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Phospholipase Cdelta4 is required for Ca2+ mobilization essential for acrosome reaction in sperm.

Fukami K, Yoshida M, Inoue T, Kurokawa M, Fissore RA, Yoshida N, Mikoshiba K, Takenawa T - J. Cell Biol. (2003)

Bottom Line: Progesterone, another physiological inducer of the acrosome reaction, failed to induce sustained [Ca2+]i increases in PLCdelta4-/- sperm, and consequently the acrosome reaction was partially inhibited.Furthermore, store-operated channel (SOC) activity was severely impaired in PLCdelta4-/- sperm.These results indicate that PLCdelta4 is an important enzyme for intracellular [Ca2+]i mobilization in the ZP-induced acrosome reaction and for sustained [Ca2+]i increases through SOC induced by ZP and progesterone in sperm.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan. kfukami@ims.u-tokyo.ac.jp

ABSTRACT
Zona pellucida (ZP)-induced acrosome reaction in sperm is a required step for mammalian fertilization. However, the precise mechanism of the acrosome reaction remains unclear. We previously reported that PLCdelta4 is involved in the ZP-induced acrosome reaction in mouse sperm. Here we have monitored Ca2+ responses in single sperm, and we report that the [Ca2+]i increase in response to ZP, which is essential for driving the acrosome reaction in vivo, is absent in PLCdelta4-/- sperm. Progesterone, another physiological inducer of the acrosome reaction, failed to induce sustained [Ca2+]i increases in PLCdelta4-/- sperm, and consequently the acrosome reaction was partially inhibited. In addition, we observed oscillatory [Ca2+]i increases in wild-type sperm in response to these acrosome inducers. Calcium imaging studies revealed that the [Ca2+]i increases induced by exposure to ZP and progesterone started at different sites within the sperm head, indicating that these agonists induce the acrosome reaction via different Ca2+ mechanisms. Furthermore, store-operated channel (SOC) activity was severely impaired in PLCdelta4-/- sperm. These results indicate that PLCdelta4 is an important enzyme for intracellular [Ca2+]i mobilization in the ZP-induced acrosome reaction and for sustained [Ca2+]i increases through SOC induced by ZP and progesterone in sperm.

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Changes in cholesterol content and tyrosine phosphorylation during capacitation. (A) Decrease in cholesterol content during capacitation is shown as percent decrease compared with the levels before capacitation. Approximately 2 × 106 sperm from PLCδ4+/+ (+/+) and PLCδ4−/− (−/−) mice were capacitated in HS buffer for the indicated times, and the content of cholesterol was measured as described in the Materials and methods. The data represent the average of four independent experiments. Error bars show standard errors. (B) Levels of tyrosine phosphorylation during capacitation. The sperm were capacitated for the indicated times and subjected to a 7.5% SDS-PAGE, and Western blot analysis was performed using the antibody 4G10. Arrowheads show the specific proteins that underwent tyrosine phosphorylation.
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fig3: Changes in cholesterol content and tyrosine phosphorylation during capacitation. (A) Decrease in cholesterol content during capacitation is shown as percent decrease compared with the levels before capacitation. Approximately 2 × 106 sperm from PLCδ4+/+ (+/+) and PLCδ4−/− (−/−) mice were capacitated in HS buffer for the indicated times, and the content of cholesterol was measured as described in the Materials and methods. The data represent the average of four independent experiments. Error bars show standard errors. (B) Levels of tyrosine phosphorylation during capacitation. The sperm were capacitated for the indicated times and subjected to a 7.5% SDS-PAGE, and Western blot analysis was performed using the antibody 4G10. Arrowheads show the specific proteins that underwent tyrosine phosphorylation.

Mentions: Before successful acrosome reaction can take place, the sperm have to undergo capacitation, a series of poorly defined molecular and biochemical changes including efflux of cholesterol, changes in the phospholipid content of the plasma membrane, hyperpolarization, and tyrosine phosphorylation (Arnoult et al., 1996; Nolan and Hammerstedt, 1997; Darszon et al., 2001). Capacitation occurs in vivo during sperm transit through the female tract and is mimicked in vitro by incubation of epididymal sperm in medium containing HCO3−, BSA, and Ca2+ (Breitbart, 2002). Several putative assays, such as assessment of cholesterol efflux, cAMP concentration, and protein tyrosine phosphorylation, can be used to know whether or not capacitation has occurred (Visconti et al., 1995, 1999; Iborra et al., 2000). To determine whether defects in capacitation may explain, at least in part, the abnormal acrosome reaction in PLCδ4−/− sperm, we measured the content of cholesterol during capacitation. The decrease in cholesterol levels began to be observed 15 min after capacitation in PLCδ4+/+ sperm and by 30 min in PLCδ4−/− sperm, although the overall decrease in cholesterol content by 90 min of capacitation was similar in both groups (Fig. 3 A). In addition, time-dependent specific protein tyrosine phosphorylation began to be detected in both PLCδ4+/+ and PLCδ4−/− after ∼60 min of incubation (Fig. 3 B, arrowheads). From these data, we conclude that PLCδ4 is likely not involved in the capacitation process.


Phospholipase Cdelta4 is required for Ca2+ mobilization essential for acrosome reaction in sperm.

Fukami K, Yoshida M, Inoue T, Kurokawa M, Fissore RA, Yoshida N, Mikoshiba K, Takenawa T - J. Cell Biol. (2003)

Changes in cholesterol content and tyrosine phosphorylation during capacitation. (A) Decrease in cholesterol content during capacitation is shown as percent decrease compared with the levels before capacitation. Approximately 2 × 106 sperm from PLCδ4+/+ (+/+) and PLCδ4−/− (−/−) mice were capacitated in HS buffer for the indicated times, and the content of cholesterol was measured as described in the Materials and methods. The data represent the average of four independent experiments. Error bars show standard errors. (B) Levels of tyrosine phosphorylation during capacitation. The sperm were capacitated for the indicated times and subjected to a 7.5% SDS-PAGE, and Western blot analysis was performed using the antibody 4G10. Arrowheads show the specific proteins that underwent tyrosine phosphorylation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172882&req=5

fig3: Changes in cholesterol content and tyrosine phosphorylation during capacitation. (A) Decrease in cholesterol content during capacitation is shown as percent decrease compared with the levels before capacitation. Approximately 2 × 106 sperm from PLCδ4+/+ (+/+) and PLCδ4−/− (−/−) mice were capacitated in HS buffer for the indicated times, and the content of cholesterol was measured as described in the Materials and methods. The data represent the average of four independent experiments. Error bars show standard errors. (B) Levels of tyrosine phosphorylation during capacitation. The sperm were capacitated for the indicated times and subjected to a 7.5% SDS-PAGE, and Western blot analysis was performed using the antibody 4G10. Arrowheads show the specific proteins that underwent tyrosine phosphorylation.
Mentions: Before successful acrosome reaction can take place, the sperm have to undergo capacitation, a series of poorly defined molecular and biochemical changes including efflux of cholesterol, changes in the phospholipid content of the plasma membrane, hyperpolarization, and tyrosine phosphorylation (Arnoult et al., 1996; Nolan and Hammerstedt, 1997; Darszon et al., 2001). Capacitation occurs in vivo during sperm transit through the female tract and is mimicked in vitro by incubation of epididymal sperm in medium containing HCO3−, BSA, and Ca2+ (Breitbart, 2002). Several putative assays, such as assessment of cholesterol efflux, cAMP concentration, and protein tyrosine phosphorylation, can be used to know whether or not capacitation has occurred (Visconti et al., 1995, 1999; Iborra et al., 2000). To determine whether defects in capacitation may explain, at least in part, the abnormal acrosome reaction in PLCδ4−/− sperm, we measured the content of cholesterol during capacitation. The decrease in cholesterol levels began to be observed 15 min after capacitation in PLCδ4+/+ sperm and by 30 min in PLCδ4−/− sperm, although the overall decrease in cholesterol content by 90 min of capacitation was similar in both groups (Fig. 3 A). In addition, time-dependent specific protein tyrosine phosphorylation began to be detected in both PLCδ4+/+ and PLCδ4−/− after ∼60 min of incubation (Fig. 3 B, arrowheads). From these data, we conclude that PLCδ4 is likely not involved in the capacitation process.

Bottom Line: Progesterone, another physiological inducer of the acrosome reaction, failed to induce sustained [Ca2+]i increases in PLCdelta4-/- sperm, and consequently the acrosome reaction was partially inhibited.Furthermore, store-operated channel (SOC) activity was severely impaired in PLCdelta4-/- sperm.These results indicate that PLCdelta4 is an important enzyme for intracellular [Ca2+]i mobilization in the ZP-induced acrosome reaction and for sustained [Ca2+]i increases through SOC induced by ZP and progesterone in sperm.

View Article: PubMed Central - PubMed

Affiliation: Division of Biochemistry, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan. kfukami@ims.u-tokyo.ac.jp

ABSTRACT
Zona pellucida (ZP)-induced acrosome reaction in sperm is a required step for mammalian fertilization. However, the precise mechanism of the acrosome reaction remains unclear. We previously reported that PLCdelta4 is involved in the ZP-induced acrosome reaction in mouse sperm. Here we have monitored Ca2+ responses in single sperm, and we report that the [Ca2+]i increase in response to ZP, which is essential for driving the acrosome reaction in vivo, is absent in PLCdelta4-/- sperm. Progesterone, another physiological inducer of the acrosome reaction, failed to induce sustained [Ca2+]i increases in PLCdelta4-/- sperm, and consequently the acrosome reaction was partially inhibited. In addition, we observed oscillatory [Ca2+]i increases in wild-type sperm in response to these acrosome inducers. Calcium imaging studies revealed that the [Ca2+]i increases induced by exposure to ZP and progesterone started at different sites within the sperm head, indicating that these agonists induce the acrosome reaction via different Ca2+ mechanisms. Furthermore, store-operated channel (SOC) activity was severely impaired in PLCdelta4-/- sperm. These results indicate that PLCdelta4 is an important enzyme for intracellular [Ca2+]i mobilization in the ZP-induced acrosome reaction and for sustained [Ca2+]i increases through SOC induced by ZP and progesterone in sperm.

Show MeSH
Related in: MedlinePlus