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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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4-AP causes hyperactivation by mobilizing stored Ca2+.A, sustained increase in [Ca2+]i (OGB fluorescent intensity) in human sperm exposed to 2 mm 4-AP (first arrow). 4-AP was washed off at the second arrow. Shown are responses of eight cells in same experiment. B, release of Ca2+ from sacroplasmic reticulum microsomes by 4-AP. The graph shows 4-AP-induced Ca2+ release (percentage of A23187-releasable Ca2+); each point represents the mean ± S.E. (error bars) of 4–7 experiments. Inset, example trace. 2 mm 4-AP was added at the first arrow, and remaining Ca2+ was released by 25 μm A23187 (second arrow). Calibration shows time and [Ca2+]. C, effect of 2 mm 4-AP in Ca2+-depleted medium. Sperm were superfused with EGTA-buffered medium (first arrow), which caused a rapid fall in [Ca2+]i. Subsequent application of 4-AP caused a [Ca2+]i transient in a subset of cells. Shown are responses of 11 cells in the same experiment. D, time course of hyperactivation (percentage of cells; squares) induced by 2 mm 4-AP in cells suspended in sEBSS (filled symbols) and in parallel experiments where cells were resuspended in EGTA-buffered sEBSS immediately before use (open symbols). Circles show the percentage of motile cells. Each point shows mean ± S.E. of four experiments. E, sustained increase in [Ca2+]i (OGB fluorescent intensity) in human sperm exposed to 5 μm thimerosal (first arrow). Thimerosal was washed off at the second arrow, and 3 μm progesterone was applied at the third arrow. Shown are the responses of eight cells in the same experiment. Temperature was 30 °C. F, time course and concentration dependence of thimerosal-induced hyperactivation (percentage of cells). Each line shows mean ± S.E. of four experiments.
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Figure 3: 4-AP causes hyperactivation by mobilizing stored Ca2+.A, sustained increase in [Ca2+]i (OGB fluorescent intensity) in human sperm exposed to 2 mm 4-AP (first arrow). 4-AP was washed off at the second arrow. Shown are responses of eight cells in same experiment. B, release of Ca2+ from sacroplasmic reticulum microsomes by 4-AP. The graph shows 4-AP-induced Ca2+ release (percentage of A23187-releasable Ca2+); each point represents the mean ± S.E. (error bars) of 4–7 experiments. Inset, example trace. 2 mm 4-AP was added at the first arrow, and remaining Ca2+ was released by 25 μm A23187 (second arrow). Calibration shows time and [Ca2+]. C, effect of 2 mm 4-AP in Ca2+-depleted medium. Sperm were superfused with EGTA-buffered medium (first arrow), which caused a rapid fall in [Ca2+]i. Subsequent application of 4-AP caused a [Ca2+]i transient in a subset of cells. Shown are responses of 11 cells in the same experiment. D, time course of hyperactivation (percentage of cells; squares) induced by 2 mm 4-AP in cells suspended in sEBSS (filled symbols) and in parallel experiments where cells were resuspended in EGTA-buffered sEBSS immediately before use (open symbols). Circles show the percentage of motile cells. Each point shows mean ± S.E. of four experiments. E, sustained increase in [Ca2+]i (OGB fluorescent intensity) in human sperm exposed to 5 μm thimerosal (first arrow). Thimerosal was washed off at the second arrow, and 3 μm progesterone was applied at the third arrow. Shown are the responses of eight cells in the same experiment. Temperature was 30 °C. F, time course and concentration dependence of thimerosal-induced hyperactivation (percentage of cells). Each line shows mean ± S.E. of four experiments.

Mentions: 4-Aminopyridine strongly hyperactivates mammalian sperm (28, 41), an effect that is correlated with fertilization rate in in vitro fertilization (42). Application of 2 mm 4-AP to cells bathed in sEBSS rapidly raised [Ca2+]i (ΔFmean = 32 ± 4%; n = 7 experiments; p < 0.0001; Fig. 3A) and recruited 26 ± 2% of cells into the hyperactivated population (n = 48; p < 10−19; Fig. 2, A and G). In cells suspended in STF, a similar hyperactivating effect of 4-AP was seen (n = 63; p < 10−33; Fig. 2, B–D). 4-AP is a weak base and in mouse sperm causes hyperpolarization of membrane potential (Em) (43) due to activation of a 4-AP-insensitive, pH-regulated K+ channel (44, 45). Hyperactivation might thus reflect alkalinization-induced activation of CatSper (28, 43). 2 mm 4-AP increased pHi by 0.27 ± 0.04 units, n = 7, an effect similar to that seen with 25 mm NH4Cl (p > 0.9) and only half that seen with 20 mm TMA (p < 0.003), yet stimulation of hyperactivation by 4-AP was strikingly more potent than either NH4Cl or TMA (in sEBSS cells, cf. NH4Cl (p < 10−7) and 20 mm TMA (p < 10−4); in STF cells, cf. NH4Cl (p < 10−14) and 20 mm TMA (p < 0.002)). 4-AP also raised pHo (∼1 unit), which was left uncorrected in order to enhance cytoplasmic entry of the drug (46, 47). Control experiments showed that elevation of pHo to 8.5 raised pHi by 0.13 ± 0.05 units (p < 0.005; n = 13, paired t test) but did not significantly increase spontaneous hyperactivation (p = 0.09; n = 20), 4-AP-induced hyperactivation (p = 0.17; n = 20), or 4-AP-induced alkalinization (p > 0.5; n = 6). 4-AP is commonly used as a K+ channel blocker, but the sperm K+ channel Slo3 shows very low sensitivity to the drug (44), and it does not depolarize human sperm,6 and the effects of 4-AP were not inhibited by clamping Em to EK with 1 μm valinomycin (data not shown). We conclude that the potent hyperactivating effect of 4-AP on human sperm is not exerted through alkalinization or depolarization of the cell.


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)

4-AP causes hyperactivation by mobilizing stored Ca2+.A, sustained increase in [Ca2+]i (OGB fluorescent intensity) in human sperm exposed to 2 mm 4-AP (first arrow). 4-AP was washed off at the second arrow. Shown are responses of eight cells in same experiment. B, release of Ca2+ from sacroplasmic reticulum microsomes by 4-AP. The graph shows 4-AP-induced Ca2+ release (percentage of A23187-releasable Ca2+); each point represents the mean ± S.E. (error bars) of 4–7 experiments. Inset, example trace. 2 mm 4-AP was added at the first arrow, and remaining Ca2+ was released by 25 μm A23187 (second arrow). Calibration shows time and [Ca2+]. C, effect of 2 mm 4-AP in Ca2+-depleted medium. Sperm were superfused with EGTA-buffered medium (first arrow), which caused a rapid fall in [Ca2+]i. Subsequent application of 4-AP caused a [Ca2+]i transient in a subset of cells. Shown are responses of 11 cells in the same experiment. D, time course of hyperactivation (percentage of cells; squares) induced by 2 mm 4-AP in cells suspended in sEBSS (filled symbols) and in parallel experiments where cells were resuspended in EGTA-buffered sEBSS immediately before use (open symbols). Circles show the percentage of motile cells. Each point shows mean ± S.E. of four experiments. E, sustained increase in [Ca2+]i (OGB fluorescent intensity) in human sperm exposed to 5 μm thimerosal (first arrow). Thimerosal was washed off at the second arrow, and 3 μm progesterone was applied at the third arrow. Shown are the responses of eight cells in the same experiment. Temperature was 30 °C. F, time course and concentration dependence of thimerosal-induced hyperactivation (percentage of cells). Each line shows mean ± S.E. of four experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: 4-AP causes hyperactivation by mobilizing stored Ca2+.A, sustained increase in [Ca2+]i (OGB fluorescent intensity) in human sperm exposed to 2 mm 4-AP (first arrow). 4-AP was washed off at the second arrow. Shown are responses of eight cells in same experiment. B, release of Ca2+ from sacroplasmic reticulum microsomes by 4-AP. The graph shows 4-AP-induced Ca2+ release (percentage of A23187-releasable Ca2+); each point represents the mean ± S.E. (error bars) of 4–7 experiments. Inset, example trace. 2 mm 4-AP was added at the first arrow, and remaining Ca2+ was released by 25 μm A23187 (second arrow). Calibration shows time and [Ca2+]. C, effect of 2 mm 4-AP in Ca2+-depleted medium. Sperm were superfused with EGTA-buffered medium (first arrow), which caused a rapid fall in [Ca2+]i. Subsequent application of 4-AP caused a [Ca2+]i transient in a subset of cells. Shown are responses of 11 cells in the same experiment. D, time course of hyperactivation (percentage of cells; squares) induced by 2 mm 4-AP in cells suspended in sEBSS (filled symbols) and in parallel experiments where cells were resuspended in EGTA-buffered sEBSS immediately before use (open symbols). Circles show the percentage of motile cells. Each point shows mean ± S.E. of four experiments. E, sustained increase in [Ca2+]i (OGB fluorescent intensity) in human sperm exposed to 5 μm thimerosal (first arrow). Thimerosal was washed off at the second arrow, and 3 μm progesterone was applied at the third arrow. Shown are the responses of eight cells in the same experiment. Temperature was 30 °C. F, time course and concentration dependence of thimerosal-induced hyperactivation (percentage of cells). Each line shows mean ± S.E. of four experiments.
Mentions: 4-Aminopyridine strongly hyperactivates mammalian sperm (28, 41), an effect that is correlated with fertilization rate in in vitro fertilization (42). Application of 2 mm 4-AP to cells bathed in sEBSS rapidly raised [Ca2+]i (ΔFmean = 32 ± 4%; n = 7 experiments; p < 0.0001; Fig. 3A) and recruited 26 ± 2% of cells into the hyperactivated population (n = 48; p < 10−19; Fig. 2, A and G). In cells suspended in STF, a similar hyperactivating effect of 4-AP was seen (n = 63; p < 10−33; Fig. 2, B–D). 4-AP is a weak base and in mouse sperm causes hyperpolarization of membrane potential (Em) (43) due to activation of a 4-AP-insensitive, pH-regulated K+ channel (44, 45). Hyperactivation might thus reflect alkalinization-induced activation of CatSper (28, 43). 2 mm 4-AP increased pHi by 0.27 ± 0.04 units, n = 7, an effect similar to that seen with 25 mm NH4Cl (p > 0.9) and only half that seen with 20 mm TMA (p < 0.003), yet stimulation of hyperactivation by 4-AP was strikingly more potent than either NH4Cl or TMA (in sEBSS cells, cf. NH4Cl (p < 10−7) and 20 mm TMA (p < 10−4); in STF cells, cf. NH4Cl (p < 10−14) and 20 mm TMA (p < 0.002)). 4-AP also raised pHo (∼1 unit), which was left uncorrected in order to enhance cytoplasmic entry of the drug (46, 47). Control experiments showed that elevation of pHo to 8.5 raised pHi by 0.13 ± 0.05 units (p < 0.005; n = 13, paired t test) but did not significantly increase spontaneous hyperactivation (p = 0.09; n = 20), 4-AP-induced hyperactivation (p = 0.17; n = 20), or 4-AP-induced alkalinization (p > 0.5; n = 6). 4-AP is commonly used as a K+ channel blocker, but the sperm K+ channel Slo3 shows very low sensitivity to the drug (44), and it does not depolarize human sperm,6 and the effects of 4-AP were not inhibited by clamping Em to EK with 1 μm valinomycin (data not shown). We conclude that the potent hyperactivating effect of 4-AP on human sperm is not exerted through alkalinization or depolarization of the cell.

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