Name: a standardized approach for multispecies purification of mammalian male germ cells by mechanical tissue dissociation and flow cytometry
Uploaded: 2017-07-12T14:00:00
Description: Watch this Scientific Journal Video about A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry at JoVE.com

The authors thank the Hillside Animal Hospital (St. Louis, MO) for dog testes; Jason Arand and Dr. Ted Cicero&#39;s lab at Washington University in St. Louis (WashU) for providing rat testes and Brianne Tabers for assisting with the collection; Jared Hartsock and Dr. Salt&#39;s Lab at WashU for the guinea pig testes; and Dr. Michael Talcott at the Division of Comparative Medicine at WashU for the miniature pig testis. The authors also acknowledge the Alvin J. Siteman Cancer Center at Washington University School of Medicine and Barnes-Jewish Hospital in St. Louis, MO, for the use of the Siteman Flow Cytometry Core, which provided staff-operated cell sorting service. The Siteman Cancer Center is supported in part by NCI Cancer Center Support Grant #P30 CA91842.
This research was funded by an FCT doctoral fellowship [SFRH/BD/51695/2011 to ACL], grants from the United States National Institutes of Health [R01HD078641 and R01MH101810 to DFC] and an FCT research contract [IF/01262/2014 to AML].

All procedures described below complied with regulations of the Animal Studies Committee at Washington University in St. Louis.
1. Preparations for Mechanical Dissociation Protocol
<ol>
	<li>Pre-wet a 50 &#181;m disposable tissue disaggregation cartridge for dissociation of a whole murine testis. 
	NOTE: This disposable tissue disaggregation cartridge contains microblades designed for cutting of tissues and an immobile steel mesh with approximately 100 hexagonal holes. ...

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+integrity+is+a+critical+molecular+indicator+for+the+assessment+of+male+infertility.">DNA integrity is a critical molecular indicator for the assessment of male infertility.</a> Mol Med Rep. 7 (5), 1631-1635 (2013).</li><li>Rodriguez-Casuriaga, R., Folle, G. A., Santinaque, F., Lopez-Carro, B., Geisinger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+efficient+technique+for+the+preparation+of+testicular+cell+suspensions.">Simple and efficient technique for the preparation of testicular cell suspensions.</a> J Vis Exp. (78), (2013).</li><li>Rodriguez-Casuriaga, R., Geisinger, A., Santinaque, F. F., Lopez-Carro, B., Folle, G. 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A., Renato de Franca, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spermatogenesis+and+cycle+of+the+seminiferous+epithelium.">Spermatogenesis and cycle of the seminiferous epithelium.</a> Adv Exp Med Biol. 636, 1-15 (2008).</li><li>Zhao, H., Traganos, F., Dobrucki, J., Wlodkowic, D., Darzynkiewicz, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+DNA+damage+response+by+the+supravital+probes+of+nucleic+acids.">Induction of DNA damage response by the supravital probes of nucleic acids.</a> Cytometry A. 75 (6), 510-519 (2009).</li><li>Gassei, K., Ehmcke, J., Dhir, R., Schlatt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+activated+cell+sorting+allows+isolation+of+spermatogonia+from+adult+primate+testes+and+reveals+distinct+GFRa1-positive+subpopulations+in+men.">Magnetic activated cell sorting allows isolation of spermatogonia from adult primate testes and reveals distinct GFRa1-positive subpopulations in men.</a> J Med Primatol. 39 (2), 83-91 (2010).</li><li>Hayama, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+selection+methods+for+rat+and+mouse+round+spermatids+without+DNA+staining+by+flow+cytometric+cell+sorting.">Practical selection methods for rat and mouse round spermatids without DNA staining by flow cytometric cell sorting.</a> Mol Reprod Dev. 83 (6), 488-496 (2016).</li><li>Zhu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FACS+selection+of+valuable+mutant+mouse+round+spermatids+and+strain+rescue+via+round+spermatid+injection.">FACS selection of valuable mutant mouse round spermatids and strain rescue via round spermatid injection.</a> Zygote. 23 (3), 336-341 (2015).</li></ol>

Considering the highly-conserved chromatin dynamics during spermatogenesis in mammals, the goal of this work was to develop a protocol to isolate male germ cells in distinct stages of differentiation from different mammalian testicular tissues (Figure 1). One of the major obstacles in the application of a single workflow to different animal models is the need of species-specific adjustments, especially in regard to tissue dissociation protocols. Current methods mostly rely on enzymatic tissu...

Given the lack of an in vitro system representative of spermatogenesis progression, and the presence of great cellular heterogeneity in testis, studies of male germ cell biology require robust techniques to isolate enriched populations of specific cell types. Fluorescence-activated cell sorting (FACS) has been widely used for this purpose 1,2,3,4,5, as it provides high yield and purity, and surpasses other isolation methods in the number of germ cell types that it can identify and select 6,7,8. The principle of flow cytometry analysis is based on the detection of differential light patterns following laser beam excitation of single cells. As a cell passes through the laser it reflects/scatters light at all angles, proportional to cell size (forward scatter; FSC) and to intracellular complexity (side scatter; SSC). See Ormerod 9 for detailed information on flow cytometry.
Male germ cells undergo specific modifications in DNA content, chromatin structure, size and shape throughout different stages of spermatogenesis. Thus, distinct cell populations can be identified and separated by combining light scattering and DNA staining with fluorescent dyes 10,11. Several dyes can be used for this purpose (reviewed in Geisinger and Rodriguez-Casuriaga 3), such as Hoechst-33342 (Hoechst) which has been frequently used in flow cytometry analysis of testicular cells for the past decade 1,2,4,10,12. Upon excitation with UV-light, Hoechst emits blue fluorescence proportional to the cellular DNA content whereas far red fluorescence reflects variability in chromatin structure and compaction 1,13,14. As a result, male germ cells in different stages of differentiation exhibit specific patterns during FACS of Hoechst-stained single cell suspensions (Ho-FACS; 1,12). Interestingly, due to a mechanism of dye efflux that is only active during the spermatogonial stage, intensity of Hoechst blue fluorescence is not proportional to chromatin content in these cells, and they cluster as a side population during Ho-FACS 15. Additionally, combining Hoechst staining with the non-permeant dye propidium iodide (PI) allows users to discriminate live (PI negative) from dead (PI positive) cells during FACS 1,2,10,12. This strategy has been previously used in flow cytometric analyses of testicular germ cells and optimized extensively in the mouse to discriminate up to 9 germ cell types, including cells in 4 different stages of meiosis I 1,2,4,16. For the purpose of this work, Hoechst staining has three main advantages. First, Ho-FACS has been successfully applied to the isolation of male germ cells in the mouse model 1,2,12, and other rodents such as rat and guinea pig 17,18,19. Second, Hoechst is a cell-permeant dye and does not require membrane permeabilization, so it preserves cell integrity. Finally, no RNase treatment is required since Hoechst binds preferentially to poly(d[AT]) DNA sequences 1,20, which means that RNA is preserved and, in addition to DNA and proteins, can be used for further downstream molecular studies of germ cell differentiation.
Despite the similarity in DNA ploidy and/or stainability observed in flow cytometry analyses of mammalian species (reviewed in Geisinger and Rodriguez-Casuriaga 3), there has been a good deal of variability in the protocols described for male germ cell isolation by flow cytometry. Different studies have employed specific protocols for tissue dissociation, and used distinct DNA-binding dyes (alone or in combination) and FACS gating strategies in different model organisms, mainly the mouse, rat and guinea pig. Hence, direct comparison of data collected for different species can be affected by unaccounted technical artifacts resulting from variability between methodologies. Importantly, the striking conservation of chromatin dynamics throughout mammalian spermatogenesis (2N-4N-2N-1N) suggests that a standardized protocol could be transversely applied to a variety of mammalian species.
The goal of this study was to develop a single workflow that is applicable to different mammalian species, by combining and adapting previously published techniques 2,20. Standardization of a method for tissue processing was achieved by performing mechanical dissociation to overcome the need for species-specific adjustments required for enzymatic digestion 5. It is noteworthy that mechanical dissociation of rodent testicular tissue has been shown to perform better than enzymatic tissue digestion 20 and Ho-FACS of single cell suspensions generated by both methods exhibit comparable results 5. As proof of principle, this protocol describes the settings used to isolate up to 5 germ cell populations: spermatogonia (SPG); primary (SPC I) and secondary spermatocytes (SPC II), and spermatids (SPD) &#8211; round (rSPD) and elongating (eSPD). Importantly, it is easy to implement in the lab, with the main requirements being the system for tissue dissociation and access to a cell sorter equipped with a UV laser. This workflow (Figure 1) is fast and straightforward and allows the simultaneous isolation of 4 germ cell populations from fresh testicular tissue in less than 2 h. The reduced processing time is crucial to maintain cellular integrity for further downstream procedures. Moreover, its successful performance in 5 different species suggests it could be broadly applied within the mammalian clade, making it the ideal method to isolate germ cells for comparative studies of mammalian male reproductive biology.
This protocol is composed of three major sections, aside from preparatory steps: (1) the mechanical dissociation of testicular tissue and (2) staining of testicular cells with Hoechst and PI, followed by (3) FACS sorting of relevant spermatogenic cells. Once collected, these enriched populations of different mammalian testicular germ cells can be used for a wide range of applications. This protocol describes a &#34;one-size fits all&#34; dissociation method to purify male germ cells from many different mammalian species. Depending on the type of study users wish to conduct with the isolated germ cells, other media or buffers can be used. The following protocol steps are for generating single-cell suspensions from one whole murine testis.

<table border="0" cellpadding="0" cellspacing="0" width="731">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:239px;">4% paraformaldehyde</td>
			<td style="width:115px;">VWR</td>
			<td style="width:101px;">#15710</td>
			<td style="width:279px;">4% PFA, For fixing sorted cells&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Medimachine</td>
			<td>BD Biosciences</td>
			<td>#340588;</td>
			<td>System for testis dissociation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1X Dulbecco modified Eagle medium</td>
			<td>Life Technologies</td>
			<td>#31053</td>
			<td>DMEM, for testis dissociation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Medicon</td>
			<td>BD Biosciences</td>
			<td>#340591</td>
			<td>Disaggregation cartridge for testis dissociation&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>Thermo Scientific</td>
			<td>#10082139</td>
			<td>FBS, for FACS ceollction</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>#H3570</td>
			<td>Hoecsht, For FACS staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">40&#181;m cell strainer</td>
			<td>GREINER BIO-ONE</td>
			<td>#89508-342</td>
			<td>For testis dissociation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100x 15mm petri dish</td>
			<td>Falcon</td>
			<td>#351029</td>
			<td>For testis dissection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5mL polypropylene round-bottom tubes</td>
			<td>Falcon</td>
			<td>#352063</td>
			<td>For FACS collection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5mL Eppendorf tubes</td>
			<td>VWR</td>
			<td>#20170-650</td>
			<td>For FACS collection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3mL needless syringe</td>
			<td>BD Biosciences</td>
			<td>#309657</td>
			<td>For testis dissociation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50mL conical tube</td>
			<td>MIDSCI</td>
			<td>#C50R</td>
			<td>For testis dissociation&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Propidium Iodide</td>
			<td>Invitrogen</td>
			<td>#L7011</td>
			<td>PI, For FACS staining</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MoFlo&#160;Legacy cell sorter</td>
			<td>Beckman Coulter&#160;</td>
			<td>#ML99030</td>
			<td>For FACS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Summit Cell Sorting software</td>
			<td>Beckman Coulter&#160;</td>
			<td>#ML99030</td>
			<td>For FACS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>#AM9625</td>
			<td>PBS, For microscopy</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscopic glass slide</td>
			<td>Fisher Scientific</td>
			<td>#12-544-4</td>
			<td>For microscopy&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass coverslip</td>
			<td>Fisher Scientific&#160;</td>
			<td>#12-548-87</td>
			<td>For microscopy</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Disposable transfer pipette (3mL)&#160;</td>
			<td>Samco Scientific</td>
			<td>#225</td>
			<td>For testis dissociation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNase I&#160;</td>
			<td>Roche</td>
			<td>#10104159001</td>
			<td>For testis dissociation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carbon steel surgical blade</td>
			<td>Miltex</td>
			<td>#4-111</td>
			<td>For testis dissection&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Countess automated cell counter&#160;</td>
			<td>Invitrogen</td>
			<td>#C10227&#160;</td>
			<td>For automatic cell counting&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypan blue solution (0.4%)</td>
			<td>Sigma</td>
			<td>#T8154</td>
			<td>For viability staining&#160;</td>
		</tr>
	</tbody>
</table>

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.

Single cell suspensions from mechanical dissociation of testicular tissue
Figure 2 compares single cell suspensions obtained by mechanical dissociation of mouse testicular tissue under different conditions. Samples obtained by processing fresh tissue, unstained (Figure 2A) or stained with Hoechst (Figure 2B), show the presence of single cells ...

Watch this Scientific Journal Video about A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry at JoVE.com

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

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. ...

The overall goal of this standardized procedure is to isolate distinct male germ cell types from different mammalian species using a single protocol. This method can help facilitate the molecular study of spermatogenesis by the high-throughput analysis of different germ cell types. The main advantage of this technique is that a single set of reagents can be applied to various mammalian species for the simultaneous isolation of four different male germ cell populations.We first had the idea for this method when we decided to investigate the evolution of specific patterns of gene expression regulation in different mammals. Chris Holley, a medical technologist from the Siteman Flow Cytometry Core, will be demonstrating how to operate the cell sorter and set the gates. Begin by decapsulating a single murine testis and cutting the tissue into small pieces.Then use forceps to transfer small tissue pieces onto a pre-wetted 50 micrometer tissue disaggregation cartridge. Add one milliliter of DMEM medium to the cartridge, and load the tissue disaggregation cartridge onto the tissue disaggregation system. Turn the knob from standby to run, and process the tissue for five minutes.Then remove the cartridge, and use a disposable, three milliliter syringe to aspirate the cell suspension from the syringe port. When all of the liquid has been recovered, filter the cells through two individual, disposal, pre-wetted 40 micrometer filters to remove any cell aggregates, and return the suspension to the disaggregation cartridge. Then process the cells for another five minutes.To stain the cells for fluorescence-activated cell sorting, collect the dissociated cell suspension in a 1.5 milliliter tube. Then transfer 150 microliters of cells into each of three new 1.5 milliliter tubes, adding the remaining volume to a fourth 1.5 milliliter tube. Setting aside the first tube as an unstained control, add 2.5 microliters of Hoechst to the second tube, one microliter of propidium iodide to the third tube, and both Hoechst and propidium iodide to the fourth tube.After 30 minutes at room temperature in the dark with gentle rotation, filter the cell suspensions through individual, pre-wetted 40 micron strainers into five milliliter round-bottom tubes. Next place four collection tubes containing DMEM supplemented with FBS under the sorting spouts of the flow cytometer, and load the unstained control tube for analysis. Use a forward versus side scatter plot to exclude the cell debris, and adjust the forward scatter threshold and pulse width to allow gating of the single cells.Stringent gating is crucial to optimize the quality of the collected sample. Ensure selection of single cells by excluding cellular debris and multicellular aggregates. Use the single-stained cells to establish the threshold for Hoechst and propidium iodide fluorescence intensity in relation to the unstained sample.Then load the double-stained experimental sample, and set the three parent gates as just demonstrated. When the three parent gates have been set, select live cells based on their lack of propidium iodide staining, and set the DNA content by plotting a histogram of cell counts based on the Hoechst blue fluorescence. Set a gate for each of the three peaks of increasing Hoechst blue fluorescence concentration, representing the haploid, diploid, and tetraploid cells, and observe at least 500, 000 events on the forward versus side scatter plot.Plot the propidium iodide-negative cells according to their Hoechst blue and red fluorescence intensities. The spermatogonia will appear as a side population. Hoechst blue fluorescence intensity correlates with DNA content.Therefore, setting this gate prior to generating Hoechst blue and red plots ensures selection of the cells with desired ploidy. For spermatid isolation, gate the haploid peak and select the population that appears when Hoechst blue and red fluorescence are plotted. For spermatocytes II isolation, gate the diploid peak and select the population that appears when Hoechst blue and red fluorescence are plotted.For spermatocytes I isolation, gate the tetraploid peak and select the population that appears when Hoechst blue and red fluorescence are plotted. Then sort the selected subpopulations into the individual collection tubes to collect approximately 0.5 to six times 10 to the six cells for each cell subset. Cell suspensions obtained by mechanical dissociation of fresh tissue unstained or stained with Hoechst demonstrate the presence of single cells in various stages of differentiation.Importantly, the cellular structures appear to be preserved, including the flagella of the spermatozoa. After cellular debris exclusion, 95 to 98%of cells are singlets, 86 to 93%of which are alive. Plotting the function of blue red Hoechst fluorescence during FACS facilitates the identification of specific testicular germ cell populations.As expected, the haploid spermatid population is the most abundant, followed by diploid spermatocyte II cells and tetraploid primary spermatocytes, a pattern that is preserved across species. Microscopic analysis of the sorted cells reveals that spermatogonia display distinct, small, round, pericentric heterochromatin that stains brightly for Hoechst. Spermatocytes are larger, granulated cells with easily identifiable nuclei that exhibit chromatin variations characteristic of meiotic cells in the spermatocyte I subpopulation and by nucleated or diakinetic nuclei in the spermatocyte II subpopulations.Spermatids are small haploid cells with a round or elongated shape that can be distinguished from spermatogonia by the presence of localized chromocenters. One mastered, this technique can be completed in two hours if it is performed properly. While attempting this procedure, it is important to remember that Hoechst is both highly sensitive and genotoxic and that the resolution of the cell populations can be improved by longer incubation periods as needed.Following this procedure, high-throughput omics methods can be performed to answer questions regarding epigenetic modifications or transcript and protein abundance and diversity within mammalian species in an evolutionary context. After its development, this technique paved the way for researchers in the field of male reproductive biology to explore the regulatory mechanisms driving spermatogenesis progression in several mammalian species. After watching this video, you should have a good understanding of how to process testicular samples for Hoechst FACS and how to identify and isolate different male germ cell type based on their Hoechst signal.DNA-binding dyes such as Hoechst and propidium iodide can be extremely hazardous. Always wear personal protective equipment when handling these reagents.

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