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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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CatSper activity enhances penetration into viscous medium and contributes to spontaneous hyperactivation.A and B, increment in cell density (percentage of control) 1 cm (A) and 2 cm (B) into methylcellulose. Cells were stimulated with 25 mm NH4Cl (dark blue), 10 mm TMA (light blue), 2 mm 4-AP (green), 3 μm progesterone (yellow), and 5 μm thimerosal (red). Bars show the mean ± S.E. (error bars) of 10–20 experiments except for TMA (4). C, increment in cell density (percentage of control) 2 cm into methylcellulose of cells stimulated with 2 mm 4-AP (green), 3 μm progesterone (yellow), and 25 mm NH4Cl (dark blue) and in parallel incubations pretreated with 10 μm NNC (black). Bars, mean ± S.E. of 8–20 experiments except for TMA (4). D, inhibition of spontaneous hyperactivation upon exposure of STF-capacitated cells to NNC (difference between control and NNC-treated cells; μm NNC) is dependent upon the level of spontaneous hyperactivation prior to application of the drug (r = 0.75, n = 19). E, increment in hyperactivation (percentage of cells) in response to 2 mm 4-AP (green), 3 μm progesterone (yellow), 25 mm NH4Cl (dark blue), and 5 μm thimerosal (red). Black bars, responses in parallel 10 μm NNC-pretreated experiments. Each bar shows mean ± S.E. of 9–20 experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 compared with control (A and B) or NNC alone (C and E). NS, not significant.
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Figure 4: CatSper activity enhances penetration into viscous medium and contributes to spontaneous hyperactivation.A and B, increment in cell density (percentage of control) 1 cm (A) and 2 cm (B) into methylcellulose. Cells were stimulated with 25 mm NH4Cl (dark blue), 10 mm TMA (light blue), 2 mm 4-AP (green), 3 μm progesterone (yellow), and 5 μm thimerosal (red). Bars show the mean ± S.E. (error bars) of 10–20 experiments except for TMA (4). C, increment in cell density (percentage of control) 2 cm into methylcellulose of cells stimulated with 2 mm 4-AP (green), 3 μm progesterone (yellow), and 25 mm NH4Cl (dark blue) and in parallel incubations pretreated with 10 μm NNC (black). Bars, mean ± S.E. of 8–20 experiments except for TMA (4). D, inhibition of spontaneous hyperactivation upon exposure of STF-capacitated cells to NNC (difference between control and NNC-treated cells; μm NNC) is dependent upon the level of spontaneous hyperactivation prior to application of the drug (r = 0.75, n = 19). E, increment in hyperactivation (percentage of cells) in response to 2 mm 4-AP (green), 3 μm progesterone (yellow), 25 mm NH4Cl (dark blue), and 5 μm thimerosal (red). Black bars, responses in parallel 10 μm NNC-pretreated experiments. Each bar shows mean ± S.E. of 9–20 experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 compared with control (A and B) or NNC alone (C and E). NS, not significant.

Mentions: A crucial aspect of sperm motility is the ability to penetrate viscous medium. To assess the significance of CatSper and store-mediated [Ca2+]o signals in regulating this aspect of sperm behavior, we assessed penetration of STF-prepared sperm into methylcellulose. In this functional test, efficacy of the various stimuli was reversed compared with their ability to induce hyperactivated motility. 25 mm NH4Cl and 10 mm TMA, 2 mm 4-AP, and 3 μm progesterone all enhanced sperm penetration into methylcellulose medium, cell numbers at 1 and 2 cm being significantly increased over those in parallel controls (Fig. 4, A and B). 20 mm TMA was less effective (p > 0.5; n = 3). In contrast, 5 μm thimerosal, the most powerful inducer of hyperactivated motility (see above), caused negligible enhancement of penetration into methylcellulose (Fig. 4, A and B). To distinguish between effects on penetration into and progression through viscous medium, we assessed kinematics of cells swimming in methylcellulose. Similarly to rodent sperm (54), methylcellulose greatly reduced speed and lateral head movement. Inclusion of 5 μm thimerosal in the methylcellulose significantly enhanced ALH and decreased beat frequency (p < 0.05; n = 6), but this effect was small. None of the stimuli caused a significant change in speed of progression through methylcellulose (data not shown), showing that stimulation of CatSper activity, either directly (progesterone) or via pHi elevation, enhances entry of sperm into methylcellulose rather than their progress through it.


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)

CatSper activity enhances penetration into viscous medium and contributes to spontaneous hyperactivation.A and B, increment in cell density (percentage of control) 1 cm (A) and 2 cm (B) into methylcellulose. Cells were stimulated with 25 mm NH4Cl (dark blue), 10 mm TMA (light blue), 2 mm 4-AP (green), 3 μm progesterone (yellow), and 5 μm thimerosal (red). Bars show the mean ± S.E. (error bars) of 10–20 experiments except for TMA (4). C, increment in cell density (percentage of control) 2 cm into methylcellulose of cells stimulated with 2 mm 4-AP (green), 3 μm progesterone (yellow), and 25 mm NH4Cl (dark blue) and in parallel incubations pretreated with 10 μm NNC (black). Bars, mean ± S.E. of 8–20 experiments except for TMA (4). D, inhibition of spontaneous hyperactivation upon exposure of STF-capacitated cells to NNC (difference between control and NNC-treated cells; μm NNC) is dependent upon the level of spontaneous hyperactivation prior to application of the drug (r = 0.75, n = 19). E, increment in hyperactivation (percentage of cells) in response to 2 mm 4-AP (green), 3 μm progesterone (yellow), 25 mm NH4Cl (dark blue), and 5 μm thimerosal (red). Black bars, responses in parallel 10 μm NNC-pretreated experiments. Each bar shows mean ± S.E. of 9–20 experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 compared with control (A and B) or NNC alone (C and E). NS, not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: CatSper activity enhances penetration into viscous medium and contributes to spontaneous hyperactivation.A and B, increment in cell density (percentage of control) 1 cm (A) and 2 cm (B) into methylcellulose. Cells were stimulated with 25 mm NH4Cl (dark blue), 10 mm TMA (light blue), 2 mm 4-AP (green), 3 μm progesterone (yellow), and 5 μm thimerosal (red). Bars show the mean ± S.E. (error bars) of 10–20 experiments except for TMA (4). C, increment in cell density (percentage of control) 2 cm into methylcellulose of cells stimulated with 2 mm 4-AP (green), 3 μm progesterone (yellow), and 25 mm NH4Cl (dark blue) and in parallel incubations pretreated with 10 μm NNC (black). Bars, mean ± S.E. of 8–20 experiments except for TMA (4). D, inhibition of spontaneous hyperactivation upon exposure of STF-capacitated cells to NNC (difference between control and NNC-treated cells; μm NNC) is dependent upon the level of spontaneous hyperactivation prior to application of the drug (r = 0.75, n = 19). E, increment in hyperactivation (percentage of cells) in response to 2 mm 4-AP (green), 3 μm progesterone (yellow), 25 mm NH4Cl (dark blue), and 5 μm thimerosal (red). Black bars, responses in parallel 10 μm NNC-pretreated experiments. Each bar shows mean ± S.E. of 9–20 experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 compared with control (A and B) or NNC alone (C and E). NS, not significant.
Mentions: A crucial aspect of sperm motility is the ability to penetrate viscous medium. To assess the significance of CatSper and store-mediated [Ca2+]o signals in regulating this aspect of sperm behavior, we assessed penetration of STF-prepared sperm into methylcellulose. In this functional test, efficacy of the various stimuli was reversed compared with their ability to induce hyperactivated motility. 25 mm NH4Cl and 10 mm TMA, 2 mm 4-AP, and 3 μm progesterone all enhanced sperm penetration into methylcellulose medium, cell numbers at 1 and 2 cm being significantly increased over those in parallel controls (Fig. 4, A and B). 20 mm TMA was less effective (p > 0.5; n = 3). In contrast, 5 μm thimerosal, the most powerful inducer of hyperactivated motility (see above), caused negligible enhancement of penetration into methylcellulose (Fig. 4, A and B). To distinguish between effects on penetration into and progression through viscous medium, we assessed kinematics of cells swimming in methylcellulose. Similarly to rodent sperm (54), methylcellulose greatly reduced speed and lateral head movement. Inclusion of 5 μm thimerosal in the methylcellulose significantly enhanced ALH and decreased beat frequency (p < 0.05; n = 6), but this effect was small. None of the stimuli caused a significant change in speed of progression through methylcellulose (data not shown), showing that stimulation of CatSper activity, either directly (progesterone) or via pHi elevation, enhances entry of sperm into methylcellulose rather than their progress through it.

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