Limits...
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.

Show MeSH

Related in: MedlinePlus

[Ca2+]i mobilization is impaired in PLCδ4−/− sperm during acrosome reaction. Capacitated sperm were loaded with 4 μM fluo4-AM for 15 min, and Ca2+ responses were monitored every 2 s. (A) Ca2+ images of PLCδ4+/+ (+/+) and PLCδ4−/− (−/−) sperm before and after (10–30 s) treatment with 3 Zp/μl solubilized mouse ZP (ZP), 100 μM progesterone (PG), 5 μM thapsigargin (TG), or 5 μM ionomycin (Iono). The same microscopic fields are shown before and after the stimulus. The experiments were done at 32°C. Bar, 10 μm. (B) Ca2+ patterns of five sperm representative of the responses induced by the agonists used here. Levels of [Ca2+]i are expressed as F/F0 ratios. The data are representative from five (ZP), nine (PG), eight (TG), and three (Iono) independent experiments. Progesterone induced repetitive [Ca2+]i increases.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172882&req=5

fig4: [Ca2+]i mobilization is impaired in PLCδ4−/− sperm during acrosome reaction. Capacitated sperm were loaded with 4 μM fluo4-AM for 15 min, and Ca2+ responses were monitored every 2 s. (A) Ca2+ images of PLCδ4+/+ (+/+) and PLCδ4−/− (−/−) sperm before and after (10–30 s) treatment with 3 Zp/μl solubilized mouse ZP (ZP), 100 μM progesterone (PG), 5 μM thapsigargin (TG), or 5 μM ionomycin (Iono). The same microscopic fields are shown before and after the stimulus. The experiments were done at 32°C. Bar, 10 μm. (B) Ca2+ patterns of five sperm representative of the responses induced by the agonists used here. Levels of [Ca2+]i are expressed as F/F0 ratios. The data are representative from five (ZP), nine (PG), eight (TG), and three (Iono) independent experiments. Progesterone induced repetitive [Ca2+]i increases.

Mentions: As it has been reported that Ca2+ has a primary role in the execution of the acrosome reaction (Patrat et al., 2000; Barratt and Publicover, 2001; Darszon et al., 2001; Breitbart, 2002), we next examined the Ca2+ responses in single sperm treated with ZP, progesterone, thapsigargin, or ionomycin (Fig. 4 A). We selected a single excitation calcium indicator fluo-4, because we could obtain calcium images of high resolution with this dye, and the images were temporally and spatially comparable to those obtained with the ratiometric dye fura-2 (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200210057/DC1). PLCδ4+/+ sperm preloaded with fluo-4 and treated with ZP exhibited a continuous [Ca2+]i increase (Fig. 4 B). This [Ca2+]i rise peaked at ∼30–60 s after the stimulation and lasted for more than 3–4 min before slowly returning to baseline. Approximately 50% of the sperm exhibited the described response (n = 120), and some sperm even showed repetitive [Ca2+]i increases after returning to baseline. In contrast, the addition of ZP induced a minor [Ca2+]i increase in PLCδ4−/− sperm, suggesting a role for PLCδ4 in the Ca2+ response during the ZP-induced acrosome reaction.


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)

[Ca2+]i mobilization is impaired in PLCδ4−/− sperm during acrosome reaction. Capacitated sperm were loaded with 4 μM fluo4-AM for 15 min, and Ca2+ responses were monitored every 2 s. (A) Ca2+ images of PLCδ4+/+ (+/+) and PLCδ4−/− (−/−) sperm before and after (10–30 s) treatment with 3 Zp/μl solubilized mouse ZP (ZP), 100 μM progesterone (PG), 5 μM thapsigargin (TG), or 5 μM ionomycin (Iono). The same microscopic fields are shown before and after the stimulus. The experiments were done at 32°C. Bar, 10 μm. (B) Ca2+ patterns of five sperm representative of the responses induced by the agonists used here. Levels of [Ca2+]i are expressed as F/F0 ratios. The data are representative from five (ZP), nine (PG), eight (TG), and three (Iono) independent experiments. Progesterone induced repetitive [Ca2+]i increases.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: [Ca2+]i mobilization is impaired in PLCδ4−/− sperm during acrosome reaction. Capacitated sperm were loaded with 4 μM fluo4-AM for 15 min, and Ca2+ responses were monitored every 2 s. (A) Ca2+ images of PLCδ4+/+ (+/+) and PLCδ4−/− (−/−) sperm before and after (10–30 s) treatment with 3 Zp/μl solubilized mouse ZP (ZP), 100 μM progesterone (PG), 5 μM thapsigargin (TG), or 5 μM ionomycin (Iono). The same microscopic fields are shown before and after the stimulus. The experiments were done at 32°C. Bar, 10 μm. (B) Ca2+ patterns of five sperm representative of the responses induced by the agonists used here. Levels of [Ca2+]i are expressed as F/F0 ratios. The data are representative from five (ZP), nine (PG), eight (TG), and three (Iono) independent experiments. Progesterone induced repetitive [Ca2+]i increases.
Mentions: As it has been reported that Ca2+ has a primary role in the execution of the acrosome reaction (Patrat et al., 2000; Barratt and Publicover, 2001; Darszon et al., 2001; Breitbart, 2002), we next examined the Ca2+ responses in single sperm treated with ZP, progesterone, thapsigargin, or ionomycin (Fig. 4 A). We selected a single excitation calcium indicator fluo-4, because we could obtain calcium images of high resolution with this dye, and the images were temporally and spatially comparable to those obtained with the ratiometric dye fura-2 (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200210057/DC1). PLCδ4+/+ sperm preloaded with fluo-4 and treated with ZP exhibited a continuous [Ca2+]i increase (Fig. 4 B). This [Ca2+]i rise peaked at ∼30–60 s after the stimulation and lasted for more than 3–4 min before slowly returning to baseline. Approximately 50% of the sperm exhibited the described response (n = 120), and some sperm even showed repetitive [Ca2+]i increases after returning to baseline. In contrast, the addition of ZP induced a minor [Ca2+]i increase in PLCδ4−/− sperm, suggesting a role for PLCδ4 in the Ca2+ response during the ZP-induced acrosome reaction.

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