Homogeneity of cell populations is a prerequisite for the analysis of biochemical and molecular events during male gamete differentiation. Given the complex organization of the mammalian testicular tissue, various methods have been used to obtain enriched or purified cell populations, including flow cell sorting. Current protocols are usually time-consuming and may imply loss of short-lived RNAs, which is undesirable for expression profiling. We describe an optimized method to speed up the preparation of suitable testicular cell suspensions for cytometric analysis of different spermatogenic stages from rodents. The procedure takes only 15 min including testis dissection, tissue cutting, and processing through the Medimachine System (Becton Dickinson). This method could be a substitute for the more tedious and time-consuming cell preparation techniques currently in use.
Key Words: Flow cytometry - Gene expression - Spermatogenesis
Introduction
Spermatogenesis is a complex differentiation process essential for all the species with sexual reproduction, which leads to the formation of male gametes. In spite of its importance, a deeper comprehension of the molecular bases is still required for the understanding of the fundamentals of normal sexual reproduction, as well as for the treatment of testicular pathologies.
Spermatogenesis can be divided into three phases: mitotic proliferation of spermatogonia, meiotic divisions of spermatocytes, and spermiogenesis. Meiosis is a successful evolutionary widespread mechanism present in all eukaryotic species from fungi to superior plants and mammals. In sexually reproducing organisms, meiosis mediates the reduction in the DNA content of gametes, therefore compensating for doubling at fertilization (1).
The process of meiosis is mainly related to the behavior of chromosomes during prophase and anaphase of the first meiotic division, with highly significant events taking place (i.e., homologous synapsis, recombination, and segregation). Particularly, the homologous recombination that occurs during meiosis (crossing over) has vital importance as a means of genetic variability (2) and therefore constitutes a main source of biodiversity.
Different spermatogenic cell types with C (round spermatids, elongating and elongated spermatids, sperm), 2C (G1 spermatogonia, secondary spermatocytes), and 4C (different stages of primary spermatocytes, G2 spermatogonia) DNA content, coexist with somatic testicular cells (e.g., Leydig and Sertoli cells) in the testes of adult mammals. Cellular heterogeneity represents one of the major problems concerning the study of the molecular basis of mammalian spermatogenesis (3), together with the lack of spermatogenic cell lines for in vitro culture.
A strategy used to allow the analysis of gene expression along spermatogenesis has been the use of whole testes of prepubertal specimens, naturally enriched in early spermatogenic stages (4, 5). However, this approach does not precisely allow the assignment of specific transcripts to individual cell types. A sophisticated approach uses the differential light absorption pattern of the seminiferous tubule to isolate specific differentiation stages under a dissection microscope (6). Although this approach has pioneered cellular and molecular analyses, it requires high expertise and does not separate the diverse cell types comprising each differentiation stage.
A different strategy has been the enrichment of cellular populations by Staput (7, 8) or elutriation (3). More recently, the identification and sorting of different spermatogenic cell subpopulations by flow cytometry have been described (9–12). Cell separation techniques precisely allow studying which transcripts are present in a certain cell type, but have the disadvantage of involving laborious cell preparation procedures, and therefore, in some cases, expression levels may change to a certain extent during the purification process. Moreover, the duration of the process is especially critical for the representation of some RNAs and proteins with short half lives (reviewed in (13)).
The first step for any cell separation method is the preparation of a cellular suspension. The main goal of a cell suspension method is to provide a rich and representative sample of the different cellular subpopulations. Moreover, it is also important to maximize the number of viable cells and to prevent cell clumps. In the case of testicular cell suspensions, it is critical to avoid selective damage of specific cell types and to minimize the formation of multinucleate cells (3) which tend to form because of the syncitial nature of the seminiferous epithelium. Besides, when the cell suspensions are prepared for downstream applications such as gene expression studies, the duration of the process and the handling involved must be taken into consideration, in order to prevent degradation or loss of macromolecules of interest.
Various protocols using mechanical dissociation for the preparation of testicular cell suspensions have been described (7). However, suspensions prepared by these methods tend to aggregate very quickly, and the yield of viable cells is at most 80% (generally less). Even more important, certain cell types may be selectively damaged (3). Additionally, since manual mechanical methods are operator-dependent, results are not highly reproducible. Methods involving a combination of gentle mechanical action with enzymatic treatment have rendered better results in terms of yield and cell type representation, while minimizing cell aggregation (3, 9). Nevertheless, they are time-consuming and involve a lot of handling.
Here, we present a very simple and efficient protocol to rapidly obtain a cellular suspension from testis material for flow cytometric analysis. This protocol eliminates steps between animal sacrifice and spermatogenic stage-specific molecular studies, aiming to optimize macromolecule preservation.
Materials and Methods
Animals
Male CD1-Swiss outbred stock mice (7–9 weeks old), Sprague–Dawley rats (8–10 weeks old), and Dunkin Hartley guinea pigs (12–14 weeks old) were used as a source of normal adult testes for all the experiments. Testis material from one specimen was used for each experiment. Between seven and nine adult individuals of each species were analyzed while two 21-day-old rat pups were processed for immature testis studies. Animals were sacrificed by cervical dislocation (rats and mice) or by administration of an overdose of sodium pentobarbital (guinea pigs), following the recommendations of the Uruguayan National Commission of Animal Experimentation (CHEA).
Preparation of Cellular Suspensions
Testes were dissected into 96 mm glass Petri dishes containing ice-cold separation medium [10% fetal calf serum in Dulbecco’s Modified Eagle’s medium, containing high glucose and l-glutamine], and cut into 2–3 mm3 pieces after removal of the tunica albuginea. Four to five of these pieces were immediately placed into a disposable disaggregator Medicon™ with 50 µm separator mesh (Becton Dickinson) plus 1 mL of ice-cold separation medium and processed for 50 s in the Medimachine System (Becton Dickinson). Each Medicon unit contains a fixed stainless-steel screen with about 100 hexagonal holes surrounded by six microblades. The tissue is brought to each hole by a metal rotor inside the Medicon chamber and disaggregated by passing over the sharpened holes and through the metal screen, while a micropump under the screen supplies liquid and flushes out the holes.
The cell suspension was recovered from the Medicon unit with a 5-mL disposable syringe, subsequently filtered through a 50-μm Filcon (Becton Dickinson) and 25 μm nylon mesh, and placed on ice. Cells were counted by means of a Neubauer chamber and diluted to a concentration of 1–2 × 107 cells/mL in separation medium. 2-Naphthol-6,8-disulfonic acid, dipotassium salt (NDA; Chemos GmBH, Regenstauf, Germany) was added to the suspension to a final concentration of 0.2% in order to prevent cell clumping. Cell viability of the testicular cell suspensions was measured with the trypan blue dye exclusion test and the LIVE/DEAD viability kit for animal cells (Molecular Probes, Eugene, OR, USA) according to manufacturer’s instructions.
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