Name: isolation of murine spermatogenic cells using a violet-excited cell-permeable dna binding dye
Uploaded: 2021-01-14T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye at JoVE.com

We thank members of the Namekawa, Yoshida, and Maezawa laboratories for their help; Katie Gerhardt for editing the manuscript; Mary Ann Handel for sharing the H1T antibody, the Cincinnati Children&#8217;s Hospital Medical Center (CCHMC) Research Flow Cytometry Core for sharing the FACS equipment supported by NIH S10OD023410; Grant-in-Aid for Scientific Research (KAKENHI; 17K07424) to T.N.; Lalor Foundation Postdoctoral Fellowship to A.S.; AMED-CREST (JP17gm1110005h0001) to S.Y.; the Research Project Grant by the Azabu University Research Services Division, Ministry of Education, Culture, Sports, Science and Technology (MEXT)-Supported Program for the Private University Research Branding Project (2016&#8211;2019), Grant-in-Aid for Research Activity Start-up (19K21196), the Takeda Science Foundation (2019), and the Uehara Memorial Foundation Research Incentive Grant (2018) to S.M.; National Institute of Health R01 GM122776 to S.H.N.

This protocol follows the guidelines of the Institutional Animal Care and Use Committee (protocol no. IACUC2018-0040) at Cincinnati Children&#8217;s Hospital Medical Center.
1. Equipment and reagent setup for the preparation of testicular cell suspension
<ol>
	<li>Prepare each enzyme stock in 1x Hanks&#8217; Balanced Salt Solution (HBSS) and store at -20 &#176;C. (Table1). 
	NOTE: Prepare any time before the experiment.</li>
	<li>One day before the experiment: Coat coll...

<ol>
	<li>Griswold, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spermatogenesis:+The+Commitment+to+Meiosis.">Spermatogenesis: The Commitment to Meiosis.</a> Physiological Reviews. 96 (1), 1-17 (2016).</li><li>Yoshida, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Spermatogenesis+Reflects+the+Unity+and+Diversity+of+Tissue+Stem+Cell+Niche+Systems.">Mouse Spermatogenesis Reflects the Unity and Diversity of Tissue Stem Cell Niche Systems.</a> Cold Spring Harbor Perspectives in Biology. , 036186 (2020).</li><li>Rathke, C., Baarends, W. M., Awe, S., Renkawitz-Pohl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+dynamics+during+spermiogenesis.">Chromatin dynamics during spermiogenesis.</a> Biochimica et Biophysica Acta. 1839 (3), 155-168 (2014).</li><li>Maezawa, S., 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=SCML2+promotes+heterochromatin+organization+in+late+spermatogenesis.">SCML2 promotes heterochromatin organization in late spermatogenesis.</a> Journal of Cell Science. 131 (17), 217125 (2018).</li><li>Maezawa, S., Yukawa, M., Alavattam, K. G., Barski, A., Namekawa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+reorganization+of+open+chromatin+underlies+diverse+transcriptomes+during+spermatogenesis.">Dynamic reorganization of open chromatin underlies diverse transcriptomes during spermatogenesis.</a> Nucleic Acids Research. 46 (2), 593-608 (2018).</li><li>Bellve, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification,+culture,+and+fractionation+of+spermatogenic+cells.">Purification, culture, and fractionation of spermatogenic cells.</a> Methods in Enzymology. 225, 84-113 (1993).</li><li>Bryant, J. M., Meyer-Ficca, M. L., Dang, V. M., Berger, S. L., Meyer, R. 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F., 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=Revealing+stage-specific+expression+patterns+of+long+noncoding+RNAs+along+mouse+spermatogenesis.">Revealing stage-specific expression patterns of long noncoding RNAs along mouse spermatogenesis.</a> RNA Biology. 17 (3), 350-365 (2020).</li><li>Telford, W. G., Bradford, J., Godfrey, W., Robey, R. W., Bates, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Side+population+analysis+using+a+violet-excited+cell-permeable+DNA+binding+dye.">Side population analysis using a violet-excited cell-permeable DNA binding dye.</a> Stem Cells. 25 (4), 1029-1036 (2007).</li><li>Alavattam, K. G., Abe, H., Sakashita, A., Namekawa, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+Spread+Analyses+of+Meiotic+Sex+Chromosome+Inactivation.">Chromosome Spread Analyses of Meiotic Sex Chromosome Inactivation.</a> Methods in Molecular Biology. 1861, 113-129 (2018).</li><li>Maezawa, S., 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=Super-enhancer+switching+drives+a+burst+in+gene+expression+at+the+mitosis-to-meiosis+transition.">Super-enhancer switching drives a burst in gene expression at the mitosis-to-meiosis transition.</a> Nature Structural &amp; Molecular Biology. , (2020).</li><li>Sakashita, A., 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=Endogenous+retroviruses+drive+species-specific+germline+transcriptomes+in+mammals.">Endogenous retroviruses drive species-specific germline transcriptomes in mammals.</a> Nature Structural &amp; Molecular Biology. , (2020).</li><li>Agrimson, K. S., 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=Characterizing+the+Spermatogonial+Response+to+Retinoic+Acid+During+the+Onset+of+Spermatogenesis+and+Following+Synchronization+in+the+Neonatal+Mouse+Testis.">Characterizing the Spermatogonial Response to Retinoic Acid During the Onset of Spermatogenesis and Following Synchronization in the Neonatal Mouse Testis.</a> Biology of Reproduction. 95 (4), 81 (2016).</li><li>Hogarth, C. A., 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=Turning+a+spermatogenic+wave+into+a+tsunami:+synchronizing+murine+spermatogenesis+using+WIN+18,446.">Turning a spermatogenic wave into a tsunami: synchronizing murine spermatogenesis using WIN 18,446.</a> Biology of Reproduction. 88 (2), 40 (2013).</li><li>Patel, 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=Dynamic+reorganization+of+the+genome+shapes+the+recombination+landscape+in+meiotic+prophase.">Dynamic reorganization of the genome shapes the recombination landscape in meiotic prophase.</a> Nature Structural &amp; Molecular Biology. 26 (3), 164-174 (2019).</li><li>Alavattam, K. 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Here we present a practical and simple protocol to isolate subpopulations of spermatocytes and spermatids from a single adult male mouse. To ensure the reproducibility of this protocol, there are some critical steps that need attention. Before enzyme digestion, wash step aims to remove interstitial cells; after digestion, this step helps to remove spermatozoa and debris. Washing/centrifuging 3 times is important. In our dissociation buffer recipe, the combination of several different enzymes facilitates the dissociation ...

Spermatogenesis is a complex process wherein a small population of spermatogonial stem cells sustain continuous production of a large number of sperm throughout adult life1,2. During spermatogenesis, dynamic chromatin remodeling takes place when spermatogenic cells undergo meiosis to produce haploid spermatids3,4,5. Isolation of meiotic spermatocytes is essential for molecular investigation, and several different approaches to isolate meiotic spermatocytes have been established, including sedimentation-based separation6,7 and fluorescence-activated cell sorting (FACS)8,9,10,11,12,13,14,15,16,17. However, these methods have technical limitations. While sedimentation-based separation yields a large number of cells5,6,7, it is labor intensive. The established FACS-based method uses Hoechst 33342 (Ho342) to separate meiotic spermatocytes based on DNA content and light scattering properties and requires FACS cell sorters equipped with an ultraviolet (UV) laser8,9,10,11. Alternative FACS-based methods require transgenic mouse lines that express florescent proteins, synchronization of spermatogenesis12, or cell fixation and antibody labeling that is not compatible with isolation of live cells13. While there is another alternative method using a cell-permeable DNA binding dye, DyeCycle Green stain14,15,16,17, this method is recommended for the isolation of spermatogenic cells from juvenile testis. Therefore, there is a critical need to develop a simple and robust isolation method for live meiotic spermatocytes that can be applied to any mouse strain of any age and that can be performed using any FACS cell sorter.
Here we describe such a long-sought cell isolation protocol using the DyeCycle Violet (DCV) stain. DCV is a low cytotoxicity, cell-permeable DNA binding dye structurally similar to Ho342 but with an excitation spectrum shifted toward the violet range18. In addition, DCV has a broader emission spectrum compared to DCG. Thus, it can be excited by both UV and violet lasers, which improves the flexibility of equipment, allowing the use of an FACS cell sorter not equipped with a UV laser. The DCV protocol presented here uses two-dimensional separation with DCV blue and DCV red, mimicking the advantage of the Ho342 protocol. With this advantage, our DCV protocol allows us to isolate highly enriched germ cells from the adult testis. We provide a detailed gating protocol to isolate live spermatogenic cells from adult mouse testes of one mouse (from two testes). We also describe an efficient and quick protocol to prepare single-cell suspension from mouse testes that can be used for this cell isolation. The procedure requires a short time to complete (preparation of single cell suspension - 1 hour, dye staining - 30 min, and cell sorting - 2-3 hours: total - 4-5 hours depending on the number of needed cells; Figure 1). Following cell isolation, a wide range of downstream applications including RNA-seq, ATAC-seq, ChIP-seq, and cell culture can be completed.

<table>
	<tbody>
		<tr>
			<td>1.5 ml tube</td>
			<td>Watson</td>
			<td>131-7155C</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Petri dish</td>
			<td>Corning, Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Centrifuge tube</td>
			<td>Watson</td>
			<td>1332-015S</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml polystyrene tube with cell strainer snap cap (35 &#181;m nylon mesh)</td>
			<td>Corning, Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge tube</td>
			<td>Watson</td>
			<td>1342-050S</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dish</td>
			<td>Corning, Falcon</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m nylon mesh</td>
			<td>Corning, Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell sorter</td>
			<td>Sony</td>
			<td>SH800S</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, recombinant, Animal-derived-free</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>036-23141</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type 1</td>
			<td>Worthington</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Fisher</td>
			<td>12-544-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytospin 3</td>
			<td>Shandon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Fisher</td>
			<td>D1306</td>
			<td>working concentration: 0.1 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Sigma</td>
			<td>D5025-150KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma</td>
			<td>S30-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s phosphate-buffered saline (DPBS)</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11885076</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Hostone H1T antibody</td>
			<td>gift from Mary Ann Handel</td>
			<td></td>
			<td>1/2000 diluted</td>
		</tr>
		<tr>
			<td>Hank&#8217;s balanced salt solution (HBSS)</td>
			<td>Gibco</td>
			<td>14175095</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase from bovine testes</td>
			<td>Sigma</td>
			<td>H3506-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>P5493-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettemen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Fisher</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>rH2AX antibody</td>
			<td>Millipore</td>
			<td>05-635</td>
			<td>working concentration: 2 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Sperm Fertilization Protein 56 (Sp56) antibody</td>
			<td>QED Bioscience</td>
			<td>55101</td>
			<td>working concentration: 0.5 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Sterilized forceps and scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost /Plus Microscope Slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>SYCP3 antibody</td>
			<td>Abcam</td>
			<td>ab205846</td>
			<td>working concentration: 5 &#956;g/mL</td>
		</tr>
		<tr>
			<td>TWEEN 20 (Polysorbate 20)</td>
			<td>Sigma</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr>
			<td>Vybrant DyeCycle Violet Stain (DCV)</td>
			<td>Invitrogen</td>
			<td>V35003</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A representative result of this sorting protocol is shown in Figure 3. The total sorting time of two testes (one mouse) is usually around 3 hours, which is dependent on the concentration of cell suspension and the sorting speed. After sorting, the purity of spermatocytes is confirmed by immunostaining of SYCP3 and &#947;H2AX&#160;(Figure 3A). The representative purity of sorted L/Z, P, D spermatocyte fractions are around 80.4%, 90.6%, and 87.6%, respectively (<s...

Watch this Scientific Journal Video about Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye at JoVE.com

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

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 ...

This new efficient method for isolating subpopulations of spermatocytes and spermatids from adult mice can be used to investigate the molecular mechanisms underlying meiosis and spermatogenesis. This method uses a low cytotoxicity DNA binding dye with a wide excitation spectrum that can be applied to most currently used FACS orders. This method is very straightforward.The most challenging aspect is the flow cytometry gating but the detailed gating strategy should help with adapting the protocol to other cells sorters. Demonstrating the procedure will be Yu-Han Yeh, a research assistant from Satoshi Namekawa's Laboratory. After harvesting both testes from an eight week old male mouse, place the tissues in a 60 millimeter Petri dish containing two milliliters of ice cold PBS and remove the tunica albuginea.Gently separate the testes with forceps to slightly disperse the seminiferous tubules. And transfer the tubules into a drop of fresh ice cold PBS in a 100 millimeter Petri dish. Use the forceps to gently untangle the tubules and wash the tubules in three additional droplets of PBS as just demonstrated.After the last wash, place the untangled seminiferous tubules into a 15 milliliter tube, containing two milliliters of dissociation buffer for a 20-minute incubation at 37 degrees Celsius. At the end of the incubation, use a 1000 microliter pipette to gently pipette the tubules 20 times, before returning the tube to the incubator for an additional six minutes. At the end of the incubation, pipette the tissues an additional 20 times, before returning the tube to the incubator.After three minutes, gently pipette the tubules 10 times or until no visible tissue pieces remain, before stopping the dissociation with 10 milliliters of FACS buffer. Then, centrifuge to the tissue suspension two times using an additional 10 milliliters for fresh FACS buffer for the second wash to remove the spermatozoa and as much debris as possible. To stain the cells for flow cytometric analysis, re-suspend the pellet in three milliliters of FACS buffer.And filter the cell suspension through a 70 micron nylon cell strainer, into a 50 milliliter tube. After counting, transfer 10%of the cells into a new tube on ice as the unstained negative control. And thoroughly mix six microliters of DCV stain into the remaining cell suspension.After a 30-minute incubation at 37 degrees Celsius in the dark with gentle shaking every 10 minutes, add five microliters of DNase I to the cells. And filter the cells through a 35 micron nylon mesh strainer into a five milliliter FACS tube on ice. For flow cytometric analysis of the cells, create a new experiment in the flow analysis software, and under the worksheet tools menu, click new density to create a forward scatter area versus backscatter area density plot on a linear scale.Click new histogram to create a DCV blue histogram plot on a logarithmic scale. And click start and record to begin processing the unstained sample. While the sample is running, click detector and threshold settings to adjust both the forward and side scatter PMT voltages to place the unstained cells on the scale of the forward and back scatter plot.Adjust the PMT voltage for the jappy channel to locate the position of the DCV negative population in the first decade of the DCV blue histogram logarithmic plot. Then, click stop to unload the unstained sample. After briefly vortexing and loading the stained sample, click next tube to create a new worksheet for the sample.Set the cytometer to acquire at least one times 10 to the six events, and click start and record. Next, click new density to create forward scatter height versus forward scatter width. And DCV blue versus DCV red density plots on a linear scale.On the forward scatter area versus backscatter area density plot, use the polygon tool to draw a gate called cells to include most of the cells and to exclude small debris. Apply this gate to the forward scatter height versus forward scatter width plot and use the rectangle tool to draw a single cell's gate to exclude non single cells. Apply the single cell's gate to the DCV blue versus DCV red density plot and adjust the scale to capture an extended profile.Use the polygon tool to draw a DCV gate to exclude the unstained cells and side population, and apply the gate to a DCV blue histogram plot on a linear scale. The three major peaks refer to the different 1C, 2C and 4C DNA contents. Create a second DCV blue versus DCV red density plot on a linear scale.And back gate the DCV gate onto the plot to locate the 1C and 4C populations. Create another DCV blue versus DCV red density plot on a linear scale. And use the ellipse tool to draw a gate on the 1C population.Use the polygon tool to gate the 4C population with a continuous curve. And create another DCV blue versus DCV red density plot on a linear scale. Use the polygon tool to create a 4C one gate.In a new forward by side scatter plot, use the ellipse tool to create pachytene, diplotene and leptotene, zygotene spermatocyte gates. When all of the gates have been drawn, create a new DCV blue versus DCV red color dot plot on a linear scale to apply the for 4C gate to ensure that the three populations are in a continuous order within the gate. Next, create a new forward scatter area versus backscatter area density plot on a linear scale, and select the unified size of cells as a pure round spermatid population.After sorting, the representative purities of the separated leptotene, zygotene, pachytene and diplotene spermatocyte fractions are typically between 80%to 90%as confirmed by SYCP3 and Gamma H2AX immunostaining. The purity of the round spermatid faction can be confirmed by nucleus staining with DCV in combination with Sp56 and H1t staining, and is also typically around 90%The viability of the isolated cell populations is usually over 95%And the average total yields of each fraction from a single adult mouse is typically sufficient for various downstream analysis. The sorted cells can be used for various next generation sequencing analysis to better explore gene expression and regulation during spermatogenesis.

Research

JoVE Journal

Developmental Biology

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