。因此，横切面上生殖细胞类型的组成是恒定的，这些细胞组合也被称为生精上皮周期的阶段（I - XII）。重要的是，根据透照显示的不同光吸收/散射特性，以及这些阶段沿小管按数字顺序依次排列的事实，也可以从完整的生精小管中识别出这些阶段。本文描述了一种透照辅助显微切割方法，用于分离代表小鼠生精上皮周期特定阶段的生精小管段。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段并用于下游应用。在这里，我们描述了针对特定阶段压片标本和完整小管段的免疫染色方案。该方法使研究人员能够专注于精子发生特定阶段发生的生物学事件，从而为生精及其潜在分子机制的发育、毒理学和细胞学研究提供了一个独特的工具。
