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

Measurement of [Ca2+]i increase in sperm responded to various acrosome inducers by mass level. Capacitated cauda epidermal sperm were loaded with 4 μM fluo4-AM for 15 min at 37°C. Approximately 1 × 106 sperm in a 96-well plate were treated with ZP, progesterone (PG), thapsigargin (TG), or ionomycin (Iono). Increasing levels of intracellular Ca2+ are expressed as an F/F0 ratio. The data were collected from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172882&req=5

fig7: Measurement of [Ca2+]i increase in sperm responded to various acrosome inducers by mass level. Capacitated cauda epidermal sperm were loaded with 4 μM fluo4-AM for 15 min at 37°C. Approximately 1 × 106 sperm in a 96-well plate were treated with ZP, progesterone (PG), thapsigargin (TG), or ionomycin (Iono). Increasing levels of intracellular Ca2+ are expressed as an F/F0 ratio. The data were collected from three independent experiments.

Mentions: To further understand the role of PLCδ4 in sperm [Ca2+]i mobilization and to confirm the validity of the Ca2+ responses obtained using single-sperm measurements, we examined at the population level the [Ca2+]i changes induced by the same agonists using a fluorescence microplate reader corresponding to a 96-well plate. We detected comparatively weak and slow increases in fluorescence in PLCδ4+/+ sperm after addition of ZP (Fig. 7 A), reflecting, perhaps, the fact that the response to ZP may start over a course of several minutes within a population of sperm, as shown by others. In contrast, progesterone triggered a very rapid and large [Ca2+]i increase and so did ionomycin and thapsigargin (Fig. 7 B). This pattern of [Ca2+]i increase was also observed in PLCδ4−/− sperm; however, the maximum intensity of [Ca2+]i uptake was about half of that observed in PLCδ4+/+ sperm, which agrees with the results reported in Figs. 4 and 5. Because thapsigargin appears to promote SOC channel activity but not generation of IP3 (Sabala et al., 1993), and ionomycin induces Ca2+ influx through the formation of synthetic Ca2+ channels, these results raise the possibility that PLCδ4 may play in sperm a novel functional role in the regulation of Ca2+ influx.


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)

Measurement of [Ca2+]i increase in sperm responded to various acrosome inducers by mass level. Capacitated cauda epidermal sperm were loaded with 4 μM fluo4-AM for 15 min at 37°C. Approximately 1 × 106 sperm in a 96-well plate were treated with ZP, progesterone (PG), thapsigargin (TG), or ionomycin (Iono). Increasing levels of intracellular Ca2+ are expressed as an F/F0 ratio. The data were collected from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Measurement of [Ca2+]i increase in sperm responded to various acrosome inducers by mass level. Capacitated cauda epidermal sperm were loaded with 4 μM fluo4-AM for 15 min at 37°C. Approximately 1 × 106 sperm in a 96-well plate were treated with ZP, progesterone (PG), thapsigargin (TG), or ionomycin (Iono). Increasing levels of intracellular Ca2+ are expressed as an F/F0 ratio. The data were collected from three independent experiments.
Mentions: To further understand the role of PLCδ4 in sperm [Ca2+]i mobilization and to confirm the validity of the Ca2+ responses obtained using single-sperm measurements, we examined at the population level the [Ca2+]i changes induced by the same agonists using a fluorescence microplate reader corresponding to a 96-well plate. We detected comparatively weak and slow increases in fluorescence in PLCδ4+/+ sperm after addition of ZP (Fig. 7 A), reflecting, perhaps, the fact that the response to ZP may start over a course of several minutes within a population of sperm, as shown by others. In contrast, progesterone triggered a very rapid and large [Ca2+]i increase and so did ionomycin and thapsigargin (Fig. 7 B). This pattern of [Ca2+]i increase was also observed in PLCδ4−/− sperm; however, the maximum intensity of [Ca2+]i uptake was about half of that observed in PLCδ4+/+ sperm, which agrees with the results reported in Figs. 4 and 5. Because thapsigargin appears to promote SOC channel activity but not generation of IP3 (Sabala et al., 1993), and ionomycin induces Ca2+ influx through the formation of synthetic Ca2+ channels, these results raise the possibility that PLCδ4 may play in sperm a novel functional role in the regulation of Ca2+ influx.

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