Revisiting testicular cell suspensions and meiocytes sorting.

Abstract

In the June 2014 issue of Cytometry A (page 556), Gaysinskaya et al. reported an optimized method for the isolation of early adult mouse meiocytes by means of Hoechst 33342 vital staining and flow cytometry 1. They present an improved procedure of testis dissociation and enzyme treatment (collagenase, trypsin, and DNAse I) for the analysis and sorting with high cell count and viability (>94%). The technique combines DNA content estimation and specific gating strategies to maximize detection of meiotic prophase I (MPI) substages and minimize contamination from non-meiotic cells. They claim to obtain high purity subpopulations of pre-leptotene (PL), leptotene (L), zygotene (Z), pachytene (P), and diplotene (D) stages of MPI spermatocytes. The identity of sorted cell populations was assessed through immunolabeling with antibodies against phosphorylated H2AX (γH2AX) and synaptonemal complex protein 3 (SYCP3). However, as shown in Table 1 1, PL, L, Z, and P sorted populations exhibit cross contamination from other MPI stages with subpopulations purities averaging 75–92% (PL), 60–80% (L), 75–90% (Z), and 81–95% (P). Our research group has developed a rapid technique for the preparation of testicular cell suspensions for flow cytometric analysis of early meiocytes, based on mechanical testis dissociation (Medimachine) which also ensures high cell viability (>90%), is much simpler, and considerably shorter in time (15 min) compared to enzyme-mediated procedures. This machine-based technique is highly reproducible and to a great extent is operator independent, providing high quality cell suspensions 2, 3. The little manipulation and brevity of the method strongly benefits the preservation of macromolecules—especially RNAs—that may be affected by dissociation-derived deleterious conditions. While Gaysinskaya et al. state that “a challenge, still, is to obtain suspensions of consistently high quality between the experiments,” it calls our attention that they do not mention our protocol for testicular cell suspensions preparation. We have compared the method of Gaysinskaya et al. and our own taking into account cell yield, viability (Trypan blue and PI staining), and quality of cell suspensions (phase contrast microscopy and amount of debris in FSC/SSC plots) in parallel from testes obtained from the same specimens, and both methods rendered similar results. Mechanical cell detachment combined with different fluorochromes including Vybrant staining (Invitrogen/Life Technologies, Carlsbad, CA) allowed sharp flow cytometry discrimination of MPI stages. In this connection, we were the first to describe the presence of a bimodal DNA content peak in 4C cell populations, and to sort guinea pig L/Z and P MPI stages with minimal cross-contamination as shown by immunodetection using anti-SYCP3 antibodies 4. Recently, we have been able to achieve similar results analyzing mouse early meiocytes with Vybrant dye (Rodríguez-Casuriaga et al., Rapid preparation of rodent testicular cell suspensions and spermatogenic stages purification by flow cytometry using a novel blue-laser-excitable vital dye, Submitted). Despite the fact that we have not yet attempted to sort L from Z cells, our L/Z and P fractions exhibit consistent high purity (>98%), thus affording excellent material to unveil specific gene expression profiles along the different MPI stages (4), [Rodríguez-Casuriaga et al., Rapid preparation of rodent testicular cell suspensions and spermatogenic stages purification by flow cytometry using a novel blue-laser-excitable vital dye; Submitted], as cross-contaminated fractions could provide misleading results. An additional advantage is the use of a 488 nm laser as excitation source for the Vybrant dye, which minimizes the potential damage to nucleic acids as may occur when bisbenzimidazole dyes such as Hoechst 33342 are excited with UV lasers 5. In that sense, although in the paper by Gaysinskaya et al. the issue of nucleic acids purification is not addressed, we have succeeded to obtain good quality RNAs after Medimachine-processing, Vybrant staining, and sorting, which has allowed high quality transcriptome comparative analyses of purified cell populations (manuscript in preparation). Adriana Geisinger* Departamento de Biología Molecular Instituto de Investigaciones Biológicas Clemente Estable (IIBCE) Montevideo, Uruguay Sección Bioquímica, Facultad de Ciencias Universidad de la República, Uruguay Rosana Rodríguez-Casuriaga Departamento de Biología Molecular Instituto de Investigaciones Biológicas Clemente Estable (IIBCE) Montevideo, Uruguay Federico F. Santiñaque Servicio de Citometría de Flujo y Clasificación Celular (SECIF) IIBCE, Montevideo, Uruguay Gustavo A. Folle Servicio de Citometría de Flujo y Clasificación Celular (SECIF) IIBCE, Montevideo, Uruguay