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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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SOC activity is impaired in PLCδ4−/− sperm. (A) PLCδ4+/+ and PLCδ4−/− sperm were incubated in Ca2+-free HS buffer containing 1 mM EGTA; the Ca2+ stores were depleted by treatment with 5 μM thapsigargin (TG). SOC activity was measured by addition of 1 mM extracellular Ca2+. Ca2+ patterns of two representative sperm responses are shown. The arrowhead denoted the time of acrosome reaction. (B) Changes in [Ca2+]i increases in different areas within a sperm are shown by pseudo-color images and time course plots. Two spots of post-acrosome (1) and equatorial segment (2) were selected for images and time course plots. The images shown were collected 6 s after addition of thapsigargin (TG) and immediately after (2 s) addition of 1 mM extracellular Ca2+ (Ca2+). The fluorescence intensity of images and plots is shown with different ratio scales.
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fig8: SOC activity is impaired in PLCδ4−/− sperm. (A) PLCδ4+/+ and PLCδ4−/− sperm were incubated in Ca2+-free HS buffer containing 1 mM EGTA; the Ca2+ stores were depleted by treatment with 5 μM thapsigargin (TG). SOC activity was measured by addition of 1 mM extracellular Ca2+. Ca2+ patterns of two representative sperm responses are shown. The arrowhead denoted the time of acrosome reaction. (B) Changes in [Ca2+]i increases in different areas within a sperm are shown by pseudo-color images and time course plots. Two spots of post-acrosome (1) and equatorial segment (2) were selected for images and time course plots. The images shown were collected 6 s after addition of thapsigargin (TG) and immediately after (2 s) addition of 1 mM extracellular Ca2+ (Ca2+). The fluorescence intensity of images and plots is shown with different ratio scales.

Mentions: Having noticed a difference in the amplitude of the [Ca2+]i increase between PLCδ4+/+ and PLCδ4−/− sperm in response to thapsigargin, we examined directly whether SOC activity was deficient in PLCδ4−/− sperm. To evaluate SOC activity, the sperm's Ca2+ stores were depleted by exposure to thapsigargin in Ca2+-free medium, followed by the addition of 1 mM extracellular Ca2+ (Fig. 8 A). The addition of thapsigargin induced a small [Ca2+]i increase in both wild-type and mutant sperm, suggesting that the content of the Ca2+ stores is comparable in both sperm populations. However, addition of extracellular Ca2+ induced a large Ca2+ influx and [Ca2+]i increase in PLCδ4+/+ sperm that decreased after driving the acrosome reaction (Fig. 8 A, top, arrowhead), whereas it produced a weak and transient [Ca2+]i increase in PLCδ4−/− sperm, demonstrating that the absence of PLCδ4 significantly affected the function of SOC channels.


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)

SOC activity is impaired in PLCδ4−/− sperm. (A) PLCδ4+/+ and PLCδ4−/− sperm were incubated in Ca2+-free HS buffer containing 1 mM EGTA; the Ca2+ stores were depleted by treatment with 5 μM thapsigargin (TG). SOC activity was measured by addition of 1 mM extracellular Ca2+. Ca2+ patterns of two representative sperm responses are shown. The arrowhead denoted the time of acrosome reaction. (B) Changes in [Ca2+]i increases in different areas within a sperm are shown by pseudo-color images and time course plots. Two spots of post-acrosome (1) and equatorial segment (2) were selected for images and time course plots. The images shown were collected 6 s after addition of thapsigargin (TG) and immediately after (2 s) addition of 1 mM extracellular Ca2+ (Ca2+). The fluorescence intensity of images and plots is shown with different ratio scales.
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Related In: Results  -  Collection

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

fig8: SOC activity is impaired in PLCδ4−/− sperm. (A) PLCδ4+/+ and PLCδ4−/− sperm were incubated in Ca2+-free HS buffer containing 1 mM EGTA; the Ca2+ stores were depleted by treatment with 5 μM thapsigargin (TG). SOC activity was measured by addition of 1 mM extracellular Ca2+. Ca2+ patterns of two representative sperm responses are shown. The arrowhead denoted the time of acrosome reaction. (B) Changes in [Ca2+]i increases in different areas within a sperm are shown by pseudo-color images and time course plots. Two spots of post-acrosome (1) and equatorial segment (2) were selected for images and time course plots. The images shown were collected 6 s after addition of thapsigargin (TG) and immediately after (2 s) addition of 1 mM extracellular Ca2+ (Ca2+). The fluorescence intensity of images and plots is shown with different ratio scales.
Mentions: Having noticed a difference in the amplitude of the [Ca2+]i increase between PLCδ4+/+ and PLCδ4−/− sperm in response to thapsigargin, we examined directly whether SOC activity was deficient in PLCδ4−/− sperm. To evaluate SOC activity, the sperm's Ca2+ stores were depleted by exposure to thapsigargin in Ca2+-free medium, followed by the addition of 1 mM extracellular Ca2+ (Fig. 8 A). The addition of thapsigargin induced a small [Ca2+]i increase in both wild-type and mutant sperm, suggesting that the content of the Ca2+ stores is comparable in both sperm populations. However, addition of extracellular Ca2+ induced a large Ca2+ influx and [Ca2+]i increase in PLCδ4+/+ sperm that decreased after driving the acrosome reaction (Fig. 8 A, top, arrowhead), whereas it produced a weak and transient [Ca2+]i increase in PLCδ4−/− sperm, demonstrating that the absence of PLCδ4 significantly affected the function of SOC channels.

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