This work was partly supported by CSIC (I+D project C022) and PEDECIBA. The authors want to thank Mariela Bollati and Valentina Porro from the Cell Biology Unit (Institut Pasteur de Montevideo) for their generous collaboration concerning the MoFlo cell sorter, and Merial-Montevideo for gently providing all the guinea pig specimens used in this project. Figure 1 was reproduced with permission of BioMed Central; Figures 2 and 3, and Table 1 with permission of S. Karger AG, Basel; and Figures 4 and 5 were reproduced or adapted with permission of John Wiley &amp; Sons, Inc (copyright owned by Wiley-Blackwell, 2011).

1.	Preparation of Cell Suspensions
<ol>
 <li>Sacrifice the specimen to be used following the recommendations of the specialized committees such as IACUC or equivalent (in Uruguay, National Commission for Animal Experimentation [CNEA]). In our case, an overdose of pentobarbital was administrated. </li>
 <li>Dissect the testes following standard approved procedures and place them in a 96 mm glass Petri dish on ice, containing 10 ml of ice-cold DMEM supplemented with 10% fetal calf serum. </li>
 <li>Remove the tunica albuginea and cut the decapsulated testes into square pieces of 2 - 3 mm on each side. </li>
 <li>Those pieces are then processed in a Medimachine, an automated mechanical grinder where the tissue is disaggregated inside a disposable unit containing a perforated stainless-steel screen and a metal rotor. To do so, place 1 ml of cold supplemented DMEM and 4 - 5 of these pieces in a 50 &mu;m disposable unit, switch on the disaggregator, and process for 50 sec following the simple instructions from the manufacturer. </li>
 <li>Recover the resulting cell suspension from the disaggregation unit using a 3 - 5 ml syringe without needle. </li>
 <li>Filter through a 50 &mu;m nylon mesh, previously soaked with 0.5 ml supplemented DMEM. </li>
 <li>Filter the suspension again using a soaked 25 &mu;m nylon mesh, and place on ice. </li>
 <li>Count in a Neubauer chamber and adjust cellular concentration to 1 - 2 x 107 cells/ml with supplemented DMEM. At least 4 x 107 cells/gram of testis material are usually obtained. </li>
 <li>Finally, add NDA (2-naphthol-6,8-disulfonic acid, dipotassium salt) to a final concentration of 0.2% in order to prevent cell clumping. </li>
 <li>Optional: check cell viability of the testicular cell suspensions with a commercially available viability kit for animal cells, following manufacturer's instructions. </li>
</ol>
2.	Flow Cytometric Analysis
	We have used a Becton-Dickinson FACSVantage flow cytometer equipped with a Coherent argon ion laser tuned to emit at 488 nm for the analysis of cells stained with high concentrations of propidium iodide (PI). (The issue of PI entrance into unfixed cells under stress has been addressed elsewhere9,10).
<ol>
 <li>For PI staining, add fluorochrome at a final concentration of 50 &mu;g/ml to the cell suspension, and incubate for 10 min at 0 &deg;C in the dark.</li>
 <li>Laser power is set to 100 mW and a 575/26 band pass filter is used to collect PI-emitted fluorescence in FL2. </li>
 <li>We perform FC measurements with a 70 &mu;m nozzle. For sorting spermatocyte populations, set sorting mode in Normal-R or Normal-C, using 3 sorted drops as envelope. Keep sample and collection tubes at 3 - 4 &deg;C by using a refrigeration unit. Adjust sample differential to analyze cells at a rate of 500 to 1,500 per second. </li>
 <li>Use CellQuest software (BD) to analyze the following parameters: forward scatter (FSC-H); side scatter (SSC-H); total emitted fluorescence or pulse-area (FL2-A); and duration of fluorescence emission or pulse-width (FL2-W). </li>
</ol>
Alternatively, we have employed the vital dye Hoechst 33342 to a final concentration of 5 &mu;g/ml and incubated for 10 min at 37 &deg;C in the dark. Cell analysis was performed by means of a MoFlo Cytometer (DakoCytomation) equipped with a UV excitation wavelength laser operating at 25 mW and using a 70 &mu;m nozzle. Data manipulation was performed with Summit v4.3 software (DakoCytomation).

<ol><li>Meistrich, M. L. Separation of spermatogenic cells and nuclei from rodent testes. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMeistrich%2C+ML+%22Methods+of+Cell+Biology%22">Methods of Cell Biology</a>. Prescott, D. M. 15, Academic Press. New York. 15-54 (1977).</li>
<li>Lam, D. M. K., Furrer, R., Bruce, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+separation%2C+physical+characterization%2C+and+differentiation+kinetics+of+spermatogonial+cells+of+the+mouse%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+USA%22%5BJournal%5D)">The separation, physical characterization, and differentiation kinetics of spermatogonial cells of the mouse.</a> Proc. Natl. Acad. Sci. USA. 65, 192-199 (1970).</li>
<li>Romrell, L. J., Bellve, A. R., Fawcet, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Separation+of+mouse+spermatogenic+cells+by+sedimentation+velocity%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">Separation of mouse spermatogenic cells by sedimentation velocity.</a> Dev. Biol. 19, 119-131 (1976).</li>
<li>Geisinger, A., Rodríguez-Casuriaga, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+cytometry+for+gene+expression+studies+in+mammalian+spermatogenesis%5BTitle%5D)+AND+%22Cytogenet.+Genome+Res%22%5BJournal%5D)">Flow cytometry for gene expression studies in mammalian spermatogenesis.</a> Cytogenet. Genome Res. 128, 46-56 (2010).</li>
<li>Getun, I. V., Torres, B., Bois, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+cytometry+purification+of+mouse+meiotic+cells%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Flow cytometry purification of mouse meiotic cells.</a> J. Vis. Exp. (50), e2602(2011).</li>
<li>Meistrich, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Separation+of+mouse+spermatogenic+cells+by+velocity+sedimentation%5BTitle%5D)+AND+%22J.+Cell+Physiol%22%5BJournal%5D)">Separation of mouse spermatogenic cells by velocity sedimentation.</a> J. Cell Physiol. 80, 299-312 (1972).</li>
<li>Meistrich, M. L., Bruce, W. R., Clermont, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+composition+of+fractions+of+mouse+testis+cells+following+velocity+sedimentation+separation%5BTitle%5D)+AND+%22Exp.+Cell+Res%22%5BJournal%5D)">Cellular composition of fractions of mouse testis cells following velocity sedimentation separation.</a> Exp. Cell Res. 79, 213-227 (1973).</li>
<li>Malkov, M., Fisher, Y., Don, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developmental+schedule+of+the+postnatal+rat+testis+determined+by+flow+cytometry%5BTitle%5D)+AND+%22Biol.+Rep%22%5BJournal%5D)">Developmental schedule of the postnatal rat testis determined by flow cytometry.</a> Biol. Rep. 59, 84-92 (1998).</li>
<li>Rodríguez-Casuriaga, R., Geisinger, A., Santiñaque, F., López, B., Folle, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-purity+flow+sorting+of+early+meiocytes+based+on+DNA+analysis+of+guinea+pig+spermatogenic+cells%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">High-purity flow sorting of early meiocytes based on DNA analysis of guinea pig spermatogenic cells.</a> Cytometry A. 79, 625-634 (2011).</li>
<li>Davey, H. M., Hexley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Red+but+not+dead%3F+Membranes+of+stressed+Saccharomyces+cerevisiae+are+permeable+to+propidium+iodide%5BTitle%5D)+AND+%22Environ.+Microbiol%22%5BJournal%5D)">Red but not dead? Membranes of stressed Saccharomyces cerevisiae are permeable to propidium iodide.</a> Environ. Microbiol. 13, 163-171 (2011).</li>
<li>Rodríguez-Casuriaga, R., Geisinger, A., López-Carro, B., Porro, V., Wettstein, R., Folle, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultra-fast+and+optimized+method+for+the+preparation+of+rodent+testicular+cells+for+flow+cytometric+analysis%5BTitle%5D)+AND+%22Biol.+Proced.+Online%22%5BJournal%5D)">Ultra-fast and optimized method for the preparation of rodent testicular cells for flow cytometric analysis.</a> Biol. Proced. Online. 11, 184-195 (2009).</li>
<li>Spanò, M., Evenson, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+cytometric+analysis+for+reproductive+biology%5BTitle%5D)+AND+%22Biol+Cell%22%5BJournal%5D)">Flow cytometric analysis for reproductive biology.</a> Biol Cell. 78, 53-62 (1993).</li>
</ol>

The optimized method described here enables the preparation of cell suspensions from rodent testicular tissue in a very fast and reproducible way, avoiding enzyme and detergent treatment and maintaining good cell integrity and type proportions. The brevity of the procedure (the 15 min span includes testis dissection, tissue cutting, and processing), minimal handling involved, and absence of enzymatic treatments are some of the main advantages. All these would account for the good preservation of short life macromolecules, which is critical when a representative sample of compounds present in the original cell population is required. 
Concerning the use of either PI or Hoechst 33342, we have observed no evident differences in the profiles resulting from flow cytometric analysis of testicular cell suspensions with the employment of one or the other dye. The dye choice will rather depend on the user's preferences and availabilities, and on planned further use. On one side, PI is cheaper, and of widespread application as does not require the use of flow cytometers and sorters having a UV laser. On the other, although we have successfully used PI-stained cells for ulterior sorting and differential gene expression studies (Figure 4), Hoechst 33342 or another vital dye with low cytotoxicity levels could be the preferable choices for applications where full cell viability is required, such as germ cell culture and/or transplantation4.
We have been able to sort specific cell populations achieving over 95% purity either for selected testicular populations with different DNA content4 (Figure 3), or even for subpopulations with the same DNA content but different chromatin condensation levels (Figure 4). This latter can be accomplished as long as the subpopulations of interest can be individualized in the cytometric profiles, particularly in the dot plots. For instance, up to now we have been able to sort different stages of guinea pig meiocytes I9, but not to discriminate and sort subpopulations within the 2C population simply by staining the DNA. Sorted cells have rendered good quality RNA, allowing downstream differential gene expression analyses9 (Figure 5).
As can be seen in the protocol description, it is very simple, easily reproducible, and does not require especially skilled personnel. We consider it can be adopted for a wide variety of applications involving flow cytometry or not. The former range from simple testicular content analysis for the rapid checking of spermatogenic advance, to others with preparative aims such as flow purification of specific cell populations for ulterior molecular studies, as shown here.

<table><tbody><tr><td>Medimachine system</td><td>BD</td><td>340587</td><td></td></tr><tr><td>Medicon unit (50 &amp;mu;m)</td><td>BD</td><td>340591</td><td></td></tr><tr><td>Filcon unit (50 &amp;mu;m)</td><td>BD</td><td>340603</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>430-2100</td><td></td></tr><tr><td>Fetal calf serum</td><td>PAA</td><td>A11-151</td><td></td></tr><tr><td>NDA</td><td>Chemos BmbH</td><td>277081</td><td></td></tr><tr><td>H&amp;#8211;chst 33342</td><td>Sigma-Aldrich</td><td>14533</td><td>Stock solution 5 mg/ml; used at a final concentration of 5 &amp;mu;g/ml</td></tr><tr><td>Propidium iodide</td><td>Sigma-Aldrich</td><td>287075</td><td>Stock solution 1 mg/ml; used at a final concentration of 50 &amp;mu;g/ml</td></tr></tbody></table>

simple and efficient technique for the preparation of testicular cell suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

An example of a well disaggregated cell suspension from rat testes prepared with the protocol described here is shown in Figure 1. 
In comparison to enzymatic treatments6,8 and to previously described mechanical disaggregation methods2, the one presented here is much faster, involves less handling, is easily reproducible (not operator-dependant), and renders scarce cell debris (especially compared to other mechanical methods) and very few multinucleates (which have been described to form as a consequence of extensive tissue manipulation1,2). Moreover, as opposed to enzymatic treatments, it avoids the use of RNAses, trypsin and collagenase, that could favor macromolecules degradation. 
On the other hand, although previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1, the application of the present method rendered very little clumping in the cellular suspensions, as can be seen in Figure 1. In that sense, we have found the inclusion of NDA very efficient in preventing cell clumping, and avoiding nozzle clogging during subsequent flow studies. Figure 1 also reveals the scarcity of cell debris, which is also obvious in the FC histograms depicted in Figure 2.
Besides, cell suspensions prepared with this method show an adequate representation of the different testicular cell types. This was concluded by comparing the cellular composition of testicular cell suspensions prepared by the method presented here with data reported from cell counts in cross sections of seminiferous tubules, and with cell suspensions prepared using other methods as well (Table 1). 
FC histograms obtained for suspensions prepared by the present protocol and stained either with Hoechst 33342 (Figure 2A) or PI (Figure 2B) do not substantially differ from previously reported ones8, also supporting the assumption that the procedure does not selectively damage any specific cell type. 
<table border="1" fo:keep-together.within-page="always">
<tbody>
 <tr>
 <td>Testicular cell types based on DNA content</td>
 <td>Intact testisa (%)</td>
 <td>Medimachine - prepared suspensionsb (%)</td>
 <td>Trypsinized suspensionsa 
 (%)</td>
 </tr>
 <tr>
 <td></td>
 <td colspan="3">A) Mus musculus</td>
 </tr>
 <tr>
 <td>C</td>
 <td>66.2</td>
 <td>62.5</td>
 <td>79.6</td>
 </tr>
 <tr>
 <td>2C</td>
 <td>16.4</td>
 <td>18.4</td>
 <td>7.3</td>
 </tr>
 <tr>
 <td>4C</td>
 <td>17.9</td>
 <td>18.7</td>
 <td>8.7</td>
 </tr>
 <tr>
 <td>Others</td>
 <td>-</td>
 <td>-</td>
 <td>4.6</td>
 </tr>
 <tr>
 <td></td>
 <td colspan="3">B) Cavia porcellus</td>
 </tr>
 <tr>
 <td>C</td>
 <td>66.5</td>
 <td>65.5</td>
 <td>N/D</td>
 </tr>
 <tr>
 <td>2C</td>
 <td>11.0</td>
 <td>11.5</td>
 <td>N/D</td>
 </tr>
 <tr>
 <td>4C</td>
 <td>22.5</td>
 <td>23.0</td>
 <td>N/D</td>
 </tr>
 <tr>
 <td colspan="4">a Cell counts were assessed by microscopical observation. b Cell counts were assessed by flow cytometry.</td>
 </tr>
 </tbody>
</table>
Table 1. Relative percentages of adult testicular cell populations differing in their DNA content for cell suspensions from mouse (A) and guinea pig (B) prepared by the hereby presented method, compared to intact testis and - for mouse - to trypsin-prepared suspensions (Modified from Geisinger and Rodríguez-Casuriaga, Cytogenet. Genome Res. 128, 46 (2010)). DNA contents are: C (round and elongating spermatids, spermatozoa), 2C (testicular somatic cells, spermatogonia, secondary spermatocytes), and 4C (primary spermatocytes and a few proliferating spermatogonia in G2 stage).
<img src="/files/ftp_upload/50102/50102fig1.jpg" alt="Sperm cells, microscope image; reproductive biology, cell morphology study, fertilization process." fo:content-width="4.5in" fo:src="/files/ftp_upload/50102/50102fig1highres.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Partial view of a cell suspension from adult rat testis prepared by the present protocol and visualized by phase contrast microscopy. As can be seen, cell cytoplasms are well preserved. The bar corresponds to 25 μm. Reproduced from Rodríguez-Casuriaga, et al., Biol. Proced. Online. 11, 184 (2009). 
<img src="/files/ftp_upload/50102/50102fig2.jpg" alt="Flow cytometry DNA content analysis showing cell cycle phases (C, 2C, 4C) in rodents; histogram." fo:content-width="5.75in" fo:src="/files/ftp_upload/50102/50102fig2highres.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. FC DNA content analysis of testicular cell suspensions from adult rats, mice, guinea pigs, and from 21 days post partum (dpp) rat pups, stained with the vital dye Hoechst 33342 (A) or with PI (B). In all the cases C, 2C and 4C cell populations can be easily disclosed in the histograms obtained with both dyes, as well as the apparently sub-haploid peak to the left of the C population. This latter peak has been demonstrated to contain the spermatozoa, which appear as a separate, less stained subpopulation due to their condensed chromatin state12. Note the scarcity of debris in all graphics. This is especially evident in the 21 dpp (juvenile specimens) profiles, which lack spermatids and spermatozoa. Modified from Geisinger and Rodríguez-Casuriaga, Cytogenet. Genome Res. 128, 46 (2010). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50102/50102fig2large.jpg" target="_blank"> Click here to view larger figure. </a>
<img src="/files/ftp_upload/50102/50102fig3.jpg" alt="Microscope image, cell morphology comparison, two slides, left dense, right sparse, scale bar included." fo:content-width="6in" fo:src="/files/ftp_upload/50102/50102fig3highres.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. 4C cell population sorted from a testicular cell suspension of adult rat. Sorted cells were deposited onto clean poly-L-lysine-treated slides and observed under phase-contrast microscopy (A) or bright field after Giemsa staining (B). Bar = 20 μm. Reproduced from Geisinger and Rodríguez-Casuriaga, Cytogenet. Genome Res. 128, 46 (2010). 
<img src="/files/ftp_upload/50102/50102fig4.jpg" alt="Flow cytometry results with PI fluorescence intensity; cell cycle analysis, scatter plot, fluorescence." fo:content-width="6in" fo:src="/files/ftp_upload/50102/50102fig4highres.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4. A) FC analysis of an adult guinea pig testicular cell suspension prepared with the protocol described here. (a), histogram; (b), dot plot. Note the two subpopulations in the 4C cell population. B) Analysis of sorted cells from 4C R3 (a,b) and R4 (a',b') regions. (a,a'), Epifluorescence microscope images of PI-stained cells. Note that nuclei from R3 region are comparatively smaller. Bar: 10 μm. (b,b'), Laser confocal microscopy of immunocytochemical reactions on spread cells using an antibody against Sycp3 (Synaptonemal complex [SC] protein 3, a lateral element component). The picture allows the conclusion that R3 fraction corresponds to early (lepto/zygotene) meiocytes, in which simple axes and short stretches of SCs can be seen, while R4 contains pachytene meiocytes, with completely assembled SCs. Modified from Rodríguez-Casuriaga, et al.,Cytometry A. 79, 625 (2011). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50102/50102fig4large.jpg" target="_blank"> Click here to view larger figure. </a> 
<img src="/files/ftp_upload/50102/50102fig5.jpg" alt="Electrophoresis results, DNA separation, protein bands, gel analysis, experimental comparison, molecular biology." data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5. A) Agarose gel electrophoresis of total RNAs extracted from spermatogenic flow-sorted cell fractions 2C, R3 and R4 (explanation on the two latter fractions is in Figure 4). Cell suspensions were prepared with the protocol described here. B) Autoradiogram of a denaturing polyacrylamide electrophoresis gel showing differential cDNA bands obtained by means of the "mRNA differential display" method (RNAimage; GenHunter Corporation, Nashville, TN) for one of the primer combinations from the kit. mRNAs from the same three cell populations as in A) were compared. All experiments were performed in duplicate (reverse transcription and PCR) following the instructions from the manufacturer, and run in adjacent lanes. Asterisks indicate differential cDNA bands for R3 (early meiotic prophase cells) that appear in duplicate experiments (presumably corresponding to differentially expressed genes). Reproduced from Rodríguez-Casuriaga et al., Cytometry A. 79, 625 (2011).

Watch this Scientific Journal Video about Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions at JoVE.com

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

simple-efficient-technique-for-preparation-testicular-cell

Nanyang Technological University

Research

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Biology

19.0K Views.  Instituto de Investigaciones Biológicas Clemente Estable (IIBCE). A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

Video: Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Developmental Biology

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals.

Presented here is a protocol for enriching pachytene spermatocytes, round spermatids, and elongating spermatids from adult mouse testes using a discontinuous bovine serum albumin density gradient with standard laboratory equipment.

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete ...

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.

Primary Sertoli cells are required for studying testis-immune privilege, signal transduction during inflammation or infection, and utilization of their immunoprotective properties. Here we describe an enzyme-based protocol for the isolation of highly purified primary Sertoli cells and peritubular cells from rat testes.

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of ...

Immunology and Infection

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

Here we present a simple and efficient method to isolate live meiotic and post-meiotic germ cells from adult mouse testes. Using a low-cytotoxicity, violet-excited DNA binding dye and fluorescence-activated cell sorting, one can isolate highly enriched spermatogenic cell populations for many downstream applications.

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

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 non-spermatogenic (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 testes - through 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 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 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.

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.

The material here describes a method developed to preserve the three-dimensional chromatin structure of testicular germ cells. This has been termed the three-dimensional (3D) slide method. This method improves sensitivity for detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH).

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

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A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Flow Cytometry Purification of Mouse Meiotic Cells

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells