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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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Spatio-temporal [Ca2+]i dynamics in a single sperm treated with acrosome reaction inducers. Capacitated sperm were loaded with 4 μM fluo4-AM for 15 min, and [Ca2+]i mobilization at the single-sperm level was monitored. Sperm from PLCδ4+/+ (A, C, and E) or PLCδ4−/− (B, D, and F) were treated with 3 Zp/μl solubilized mouse ZP (A and B), 100 μM progesterone (C and D), or 5 μM thapsigargin (E and F). Images were collected every 2 s. The relative [Ca2+]i change is calculated as a ratio to F0 images and shown in pseudo-color. Bar, 10 μm. Figures show time (sec) after treatment of the reagents.
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fig5: Spatio-temporal [Ca2+]i dynamics in a single sperm treated with acrosome reaction inducers. Capacitated sperm were loaded with 4 μM fluo4-AM for 15 min, and [Ca2+]i mobilization at the single-sperm level was monitored. Sperm from PLCδ4+/+ (A, C, and E) or PLCδ4−/− (B, D, and F) were treated with 3 Zp/μl solubilized mouse ZP (A and B), 100 μM progesterone (C and D), or 5 μM thapsigargin (E and F). Images were collected every 2 s. The relative [Ca2+]i change is calculated as a ratio to F0 images and shown in pseudo-color. 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)

Spatio-temporal [Ca2+]i dynamics in a single sperm treated with acrosome reaction inducers. Capacitated sperm were loaded with 4 μM fluo4-AM for 15 min, and [Ca2+]i mobilization at the single-sperm level was monitored. Sperm from PLCδ4+/+ (A, C, and E) or PLCδ4−/− (B, D, and F) were treated with 3 Zp/μl solubilized mouse ZP (A and B), 100 μM progesterone (C and D), or 5 μM thapsigargin (E and F). Images were collected every 2 s. The relative [Ca2+]i change is calculated as a ratio to F0 images and shown in pseudo-color. Bar, 10 μm. Figures show time (sec) after treatment of the reagents.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Spatio-temporal [Ca2+]i dynamics in a single sperm treated with acrosome reaction inducers. Capacitated sperm were loaded with 4 μM fluo4-AM for 15 min, and [Ca2+]i mobilization at the single-sperm level was monitored. Sperm from PLCδ4+/+ (A, C, and E) or PLCδ4−/− (B, D, and F) were treated with 3 Zp/μl solubilized mouse ZP (A and B), 100 μM progesterone (C and D), or 5 μM thapsigargin (E and F). Images were collected every 2 s. The relative [Ca2+]i change is calculated as a ratio to F0 images and shown in pseudo-color. 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