Continuous behavioural 'switching' in human spermatozoa and its regulation by Ca2+-mobilising stimuli

Abstract

Human sperm show a variety of different behaviours (types of motility) that have different functional roles. Previous reports suggest that sperm may reversibly switch between these behaviours. We have recorded and analysed the behaviour of individual human sperm (180 cells in total), each cell monitored continuously for 3-3.5 min either under control conditions or in the presence of Ca2+-mobilising stimuli. Switching between different behaviours was assessed visually (1 s bins using four behaviour categories), and was verified by fractal dimension analysis of sperm head tracks. In the absence of stimuli, ~90% of cells showed at least one behavioural transition (mean rate under control conditions = 6.4 ± 0.8 transitions.min-1). Type 1 behaviour (progressive, activated-like motility) was most common, but the majority of cells (>70%) displayed at least three behaviour types. Treatment of sperm with Ca2+-mobilising agonists had negligible effects on the rate of switching but increased the time spent in type 2 and type 3 (hyperactivation-like) behaviours (P < 2*10-8; chi-square). Treatment with 4-aminopyridine under alkaline conditions (pHo = 8.5), a highly-potent Ca2+-mobilising stimulus, was the most effective in increasing the proportion of type 3 behaviour, biasing switching away from type 1 (P < 0.005) and dramatically extending the duration of type 3 events (P < 10-16). Other stimuli, including 300 nM progesterone and 1% human follicular fluid, had qualitatively similar effects but were less potent. We conclude that human sperm observed in vitro constitutively display a range of behaviours and regulation of motility by [Ca2+]i, at the level of the single cell, is achieved not by causing cells to adopt a 'new' behaviour but by changing the relative contributions of those behaviours.

Keywords: behaviour; calcium; motility; pH; spermatozoa.

Figure 1

Behavioural switching in a free-swimming human sperm under control conditions (pH _o = 7.4). (a) Variation in behaviour type (categorised visually as type1, type 2, type 3 or type 4; see Supplementary Figure S1) of a single sperm over a period of 190 s. (b) Variation in fractal dimension (FD) over time (black trace) overlaid with the visually categorised behaviour types (red trace). Visual analysis and FD show good agreement, with no visually identified behavioural transitions that are not confirmed by FD. (c) Track of the same cell, colour coded to display variation in the fractal dimension (FD); (1 < FD ≤ 1.2 (dark blue); 1.2 < FD ≤ 1.4 (light blue); 1.4 < FD ≤ 1.6 (green); 1.6 < FD ≤ 1.8 (yellow); 1.8 < FD ≤ 2.0 (red)). Axes show distance in μm.

Figure 2

Characteristics of behavioural switching. (a) Dwell times (period during which a single behaviour type occurred) for types 1 (light blue, n = 164 events), 2 (green, n = 79), 3 (red, n = 107) and 4 (black, n = 15) behaviours in cells control cells (pH 7.4). Plots show median and interquartile range (box) and maximum/minimum values (whiskers). (b) Mean % time spent in each behaviour; type 1 (light blue), type 2 (green), type 3 (red) and type 4 (black) under control conditions (n = 18 cells) and in the presence of 300 nM P4 (n = 18 cells), 1% FF (n = 17 cells), 2 mM 4-AP (n = 21 cells) and 1 μM thimerosal (n = 17 cells). (c) Relative frequencies of transitions into type 1 (light blue), type 2 (green), type 3 (red) and type 4 (black) behaviours under control conditions (n = 347 transitions) and in the presence of progesterone (P4; 300 nM; n = 420), human follicular fluid (FF nM, 1%; n = 353), 4-aminopyridine (4-AP, 2 mM, n = 211) and thimerosal (1 μM, n = 115). Asterisks indicate significant difference from control; ^*** = P < 0.005. (d–f) Dwell times for type 1 (panel ‘d’; blue), type 2 (panel ‘e’; green) and type 3 (panel ‘f’; red) behaviours under control conditions and in the presence of 300 nM progesterone (P4), 1% human follicular fluid (FF), 2 mM 4-aminopyridine (4-AP) and 1 μM thimerosal. Plots show median and interquartile range (box) and maximum/minimum values (whiskers) of 51–167 events. Asterisks indicate significant difference from control; ^** = P < 0.01; ^*** = P < 0.001; ^**** = P < 0.0001.

Figure 3

Characteristics of behavioural switching at pH _o = 8.5. (a) Mean switching rate (±s.e.m.) for cells incubated at pH_o = 7.4 (grey bars) and pH_o = 8.5 (red bars) under control conditions (n = 18 cells) and in the presence of 300 nM P4 (n = 18 cells), 1% FF (n = 17 cells), 2 mM 4-AP (n = 21 cells) and 1 μM thimerosal (n = 17 cells). (b) Mean % time spent in each behaviour at pH_o = 8.5; type 1 (light blue), type 2 (green), type 3 (red) and type 4 (black) under control conditions (n = 18 cells) and in the presence of 300 nM P4 (n = 15 cells), 1% FF (n = 18 cells), 2 mM 4-AP (n = 20 cells) and 1 μM thimerosal (n = 16 cells). (c) Relative frequencies of transitions into type 1 (light blue), type 2 (green), type 3 (red) and type 4 (black) behaviours at pH_o = 8.5 under control conditions (n = 220 transitions) and in the presence of progesterone (P4; 300 nM; n = 194), human follicular fluid (FF nM, 1%; n = 151), 4-aminopyridine (4-AP, 2 mM, n = 47) and thimerosal (1 μM, n = 63). (d–f) Dwell times at pH_o = 8.5 for type 1 (panel ‘d’; blue), type 2 (panel ‘e’; green) and type 3 (panel ‘f’; red) behaviours under control conditions and in the presence of 300 nM progesterone (P4), 1% human follicular fluid (FF), 2 mM 4-aminopyridine (4-AP) and 1 μM thimerosal. Plots show median and interquartile range (box) and maximum/minimum values (whiskers) of 10–110 events (except thimerosal type 1 where n = 2). Asterisks indicate significant difference from control; ^* = P < 0.05; ^** = P < 0.01; ^*** = P < 0.001; ^**** = P < 0.0001.