Name: Recording Electrical Currents across the Plasma Membrane of Mammalian Sperm Cells (Video) | JoVE Journal
Uploaded: 2021-02-14T13:00:00.000+00:00
Duration: 9 min 37 s
Description: Learn the spermatozoan patch-clamp technique for recording ion channel activity in mammalian sperm cells. This method enables detailed study of CatSper and other channels to understand male fertility.

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This work was supported by NIH Grant R01GM111802, Pew Biomedical Scholars Award 00028642, Alfred P. Sloan Award FR-2015-65398, and Packer Wentz Endowment Will (to P.V.L.). This work was also supported by Deutsche Forschungsgemeinschaft (German Research Foundation) 368482240/GRK2416 (to N.M.) and by China Scholarship Council Fellowship to B.L. We thank Dr. Dan Feldman for sharing rat tissue, Katie Klooster and Stuart Meyers from UC Davis for help with primate sperm cells&#160;acquisition, and Steven Mansell for the help with data acquisition analysis from boar and bull sperm cells.

The authors have nothing to disclose.

We describe a detailed protocol to perform electrophysiological recordings from sperm cells of various species. Given the physiological significance of ion channels and electrogenic transporters for spermatozoa, this technique is a powerful tool to study sperm cell physiology as well as defects that lead to male infertility. The experimenter might find the execution of this technique challenging at first, but with perseverance and endurance, success follows.
Mammalian spermatozoa are long (usually &#62;50 &#181;m), narrow, and highly motile. The basal beat frequency (BF) of mammalian spermatozoa varies greatly with values averaging from 4 Hz (mouse 69), 7-15 Hz (boar 70,71), 11 Hz (rat 72), 11-20 Hz (bull 18), 24 Hz (rhesus macaque 23), and up to 25 Hz (human 3). The cytoplasmic droplet (CD) is the entryway for recording from sperm cells. In rodent spermatozoa the CD is often distal but moves alongside the flagellum (Figure 10), creating an additional obstacle to recording. However, in human sperm cells the CD is more commonly located near the head. The key components of a successful sperm patch-clamp are therefore excellent optics to enable a clear, sharp view of the CD and a highly precise micromanipulator system without drift or vibration. An&#160;initial high rate of failure&#160;is expected and is normal within the first several days of sperm patch clamp. We recommend routine practice involving numerous attempts per week. Achieving several recordings per day per week will establish a routine and improve motor skills.
Until recently, the identification and pharmacological characterization of sperm ion channels was hindered by an inability to study them directly. The field largely relied on immunocytochemistry studies, which often suffer from nonspecificity of antibodies and/or the lack of corresponding genetic&#160;models. To study calcium channels, the classical calcium imaging method has been widely used, which has its own advantages and limitations73,74,75,76,77. While calcium imaging is a relatively easy method that is applicable for medium-to-high throughput studies78,79,80,81 and is less invasive, it requires relatively intact cells, and hence poses a hurdle to dissect the function of ion channels decoupled from intracellular signaling cascades or to distinguish them from calcium ion exchangers. Additionally, it is difficult to control membrane potential and therefore, harder to exclude the contribution of the voltage-gated calcium channels. Among several advantages of calcium fluorometry is the use of calcium ratiometric dyes that allows precise measurement of the changes in calcium ion&#160;concentration. At the same time, one must be aware that the sensitivity of these dyes can vary based on the changes of intracellular pH.
Below we describe the critical steps within the protocol, including troubleshooting steps of the method. It is essential to use only pure reagents for the preparations of the experimental solutions, as even&#160;small contamination with undesirable ions (such as magnesium or heavy metals) can impair the detection of monovalent currents. Given the small size of the sperm cells, one can expect a relatively low number&#160;of ion channels per cell. Hence, the net current ranges from a few pA to several hundred pA. Therefore, the internal electrical noise of the rig must be minimal to ensure detection of small currents, and the use of drift-free equipment is highly recommended. In order to distinguish a specific conductance from electrical noise and background leak, the recording apparatus and grounding system must be maximized. This is achieved by properly grounding the rig to avoid any electrical interference82. The use of a Faraday cage is highly recommended to protect from electrical interference produced by a variety of electrical devices, such as building lights and in-wall electrical wiring. It is essential that all electrically powered components of the rig, including the computer keyboard and mouse radiate little or no electrical (50 Hz or 60 Hz) noise and that all components of the rig are properly grounded. The electrical noise in the whole-cell configuration when all ion channels are closed should be &#60; 0.5-1 pA.
Another important point is to monitor correct osmolarities of the working solutions. The composition of the intra- and extracellular solutions must be precisely determined and their osmolarities&#160;measured correctly. The extracellular solution must be slightly hypotonic in comparison to the pipette solution as it leads to miniscule cell swelling and prevents the pipette being clogged by the sperm membrane. Note: if the pipette solution is too hypertonic and differs from the bath solution more than 10 mOsm, excessive cell swelling, and seal rupture ensues. As a result, the cell will be fragile and the gigaseal lost within seconds after break-in. In our experience, inaccurate solution preparation is one of the most common mistakes that prevents successful patch-clamping.
Another potential obstacle to avoid is plasticizer/phthalate-containing plastic, as well as mineral oil lubricated syringes. The tubing, syringes and all plastic equipment that encounters solutions, and hence sperm cells, should not leach plasticizers or other environmental toxins or oils, since such chemicals can significantly alter ion channel activity. We use small diameter Teflon tubing as the main perfusion line. Teflon (PTFE) has few leachable compounds but is rather stiff. Flexible connections are made of high purity silicon tubing that fits over the Teflon tubing. All syringes used for the perfusion system lack any lubricant, since the mineral oil or other lubricating additives can interfere with ion channel recording.
We cannot overstate the importance of using the right glass and pulling the correct micropipette shape. Hence, the optimal fabrication of glass micropipettes is a prerequisite for successful patching. We use glass micropipettes made only from borosilicate glass&#160;containing a filament for better solution filling. The tip of the pipettes must be fire-polished to provide the ideal tight seal. Pipette tips that exceed 2 &#956;m in diameter&#160;(and hence have a resistance of 10 M&#937; or below) are generally not suitable for sperm cell patch-clamp.
Another important step is to ensure that the micropipette tip&#160;be kept clean of any debris or air bubbles before seal formation. This is a difficult task given that the micropipette is loaded into a solution full of&#160;motile cells. One factor that helps avoid accidental &#34;bumping&#34; of the pipette into free-swimming sperm cells, is to use a constant perfusion to wash away all nonadherent cells. Another tool is a home-made &#34;U-tube&#34; that allows one to switch between positive and negative pressure modes to keep the tip clean (Figure 11 and Figure 12).
As sperm cells vary greatly in the shape&#160;and size&#160;of their cytoplasmic droplets (CD), it is important to pick a&#160;droplet with suitable morphology. As shown on Figure 2, only CDs that are small (1-3 &#181;m), smooth, uniform, and not overly swollen are suitable for patch-clamp. Tiny, one-sided; &#34;bloated&#34;, fully transparent CDs produce weak or no seals. CDs that have large soluble particles inside may clog the recording pipette. When testicular mouse spermatozoa enter the epididymis, their CDs are located in the neck region, close to the head. As they travel through the epididymis, their CDs move along the midpiece and eventually arrive at the connection between the midpiece and principal piece (the annulus) when spermatozoa reach the cauda epididymis. Therefore, as mentioned above in sperm cells isolated from the corpus epididymis, the CD is usually located close to the center of the midpiece. In caudal cells, the CD can usually be found close to the annulus (Figure 2C). For human sperm, the CD is located in the neck region (Figure 2A,B).
While this is not an issue for spermatozoa isolated from laboratory animals, significant variability exists between human donors. Variation in sperm quality within the same donor mainly affects the quality of the sperm plasma membrane and sometimes makes seal formation relatively difficult. There is less variability in ion channel behavior and pharmacology, factors that probably correlate with individual genetics or physiology. One has to be persistent and assess samples from various donations during multiple days, as well as rely on multiple human donor participants. Working with human material requires extra patience, since donated sperm vary greatly in sperm quality within the same donor, depending on various environmental factors. We recommend assessing samples from various donation days to make a final decision on the donor status. While ejaculated purified spermatozoa are generally suitable for electrophysiology within hours (up to 12 hours after isolation for human sperm), epididymal murine sperm cells are suitable for patching only within a 2-hour window after isolation.
And last, but not&#160;least, gigaseal formation differs among sperm cells. For murine/rodent sperm cells, gigaseal formation happens almost instantaneously, while several seconds (and sometimes up to a minute) are required to form a gigaseal with a human sperm. Often the initial suction results in an input resistance ranging from 200 M&#937; to 800 M&#937;. Switching holding potential to -60 mV and providing &#34;Membrane Test&#34; short pulses up to 10mV often helps rescue gigaseal formation (through voltage field induced movement of the membrane in the pipette).
The sperm cell patch clamp technique enables the detailed study of specific ion channels in their natural expression system. The success of the technique depends on proper equipment, high quality viable sperm cells, pure reagents,&#160;basic electrophysiology skills, patience,&#160;and persistence. The method opens new frontiers in sperm physiology by studying ion channel evolutionary diversity, mechanisms of their regulation, and alterations in their function as they move from the male to the female reproductive tract and are altered by exogenous conditions, such as pH and ligands.

Similar to the traditional patch clamp invented by Erwin Neher and Bert Sakmann1, sperm cell patch clamp enables interrogation of individual ion channel activity, as well as recording from the activity of the entire ion channel population within the single cell2,3. The method allows for the identification of a specific ion channel type under degrees of decoupling from enzymatic intracellular processes. This method is crucial for the determination of ion channel activity based on its electrophysiological and pharmacological fingerprints, and hence, provides a reliable identification strategy. The downside of the method is its inability to detect non-electrogenic transporters. Additionally, basic electrophysiological training is helpful to understand the nuances of the protocol. To master the patch clamp technique and apply it to mammalian spermatozoa, we recommend studying basic patch clamp literature4,5. In this paper we provide a detailed step-by-step procedure and highlight unique practices that make this technique easy to understand and available to anyone who wishes to practice sperm cell electrophysiology.
Ion homeostasis is an essential physiological function of sperm cells that heavily relies on ion channels and ion transporters to maintain physiologically important ion gradients, vary intracellular calcium, and change transmembrane voltage. Ion channels and ion transporters regulate essential sperm cell functions such as motility, navigation in the female reproductive tract, spermatozoan maturation, and in marine organisms, chemotaxis toward the egg6,7,8,9,10,11,12. Sperm motility is a gradually acquired process. Sperm cells are mainly quiescent during their maturation in the testis and during their consequent passage through epididymis. Their motility is restrained by an acidic epididymal environment that leads to an internal acidification of the sperm cell. This impairs the function of the axoneme since it is unable to function below pH 6.013,14. However, upon exposure to the seminal fluids or a more alkaline environment, sperm intracellular ion concentrations and cytoplasmic pH undergo major changes and the spermatozoon becomes motile15,16,17. The movement of the sperm flagellum is powered by ATP hydrolysis that supports sliding of axonemal microtubules18 and this process is highly pH-dependent14. Additionally, flagellar movement is also controlled by an elevation of intraflagellar calcium and cAMP13,19,20,21,22,23,24. These factors i.e., sperm intracellular calcium concentration [Ca2+]i ,pH, ATP and cAMP are the main regulatory mechanisms allowing for motility changes and their concentrations are tightly regulated by the sperm ion channels and transporters.
Sperm cells are unique in that they express a number of proteins that cannot be found anywhere else in the body. Notable examples are sperm ion channels, such as the potassium channel, Slo325,26,27,28,29 and the Cationic channel of Sperm, CatSper2,30,31,32. The latter is the principal calcium channel of mammalian spermatozoa31, and is regulated by intracellular alkalization2,30,31,32,33,34. CatSper is also regulated by species-specific cues7,35 and is organized in quadrilateral longitudinal nanodomains along the sperm flagellum36,37,38. In primates, CatSper is activated by a combination of&#160;flagellar alkalinity, membrane depolarization&#160;and progesterone3,39,40,41, while for&#160;murine&#160;CatSper activation&#160;progesterone is not required2,39. Another specific feature of this channel is its multisubunit organization: CatSper is a complex of at least 10 different subunits31,32,34,37,38,42,43,44,45,46,47. Such sophisticated structure and specifics of its regulation hindered recombinant expression of CatSper in any known heterologous expression system, and hence physiological characterization of CatSper has been restricted to its native system of expression- the sperm cell. While molecular characterization of CatSper protein was achieved in seminal paper in 2000 by D. Ren et. al.31, the ultimate proof that CatSper is a bona fide ion channel was possible only after the introduction of the sperm patch clamp method in 20062. Since then this technique allowed for precise characterization of many ion conducting pathways in sperm cells9,28,37,39,40,44,46,48-54.
The classical and the most straightforward method to study ion channel characteristics- the patch clamp technique- was believed to be inapplicable to sperm cells due to their motility and specific morphology (Figure 1A). Specifically, the miniscule volume of the sperm cytoplasm and sperm plasma membrane&#39;s tight attachment to the rigid intracellular structures such as sperm&#39;s fibrous sheath and&#160;nucleus were the main challenges55. These two structural features result in a slim, arrow-shaped cell that is designed to penetrate through highly viscous environments such as eggs&#39; protective vestments, without significant deformation or damage to the plasma membrane.
The first step of the patch clamp method is&#160;establishment of the tight seal between a recording pipette (a glass micropipette) and the cell plasma membrane. In order to achieve this, one has to pull enough plasma membrane inside the recording pipette for a mechanically stable gigaseal to form between the plasma membrane and glass. The plasma membrane must be flexible and not rigid (Figure 1B). As mentioned above, the entire surface of the sperm plasma membrane is rather tightly adhered, except for the region known as the cytoplasmic droplet (Figure 1A and Figure 2). Hence, the rigid nature of sperm&#39;s plasma membrane was considered to be a main obstacle in obtaining the tight seal or &#39;gigaseal&#39;, so named because &#62;109 ohms are required for good recordings. However, the introduction of the sperm patch clamp technique in 20062 removed this barrier and this method could be successfully applied to sperm cells of several mammalian species2,41,51,56. This breakthrough has been achieved by focusing on the cytoplasmic droplet (CD)2,8, a tiny structure found along the midpiece of the sperm (Figure 1A and Figure 2), and is simply the remnant of the elongated spermatid- a sperm cell precursor from which the head and the tail develop. Functionally, it may help the cell adapt to changes in extracellular osmolarity during ejaculation. The important feature is that the plasma membrane within the CD is flexible enough to be drawn into the pipette to form a gigaohm seal. Thus, the sperm CD is the best part on the sperm surface through which one can achieve a successful gigaseal formation and transition to a whole-cell mode which ultimately electrically couples the sperm cell to a patch-clamp amplifier2,8. It is worth noting, that previous publications reported successful gigaseal formation at the sperm head, which enables recording in the cell-attached configuration54,57,58,59. However, the recordings in whole-cell configuration have so far only been reported by performing gigaseal formation at the CD region. This whole-cell mode allows the electrical access to the entire volume of the sperm cells, and therefore, allows detection of ion channel activities located on the sperm flagellum, as well as on the sperm head. For only a few years since its development, the sperm patch clamp technique has resulted in tremendous progress in our understanding of the sperm ion channels and is so far one of the most robust techniques to directly investigate the functionality of the sperm ion channels9,28,37,39,40,44,46,48,49,50,51,52,53 (Figure 1).
Sperm patch clamp varies in some details from the classical patch clamp technique as outlined below. First, most of the sperm plasma membrane is tightly attached to the rigid intracellular structure and hence, spermatozoa have almost no &#34;spare&#34; plasma membrane to be drawn into the pipette. The only region that is flexible is the CD&#39;s membrane that resembles plasma membrane of many somatic cells, and therefore, can be easily drawn into the pipette. To form a gigaohm seal with the CD, negative pressure is created by light suction at the top of the pipette in order to draw a small portion of the sperm plasma membrane into the tip of the micropipette (Figure 1B). This portion of the membrane forms a &#937;-shaped invagination into the tip of the pipette and establishes a tight seal with its internal walls.
Second, the cytoplasmic droplet in human and mouse spermatozoa is between 1 and 2 &#181;m (Figure 1 and Figure 2). Hence, the application of the patch-clamp technique to such a small object requires high-resolution optics. Most sperm patch-clamp rigs are equipped with an inverted microscope with a differential interference contrast (DIC) or Nomarski optical components (Figure 2 and Figure 3). Using a microscope equipped with DIC optics for sperm patch clamp is highly recommended over more conventional phase-contrast optics, since the spatial information seen in DIC helps achieve superior precision in positioning a patch pipette onto the tiny CD. We also suggest using a 60x water immersion objective or similar lens, with numerical aperture of 1.2. This objective has a long working distance (0.28 mm), which allows observation of free-swimming sperm cells in solution (Figure 2). The objective also has an adjustment collar to adjust to the thickness of the cover slip (variable from 0.13 to 0.21 mm). This combination of the long working distance and adjustment collar enables observation through two 0.13 mm cover slips; one cover slip serves as the glass bottom of the recording chamber, and the 5 mm coverslip with deposited sperm cells is placed on top. As discussed below, depositing sperm cells on easily exchangeable round 5 mm coverslips, rather than on the bottom of the recording chamber directly, is a convenient way to load fresh sperm cells into the recording chamber.
Third, the sperm patch clamp rig must be equipped with a low noise patch-clamp amplifier and a digitizer to record tiny (picoampere range) electrical currents and miniscule changes in membrane potential. This equipment must ensure the lowest amplifier noise. The absence of vibration is an essential part of a successful patch clamp recording. Sperm patch clamping requires a drift-free precise micromanipulator that can be attached to the inverted microscope with a micromanipulator platform to ensure better stability than an independent micromanipulator stand (Figure 3A). To test the setup, one should not see any movement of the pipette tip (under 60x magnification) even when a person jumps up and down on the floor near the vibration-isolation table.

<table><tbody><tr><td>IX71 with DIC optics</td><td>Olympus Inc</td><td>IX71</td><td>&lt;em&gt;Nikon TiU microscope can be used as well&lt;/em&gt;</td></tr><tr><td>UplanSApo 60x</td><td>Olympus Inc</td><td></td><td>&lt;em&gt;water immersion objective&lt;/em&gt;</td></tr><tr><td>Vibration-damping air table</td><td>Newport Inc</td><td></td><td>&lt;em&gt;TMC airtables or similar can be used&lt;/em&gt;</td></tr><tr><td>Axopatch&amp;trade; 200B amplifier</td><td>Axon&amp;trade;&amp;nbsp; /Molecular Devices</td><td></td><td>&lt;em&gt;Sutter IPA&amp;reg;/Double IPA&amp;reg; Integrated patch clamp system is also an excellent amplifier&lt;/em&gt;</td></tr><tr><td>Axon Digidata analog to digital converter</td><td>Axon&amp;trade;&amp;nbsp; /Molecular Devices</td><td>1440 or 1550</td><td>&lt;em&gt;can be used as well&lt;/em&gt;</td></tr><tr><td>Vapor pressure osmometer</td><td>Wescor</td><td>model 5600</td><td></td></tr><tr><td>MPC 385 micromanipulator</td><td>Sutter Instruments, Novato CA</td><td>MPC 385</td><td>&lt;em&gt;The Eppendorf micromanipulator TransferMan series can be also used&lt;/em&gt;</td></tr><tr><td>Micropipette puller</td><td>Sutter Instruments, Novato CA</td><td>P1000</td><td>&lt;em&gt;P97 can be used&lt;/em&gt;</td></tr><tr><td>MicroForge</td><td>Narishige</td><td>MF-830</td><td>&lt;em&gt;Should be equiped with Nikon MPlan 100/0.80 ELWD 210/0 objective&lt;/em&gt;</td></tr><tr><td>Faraday cage</td><td>Homemade</td><td></td><td>&lt;em&gt;to shield the setup from ambient electrical interference&lt;/em&gt;</td></tr><tr><td>&amp;nbsp;5 mm glass Cover Slips</td><td>WPI</td><td>&amp;nbsp;#502040</td><td></td></tr><tr><td>Perfusion chamber</td><td>Warner Instruments, Inc</td><td>&amp;nbsp;RC-24E</td><td></td></tr><tr><td>Borosilicate glass capilary</td><td>Sutter Instruments, Novato CA</td><td>BF 150-85-7.5</td><td>&lt;em&gt;outer diameter 1.5 mm, inner diameter 0.86 mm and an internal filament&lt;/em&gt;</td></tr><tr><td>Teflon manifold MP-8</td><td>Warner Instruments, Inc</td><td>64-0211</td><td>&lt;em&gt;Teflon 8-position perfusion manifold&lt;/em&gt;</td></tr><tr><td>Nunc 4-well plate</td><td>Nunc</td><td>#179820</td><td></td></tr><tr><td>1 X HTF buffer</td><td>EmbryoMax</td><td>MR-070-D</td><td>&lt;em&gt;capacitation solution&lt;/em&gt;</td></tr><tr><td>SA-Oly/2 stage adapter</td><td>Warner Instruments, Inc</td><td>for series 20 platforms</td><td>&lt;em&gt;only for Olympus microscope&lt;/em&gt;</td></tr><tr><td>Magnetic heated platform</td><td>Warner Instruments, Inc</td><td>PM-1 or similar series</td><td>&lt;em&gt;to hold RC-24E chamber&lt;/em&gt;</td></tr><tr><td>MAG-1 magnetic clamp</td><td>Warner Instruments, Inc</td><td>#64-0358</td><td></td></tr><tr><td>Microelectrode holder with 2mm Ag/AgCl pellet</td><td>WPI</td><td>MEH3F4515</td><td></td></tr><tr><td>Stopcock with Luer connections; 4-way; male lock</td><td>Cole-Parmer, Inc</td><td>EW-30600&amp;ndash;09</td><td></td></tr><tr><td>female luer hose barb adapter, 1/16&amp;rdquo;</td><td>Cole-Parmer, Inc</td><td>EW-45508&amp;ndash;00</td><td></td></tr><tr><td>Polytetrafluoroethylene (PTFE) perfusion tubing</td><td>Cole-Parmer, Inc</td><td>EW-06417&amp;ndash;21</td><td>&lt;em&gt;(Microbore PTFE Tubing, 0.022&amp;rdquo; ID &amp;times; 0.042&amp;rdquo; OD)&lt;/em&gt;</td></tr><tr><td>Silicone connector tubing (platinum-cured silicone tubing, 1/32&amp;rdquo; ID &amp;times; 3/32&amp;rdquo; OD)</td><td>Cole-Parmer, Inc</td><td>EW-95802&amp;ndash;01</td><td></td></tr><tr><td>Manifold connector tubing (PTFE Tubing, 1/32&amp;rdquo; ID &amp;times; 1/16&amp;rdquo; OD)</td><td>Cole-Parmer, Inc</td><td>EW-06407&amp;ndash;41</td><td></td></tr><tr><td>male Luer series barb adapter, 1/16&amp;rdquo;</td><td>Cole-Parmer, Inc</td><td>45518&amp;ndash;00</td><td></td></tr><tr><td>Male Luer integral lock adapter 1/8&amp;rdquo;</td><td>Cole-Parmer, Inc</td><td>45-503-04</td><td></td></tr><tr><td>Silicone connector tubing</td><td>Dow Silicone Corporation;&amp;nbsp; MI</td><td>#508-008</td><td></td></tr><tr><td>Syringes</td><td>Fisher Scientific or VWR</td><td>Air-Tite, Norm-Ject Luer</td><td>&lt;em&gt;1 mL, 3mL, and 20 mL&lt;/em&gt;</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>KH2PO4</td><td>Sigma-Aldrich</td><td>60216</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4 &lt;/sub&gt;x 7&lt;sup&gt;.&lt;/sup&gt;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>63140</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2 &lt;/sub&gt;x 2&lt;sup&gt;.&lt;/sup&gt;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>21097</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H7523-250G</td><td></td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>Sodium lactate (60% w/w)</td><td>Sigma-Aldrich</td><td>L7900</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P2256</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>BCBG2421V</td><td></td></tr><tr><td>CsMeSO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C1426</td><td>&lt;em&gt;Cesium methanesulfonate&lt;/em&gt;</td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>P217</td><td></td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>BCBF5871V</td><td></td></tr><tr><td>Tris-HCl</td><td>Quality Biological</td><td>315-006-721</td><td>&lt;em&gt;1 M solution of similar&lt;/em&gt;</td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>221465</td><td></td></tr><tr><td>CsOH</td><td>Sigma-Aldrich</td><td>232041</td><td></td></tr><tr><td>Embryomax Human Tubal Fluid medium:</td><td>Millipore</td><td>MR-070-D</td><td>&lt;em&gt;capacitation medium for murine sperm cells&lt;/em&gt;</td></tr><tr><td>(Embryomax-HTF)</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Animals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Male Wistar rats</td><td>Wistar</td><td>Harlan Laboratories (Livermore, CA)</td><td>&lt;em&gt;adult rats&lt;/em&gt;</td></tr><tr><td>Male C57BL/6 mice</td><td>C57BL/6 mice</td><td>Harlan Laboratories (Livermore, CA)</td><td>&lt;em&gt;3-6 month old&lt;/em&gt;</td></tr></tbody></table>

recording electrical currents across the plasma membrane of mammalian sperm cells

Recording Electrical Currents across the Plasma Membrane of Mammalian Sperm Cells

Recording of the electrical activity from one of the smallest cells of a mammalian organism- a sperm cell- has been a challenging task for electrophysiologists for many decades. The method known as "spermatozoan patch clamp" was introduced in 2006. It has enabled the direct recording of ion channel activity in whole-cell and cell-attached configurations and has been instrumental in describing sperm cell physiology and the molecular identity of various calcium, potassium, sodium, chloride, and proton ion channels. However, recording from single spermatozoa requires advanced skills and training in electrophysiology. This detailed protocol summarizes the step-by-step procedure and highlights several 'tricks-of-the-trade' in order to make it available to anyone who wishes to explore the fascinating physiology of the sperm cell. Specifically, the protocol describes recording from human and murine sperm cells but can be adapted to essentially any mammalian sperm cell of any species. The protocol covers important details of the application of this technique, such as isolation of sperm cells, selection of reagents and equipment, immobilization of the highly motile cells, formation of the tight (Gigaohm) seal between a recording electrode and the plasma membrane of the sperm cells, transition into the whole-spermatozoan mode (also known as break-in), and exemplary recordings of the sperm cell calcium ion channel, CatSper, from six mammalian species. The advantages and limitations of the sperm patch clamp method, as well as the most critical steps, are discussed.

This protocol describes how to perform electrical recordings from mammalian sperm cells in a whole-cell configuration, with the goal of directly recording ion channel activity. The method has been instrumental in describing the electrophysiological profiles of several sperm ion channels and helped to reveal their molecular identity and regulation.

Recording of the electrical activity from one of the smallest cells of a mammalian organism- a sperm cell- has been a challenging task for ...

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.

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Research

JoVE Journal

Biology

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

Here, we present methodologies to evaluate spermatozoan membrane integrity, a cellular feature associated with sperm fertilization competence. We describe three techniques for the fluorimetric assessment of sperm membranes: simultaneous staining with specific fluorescent probes, fluorescence microscopy, and advanced sperm-dedicated flow cytometry. Examples of combining the methodologies are also presented.

Fluorimetric Techniques for the Assessment of Sperm Membranes

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

We describe the application of an extracellular flux analyzer to monitor real-time changes in glycolysis and oxidative phosphorylation during mouse sperm capacitation.

Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation

The AcroSensE mouse model and live cell imaging methods described here provide a new approach to studying calcium dynamics in the subcellular compartment of the sperm acrosome and how they regulate intermediate steps leading to membrane fusion and acrosome exocytosis.

Real-Time Imaging of Acrosomal Calcium Dynamics and Exocytosis in Live Mouse Sperm

External electric field induces a voltage on the membrane of a cell, termed the induced membrane voltage (ΔΦ). By using the potentiometric dye di-8-ANEPPS, it is possible to measure the ΔΦ noninvasively. This video shows the protocol for measuring ΔΦ using di-8-ANEPPS.

Measuring the Induced Membrane Voltage with Di-8-ANEPPS

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Learn the spermatozoan patch-clamp technique for recording ion channel activity in mammalian sperm cells. This method enables detailed study of CatSper and other channels to understand male fertility.

Recording Electrical Currents across the Plasma Membrane of Mammalian Sperm Cells

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