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

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 and germline stem cells. We achieve ∼90% purity at each step of development from the germline stem cell pool through late meiotic prophase. Utilizing this “3S” 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. The 3S method should enable detailed characterization of molecular changes that occur during the mitotic and meiotic phases of spermatogenesis.