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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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Magnification of the initial images of sperm treated with acrosome reaction inducers. Sperm from PLCδ4+/+ (A, C, and E) or PLCδ4−/− (B, D, and F) were treated with ZP (A and B), progesterone (C and D), or thapsigargin (E and F). Bar, 10 μm. Figures show time (sec) after treatment of the reagents.
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fig6: Magnification of the initial images of sperm treated with acrosome reaction inducers. Sperm from PLCδ4+/+ (A, C, and E) or PLCδ4−/− (B, D, and F) were treated with ZP (A and B), progesterone (C and D), or thapsigargin (E and F). Bar, 10 μm. Figures show time (sec) after treatment of the reagents.

Mentions: Typical [Ca2+]i increase patterns of sperm in response to ZP, progesterone, or thapsigargin over time are shown in Figs. 5 and 6 and Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200210057/DC1). When PLCδ4+/+ sperm preloaded with fluo-4 were treated with ZP, the [Ca2+]i increase was first detected in the acrosome area (2 s), then in the equatorial segment (4 s), and finally extended to the whole sperm head (around 14 s) (Fig. 6 A). Although there are a few reports that describe the global [Ca2+]i elevations in bovine or hamster sperm head during the ZP-induced acrosome reaction (Florman, 1994; Shirakawa and Miyazaki, 1999), this is the first observation that clearly notes the sequential [Ca2+]i mobilization induced by ZP. These data support the notion that the acrosome vesicle serves as the intracellular Ca2+ store and that ZP mobilizes [Ca2+]i before promoting Ca2+ influx during acrosome reaction. Interestingly, the acrosome reaction always occurred after peak [Ca2+]i values were attained, and the occurrence of the reaction was confirmed by simultaneous monitoring of fluo-4 and Alexa Fluor®594–labeled SBTI (unpublished data). As expected, only a minor [Ca2+]i increase was detected in PLCδ4−/− sperm after the addition of ZP (Fig. 5 B; Fig. 6 B).


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)

Magnification of the initial images of sperm treated with acrosome reaction inducers. Sperm from PLCδ4+/+ (A, C, and E) or PLCδ4−/− (B, D, and F) were treated with ZP (A and B), progesterone (C and D), or thapsigargin (E and F). Bar, 10 μm. Figures show time (sec) after treatment of the reagents.
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Related In: Results  -  Collection

Show All Figures
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fig6: Magnification of the initial images of sperm treated with acrosome reaction inducers. Sperm from PLCδ4+/+ (A, C, and E) or PLCδ4−/− (B, D, and F) were treated with ZP (A and B), progesterone (C and D), or thapsigargin (E and F). Bar, 10 μm. Figures show time (sec) after treatment of the reagents.
Mentions: Typical [Ca2+]i increase patterns of sperm in response to ZP, progesterone, or thapsigargin over time are shown in Figs. 5 and 6 and Video 1 (available at http://www.jcb.org/cgi/content/full/jcb.200210057/DC1). When PLCδ4+/+ sperm preloaded with fluo-4 were treated with ZP, the [Ca2+]i increase was first detected in the acrosome area (2 s), then in the equatorial segment (4 s), and finally extended to the whole sperm head (around 14 s) (Fig. 6 A). Although there are a few reports that describe the global [Ca2+]i elevations in bovine or hamster sperm head during the ZP-induced acrosome reaction (Florman, 1994; Shirakawa and Miyazaki, 1999), this is the first observation that clearly notes the sequential [Ca2+]i mobilization induced by ZP. These data support the notion that the acrosome vesicle serves as the intracellular Ca2+ store and that ZP mobilizes [Ca2+]i before promoting Ca2+ influx during acrosome reaction. Interestingly, the acrosome reaction always occurred after peak [Ca2+]i values were attained, and the occurrence of the reaction was confirmed by simultaneous monitoring of fluo-4 and Alexa Fluor®594–labeled SBTI (unpublished data). As expected, only a minor [Ca2+]i increase was detected in PLCδ4−/− sperm after the addition of ZP (Fig. 5 B; Fig. 6 B).

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