A1 Refereed original research article in a scientific journal

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment




AuthorsDa Ros M., Lehtiniemi T., Olotu O., Meikar O., Kotaja N.

PublisherJOURNAL OF VISUALIZED EXPERIMENTS

Publication year2019

JournalJournal of Visualized Experiments

Journal name in sourceJOVE-JOURNAL OF VISUALIZED EXPERIMENTS

Journal acronymJOVE-J VIS EXP

Article numberARTN e60271

Issue151

Number of pages10

ISSN1940-087X

DOIhttps://doi.org/10.3791/60271

Web address 10.3791/60271

Self-archived copy’s web addresshttps://research.utu.fi/converis/portal/detail/Publication/43810693


Abstract
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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