Electrophysiology of Individual Spermatozoa

The electrophysiological properties of individual spermatozoa were investigated using the whole cell recording technique (Hamill et al., 1981 (link); Kirichok et al., 2006 (link); Lishko et al., 2011a (link)). The recording pipettes (10–18 MΩ) were fabricated from borosilicate glass and normally filled with standard pipette solution. Gigaohm seals were obtained by bringing the pipette tip into gentle contact with the cytoplasmic droplet, which lies just behind the sperm head, and the patch of membrane spanning the pipette tip then ruptured by applying suction in conjunction with 1 ms voltage pulses (see Lishko et al., 2010 (link)). Our standard recording conditions were designed to preserve physiologically relevant Na⁺, K⁺ and Cl⁻ gradients and V_m was held (pClamp 10 Software, Axon Instruments) at a hyperpolarized value (−92 mV) between test pulses. Initial experiments were undertaken by recording the membrane currents (I_m) evoked by ramping (250 ms) V_m from −92 mV to 68 mV at 1 Hz. To analyse the results of such experiments, I_m was first normalized to input capacitance (i.e. expressed as pA pF⁻¹) to ensure that variations between the sizes of different spermatozoa did not contribute to the variability in the presented data. All cited values of V_m were corrected for the liquid junction potential between the pipette/bath solutions (E_L), and for the voltage drop across the access resistance (R_a, 62.8 ± 0.8 MΩ, n = 476 cells from 29 donors). The latter correction was applied retrospectively using the expression V_m = V_Pip − R_a·I_m, where V_Pip is the pipette potential. Since the bath was grounded via a 4% agar/3 M KCl, bridge, the bath solution changes imposed during the present study had negligible effects upon E_L. Plots showing the relationship between I_m and V_m were constructed and, unless otherwise stated, cited values of membrane conductance (G_m, pS pF⁻¹) are derived by regression analysis (i.e. ΔI_m/ΔV_m) of data recorded at positive potentials. Resting V_m was either inferred from the reversal potential (V_Rev, i.e. the value of V_m at which I_m is zero, voltage clamp experiments) or measured directly by monitoring (5 KHz, data low pass filtered at 3 KHz) the zero current potential (see Hamill et al., 1981 (link)). Experiments that quantified the responses to step changes in V_m were undertaken using an experimental design that employed the standard features of pClamp software (V/4 protocol) to subtract leak/capacitive currents from all recorded data. The statistical significance of differences between control/experimental values were determined tested using Student's paired (repeated measurements on the same cells) or unpaired (comparison between different groups of cells) t-test. The results of experiments that followed more complex protocols were analysed by one way analysis of variance (ANOVA)/Dunnet's post hoc test. Data are cited as mean ± s.e.m. and values of n refer to the number of spermatozoa in each group. All observations were confirmed using spermatozoa from at least three different donors.