A1 Refereed original research article in a scientific journal
Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses
Authors: Mäkelä Juho-Antti, Cisneros-Montalvo Sheyla, Lehtiniemi Tiina, Olotu Opeyemi, La Hue M, Toppari Jorma, Hobbs Robin M, Parvinen Martti, Kotaja Noora
Publisher: JOURNAL OF VISUALIZED EXPERIMENTS
Publication year: 2020
Journal: Journal of Visualized Experiments
Journal name in source: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Journal acronym: JOVE-J VIS EXP
Article number: ARTN e61800
Issue: 164
Number of pages: 18
ISSN: 1940-087X
eISSN: 1940-087X
DOI: https://doi.org/10.3791/61800
Self-archived copy’s web address: https://research.utu.fi/converis/portal/detail/Publication/51141759
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.
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