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 collecting tubes (1.5 mL tubes) with Fetal bovine serum (FBS) at 4 &#176;C overnight.</li>
	<li>On the day of the experiment: Set the water bath to 37 &#176;C.</li>
	<li>Pre-warm Dulbecco&#39;s Modified Eagle Medium (DMEM) at 37 &#176;C and prepare 2 mL of 1x dissociation buffer for each sample right before use (Table 1) in 15 mL centrifuge tube. 
	NOTE: 2 mL dissociation buffer is prepared for two testes. For the dissociation buffer recipe, please refer to Table 1.</li>
</ol>
2. Animal dissection and preparation of testicular cell suspension
<ol>
	<li>Sacrifice an 8-week-old male mouse by leaving in a carbon dioxide chamber for at least 10 min.</li>
	<li>Remove both testes and place on a 60 mm Petri dish containing 2 mL of ice-cold phosphate-buffered saline (PBS).</li>
	<li>Remove the tunica albuginea from the testes. Slightly disperse the seminiferous tubules by gently separating with forceps.</li>
	<li>Transfer the seminiferous tubules to a 100 mm Petri dish with a new drop of ice-cold PBS and untangle seminiferous tubules gently with forceps. Repeat this wash 3 times to remove interstitial cells as much as possible.</li>
	<li>Incubate untangled seminiferous tubules in a 15 mL tube containing 2 mL of dissociation buffer at 37 &#176;C for 20 min.</li>
	<li>Gently pipette the tubules 20 times using a 1000 &#181;L micropipette.</li>
	<li>Incubate for 6 min. Repeat gentle pipetting 20 times.</li>
	<li>Incubate for 3 min. Repeat gentle pipetting 10 times until no visible chunks remain.</li>
	<li>Add 10 mL of FACS buffer to the suspension. Centrifuge at 300 x g for 5 min at room temperature and discard the supernatant.</li>
	<li>Repeat step 2.9 twice to remove the spermatozoa and as much debris as possible.</li>
</ol>
3. Cell staining
<ol>
	<li>Resuspend the cell pellet with 3 mL of FACS buffer (Table 1) and filter the cell suspension through a 70 &#181;m nylon cell strainer into a 50 mL tube. 
	NOTE: The expected cell yield is approximately 100 million cells per two testicles (from an 8-week-old B6 wild-type mouse).</li>
	<li>Count the cell number and split 10% of the cells into a new tube as the unstained negative control and leave on ice.</li>
	<li>Add 6 &#181;L of DCV stain (the original concentration is 5 mM) to the remaining cell suspension and mix well. The final concentration is 10 &#181;M, and the capacity is approximately 100 million cells.</li>
	<li>Incubate at 37 &#176;C for 30 min in the dark. Gently shake the cell suspension every 10 min.</li>
	<li>After incubation, without a wash step, directly add 5 &#181;L of DNase I (the stock concentration is 10 mg/mL) to the cell suspension and filter the cells into a 5 mL FACS tube through the 35 &#181;m nylon mesh cap. Keep the samples on ice until sorting.</li>
</ol>
4. Flow cytometry and experimental gates
<ol>
	<li>Prepare the FACS cell sorter. Ensure that FACS cell sorter is equipped with Excitation optics: Violet (405-nm) laser; Detection optics: &#64257;lter combination of 450/50 bandpass [same filter set of 4&#8242;,6-diamidino-2-phenylindole (DAPI) for DCV-blue detection] and 665/30 bandpass [same filter set of Allophycocyanin (APC) for DCV-red detection]. Here we use Sony SH800S cell sorter as an example.</li>
	<li>Create a new experiment and set up the following working plots displaying parameters to be optimized: Click &#8220;New Density&#8221; on the &#8220;Worksheet Tools&#8221; menu bar to create a forward scatter - area (FSC-A) vs. back scatter &#8211; area (BSC-A) density plot on a linear scale. Click &#8220;New Histogram&#8221; to create a DCV-blue histogram plot on logarithmic scale.</li>
	<li>Briefly vortex the unstained negative control (from step 3.2) and load the sample.</li>
	<li>Click &#8220;Start&#8221; and &#8220;Record&#8221; to begin processing the unstained sample. While the sample is running, click &#8220;Detector &#38; Threshold Settings&#8221; to optimize FSC and BSC voltages by adjusting both the photomultiplier tube (PMT) voltages up or down to place the unstained cells on the scale of the FSC/BSC plot.</li>
	<li>Adjust the PMT voltage up and down on &#8220;FL1: DAPI&#8221; while the sample is running to locate the position of the DCV-negative population in the &#64257;rst decade of the DCV-blue histogram logarithmic plot (Figure 2A). After completing the PMT voltage adjustment, click &#8220;Stop&#8221; to unload the unstained sample.</li>
	<li>Briefly vortex the sample (from step 3.5) and load the sample.</li>
	<li>Click &#8220;Next Tube&#8221; to create a new worksheet for the DCV-stained sample; and click &#8220;Start&#8221; and &#8220;Record&#8221; to acquire the DCV-stained sample, record &#8805; 1 x 106 events. Add the following working plots: Click &#8220;New Density&#8221; on the &#8220;Worksheet Tools&#8221; menu bar to create an FSC-H (height) vs. FSC-W (width) density plot on a linear scale; Click &#8220;New Density&#8221; to create a DCV-blue vs. DCV-red density plot on a linear scale. After recording &#8805; 1 x 106 events, click &#8220;Stop&#8221; and unload the sample.</li>
	<li>On the FSC-A vs. BSC-A density plot, click &#8220;Polygon&#8221; on the &#8220;Plot Tools&#8221; menu bar to draw a big gate &#8220;Cells&#8221; to include most cells and exclude small debris (Figure 2B). Apply the gate &#8220;Cells&#8221; to FSC-H vs. FSC-W density plot. Click &#8220;Rectangle&#8221; to draw a &#8220;Single Cells&#8221; gate to exclude non-single cells (Figure 2C).</li>
	<li>Apply &#8220;Single Cells&#8221; gate to DCV-blue vs. DCV-red density plot and adjust the scale to capture an extended profile as shown in Figure 2D. Click &#8220;Polygon&#8221; on the &#8220;Plot Tools&#8221; menu bar to draw a &#8220;DCV&#8221; gate to exclude the unstained cells and side population (Figure 2D).</li>
	<li>Apply &#8220;DCV&#8221; gate to DCV-blue histogram plot on a linear scale. The three major peaks refer to different DNA content: 1C, 2C, and 4C (Figure 2E).</li>
	<li>Click &#8220;New Density&#8221; to create a DCV-blue vs. DCV-red density plot on a linear scale and apply &#8220;DCV&#8221; gate to perform back gating from Figure 2E to locate the 1C and 4C populations (Figure 2F). Click &#8220;Ellipse&#8221; to draw a gate on the 1C population, which is within a condensed area (gate 1C). Click &#8220;Polygon&#8221; to draw a gate on the 4C population which is a continuous curve (gate 4C).</li>
	<li>Click &#8220;New Density&#8221; to create a DCV-blue vs. DCV-red density plot on a linear scale and apply &#8220;4C&#8221; gate; adjust the scale to zoom in and click &#8220;Polygon&#8221; to draw a &#8220;4C_1&#8221; gate for more precise selection (Figure 2G).</li>
	<li>Click &#8220;New Density&#8221; to create a new FSC-A vs. BSC-A density plot on a linear scale and apply &#8220;4C_1&#8221; gate; there will be three enriched populations separated by size corresponding to leptotene (L) /zygotene (Z), pachytene (P) and diplotene (D) spermatocytes. Click &#8220;Polygon&#8221; or &#8220;Ellipse&#8221; to draw 3 gates: &#8220;L/Z&#8221;, &#8220;P&#8221;, and &#8220;D&#8221; based on the growing size (Figure 2H).</li>
	<li>Click &#8220;New Dot Plot&#8221; to create a DCV-blue vs. DCV-red color dot plot on a linear scale to apply gate and ensure the three populations are in a continuous order within the &#8220;4C_1&#8221; gate (Figure 2I).</li>
	<li>Similarly, for the 1C population, click &#8220;New Density&#8221; to create a new FSC-A vs. BSC-A density plot on a linear scale and apply &#8220;1C&#8221; gate, select the unified size of cells as pure round spermatid population, and click &#8220;Ellipse&#8221; to draw an &#8220;RS&#8221; gate (Figure 2J).</li>
</ol>
5. Sort male germ cell subpopulations
<ol>
	<li>Prepare 1.5 mL tubes containing 500 &#956;L 50% FBS for cell collection and load the collection tube into the collector and click &#8220;Load Collection&#8221;.</li>
	<li>Click &#8220;Next Tube&#8221;, &#8220;Start&#8221;, &#8220;Record&#8221; and &#8220;Sort Start&#8221;. For using a two-way system that allows two populations of interest to be sorted at the same time into the collection device, follow pairwise combinations: leptotene (L) /zygotene (Z) and pachytene (P) spermatocytes based on L/Z and P back-gates; Round spermatids (RS) and diplotene (D) spermatocytes based on RS and D back-gates.</li>
	<li>While the sample is running, adjust the flow rate to ~3000 events/s to get the most efficient sorting.</li>
</ol>
6. Purity analysis of sorted cells
<ol>
	<li>Collect &#8805; 10,000 cells/each population. Perform cell immunostaining to confirm the substage.</li>
	<li>Centrifuge at 300 x g for 5 min at 4 &#176;C and carefully discard the supernatant, keeping around 110 &#181;L of the liquid in the bottom of the tube.</li>
	<li>Take a 10 &#181;L drop of cell suspension to observe under microscope and evaluate the overview cell morphology and number.</li>
	<li>Apply 100 &#181;L of cell suspension to each of the sample chamber slides (see Table of Materials), and load the chambers to the Cytospin.</li>
	<li>Spin the samples at 30 x g for 5 min at room temperature. Draw a circle around the cell with a hydrophobic pen. Dry slides on lab bench for a few minutes at room temperature.</li>
	<li>Drop 50 &#181;L of PBS in the circle of the slide and tap off.</li>
	<li>Add primary antibody solution (Dilute primary antibodies with 5% Donkey Serum in PBS with 0.02% Polysorbate 20) in the circle of the slide and incubate at 4 &#176;C overnight. 
	NOTE: To judge the substage of meiotic spermatocytes, SYCP3 was detected, which is a marker of meiotic chromosome axes, and &#947;H2AX, which is a marker of DNA damage response. (For the antibody working concentration, please refer to the Table of Materials).</li>
	<li>Tap off the primary antibody solution from the slides.</li>
	<li>Drop 50 &#181;L of PBS in the circle of the slide and tap off. Repeat one time.</li>
	<li>Add secondary antibody solution (dilute secondary antibodies in PBS with 0.02% Polysorbate 20) in the circle of the slide and incubate at room temperature for 1 h in dark.</li>
	<li>Drop 50 &#181;L of PBS in the circle of the slide and tap off. Repeat one time.</li>
	<li>Add 50 &#181;L of DAPI (stock concentration is 0.1 &#956;g/mL) stain for 5 min and tap off.</li>
	<li>Add 1-2 drops of mounting media on the slides. Carefully cover the mounting media with a cover glass and gently press the cover glass to remove extra mounting media and air bubble. The slides are ready for microscopy evaluation.</li>
</ol>

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</ol>

The authors declare that they have no competing interests.

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 &amp;micro;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 &amp;micro;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&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Fisher</td><td>D1306</td><td>working concentration: 0.1 &amp;mu;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&amp;rsquo;s phosphate-buffered saline (DPBS)</td><td>Gibco</td><td>14190144</td><td></td></tr><tr><td>Dulbecco&#039;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>Histone H1t antibody</td><td>gift from Mary Ann Handel</td><td></td><td>1/2000 diluted</td></tr><tr><td>Hank&amp;rsquo;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 &amp;mu;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 &amp;mu;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 &amp;mu;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...

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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 ...

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Research

JoVE Journal

Developmental Biology

5.8K Views.  Cincinnati Children's Hospital Medical Center. Deze nieuwe efficiënte methode voor het isoleren van subpopulaties van spermatocyten en spermatids van volwassen muizen kan worden gebruikt om de moleculaire mechanismen die ten grondslag liggen aan meiose en spermatogenese te onderzoeken. Deze methode maakt gebruik van een lage cytotoxiciteit DNA bindende kleurstof met een breed excitatiespectrum dat kan worden toegepast op de meeste momenteel gebruikte FACS orders. Deze methode is zeer eenvoudig.Het meest uitdagende aspect is de str...

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

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

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. ...

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 ...

Transillumination-Assisted Dissection of Specific Stages of the Mouse Seminiferous Epithelial Cycle for Downstream Immunostaining Analyses

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. Differentiation of germ cells takes place in the cytoplasmic pockets of somatic Sertoli cells that host 4 to 5 generations of germ cells simultaneously and coordinate and synchronize their development. Therefore, the composition of germ cell types within a cross-section is constant, and these cell associations are also known as stages (I&#8211;XII) of the seminiferous epithelial cycle. Importantly, stages can also be identified from intact seminiferous tubules based on their differential light absorption/scatter characteristics revealed by transillumination, and the fact that the stages follow each other along the tubule in a numerical order. This article describes a transillumination-assisted microdissection method for the isolation of seminiferous tubule segments representing specific stages of mouse seminiferous epithelial cycle. The light absorption pattern of seminiferous tubules is first inspected under a dissection microscope, and then tubule segments representing specific stages are cut and used for downstream applications. Here we describe immunostaining protocols for stage-specific squash preparations and for intact tubule segments. This method allows a researcher to focus on biological events taking place at specific phases of spermatogenesis, thus providing a unique tool for developmental, toxicological, and cytological studies of spermatogenesis and underlying molecular mechanisms.

This protocol describes transillumination-assisted microdissection of segments of adult mouse seminiferous tubules representing specific stages of seminiferous epithelial cycle, and cell types therein, and subsequent immunostaining of squash preparations and intact tubule segments.

Spermatogenesis is a unique differentiation process that ultimately gives rise to one of the most distinct cell types of the body, the sperm. ...

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 ...

Biology

Murine Round Spermatid Injection Procedure to Study Embryonic Development

Mouse Round Spermatid Injection

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of round spermatid injection (ROSI), oocytes can be successfully fertilized and developed into viable fetuses. In a groundbreaking milestone achieved in 1995, the first mouse fetus was born through ROSI technology. ROSI has since emerged as a pivotal tool for unraveling the intricate mechanisms governing embryonic development and holds significant potential in various applications, including the acceleration of mouse generation and the production of genetically modified mice. In 1996, a milestone was reached when the first human fetus was born through ROSI technology. However, the clinical applications of this method have shown a fluctuating pattern of success and failure. To date, ROSI technology has not found widespread application in clinical practice, primarily due to its low birth efficiency and insufficient validation of fetal safety. This article provides a comprehensive account of the precise methods of performing ROSI in mice, aiming to shed new light on basic research and its potential clinical applications.

Here, we introduce a method for performing round spermatid injection (ROSI) in mice, a technique with promising clinical applications and utility for investigating the mechanisms underlying embryonic development.

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of ...

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 .

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 ...

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 ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...

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

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