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

The simple and inexpensive method that we call MDR allows you to isolate enriched populations of pachytene spermatocytes, round spermatids and elongating spermatids from mouse testes. The main advantage of the MDR method is its simplicity. You need only standard laboratory equipment that are available in most biomedical research laboratories.

The MDR protocol is great for researchers doing functional studies on male germ cells. For example, groups that are studying spermatogenesis, factors affecting male fertility, as well as paternal epigenetic inheritance. Minimal starting material is required for the MDR method.

And in fact, we routinely obtain good amount of cells using just one adult male mouse. Begin by setting up the necessary equipment and reagents. Set a water bath to 37 degrees Celsius and a cell culture incubator the 34 degrees Celsius, 5%carbon dioxide and 95%humidity.

Place a tube rotator inside the incubator. Prepare and label the appropriate amount of microscopy glass slides, then draw a ring of about one centimeter in diameter with a grease pen, and let the grease dry. When ready to dissect the animal, spray the ventral abdomen of the mouse with 70%ethanol and use scissors to make a V shape opening in the abdominal pelvic cavity.

Pull on the epididymal fat pad with forceps, locate the testes and remove them with scissors, making sure to avoid disturbing the tunica albuginea. Place the testes on a six centimeter Petri dish containing 1X KREBS. Decapsulate the testes and discard the tunica albuginea, then slightly disperse the seminiferous tubules by gently teasing them apart with forceps.

Transfer them into a 50 milliliter conical tube containing two milliliters of freshly prepared collagenase solution. Incubate the tubules in the 37 degrees Celsius water bath for three minutes, agitating them gently by rocking. Then add at least 40 milliliters of warm 1X KREBS and allow the tubules to sediment at room temperature.

Remove the supernatant and repeat the wash once more. Add 25 milliliters of freshly prepared trypsin solution. Place the tube on the rotator inside the cell culture incubator, and leave it there for 15 to 20 minutes, rotating at approximately 15 rpm.

Sporadically check the tubules. Once the solution becomes cloudy and only small pieces of the tubules remain, place the tube on ice and proceed to the next step. Filter the solution through a 40 micrometer cell strainer into a new 50 milliliter conical tube on ice.

Then centrifuge it at 600 x g for five minutes at four degrees Celsius to pellet the cells. Carefully pour out the supernatant and tap the cell pellet to resuspend the cells in the remainder of the 1X KREBS. Add at least 40 milliliters of cold 1X KREBS to the resuspended cells and repeat the centrifugation.

Resuspend the cells by tapping the tube. Then mount a pipette tip on to a one milliliter pipette and cut it so that the aperture is about three millimeters in diameter. Add up to three milliliters of 0.5%BSA in KREBS.

Resuspend the cells by pipetting up and down, making sure to avoid bubbles. Filter the cell suspension through a 40 micrometer strainer and proceed immediately to loading the cells on the BSA gradient. Your must obtain a homogeneous single cell suspension before loading the cells on to the gradient.

Clumped cells will sediment faster, disrupting your gradient and contaminating the fractions. Set up a 50 milliliter tube vertically on ice, making sure that it is possible to see one side of the tube. Then cut about five to ten millimeters from the tip of a ten milliliter serological pipette and mount it on a pipette controller.

Pipette five milliliters of the 5%BSA solution into the bottom of the 50 milliliter tube. Gently touch the surface of the 5%solution with the cut tip of the pipette and slowly layer five milliliters of 4%BSA solution on top of the 5%solution. Repeat this procedure with other BSA solutions to obtain a gradient of 5%to 1%BSA.

A clear line should be visible between the layers. Then carefully load the single cell suspension on top of the gradient without disturbing it. Let the cells sediment through the gradient for one and a half hours.

Using a cut pipette tip, carefully collect the one milliliter fractions into separate 1.5 milliliter tubes and store them on ice, making sure to number the tubes in the order that they were collected. Centrifuge the germ cell fractions at 600 x g for ten minutes at four degrees Celsius. Then taking care not to disturb the pellet, discard most of the supernatant and resuspend the cells by flicking the tube.

Add one milliliter of ice cold 1X KREBS buffer to each tube and repeat the centrifugation. Discard most of the supernatant, leaving about 100 microliters and resuspend the cell pellet carefully. To analyze the cell fractions, start by pipetting 20 microliters of 4%paraformaldehyde inside each grease pen ring on a numbered microscopy slide.

Immediately add two microliters of the resuspended cells from the corresponding fraction. Then dry the slides at room temperature for at least one hour. Rinse the slides once with PBS and use a mounting medium with DAPI to mount the slides.

Analyze each slide under a fluorescence microscope to estimate which particular germ cell type is enriched in each fraction. Once a sample from each fraction has been taken for microscopy, add one milliliter of ice cold 1X KREBS to each fraction and centrifuge the cells down at 600 to 13000 x g for ten minutes at four degrees Celsius. Remove and discard the supernatant and continue with the preferred downstream analysis.

This protocol works particularly well for enriching round spermatids. Enrichment above 90%was achieved in fractions two, three and four. Elongating spermatids tend to stay on top of the gradient and were collected with the first fraction.

Due to their large size, pachytene spermatocytes sediment faster and were collected last. Enrichment was around 75%in fractions 14 and 15. The total RNA obtained from the majority of fractions ranged from 0.5 to 2.5 micrograms, enough for downstream RNA analyzes.

The amount of protein obtained from each fraction typically ranged from 20 to 140 micrograms, which is sufficient for several Western blots. In this protocol, 10%of the protein lysates derived from single fractions was sufficient to clearly detect DDX4, PIWIL1 and PIWIL2 proteins on a standard Western blot. The amount of protein in one fraction was also sufficient for immunoprecipitation using an antibody against PIWIL1 as well as for the detection of coimmunoprecipitated PIWIL2.

Even for a first timer, this protocol works very well as long as you make sure that the pre-treatment of your cells is good, and nothing disturbs your gradient during the sedimentation. From a single mouse, you get highly enriched cells, which can be used for different downstream analyzes such as RT-PCR, RNA sequencing, immunoprecipitation and Western blotting. While MDR is not the only method for enriching male germ cells, it is a very convenient tool because it does not require any specialized equipments or extensive training.