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Simultaneous knockout of Slo3 and CatSper1 abolishes all alkalization- and voltage-activated current in mouse spermatozoa.

Zeng XH, Navarro B, Xia XM, Clapham DE, Lingle CJ - J. Gen. Physiol. (2013)

Bottom Line: Capacitation involves a sequence of changes in biochemical and electrical properties, the onset of a hyperactivated swimming behavior, and development of the ability to undergo successful fusion and penetration with an egg.After genetic deletion of the Slo3 gene, KSPER current is abolished, but there remains a small voltage-activated K(+) current hypothesized to reflect monovalent flux through CATSPER.Here, we address two questions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Life Science, Nanchang University, Nanchang, Jiangxi 330031, China.

ABSTRACT
During passage through the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that renders sperm competent to produce fertilization. Capacitation involves a sequence of changes in biochemical and electrical properties, the onset of a hyperactivated swimming behavior, and development of the ability to undergo successful fusion and penetration with an egg. In mouse sperm, the development of hyperactivated motility is dependent on cytosolic alkalization that then results in an increase in cytosolic Ca(2+). The elevation of Ca(2+) is thought to be primarily driven by the concerted interplay of two alkalization-activated currents, a K(+) current (KSPER) composed of pore-forming subunits encoded by the Kcnu1 gene (also termed Slo3) and a Ca(2+) current arising from a family of CATSPER subunits. After deletion of any of four CATSPER subunit genes (CATSPER1-4), the major remaining current in mouse sperm is alkalization-activated KSPER current. After genetic deletion of the Slo3 gene, KSPER current is abolished, but there remains a small voltage-activated K(+) current hypothesized to reflect monovalent flux through CATSPER. Here, we address two questions. First, does the residual outward K(+) current present in the Slo3 (-/-) sperm arise from CATSPER? Second, can any additional membrane K(+) currents be detected in mouse sperm by patch-clamp methods other than CATSPER and KSPER? Here, using mice bred to lack both SLO3 and CATSPER1 subunits, we show conclusively that the voltage-activated outward current present in Slo3 (-/-) sperm is abolished when CATSPER is also deleted. Any leak currents that may play a role in setting the resting membrane potential in noncapacitated sperm are likely smaller than the pipette leak current and thus cannot be resolved within the limitation of the patch-clamp technique. Together, KSPER and CATSPER appear to be the sole ion channels present in mouse sperm that regulate membrane potential and Ca(2+) influx in response to alkalization.

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Examples of ramp-activated currents recorded at high gain. (A) Currents activated over the range of −90 to 90 mV are shown at high gain for one sperm. The line corresponds to a linear fit to current over the range of −30 to 30 mV. (B) Similar currents and the linear fit are shown for a dKO sperm. The full ramp protocol is shown on the top. Currents were filtered at 2 kHz. (C) The sweep shows currents from the Slo3 −/− sperm with pHi 8.0 in A, but after subtraction of the linear component. The time base in A also applies to B–D and F. (D) Subtracted currents from the dKO sperm in B with pHi 8.0. (E) Filtered subtracted currents from one dKO sperm with pHi 6.0. (F) Currents from a dKO sperm at pHi 8.0 before and during application of 100 µM quinidine. The dashed lines represent 0 current level.
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fig3: Examples of ramp-activated currents recorded at high gain. (A) Currents activated over the range of −90 to 90 mV are shown at high gain for one sperm. The line corresponds to a linear fit to current over the range of −30 to 30 mV. (B) Similar currents and the linear fit are shown for a dKO sperm. The full ramp protocol is shown on the top. Currents were filtered at 2 kHz. (C) The sweep shows currents from the Slo3 −/− sperm with pHi 8.0 in A, but after subtraction of the linear component. The time base in A also applies to B–D and F. (D) Subtracted currents from the dKO sperm in B with pHi 8.0. (E) Filtered subtracted currents from one dKO sperm with pHi 6.0. (F) Currents from a dKO sperm at pHi 8.0 before and during application of 100 µM quinidine. The dashed lines represent 0 current level.

Mentions: As a final evaluation of this issue, we examined ramp-activated currents over the range of −90 to 90 mV at high gain (Fig. 3). Such traces were filtered at 2 kHz, and a linear leak current (Fig. 3, A and B) defined by the slope of the currents over the range of −30 to 30 mV was subtracted from the records. According to this procedure, asymmetries around the 0 current level would be potentially indicative of additional voltage-dependent conductances. Close inspection of raw traces suggested that in the dKO sperm, some sperm exhibited a small nonlinear increase in current at both the most negative and most positive voltages (Fig. 3, A and B). After filtering and subtraction, currents from Slo3 −/− sperm at pHi 8.0 (Fig. 3 C) show the upward curvature associated with monovalent flux through CATPSER channels, while also showing a small asymmetric increase in current variance at the most negative potentials. For dKO sperm at pHi 8.0 (Fig. 3 D), there remained some small asymmetric current at both positive and negative potentials, with the currents at negative potentials indistinguishable from Slo3 −/− sperm and from those in dKO sperm at pHi 6.0 (Fig. 3 E). Whatever the origins of this asymmetric increase in current, it is not only insensitive to pH but also to 100 µM quinidine (Fig. 3 F). We suspect that the small asymmetric currents at very negative and positive potentials may reflect recording instabilities (i.e., unstable seal leak conductance).


Simultaneous knockout of Slo3 and CatSper1 abolishes all alkalization- and voltage-activated current in mouse spermatozoa.

Zeng XH, Navarro B, Xia XM, Clapham DE, Lingle CJ - J. Gen. Physiol. (2013)

Examples of ramp-activated currents recorded at high gain. (A) Currents activated over the range of −90 to 90 mV are shown at high gain for one sperm. The line corresponds to a linear fit to current over the range of −30 to 30 mV. (B) Similar currents and the linear fit are shown for a dKO sperm. The full ramp protocol is shown on the top. Currents were filtered at 2 kHz. (C) The sweep shows currents from the Slo3 −/− sperm with pHi 8.0 in A, but after subtraction of the linear component. The time base in A also applies to B–D and F. (D) Subtracted currents from the dKO sperm in B with pHi 8.0. (E) Filtered subtracted currents from one dKO sperm with pHi 6.0. (F) Currents from a dKO sperm at pHi 8.0 before and during application of 100 µM quinidine. The dashed lines represent 0 current level.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3753610&req=5

fig3: Examples of ramp-activated currents recorded at high gain. (A) Currents activated over the range of −90 to 90 mV are shown at high gain for one sperm. The line corresponds to a linear fit to current over the range of −30 to 30 mV. (B) Similar currents and the linear fit are shown for a dKO sperm. The full ramp protocol is shown on the top. Currents were filtered at 2 kHz. (C) The sweep shows currents from the Slo3 −/− sperm with pHi 8.0 in A, but after subtraction of the linear component. The time base in A also applies to B–D and F. (D) Subtracted currents from the dKO sperm in B with pHi 8.0. (E) Filtered subtracted currents from one dKO sperm with pHi 6.0. (F) Currents from a dKO sperm at pHi 8.0 before and during application of 100 µM quinidine. The dashed lines represent 0 current level.
Mentions: As a final evaluation of this issue, we examined ramp-activated currents over the range of −90 to 90 mV at high gain (Fig. 3). Such traces were filtered at 2 kHz, and a linear leak current (Fig. 3, A and B) defined by the slope of the currents over the range of −30 to 30 mV was subtracted from the records. According to this procedure, asymmetries around the 0 current level would be potentially indicative of additional voltage-dependent conductances. Close inspection of raw traces suggested that in the dKO sperm, some sperm exhibited a small nonlinear increase in current at both the most negative and most positive voltages (Fig. 3, A and B). After filtering and subtraction, currents from Slo3 −/− sperm at pHi 8.0 (Fig. 3 C) show the upward curvature associated with monovalent flux through CATPSER channels, while also showing a small asymmetric increase in current variance at the most negative potentials. For dKO sperm at pHi 8.0 (Fig. 3 D), there remained some small asymmetric current at both positive and negative potentials, with the currents at negative potentials indistinguishable from Slo3 −/− sperm and from those in dKO sperm at pHi 6.0 (Fig. 3 E). Whatever the origins of this asymmetric increase in current, it is not only insensitive to pH but also to 100 µM quinidine (Fig. 3 F). We suspect that the small asymmetric currents at very negative and positive potentials may reflect recording instabilities (i.e., unstable seal leak conductance).

Bottom Line: Capacitation involves a sequence of changes in biochemical and electrical properties, the onset of a hyperactivated swimming behavior, and development of the ability to undergo successful fusion and penetration with an egg.After genetic deletion of the Slo3 gene, KSPER current is abolished, but there remains a small voltage-activated K(+) current hypothesized to reflect monovalent flux through CATSPER.Here, we address two questions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Life Science, Nanchang University, Nanchang, Jiangxi 330031, China.

ABSTRACT
During passage through the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that renders sperm competent to produce fertilization. Capacitation involves a sequence of changes in biochemical and electrical properties, the onset of a hyperactivated swimming behavior, and development of the ability to undergo successful fusion and penetration with an egg. In mouse sperm, the development of hyperactivated motility is dependent on cytosolic alkalization that then results in an increase in cytosolic Ca(2+). The elevation of Ca(2+) is thought to be primarily driven by the concerted interplay of two alkalization-activated currents, a K(+) current (KSPER) composed of pore-forming subunits encoded by the Kcnu1 gene (also termed Slo3) and a Ca(2+) current arising from a family of CATSPER subunits. After deletion of any of four CATSPER subunit genes (CATSPER1-4), the major remaining current in mouse sperm is alkalization-activated KSPER current. After genetic deletion of the Slo3 gene, KSPER current is abolished, but there remains a small voltage-activated K(+) current hypothesized to reflect monovalent flux through CATSPER. Here, we address two questions. First, does the residual outward K(+) current present in the Slo3 (-/-) sperm arise from CATSPER? Second, can any additional membrane K(+) currents be detected in mouse sperm by patch-clamp methods other than CATSPER and KSPER? Here, using mice bred to lack both SLO3 and CATSPER1 subunits, we show conclusively that the voltage-activated outward current present in Slo3 (-/-) sperm is abolished when CATSPER is also deleted. Any leak currents that may play a role in setting the resting membrane potential in noncapacitated sperm are likely smaller than the pipette leak current and thus cannot be resolved within the limitation of the patch-clamp technique. Together, KSPER and CATSPER appear to be the sole ion channels present in mouse sperm that regulate membrane potential and Ca(2+) influx in response to alkalization.

Show MeSH
Related in: MedlinePlus