Recording Electrical Currents across the Plasma Membrane of Mammalian Sperm Cells

Recording the electrical activity of the sperm cell using the patch-clamp technique has been instrumental in identification of various sperm ion channels and understanding of sperm electrophysiology at the molecular level. This method excels in measuring electrophysiological and pharmacological fingerprints of ion channels and is the most reliable tool for ion channel identification. Given the physiological importance of ion channels and electrogenic transporters for male fertility, this technique is a powerful tool to understand certain sperm defects that lead to infertility.

For micropipette fabrication, start with borosilicate glass capillaries with an outer diameter of 1.5 millimeters, an inner diameter of 0.86 millimeters, and an internal filament. Ensure that the inner diameter of the pipette tip is approximately two micrometers before fire polishing and is reduced to 0.5 micrometers after proper polishing. Assemble an agar bridge to keep the environment around the reference electrode stable.

Make an L-shaped glass capillary by bending it under a small Bunsen burner fire and let it cool. Make a solution of 1%agarose in one molar potassium chloride and heat it in a microwave until the agarose melts and the solution becomes transparent. Carefully fill the L-shaped glass capillary with the agarose to avoid air bubbles and let it cool to room temperature.

Alternatively, submerge the L-shaped glass capillary in the melted agarose. Swirl the container several times and heat it again in the microwave until the agarose boils, which will expel air bubbles from the agar bridge. Open the lower abdominal area of the mouse with scissors and extract both epididymides.

Place them in a 35-millimeter cell culture dish filled with high saline or HS solution that has been prewarmed to room temperature. Transfer the epididymides into a new cell culture dish containing HS solution and thoroughly remove all residual fat. Separate the epididymides into caput, corpus, and cauda using a number 15 scalpel blade.

Transfer the corpus of each epididymis into a new cell culture dish containing HS solution. Make multiple incisions in the isolated part of the epididymis using a pointed number 11 scalpel blade. Transfer the parts of the epididymides with multiple incisions into a 1.5 milliliter microcentrifuge tube containing 1.5 milliliters of HS solution.

Briefly shake sperm cells from the epididymis into the solution using Superfine Dumont Type 5A forceps. Then discard the epididymides and leave the tube at room temperature for 10 minutes. Wait until the solid matter sediments to the bottom of the tube.

Then transfer the supernatant into another 1.5 milliliter microcentrifuge tube. Find the suitable sperm cell with a cytoplasmic droplet using 600x magnification. Ensure that the cytoplasmic droplet is oval and has a slightly elongated spindle-like shape.

Select a spermatozoon that is motile with the head attached to the cover slip such that the sperm cell is partly fixed, but the cytoplasmic droplet and the rest of the flagellan continue to move with flagella beating. Ensure that the head of the sperm is loosely attached to the cover slip. After visual selection of a sperm cell with a proper morphology, fill the micropipette with the pipette solution and secure it into the pipette holder.

In order to keep the pipette tip clean from debris, apply positive pressure to the pipette using the U-tube-shaped assembly. Lower the pipette down and immerse its tip into the bath solution. In order to clearly visualize the cell, position the tip of the pipette above the cytoplasmic droplet so that the opening of the tip is aligned diagonally toward the droplet.

Quickly lower the tip of the pipette toward the droplet so that they are in the same focal plane. As soon as the tip of the pipette touches the droplet, apply negative pressure to the pipette to move part of the droplet into the tip and form a giga-ohm seal. Then lift the spermatozoon from the cover slip.

Compensate stray capacitance transience using the amplifier's compensatory mode before transitioning to the whole cell mode. Perform a break-in and transition into the whole cell mode by applying one millisecond, gradually increasing voltage pulses combined with a very light suction. After the application of each break-in voltage pulse, launch the membrane test tool to check whether larger capacitance transience appear.

Fit the larger capacitance transience to determine the capacitance of the whole cell, as well as its access resistance. After a successful break-in, proceed with the planned whole cell patch-clamp experiments such as applying various bath solutions containing different compounds or measuring channel activities using voltage step or voltage ramp protocols. CatSper recordings were performed by establishing a high resistant seal between the patch pipette and mammalian spermatozoon at its cytoplasmic droplet.

Caesium whole cell CatSper currents'densities were recorded from caudal wild-type neuron sperm cells and CatSper deficient caudal neuron sperm cells. Sperm cells of different species are diverse in their morphology and internal regulatory pathways. Primate and human spermatozoa showed similar CatSper channel properties and regulation.

Interestingly, progesterone activation of CatSper seems to be unique for primate spermatozoa. Boar, bull, and rodent sperm did not display any progesterone-stimulated alteration of their CatSper currents. In bull and boar spermatozoa, even basal CatSper channel activity was below detectable limits, suggesting that calcium influx and consequent hyperactivation is driven by other channels or transporters or that a different natural stimulator is needed for activation of their CatSper channels.

The small, smooth, uniform, and not overly swollen cytoplasmic droplets are best suited for patch-clamp. You should avoid using those tiny one-sided, bloated, and fully transparent cytoplasmic droplets since those will result in weak or no seal. This technique enables the detailed study of specific air channels in their natural expressing system, and the success depends on high quality variable sperm cells, pure regions, basic intellectual physiology skills, patience, and most importantly, persistence.