Dual DNA staining enables isolation of multiple sub‐types of post‐replicative mouse male germ cells

Abstract

During spermatogenesis, mammalian male germ cells undergo multiple developmental processes, including meiosis and post-meiotic differentiation (spermiogenesis). To understand the transitions between different cellular states it is essential to isolate pure populations of cells at different stages of development. Previous approaches enabled the isolation of cells from different stages of meiotic prophase I, but techniques to sub-fractionate unfixed, post-meiotic spermatids have been lacking. Here we report the development of a protocol enabling simultaneous isolation of cells at different stages of meiotic prophase and post-meiotic differentiation from testes of adult mice. This approach builds on existing fluorescence activated cell sorting protocols designed to purify cells in different stages of meiotic prophase I. By utilizing the specific spectral properties that two different DNA dyes (Hoechst 33342 and SYTO 16) exhibit when bound to chromatin of different stage male germ cells, we obtain highly pure populations of cells in relatively large numbers. This FACS protocol will enable immunocytological and molecular characterization studies of fractionated meiotic and haploid germ cells from both wild type and genetically mutant animals.

De novo transcriptome assembly of mouse male germ cells reveals novel genes, stage-specific bidirectional promoter activity, and noncoding RNA expression

“…Using our newly developed FACS-based isolation strategy ( Gill et al 2022 ), we collected one to 10 million cells from 3-mo-old C57BL/6J mice from five distinct stages of postreplicative development ( Fig. 1 A): leptotene/zygotene (LZ) spermatocytes (which are in the early stages of meiotic prophase), pachytene/diplotene (PD) spermatocytes (which are in the late stages of meiotic prophase), RSs (which are early, transcriptionally active postmeiotic cells), and early and late elongating spermatids (EES and LES, respectively; which are postmeiotic cells undergoing global transcriptional silencing and histone-to-protamine exchange).…”

“…A detailed description of the cell purification is provided elsewhere ( Gill et al 2022 ). Essentially, single-cell preparations from total testes were generated by sequential digestion with collagenase followed by trypsin dissolved in Gey's balanced salt solution (GBSS).…”

“…A portion of each cell population was used to determine the purity of the fractions (reported by Gill et al 2022 ). For the two spermatocyte populations (LZ & PD), purity was ∼90% (with 5%–7% contamination for the other spermatocyte populations).…”

Isolation of stage‐specific spermatogenic cells by dynamic histone incorporation and removal in spermatogenesis

“…In contrast, post-meiotic cells (e.g., spermatids) are recognized by these stains as a single population. More recently, SYTO16 can be applied to the fine separation of spermatids because of its unique high affinity for spermatids at the later stages of differentiation [24,25]. In fixed cells, SYTO16 staining can separate spermatids into four populations (steps 1-9, 10-12, 13-14, and 15-16), each with an extremely high purity of 95%-100% [24].…”

“…In fixed cells, SYTO16 staining can separate spermatids into four populations (steps 1-9, 10-12, 13-14, and 15-16), each with an extremely high purity of 95%-100% [24]. In living cells, on the other hand, SYTO16 can separate three populations (round, early elongating, and late elongating spermatids); the latter two populations have been shown to be 10%-20% cross-contaminated with each other [25].…”