Isolation and analyses of enriched populations of male mouse germ cells by sedimentation velocity: the centrifugal elutriation

Abstract

The studies of molecular events that occur in single cell types within a tissue often require the disaggregation of the tissue into a single cell suspension, followed by isolation of distinct cell populations. The germinal epithelium of mammals is composed of several cell types, which divide mitotically, before entering meiosis. In this chapter, we describe the isolation of five mouse germ-cell fractions by centrifugal elutriation, and characterize them by their DNA content (flow cytometry), cell morphology (DAPI staining of nuclei, Giemsa staining of squashed cells) and deposition of stage-specific meiotic markers (SYCP3, H1t, SAM68) on chromosome spreads and whole cells. Within 2 h it is possible to obtain enriched populations of elongated spermatids (up to approximately 50% of the fraction), round spermatids (up to approximately 80%), primary spermatocytes (up to approximately 89%), and secondary spermatocytes (up to approximately 17%). Furthermore, most of the collected spermatocytes of the primary spermatocyte fraction are in early-mid pachytene stage as judged by chromosome spreads, enriched up to approximately 89%. Elutriation and techniques used for characterization of germ cell fractions are described.