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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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PLCδ4/ATL-II is expressed in sperm. (A) The molecular weight of PLCδ4 in brain (B) and sperm (S) was compared by Western blot analysis. 30 μg of each homogenate was subjected to SDS-PAGE. Western blot analysis was performed using an antibody against PLCδ4 raised in the laboratory. (B) The molecular weight of sperm PLCδ4 was compared with PLCδ4 and alternative splicing isoforms of PLCδ4. PLCδ4 (δ4) and splicing isoforms of PLCδ4, PLCδ4/ALT-I (δ4-I) and PLCδ4/ALT-II (δ4-II), were overexpressed in COS-7 cells. 40 μg of lysates was subjected to SDS-PAGE, and then Western blotting was performed. The arrowhead denotes that the sperm protein closely resembles in size the product from the PLCδ4/ALT-II splice variant. (C) The products of RT-PCR using testis RNA were compared in size with authentic products using PLCδ4 (491 bp), PLCδ4/ALT-I (δ4-I; 587 bp), PLCδ4/ALT-II (δ4-II; 533 bp), and PLCδ4/ALT-III (δ4-III; 398 bp) plasmids. The arrowhead denotes that the major testis PCR product coincides in size with that of the PLCδ4/ALT-II splice variant.
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fig1: PLCδ4/ATL-II is expressed in sperm. (A) The molecular weight of PLCδ4 in brain (B) and sperm (S) was compared by Western blot analysis. 30 μg of each homogenate was subjected to SDS-PAGE. Western blot analysis was performed using an antibody against PLCδ4 raised in the laboratory. (B) The molecular weight of sperm PLCδ4 was compared with PLCδ4 and alternative splicing isoforms of PLCδ4. PLCδ4 (δ4) and splicing isoforms of PLCδ4, PLCδ4/ALT-I (δ4-I) and PLCδ4/ALT-II (δ4-II), were overexpressed in COS-7 cells. 40 μg of lysates was subjected to SDS-PAGE, and then Western blotting was performed. The arrowhead denotes that the sperm protein closely resembles in size the product from the PLCδ4/ALT-II splice variant. (C) The products of RT-PCR using testis RNA were compared in size with authentic products using PLCδ4 (491 bp), PLCδ4/ALT-I (δ4-I; 587 bp), PLCδ4/ALT-II (δ4-II; 533 bp), and PLCδ4/ALT-III (δ4-III; 398 bp) plasmids. The arrowhead denotes that the major testis PCR product coincides in size with that of the PLCδ4/ALT-II splice variant.

Mentions: Western blot analysis using a specific antibody against PLCδ4 (Liu et al., 1996) revealed that an 85-kD PLCδ4 protein is expressed in brain, whereas a 90-kD protein is expressed in sperm (Fig. 1 A). There are three splicing variants, termed PLCδ4/ATL-I, PLCδ4/ATL-II, and PLCδ4/ATL-III in addition to PLCδ4 in testis (Nagano et al., 1999). PLCδ4/ATL-I and PLCδ4/ATL-II contain an additional 32 and 14 amino acids in their respective sequences in the linker region between the X and Y domains, and PLCδ4/ATL-III lacks PLC activity because the COOH-terminal region of the X domain and the linker region are substituted for 32 amino acids corresponding to the insert sequence of PLCδ4/ATL-I. To understand which splicing isoform exists in sperm and contributes to the acrosome reaction, we compared a band of molecular weight of ∼90 kD in sperm with PLCδ4 splicing variants overexpressed in COS-7 cells (Fig. 1 B). Western blot analysis showed that the 90-kD protein coincided with PLCδ4/ATL-II. There were no other PLCδ4 proteins in sperm. To confirm further the predominant role/expression of PLCδ4/ATL-II in sperm, we performed RT-PCR using total testis RNA and compared the molecular weight of the major PCR products using testis cDNA and plasmids coding for each of the splicing variants as templates. The major testis PCR product had a similar molecular weight to that produced from the PLCδ4.ATLII plasmid (Fig. 1 C, arrowhead), although a faint band corresponding to PLCδ4.ATLI was also detected under this condition. Therefore, we concluded that PLCδ4/ATL-II is the predominant PLCδ4 isoform expressed in sperm.


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)

PLCδ4/ATL-II is expressed in sperm. (A) The molecular weight of PLCδ4 in brain (B) and sperm (S) was compared by Western blot analysis. 30 μg of each homogenate was subjected to SDS-PAGE. Western blot analysis was performed using an antibody against PLCδ4 raised in the laboratory. (B) The molecular weight of sperm PLCδ4 was compared with PLCδ4 and alternative splicing isoforms of PLCδ4. PLCδ4 (δ4) and splicing isoforms of PLCδ4, PLCδ4/ALT-I (δ4-I) and PLCδ4/ALT-II (δ4-II), were overexpressed in COS-7 cells. 40 μg of lysates was subjected to SDS-PAGE, and then Western blotting was performed. The arrowhead denotes that the sperm protein closely resembles in size the product from the PLCδ4/ALT-II splice variant. (C) The products of RT-PCR using testis RNA were compared in size with authentic products using PLCδ4 (491 bp), PLCδ4/ALT-I (δ4-I; 587 bp), PLCδ4/ALT-II (δ4-II; 533 bp), and PLCδ4/ALT-III (δ4-III; 398 bp) plasmids. The arrowhead denotes that the major testis PCR product coincides in size with that of the PLCδ4/ALT-II splice variant.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: PLCδ4/ATL-II is expressed in sperm. (A) The molecular weight of PLCδ4 in brain (B) and sperm (S) was compared by Western blot analysis. 30 μg of each homogenate was subjected to SDS-PAGE. Western blot analysis was performed using an antibody against PLCδ4 raised in the laboratory. (B) The molecular weight of sperm PLCδ4 was compared with PLCδ4 and alternative splicing isoforms of PLCδ4. PLCδ4 (δ4) and splicing isoforms of PLCδ4, PLCδ4/ALT-I (δ4-I) and PLCδ4/ALT-II (δ4-II), were overexpressed in COS-7 cells. 40 μg of lysates was subjected to SDS-PAGE, and then Western blotting was performed. The arrowhead denotes that the sperm protein closely resembles in size the product from the PLCδ4/ALT-II splice variant. (C) The products of RT-PCR using testis RNA were compared in size with authentic products using PLCδ4 (491 bp), PLCδ4/ALT-I (δ4-I; 587 bp), PLCδ4/ALT-II (δ4-II; 533 bp), and PLCδ4/ALT-III (δ4-III; 398 bp) plasmids. The arrowhead denotes that the major testis PCR product coincides in size with that of the PLCδ4/ALT-II splice variant.
Mentions: Western blot analysis using a specific antibody against PLCδ4 (Liu et al., 1996) revealed that an 85-kD PLCδ4 protein is expressed in brain, whereas a 90-kD protein is expressed in sperm (Fig. 1 A). There are three splicing variants, termed PLCδ4/ATL-I, PLCδ4/ATL-II, and PLCδ4/ATL-III in addition to PLCδ4 in testis (Nagano et al., 1999). PLCδ4/ATL-I and PLCδ4/ATL-II contain an additional 32 and 14 amino acids in their respective sequences in the linker region between the X and Y domains, and PLCδ4/ATL-III lacks PLC activity because the COOH-terminal region of the X domain and the linker region are substituted for 32 amino acids corresponding to the insert sequence of PLCδ4/ATL-I. To understand which splicing isoform exists in sperm and contributes to the acrosome reaction, we compared a band of molecular weight of ∼90 kD in sperm with PLCδ4 splicing variants overexpressed in COS-7 cells (Fig. 1 B). Western blot analysis showed that the 90-kD protein coincided with PLCδ4/ATL-II. There were no other PLCδ4 proteins in sperm. To confirm further the predominant role/expression of PLCδ4/ATL-II in sperm, we performed RT-PCR using total testis RNA and compared the molecular weight of the major PCR products using testis cDNA and plasmids coding for each of the splicing variants as templates. The major testis PCR product had a similar molecular weight to that produced from the PLCδ4.ATLII plasmid (Fig. 1 C, arrowhead), although a faint band corresponding to PLCδ4.ATLI was also detected under this condition. Therefore, we concluded that PLCδ4/ATL-II is the predominant PLCδ4 isoform expressed in sperm.

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