Intracellular Ca2+ threshold reversibly switches flagellar beat off and on†

Abstract

Sperm motility is essential for fertilization. The asymmetry of flagellar beat in spermatozoa is finely regulated by intracellular calcium concentration ([Ca2+]i). Recently, we demonstrated that the application of high concentrations (10-20 μM) of the Ca2+ ionophore A23187 promotes sperm immobilization after 10 minutes, and its removal thereafter allows motility recovery, hyperactivation and fertilization. In addition, the same ionophore treatment overcomes infertility observed in sperm from Catsper1-/-, Slo3-/- and Adcy10-/-, but not PMCA4-/-, which strongly suggest that regulation of [Ca2+]i is mandatory for sperm motility and hyperactivation. In this study we found that prior to inducing sperm immobilization, high A23187 concentrations (10 μM) increase flagellar beat. While 5-10 μM A23187 substantially elevates [Ca2+]i and rapidly immobilizes sperm in a few minutes, smaller concentrations (0.5 and 1 μM) provoke smaller [Ca2+]i increases and sperm hyperactivation, confirming that [Ca2+]i increases act as a motility switch. Until now the [Ca2+]i thresholds that switch motility on and off were not fully understood. To study the relationship between [Ca2+]i and flagellar beating, we developed an automatic tool that allows the simultaneous measurement of these two parameters. Individual spermatozoa were treated with A23187 which is then washed to evaluate [Ca2+]i and flagellar beat recovery using the implemented method. We observe that [Ca2+]i must decrease below a threshold concentration range to facilitate subsequent flagellar beat recovery and sperm motility.

Capacitation increases glucose consumption in murine sperm

“…Incubating sperm in the presence of calcium ionophore for 10 min, which transiently increases intracellular Ca 2+ (Tateno et al, 2013), did not increase glucose consumption rates (Figure 4b). As described (Sanchez‐Cardenas et al, 2018; Tateno et al, 2013), while ionophore exposure completely stopped sperm motility in minutes, sperm motility was recovered immediately after its removal. Thus, although Ca 2+ plays a role in stimulating glucose consumption, the temporal increase of its intracellular concentration is not sufficient to elicit the increase in glucose consumption observed during capacitation.…”

“…Incubating sperm in the presence of calcium ionophore for 10 min, which transiently increases intracellular Ca 2+ (Tateno et al, 2013), did not increase glucose consumption rates (Figure 4b). As described (Sanchez-Cardenas et al, 2018;Tateno et al, 2013), while ionophore exposure completely stopped sperm motility in minutes, sperm motility was recovered immediately after its removal.…”

“…Once in these media, 4Br‐A 23187 was added to a final concentration of 20 µM and sperm incubated for 10 min at 37°C. After this time, sperm were immediately rendered motionless due to a massive entrance of Ca 2+ into the sperm (Sanchez‐Cardenas et al, 2018). Subsequently, spermatozoa were washed twice by centrifugations (5 min at 600 g ) in non‐cap TYH (glucose and pyruvate free) to remove 4Br‐A 23187 from the media.…”

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

“…An additional type of behaviour was occasionally observed, in which the mid-piece and anterior flagellum became tightly curved (forming a ‘J’ shape or coil) and arrested or ‘twitched’ (Supplementary Video 3, Table I). Since behaviour of this type is seen in cells treated with Ca 2+ -ionophore, where it apparently reflects excess Ca 2+ -loading that takes the cells ‘beyond’ hyperactivated motility (Sanchez-Cardenas et al , 2018), this was designated type 4 behaviour. This behaviour was typically interspersed with very brief (100–500 ms) bursts of flagellar activity such that the cell became virtually immobilised for up to 20 s (Supplementary Fig.…”

Transient Sperm Starvation Improves the Outcome of Assisted Reproductive Technologies

“…After this period, sperm suspensions (25 μL) were loaded into pre-warmed chamber slide (depth, 100 μm) (Leja slide, Spectrum Technologies) and placed on a microscope stage at 37°C. Sperm motility was examined using the CEROS computer-assisted semen analysis (CASA) system (Hamilton Thorne Research, Beverly, MA, United States) as previously described (Sanchez-Cardenas et al, 2018). The default settings include the following: frames acquired: 90; frame rate: 60 Hz; minimum cell size: 4 pixels; static head size: 0.13–2.43; static head intensity: 0.10–1.52; and static head elongation: 5–100.…”