Effects of pH manipulation, CatSper stimulation and Ca2+-store mobilization on [Ca2+]i and behaviour of human sperm

Achikanu, Cosmas Ezekaibeya; Pendekanti, Venkata; Teague, Rebecca; Publicover, Stephen J.

doi:10.1093/humrep/dey280

Cited by 25 publications

(39 citation statements)

References 34 publications

(26 reference statements)

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“…A pivotal mechanism for regulation of behaviour in human sperm is [Ca 2+ ] i (Publicover et al , 2007; Suarez, 2008). To investigate the effect of [Ca 2+ ] i elevation, we monitored the activity of cells exposed to four different stimuli (300 nM P4, 1% FF [containing ~300 nM P4], 2 mM 4-AP and 1 μM thimerosal), all of which are known to increase [Ca 2+ ] i and stimulate hyperactivation in human and other sperm, but with varying potency (Ho and Suarez, 2001; Alasmari et al , 2013b; Brown et al , 2017; Achikanu et al , 2018). CASA assessments, carried out during the period of behavioural data collection, using cells prepared under identical conditions, confirmed the efficacy and relative potency of these stimuli (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

“…DMSO in P4 and 4-AP experiments was 0.003% and 0.4%, respectively. One percent DMSO had no detectable effect on kinematic properties of sperm motility (Achikanu et al , 2018).…”

Section: Methodsmentioning

confidence: 97%

“…Hyperactivation was assessed as described previously (Achikanu et al , 2018). After addition of the stimulus, cells were loaded into pre-warmed 20 μm CASA chamber (36°C).…”

Section: Methodsmentioning

confidence: 99%

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

Achikanu

Correia

Guidobaldi

et al. 2019

Molecular Human Reproduction

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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.

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Section: Resultsmentioning

confidence: 99%

“…DMSO in P4 and 4-AP experiments was 0.003% and 0.4%, respectively. One percent DMSO had no detectable effect on kinematic properties of sperm motility (Achikanu et al , 2018).…”

Section: Methodsmentioning

confidence: 97%

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

Achikanu

Correia

Guidobaldi

et al. 2019

Molecular Human Reproduction

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“…Progesterone increases [Ca 2+ ] i in human spermatozoa at concentrations as low as 300 nM, through CatSper activation (Tesarik et al, 1992;Harper et al, 2003;Achikanu et al, 2018). Recently it was described that the progesterone receptor in these cells is a α/β hydrolase domain-containing protein (ABHD2), which depletes the endocannabinoid 2-arachinoylglycerol (2AG) from membrane and removes CatSper inactivation (Miller et al, 2016;Mannowetz et al, 2017).…”

Section: Effect Of Progesterone In Human Spermatozoamentioning

confidence: 99%

Quantitative Intracellular pH Determinations in Single Live Mammalian Spermatozoa Using the Ratiometric Dye SNARF-5F

Chávez

Darszon

Treviño

et al. 2020

Front. Cell Dev. Biol.

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Intracellular pH (pH i) plays a crucial role in mammalian sperm physiology. However, it is a challenging task to acquire quantitative single sperm pH i images due to their small size and beating flagella. In this study, we established a robust pH i imaging system using the dual-emission ratiometric pH indicator, SNARF-5F. Simultaneous good signal/noise ratio fluorescence signals were obtained exciting with a green high-power LED (532 nm) and acquiring with an EM-CCD camera through an image splitter with two band-pass filters (550-600 nm, channel 1; 630-650 nm, channel 2). After in vivo calibration, we established an imaging system that allows determination of absolute pH i values in spermatozoa, minimizing cell movement artifacts. Using this system, we determined that bicarbonate increases non-capacitated human pH i with slower kinetics than in mouse spermatozoa. This difference suggests that distinct ionic transporters might be involved in the bicarbonate influx into human and mouse spermatozoa. Alternatively, pH i regulation downstream bicarbonate influx into spermatozoa could be different between the two species.

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“…The activity of CatSper is controlled by both the membrane potential (V m ) and intracellular pH (pH i ) (2)(3)(4)6,8,9), and, in human sperm, also by oviductal steroids and prostaglandins (3,4,(10)(11)(12)(13)(14). Thereby, CatSper translates changes of the chemical microenvironment into changes of [Ca 2+ ] i and swimming behavior, which enables sperm to reach the site of fertilization, to overcome the egg´s protective vestments, and, ultimately, to fertilize the egg (1,(14)(15)(16)(17)(18)(19)(20). CatSper is, hence, absolutely required for fertilization in mice and humans (1,11,13,14,(21)(22)(23)(24).…”

Section: Introductionmentioning

confidence: 99%

The Ca2+ channel CatSper is not activated by cAMP/PKA signaling but directly affected by chemicals used to probe the action of cAMP and PKA

Wang

Young

Krenz

et al. 2020

Journal of Biological Chemistry

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The sperm-specific CatSper channel controls the influx of Ca2+ into the flagellum and, thereby, the swimming behavior of sperm. A hallmark of human CatSper is its polymodal activation by membrane voltage, intracellular pH, and oviductal hormones. Whether CatSper is also activated by signaling pathways involving an increase of cAMP and ensuing activation of protein kinase A (PKA) is, however, a matter of controversy. To shed light on this question, we used kinetic ion-sensitive fluorimetry, patch-clamp recordings, and optochemistry to study transmembrane Ca2+ flux and membrane currents in human sperm from healthy donors and from patients that lack functional CatSper channels. We find that human CatSper is neither activated by intracellular cAMP directly nor indirectly by the cAMP/PKA-signaling pathway. Instead, we show that non-physiological concentrations of cAMP and membrane-permeable cAMP analogs used to mimic the action of intracellular cAMP activate human CatSper from the outside via a hitherto unknown extracellular binding site. Finally, we demonstrate that the effects of common PKA inhibitors on human CatSper rest predominantly, if not exclusively, on off-target drug actions on CatSper itself rather than on inhibition of PKA. We conclude that the concept of an intracellular cAMP/PKA-activation of CatSper is primarily based on unspecific effects of chemical probes used to interfere with cAMP signaling. Altogether, our findings solve several controversial issues and reveal a novel ligand-binding site controlling the activity of CatSper, which has important bearings on future studies of cAMP and Ca2+ signaling in sperm.

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Effects of pH manipulation, CatSper stimulation and Ca2+-store mobilization on [Ca2+]i and behaviour of human sperm

Abstract: CA was supported by the Nigerian government (Tertiary Education Trust (TET) Fund). The authors have no conflicts of interest.

Cited by 25 publications

References 34 publications

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

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

Quantitative Intracellular pH Determinations in Single Live Mammalian Spermatozoa Using the Ratiometric Dye SNARF-5F

The Ca2+ channel CatSper is not activated by cAMP/PKA signaling but directly affected by chemicals used to probe the action of cAMP and PKA

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