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Ca2+ signals generated by CatSper and Ca2+ stores regulate different behaviors in human sperm.

Alasmari W, Costello S, Correia J, Oxenham SK, Morris J, Fernandes L, Ramalho-Santos J, Kirkman-Brown J, Michelangeli F, Publicover S, Barratt CL - J. Biol. Chem. (2013)

Bottom Line: Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pHi and mobilized Ca(2+) stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca(2+)-depleted medium and also potentiated penetration into methylcellulose.The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not.We conclude that these two components of the [Ca(2+)]i signaling apparatus have strikingly different effects on sperm motility.

View Article: PubMed Central - PubMed

Affiliation: From the Reproductive and Developmental Biology, Medical School, University of Dundee, Ninewells Hospital, Dundee DD1 9SY, Scotland, United Kingdom.

ABSTRACT
[Ca(2+)]i signaling regulates sperm motility, enabling switching between functionally different behaviors that the sperm must employ as it ascends the female tract and fertilizes the oocyte. We report that different behaviors in human sperm are recruited according to the Ca(2+) signaling pathway used. Activation of CatSper (by raising pHi or stimulating with progesterone) caused sustained [Ca(2+)]i elevation but did not induce hyperactivation, the whiplash-like behavior required for progression along the oviduct and penetration of the zona pellucida. In contrast, penetration into methylcellulose (mimicking penetration into cervical mucus or cumulus matrix) was enhanced by activation of CatSper. NNC55-0396, which abolishes CatSper currents in human sperm, inhibited this effect. Treatment with 5 μm thimerosal to mobilize stored Ca(2+) caused sustained [Ca(2+)]i elevation and induced strong, sustained hyperactivation that was completely insensitive to NNC55-0396. Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pHi and mobilized Ca(2+) stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca(2+)-depleted medium and also potentiated penetration into methylcellulose. The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not. We conclude that these two components of the [Ca(2+)]i signaling apparatus have strikingly different effects on sperm motility. Furthermore, since stored Ca(2+) at the sperm neck can be mobilized by Ca(2+)-induced Ca(2+) release, we propose that CatSper activation can elicit functionally different behaviors according to the sensitivity of the Ca(2+) store, which may be regulated by capacitation and NO from the cumulus.

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Alkalinization raises [Ca2+]i in human sperm.A, 2 mm 4-AP (green trace) and 25 mm NH4Cl (dark blue trace) cause similar changes in pHi of human sperm populations. The additions are marked by an arrow. Both aliquots were from the same ejaculate. B, amplitude of pHi increment imposed by 25 mm NH4Cl (dark blue), 10 and 20 mm TMA (light blue), 2 mm 4-AP (green), and 10 mm thimerosal (red). Each bar shows the mean of 4–10 experiments ± S.E. (error bars). C, 25 mm NH4Cl (added at the arrow) increases [Ca2+]o (OGB fluorescent intensity) in human sperm. Shown are the responses of eight cells in the same experiment. D, 20 mm TMA (first arrow) induces a large prolonged increment in pHi of human sperm. 2 mm 4-AP was added at the second arrow. E, 20 mm TMA (added at the first arrow) increases [Ca2+]o (OGB fluorescent intensity) in human sperm. The upward arrow shows TMA washout. Shown are the responses of eight cells in the same experiment. F, 3 μm progesterone (added at the arrow) causes a biphasic increase in [Ca2+]o (OGB fluorescent intensity) in human sperm. Shown are the responses of six cells in the same experiment, one of which generates [Ca2+]i oscillations after the initial transient.
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Figure 1: Alkalinization raises [Ca2+]i in human sperm.A, 2 mm 4-AP (green trace) and 25 mm NH4Cl (dark blue trace) cause similar changes in pHi of human sperm populations. The additions are marked by an arrow. Both aliquots were from the same ejaculate. B, amplitude of pHi increment imposed by 25 mm NH4Cl (dark blue), 10 and 20 mm TMA (light blue), 2 mm 4-AP (green), and 10 mm thimerosal (red). Each bar shows the mean of 4–10 experiments ± S.E. (error bars). C, 25 mm NH4Cl (added at the arrow) increases [Ca2+]o (OGB fluorescent intensity) in human sperm. Shown are the responses of eight cells in the same experiment. D, 20 mm TMA (first arrow) induces a large prolonged increment in pHi of human sperm. 2 mm 4-AP was added at the second arrow. E, 20 mm TMA (added at the first arrow) increases [Ca2+]o (OGB fluorescent intensity) in human sperm. The upward arrow shows TMA washout. Shown are the responses of eight cells in the same experiment. F, 3 μm progesterone (added at the arrow) causes a biphasic increase in [Ca2+]o (OGB fluorescent intensity) in human sperm. Shown are the responses of six cells in the same experiment, one of which generates [Ca2+]i oscillations after the initial transient.

Mentions: To activate CatSper channels, we increased pHi. Resting pHi of human sperm capacitated in sEBSS was 6.99 ± 0.06 (n = 23). 25 mm NH4Cl raised pHi by 0.27 ± 0.02 units in <1 min (p < 10−4; n = 6; Fig. 1, A and B). [Ca2+]i increased more slowly in response to NH4Cl, the mean change in fluorescence of OGB (ΔFmean) stabilizing at 22.4 ± 4.1% above control levels within 5 min (n = 4; Fig. 1C; p < 0.02). In sperm incubated in sEBSS, the proportion of cells showing hyperactivated motility, as assessed by CASA (34), was 3.6 ± 0.4% (n = 60). Stimulation with 25 mm NH4Cl recruited only a further 4.1 ± 1.3% of cells into the hyperactivated class (p < 0.01; paired t test; n = 21; Fig. 2A). These experiments were repeated using sperm incubated in STF, a medium that promotes rapid and potent sperm capacitation. In STF-incubated cells, the proportion showing “spontaneous” hyperactivated motility (recorded in the absence of stimulation) was increased >3-fold (12.2 ± 1.0%; n = 73; p < 10−11), but the effects of NH4Cl on hyperactivated motility were negligible (Fig. 2B). Frequency distributions of two key kinematic parameters measured by CASA, amplitude of side-side movement of the sperm head (ALH) and linear distance from first to last track point/total track length (linearity) confirmed this observation. Activation of CatSper by alkalinization reduced the proportion of cells showing very low ALH and very highly linear behavior (≥75%), but there was no increase in the proportion of cells showing ALH ≥9 μm or linearity ≤35% (Fig. 2, C and D).


Ca2+ signals generated by CatSper and Ca2+ stores regulate different behaviors in human sperm.

Alasmari W, Costello S, Correia J, Oxenham SK, Morris J, Fernandes L, Ramalho-Santos J, Kirkman-Brown J, Michelangeli F, Publicover S, Barratt CL - J. Biol. Chem. (2013)

Alkalinization raises [Ca2+]i in human sperm.A, 2 mm 4-AP (green trace) and 25 mm NH4Cl (dark blue trace) cause similar changes in pHi of human sperm populations. The additions are marked by an arrow. Both aliquots were from the same ejaculate. B, amplitude of pHi increment imposed by 25 mm NH4Cl (dark blue), 10 and 20 mm TMA (light blue), 2 mm 4-AP (green), and 10 mm thimerosal (red). Each bar shows the mean of 4–10 experiments ± S.E. (error bars). C, 25 mm NH4Cl (added at the arrow) increases [Ca2+]o (OGB fluorescent intensity) in human sperm. Shown are the responses of eight cells in the same experiment. D, 20 mm TMA (first arrow) induces a large prolonged increment in pHi of human sperm. 2 mm 4-AP was added at the second arrow. E, 20 mm TMA (added at the first arrow) increases [Ca2+]o (OGB fluorescent intensity) in human sperm. The upward arrow shows TMA washout. Shown are the responses of eight cells in the same experiment. F, 3 μm progesterone (added at the arrow) causes a biphasic increase in [Ca2+]o (OGB fluorescent intensity) in human sperm. Shown are the responses of six cells in the same experiment, one of which generates [Ca2+]i oscillations after the initial transient.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585060&req=5

Figure 1: Alkalinization raises [Ca2+]i in human sperm.A, 2 mm 4-AP (green trace) and 25 mm NH4Cl (dark blue trace) cause similar changes in pHi of human sperm populations. The additions are marked by an arrow. Both aliquots were from the same ejaculate. B, amplitude of pHi increment imposed by 25 mm NH4Cl (dark blue), 10 and 20 mm TMA (light blue), 2 mm 4-AP (green), and 10 mm thimerosal (red). Each bar shows the mean of 4–10 experiments ± S.E. (error bars). C, 25 mm NH4Cl (added at the arrow) increases [Ca2+]o (OGB fluorescent intensity) in human sperm. Shown are the responses of eight cells in the same experiment. D, 20 mm TMA (first arrow) induces a large prolonged increment in pHi of human sperm. 2 mm 4-AP was added at the second arrow. E, 20 mm TMA (added at the first arrow) increases [Ca2+]o (OGB fluorescent intensity) in human sperm. The upward arrow shows TMA washout. Shown are the responses of eight cells in the same experiment. F, 3 μm progesterone (added at the arrow) causes a biphasic increase in [Ca2+]o (OGB fluorescent intensity) in human sperm. Shown are the responses of six cells in the same experiment, one of which generates [Ca2+]i oscillations after the initial transient.
Mentions: To activate CatSper channels, we increased pHi. Resting pHi of human sperm capacitated in sEBSS was 6.99 ± 0.06 (n = 23). 25 mm NH4Cl raised pHi by 0.27 ± 0.02 units in <1 min (p < 10−4; n = 6; Fig. 1, A and B). [Ca2+]i increased more slowly in response to NH4Cl, the mean change in fluorescence of OGB (ΔFmean) stabilizing at 22.4 ± 4.1% above control levels within 5 min (n = 4; Fig. 1C; p < 0.02). In sperm incubated in sEBSS, the proportion of cells showing hyperactivated motility, as assessed by CASA (34), was 3.6 ± 0.4% (n = 60). Stimulation with 25 mm NH4Cl recruited only a further 4.1 ± 1.3% of cells into the hyperactivated class (p < 0.01; paired t test; n = 21; Fig. 2A). These experiments were repeated using sperm incubated in STF, a medium that promotes rapid and potent sperm capacitation. In STF-incubated cells, the proportion showing “spontaneous” hyperactivated motility (recorded in the absence of stimulation) was increased >3-fold (12.2 ± 1.0%; n = 73; p < 10−11), but the effects of NH4Cl on hyperactivated motility were negligible (Fig. 2B). Frequency distributions of two key kinematic parameters measured by CASA, amplitude of side-side movement of the sperm head (ALH) and linear distance from first to last track point/total track length (linearity) confirmed this observation. Activation of CatSper by alkalinization reduced the proportion of cells showing very low ALH and very highly linear behavior (≥75%), but there was no increase in the proportion of cells showing ALH ≥9 μm or linearity ≤35% (Fig. 2, C and D).

Bottom Line: Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pHi and mobilized Ca(2+) stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca(2+)-depleted medium and also potentiated penetration into methylcellulose.The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not.We conclude that these two components of the [Ca(2+)]i signaling apparatus have strikingly different effects on sperm motility.

View Article: PubMed Central - PubMed

Affiliation: From the Reproductive and Developmental Biology, Medical School, University of Dundee, Ninewells Hospital, Dundee DD1 9SY, Scotland, United Kingdom.

ABSTRACT
[Ca(2+)]i signaling regulates sperm motility, enabling switching between functionally different behaviors that the sperm must employ as it ascends the female tract and fertilizes the oocyte. We report that different behaviors in human sperm are recruited according to the Ca(2+) signaling pathway used. Activation of CatSper (by raising pHi or stimulating with progesterone) caused sustained [Ca(2+)]i elevation but did not induce hyperactivation, the whiplash-like behavior required for progression along the oviduct and penetration of the zona pellucida. In contrast, penetration into methylcellulose (mimicking penetration into cervical mucus or cumulus matrix) was enhanced by activation of CatSper. NNC55-0396, which abolishes CatSper currents in human sperm, inhibited this effect. Treatment with 5 μm thimerosal to mobilize stored Ca(2+) caused sustained [Ca(2+)]i elevation and induced strong, sustained hyperactivation that was completely insensitive to NNC55-0396. Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pHi and mobilized Ca(2+) stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca(2+)-depleted medium and also potentiated penetration into methylcellulose. The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not. We conclude that these two components of the [Ca(2+)]i signaling apparatus have strikingly different effects on sperm motility. Furthermore, since stored Ca(2+) at the sperm neck can be mobilized by Ca(2+)-induced Ca(2+) release, we propose that CatSper activation can elicit functionally different behaviors according to the sensitivity of the Ca(2+) store, which may be regulated by capacitation and NO from the cumulus.

Show MeSH
Related in: MedlinePlus