Elsevier

Developmental Biology

Volume 443, Issue 1, 1 November 2018, Pages 19-34
Developmental Biology

Isolating mitotic and meiotic germ cells from male mice by developmental synchronization, staging, and sorting

https://doi.org/10.1016/j.ydbio.2018.08.009Get rights and content
Under an Elsevier user license
open archive

Highlights

  • We present “3 S,” a new method for sorting germ cell populations from mouse testis.

  • “3 S” employs in vivo synchronization to reduce cellular complexity of the testis.

  • Synchronization is followed by staging to verify cell identity, and by sorting.

  • Yields ~90% purity of a wide variety of mitotic and meiotic germ cell populations.

  • Provides sufficient numbers of sorted cells for biochemical and epigenomic studies.

Abstract

Isolating discrete populations of germ cells from the mouse testis is challenging, because the adult testis contains germ cells at every step of spermatogenesis, in addition to somatic cells. We present a novel method for isolating precise, high-purity populations of male germ cells. We first synchronize germ cell development in vivo by manipulating retinoic acid metabolism, and perform histological staging to verify synchronization. We use fluorescence-activated cell sorting to separate the synchronized differentiating germ cells from contaminating somatic cells and undifferentiated spermatogonia. We achieve ~90% purity at each step of development from undifferentiated spermatogonia through late meiotic prophase. Utilizing this “3 S” method (synchronize, stage, and sort), we can separate germ cell types that were previously challenging or impossible to distinguish, with sufficient yield for epigenetic and biochemical studies. 3 S expands the toolkit of germ cell sorting methods, and should facilitate detailed characterization of molecular and biochemical changes that occur during the mitotic and meiotic phases of spermatogenesis.

Keywords

Germ cells
Testis
Spermatogenesis
Meiosis
Synchronization
Epigenomics

Cited by (0)