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

Abstract

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.

The deubiquitinase cofactor UAF1 interacts with USP1 and plays an essential role in spermiogenesis

“…Fluorescence-activated cell sorting (FACS) purification was conducted to collect round spermatids from 8-week-old WT and Uaf1 sKO mice for proteomics. 51 , 52 After removing the tunica albuginea, the testes were placed in 5 ml PBS containing collagenase type I (120 U/ml) and mixed for 10 min at 35°C. The testes were digested in 5 ml of 0.25% trypsin plus 0.1 ml of deoxyribonuclease I (5 mg/ml) at 35°C for 8 min, and the reaction was terminated by the addition of 0.5 ml fetal bovine serum.…”

Flow Cytometry of Hematological and Immunological Samples from Different Species

“…Flow cytometry is a standard tool to detect protein expression, DNA content, and other cell physiological parameters at single cell level (1–3). Since flow cytometers are capable of processing single cell suspensions, they are excellent instruments to study fluid tissue samples like blood or bone marrow aspirates.…”

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

“…Various approaches have been used to isolate testicular germ cells at specific spermatogenic stages, such as centrifugal elutriation (8,9), BSA gradient sedimentation/STA-PUT (10)(11)(12)(13)(14), and, more recently, fluorescence-activated cell sorting (FACS) (15)(16)(17)(18)(19). One of the advantages of centrifugal elutriation and BSA gradient sedimentation is that they can manage a large number of cells (>10 8 cells) within a few hours, with the purity of the isolated cells usually maintained at approximately 70-90%.…”