Simultaneous Purification of Round and Elongated Spermatids from Testis Tissue Using a FACS-Based DNA Ploidy Assay

Spermiogenesis is the final phase of spermatogenesis during which post-meiotic haploid round spermatids (rSpt) differentiate into elongated spermatozoa and includes several critical cell-specific processes like DNA condensation, formation of the acrosome, and production of the flagellum. Disturbances in this process will lead to complications in sperm development and subsequently cause infertility. As such, studying spermiogenesis has clinical relevance in investigating the etiology of male infertility and will improve our scientific understanding of male germ cell formation. Here, we were able to purify round spermatid and elongated spermatid fractions from a single cryopreserved human testicular tissues sample with an efficiency of 85.4% ± 4.9% and 97.6% ± 0.6%, respectively. We confirmed the cell types by morphology and immunohistochemistry for histone H4 and PNA protein expression. The purity was measured by manual counting of histone H4 positive (round) and negative (elongated) spermatids in both sorted 1 N cell fractions. This method can be applied to both human and rodent studies. Especially in studies with limited access to testicular tissue, this method provides a reliable means to simultaneously isolate these cell types with high purity. Our method allows for further investigation of germ cell development and the process of spermiogenesis in particular, as well as provides a tool to study the etiology of male infertility, including morphological and biochemical assessment of round and elongating spermatids from subfertile men. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.