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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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Requirement of PLCδ4 in ZP- and progesterone-induced acrosome reaction. (A) Fluorescence and phase images of sperm treated with several acrosome reaction–inducing reagents. PLCδ4+/+ (+/+) and PLCδ4−/− (−/−) sperm capacitated for 1 h were treated with 1.0% DMSO, 3 Zp/μl solubilized mouse ZP (ZP), 100 μM progesterone (PG), or 5 μM thapsigargin (TG) for 15 min at 32°C in the presence of 2 μM Alexa Fluor®594–conjugated SBTI to monitor the occurrence of the acrosome reaction. The same microscopic fields are shown for acrosome reaction (AR) and bright field images. Bar, 10 μm. (B) Percent of sperm that underwent the acrosome reaction. The numbers above each column represent the number of sperm examined. Data were obtained from total amount of sperm by four independent experiments for progesterone and ZP and three independent experiments for thapsigargin.
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fig2: Requirement of PLCδ4 in ZP- and progesterone-induced acrosome reaction. (A) Fluorescence and phase images of sperm treated with several acrosome reaction–inducing reagents. PLCδ4+/+ (+/+) and PLCδ4−/− (−/−) sperm capacitated for 1 h were treated with 1.0% DMSO, 3 Zp/μl solubilized mouse ZP (ZP), 100 μM progesterone (PG), or 5 μM thapsigargin (TG) for 15 min at 32°C in the presence of 2 μM Alexa Fluor®594–conjugated SBTI to monitor the occurrence of the acrosome reaction. The same microscopic fields are shown for acrosome reaction (AR) and bright field images. Bar, 10 μm. (B) Percent of sperm that underwent the acrosome reaction. The numbers above each column represent the number of sperm examined. Data were obtained from total amount of sperm by four independent experiments for progesterone and ZP and three independent experiments for thapsigargin.

Mentions: We have reported that PLCδ4 is an essential protein for ZP-induced acrosome reaction. To understand how PLCδ4 is involved in this process, we first examined whether PLCδ4 is required for the acrosome reaction induced by progesterone and thapsigargin. A soybean trypsin inhibitor (SBTI) was used to monitor the acrosome reaction. SBTI is known to tightly bind to acrosin (Tollner et al., 2000), which is located on the inner acrosomal membrane and only becomes exposed extracellularly after the acrosome reaction; therefore, we could easily recognize acrosome-reacted live sperm using fluorescence-labeled SBTI (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200210057/DC1). Moreover, this method enabled us to avoid spontaneously acrosome-reacted sperm before the addition of the agonists. In this study we used Alexa Fluor®594–labeled SBTI to monitor the acrosome reaction induced by ZP, progesterone, and thapsigargin (Fig. 2 A). The fluorescence signal began to be detected at ∼2–5 min after the addition of these reagents, and 31.7% of wild-type sperm completed the acrosome reaction within 15 min of addition of 3 ZP/μl, whereas only 6.2% of PLCδ4−/− sperm underwent the acrosome reaction (Fig. 2 B). 100 μM progesterone induced higher rates of acrosome reaction with 49.7% of PLCδ4+/+ sperm undergoing the reaction, whereas only 26.1% of the PLCδ4−/− sperm completed the process. These results suggest that PLCδ4 has an important role in the progesterone- and ZP-induced acrosome reaction. Nonetheless, because some of the sperm from PLCδ4−/− males underwent the acrosome reaction, we cannot exclude the possibility that other PLC isozymes might be involved in these acrosome reactions in the absence of PLCδ4, especially when progesterone is used to induce the reactions. What is more, we also cannot discount the possibility that some of these sperm might have undergone spontaneous acrosome reactions during the monitoring. There was no apparent difference in the number of wild-type and mutant sperm that underwent the acrosome reaction in response to thapsigargin (58.4 and 49.7%, respectively) (Fig. 2 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)

Requirement of PLCδ4 in ZP- and progesterone-induced acrosome reaction. (A) Fluorescence and phase images of sperm treated with several acrosome reaction–inducing reagents. PLCδ4+/+ (+/+) and PLCδ4−/− (−/−) sperm capacitated for 1 h were treated with 1.0% DMSO, 3 Zp/μl solubilized mouse ZP (ZP), 100 μM progesterone (PG), or 5 μM thapsigargin (TG) for 15 min at 32°C in the presence of 2 μM Alexa Fluor®594–conjugated SBTI to monitor the occurrence of the acrosome reaction. The same microscopic fields are shown for acrosome reaction (AR) and bright field images. Bar, 10 μm. (B) Percent of sperm that underwent the acrosome reaction. The numbers above each column represent the number of sperm examined. Data were obtained from total amount of sperm by four independent experiments for progesterone and ZP and three independent experiments for thapsigargin.
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Related In: Results  -  Collection

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fig2: Requirement of PLCδ4 in ZP- and progesterone-induced acrosome reaction. (A) Fluorescence and phase images of sperm treated with several acrosome reaction–inducing reagents. PLCδ4+/+ (+/+) and PLCδ4−/− (−/−) sperm capacitated for 1 h were treated with 1.0% DMSO, 3 Zp/μl solubilized mouse ZP (ZP), 100 μM progesterone (PG), or 5 μM thapsigargin (TG) for 15 min at 32°C in the presence of 2 μM Alexa Fluor®594–conjugated SBTI to monitor the occurrence of the acrosome reaction. The same microscopic fields are shown for acrosome reaction (AR) and bright field images. Bar, 10 μm. (B) Percent of sperm that underwent the acrosome reaction. The numbers above each column represent the number of sperm examined. Data were obtained from total amount of sperm by four independent experiments for progesterone and ZP and three independent experiments for thapsigargin.
Mentions: We have reported that PLCδ4 is an essential protein for ZP-induced acrosome reaction. To understand how PLCδ4 is involved in this process, we first examined whether PLCδ4 is required for the acrosome reaction induced by progesterone and thapsigargin. A soybean trypsin inhibitor (SBTI) was used to monitor the acrosome reaction. SBTI is known to tightly bind to acrosin (Tollner et al., 2000), which is located on the inner acrosomal membrane and only becomes exposed extracellularly after the acrosome reaction; therefore, we could easily recognize acrosome-reacted live sperm using fluorescence-labeled SBTI (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200210057/DC1). Moreover, this method enabled us to avoid spontaneously acrosome-reacted sperm before the addition of the agonists. In this study we used Alexa Fluor®594–labeled SBTI to monitor the acrosome reaction induced by ZP, progesterone, and thapsigargin (Fig. 2 A). The fluorescence signal began to be detected at ∼2–5 min after the addition of these reagents, and 31.7% of wild-type sperm completed the acrosome reaction within 15 min of addition of 3 ZP/μl, whereas only 6.2% of PLCδ4−/− sperm underwent the acrosome reaction (Fig. 2 B). 100 μM progesterone induced higher rates of acrosome reaction with 49.7% of PLCδ4+/+ sperm undergoing the reaction, whereas only 26.1% of the PLCδ4−/− sperm completed the process. These results suggest that PLCδ4 has an important role in the progesterone- and ZP-induced acrosome reaction. Nonetheless, because some of the sperm from PLCδ4−/− males underwent the acrosome reaction, we cannot exclude the possibility that other PLC isozymes might be involved in these acrosome reactions in the absence of PLCδ4, especially when progesterone is used to induce the reactions. What is more, we also cannot discount the possibility that some of these sperm might have undergone spontaneous acrosome reactions during the monitoring. There was no apparent difference in the number of wild-type and mutant sperm that underwent the acrosome reaction in response to thapsigargin (58.4 and 49.7%, respectively) (Fig. 2 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