A1 Refereed original research article in a scientific journal
Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment
Authors: Da Ros M., Lehtiniemi T., Olotu O., Meikar O., Kotaja N.
Publisher: JOURNAL OF VISUALIZED EXPERIMENTS
Publication year: 2019
Journal: Journal of Visualized Experiments
Journal name in source: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Journal acronym: JOVE-J VIS EXP
Article number: ARTN e60271
Issue: 151
Number of pages: 10
ISSN: 1940-087X
DOI: https://doi.org/10.3791/60271
Web address : 10.3791/60271
Self-archived copy’s web address: https://research.utu.fi/converis/portal/detail/Publication/43810693
To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.
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