Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Abstract

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types -both nonspermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) -studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells -in this case, from the testesthrough a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 10 8 cells/spermatogenic cell type from a starting population of 7-8 x 10 8 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator. Video LinkThe video component of this article can be found at

Humanized H19/Igf2 locus reveals diverged imprinting mechanism between mouse and human and reflects Silver–Russell syndrome phenotypes

“…We hypothesized that depletion of H3K9me3, increased enrichment of H3K4me2, or both contribute to the inability to fully establish DNA methylation at hIC1. Spermatogenic cells were fractionated by the STA-PUT method, and chromatin was isolated from a round spermatid-enriched fraction (22). ChIP-qRT-PCR analyses in round spermatids revealed that hIC1 had fivefold greater enrichment of H3K4me2 (Fig.…”

“…Round spermatid fractions of mouse spermatogenic cells were collected using STA-PUT in two independent replicates, and the purity of each fraction was verified as described previously (22,44). Each collection used both testes of 12 heterozygous (H19 hIC1/+ ) male mice.…”

Characterization of BRD4 during Mammalian Postmeiotic Sperm Development

“…As mentioned above, Brd4 null heterozygous male mice show spermatogenic defects, suggesting that BRD4 may play a role in spermatogenesis (28,29). To investigate the expression pattern of BRD4 over the course of spermatogenesis, we analyzed protein levels in different cell types obtained by STA-PUT velocity sedimentation from mature mouse testes (35). By this method, we collected four cell populations (Fig.…”

“…IF assay was performed with 8-m cryosections of testes from 10-week-old mice or cell suspensions obtained by the spermatogenic cell separation method described above (35). Testes were dissected and immediately placed in 4% paraformaldehyde (PFA; in phosphate-buffered saline [PBS]) or snap-frozen in liquid nitrogen.…”

“…Mouse spermatogenic cell fractionation. Spermatogenic cell fractionation was performed by sedimentation of cells prepared from adult mouse testes through a bovine serum albumin (BSA) gradient as previously described (35). Each fractionation experiment used approximately 22 testes.…”