Recombinant Phospholipase Cζ Has High Ca2+ Sensitivity and Induces Ca2+ Oscillations in Mouse Eggs

Abstract

Sperm-specific phospholipase Cζ (PLCζ) is known to induce intracellular Ca2+ oscillations and subsequent early embryonic development when expressed in mouse eggs by injection of RNA encoding PLCζ (Saunders, C. M., Larman, M. G., Parrington, J., Cox, L. J., Royse, J., Blayney, L. M., Swann, K., and Lai, F. A. (2002) Development 129, 3533-3544). The present study addressed characteristics of purified mouse PLCζ protein that was synthesized using the baculovirus/Sf9 cell expression system. Microinjection of recombinant PLCζ protein into mouse eggs induced serial Ca2+ spikes quite similar to those produced by the injection of sperm extract, probably because of repetitive Ca2+ release from the endoplasmic reticulum caused by continuously produced inositol 1,4,5-trisphosphate. Recombinant PLCδ1 also induced Ca2+ oscillations, but a 20-fold higher concentration was required compared with PLCζ. In the enzymatic assay of phosphatidylinositol 4,5-bisphosphate hydrolyzing activity in vitro at various calcium ion concentrations ([Ca2+]), PLCζ exhibited a significant activity at [Ca2+] as low as 10 nm and had 70% maximal activity at 100 nm [Ca2+] that is usually the basal intracellular calcium ion concentration level of cells. On the other hand, the activity of PLCδ1 increased at a [Ca2+] between 1 and 30 μm. EC50 was 52 nm for PLCζ and 5.7 μm for PLCδ1. Thus, PLCζ has an ∼100-fold higher Ca2+ sensitivity than PLCδ1. The ability of purified PLCζ protein to induce Ca2+ oscillations qualifies PLCζ as a proper candidate of the mammalian egg-activating sperm factor. Furthermore, such a high Ca2+ sensitivity of PLC activity as PLCζ that can be active in cells at the resting state is thought to be an appropriate characteristic of the sperm factor, which is introduced into the ooplasm upon sperm-egg fusion, triggers Ca2+ release first, and maintains Ca2+ oscillations. Sperm-specific phospholipase Cζ (PLCζ) is known to induce intracellular Ca2+ oscillations and subsequent early embryonic development when expressed in mouse eggs by injection of RNA encoding PLCζ (Saunders, C. M., Larman, M. G., Parrington, J., Cox, L. J., Royse, J., Blayney, L. M., Swann, K., and Lai, F. A. (2002) Development 129, 3533-3544). The present study addressed characteristics of purified mouse PLCζ protein that was synthesized using the baculovirus/Sf9 cell expression system. Microinjection of recombinant PLCζ protein into mouse eggs induced serial Ca2+ spikes quite similar to those produced by the injection of sperm extract, probably because of repetitive Ca2+ release from the endoplasmic reticulum caused by continuously produced inositol 1,4,5-trisphosphate. Recombinant PLCδ1 also induced Ca2+ oscillations, but a 20-fold higher concentration was required compared with PLCζ. In the enzymatic assay of phosphatidylinositol 4,5-bisphosphate hydrolyzing activity in vitro at various calcium ion concentrations ([Ca2+]), PLCζ exhibited a significant activity at [Ca2+] as low as 10 nm and had 70% maximal activity at 100 nm [Ca2+] that is usually the basal intracellular calcium ion concentration level of cells. On the other hand, the activity of PLCδ1 increased at a [Ca2+] between 1 and 30 μm. EC50 was 52 nm for PLCζ and 5.7 μm for PLCδ1. Thus, PLCζ has an ∼100-fold higher Ca2+ sensitivity than PLCδ1. The ability of purified PLCζ protein to induce Ca2+ oscillations qualifies PLCζ as a proper candidate of the mammalian egg-activating sperm factor. Furthermore, such a high Ca2+ sensitivity of PLC activity as PLCζ that can be active in cells at the resting state is thought to be an appropriate characteristic of the sperm factor, which is introduced into the ooplasm upon sperm-egg fusion, triggers Ca2+ release first, and maintains Ca2+ oscillations. Fertilized mammalian eggs exhibit repetitive transient rises in [Ca2+]i 1The abbreviations used are: [Ca2+]i, intracellular [Ca2+]; [Ca2+], calcium ion concentration; PLC, phospholipase C; PLCζΔEFX, PLCζ lacking three EF-hand domains and catalytic X domain; PtdInsP2, phosphatidylinositol 4,5-bisphosphate; InsP3, inositol 1,4,5-trisphosphate; PH domain, pleckstrin homology domain; s-PLCζ, short form of PLCζ. referred to as Ca2+ oscillations because of repeated Ca2+ release from the ER mainly through the InsP3 receptor (1Miyazaki S. Shirakawa H. Nakada K. Honda Y. Dev. Biol. 1993; 158: 62-78Crossref PubMed Scopus (506) Google Scholar). The [Ca2+]i rises are a pivotal signal for egg activation and are responsible for early embryogenesis (2Jones K.T. Int. J. Dev. Biol. 1998; 42: 1-10PubMed Google Scholar, 3Ducibella T. Huneau D. Angelichio E. Xu Z. Schultz R.M. Kopf G.S. Fissore R. Madoux S. Ozil J.-P. Dev. Biol. 2002; 250: 280-291Crossref PubMed Google Scholar). Accumulated evidence suggests that Ca2+ oscillations are induced by cytosolic sperm factor introduced into the ooplasm upon sperm-egg fusion (2Jones K.T. Int. J. Dev. Biol. 1998; 42: 1-10PubMed Google Scholar, 4Swann K. Rev. Reprod. 1996; 1: 33-39Crossref PubMed Scopus (76) Google Scholar). Therefore, the identification of the Ca2+ oscillation-inducing sperm factor, which is the egg-activating sperm factor, is currently being studied as a central subject to elucidate the mechanisms of fertilization. Recently, Saunders et al. (5Saunders C.M. Larman M.G. Parrington J. Cox L.J. Royse J. Blayney L.M. Swann K. Lai F.A. Development. 2002; 129: 3533-3544Crossref PubMed Google Scholar) have reported a novel type of PLC (the enzyme that produces InsP3 and diacylglycerol from membrane PtdInsP2), PLCζ, which is specifically expressed in mammalian sperm. The injection of RNA-encoding PLCζ into mouse eggs causes Ca2+ oscillations and subsequent early embryonic development by expressed PLCζ at an estimated level comparable with the content in a single sperm (5Saunders C.M. Larman M.G. Parrington J. Cox L.J. Royse J. Blayney L.M. Swann K. Lai F.A. Development. 2002; 129: 3533-3544Crossref PubMed Google Scholar). The Ca2+ oscillation-inducing activity of sperm extract (4Swann K. Rev. Reprod. 1996; 1: 33-39Crossref PubMed Scopus (76) Google Scholar, 6Oda S. Deguchi R. Mohri T. Shikano T. Nakanishi S. Miyazaki S. Dev. Biol. 1999; 209: 172-185Crossref PubMed Scopus (67) Google Scholar) is lost when pretreated with an antibody against PLCζ (5Saunders C.M. Larman M.G. Parrington J. Cox L.J. Royse J. Blayney L.M. Swann K. Lai F.A. Development. 2002; 129: 3533-3544Crossref PubMed Google Scholar). Thus, PLCζ is considered a strong candidate of the sperm factor. To assure this possibility, it is primarily necessary to examine whether purified PLCζ protein induces Ca2+ oscillations in the egg. It has been shown that PLCβ1 (7Fukami K. Nakao K. Inoue T. Kataoka U. Kurokawa M. Fissore R.A. Nakamura K. Katsuki M. Mikoshiba K. Yoshida N. Takenawa T. Science. 2001; 292: 920-923Crossref PubMed Scopus (169) Google Scholar), -γ1 (8Mehlman L.M. Carpenter G. Rhee F.G. Jaffe L.A. Dev. Biol. 1998; 203: 221-232Crossref PubMed Scopus (126) Google Scholar, 9Dupont G. McGuinness O.M. Johnson M.H. Berridge M.J. Borgese F. Biochem. J. 1996; 316: 583-591Crossref PubMed Scopus (135) Google Scholar), -γ2 (8Mehlman L.M. Carpenter G. Rhee F.G. Jaffe L.A. Dev. Biol. 1998; 203: 221-232Crossref PubMed Scopus (126) Google Scholar), -δ1 (10Lee K.L. Kim J.K. Seo M-K. Cha J.-H. Lee K.J. Rha H.K. Mi D.S. Jo Y.-H. Lee K-H. Biochem. Biophys. Res. Commun. 1999; 261: 393-399Crossref PubMed Scopus (31) Google Scholar), and -δ4 (7Fukami K. Nakao K. Inoue T. Kataoka U. Kurokawa M. Fissore R.A. Nakamura K. Katsuki M. Mikoshiba K. Yoshida N. Takenawa T. Science. 2001; 292: 920-923Crossref PubMed Scopus (169) Google Scholar) are expressed in mammalian sperm and that recombinant PLCβ1, -γ1, -γ2, and -δ1 failed to cause Ca2+ release in the ooplasm (11Jones K.T. Matsuda M. Parrington J. Katan M. Swann K. Biochem. J. 2000; 346: 743-749Crossref PubMed Scopus (86) Google Scholar). PLCζ is the smallest PLC identified to date, lacking the N-terminal PH domain (Fig. 1A) (5Saunders C.M. Larman M.G. Parrington J. Cox L.J. Royse J. Blayney L.M. Swann K. Lai F.A. Development. 2002; 129: 3533-3544Crossref PubMed Google Scholar) that is found in all isoforms of PLCβ, -γ, and -δ and is the site for interaction with membrane phospholipids (12Katan M. Biochim. Biophys. Acta. 1998; 1436: 5-17Crossref PubMed Scopus (192) Google Scholar). Because PLCζ as well as PLCδ lacks a regulatory domain such as the G protein-binding site of PLCβ or the SH domain of PLCγ for phosphorylation by tyrosine kinase, the activation mechanism of PLCζ and PLCδ is unknown. Therefore, it is also necessary to access how PLCζ undergoes the active state for production of InsP3. Here we first show that recombinant PLCζ protein induces Ca2+ oscillations in mouse eggs and that PLCζ possesses an extremely high Ca2+ sensitivity in the PtdInsP2 hydrolyzing activity to be active even at the resting state of cells. Cloning of PLCζ and PLCδ1—cDNA encoding full-length PLCζ (GenBank™ accession number AF435950) was cloned from a cDNA library originated from mouse testis mRNAs. PLCζ cDNA was amplified by PCR using Pfu polymerase and the following primers: forward, 5′-GAC AAGCGGCCCAGATCATG-3′; internal forward primer involving an EcoRI site, 5′-GGAATTCATATGGAAAGCCAACTTCATGAG-3′; reverse, 5′-CTAACGCGTCAGTTACATGCG-3′; internal reverse primer involving an XbaI site, 5′-GTCTAGATTACTCTCTGAAGTACCAAACATAAATAAAC-3′. cDNA of PLCδ1 (GenBank™ accession number AF133125) derived from the mouse testis was amplified using 5′-GGAATTCATATGGACTCCGGTCGGGAC-3′ involving an EcoRI site and 5′-GCTCTAGA TTAGTCCTGGATGGAGATCTTC-3′ involving an XbaI site. PLCζ and PLCδ1 were subcloned into pBluescript II SK(+) (Stratagene) using EcoRI and XbaI sites. A PLCζ variant lacking three EF-hand domains (GenBank™ accession number AK006672), designated as s-PLCζ, was cloned using the same primers as those for PLCζ except the internal forward primer, 5′-GGAATTCATATGGAGATCGATCACTCTGATTC-3′. A mutant PLCζ lacking three EF-hand domains and a catalytic X domain, PLCζΔEFX, was constructed using 5′-GGAATTCATATGGAGATCGATCACTCTGATT-3′ involving an EcoRI site, 5′-GTCTAGATTACTCTCTGAAGTACCAAACATAAATAAAC-3′ involving an XbaI site, 5′-GTGGGTACCTTATCTGAAACCCACGAG-3′ involving a KpnI site, and the reverse primer. The correct nucleotide sequences of PLCζ (1Miyazaki S. Shirakawa H. Nakada K. Honda Y. Dev. Biol. 1993; 158: 62-78Crossref PubMed Scopus (506) Google Scholar), s-PLCζ, PLCδ1 (10Lee K.L. Kim J.K. Seo M-K. Cha J.-H. Lee K.J. Rha H.K. Mi D.S. Jo Y.-H. Lee K-H. Biochem. Biophys. Res. Commun. 1999; 261: 393-399Crossref PubMed Scopus (31) Google Scholar), and PLCζΔEFX were confirmed using a BigDye Terminator Sequencing kit (Applied Biosystems). Synthesis and Purification of PLCζ and PLCδ1—Recombinant PLCζ or PLCδ1 was prepared by a baculovirus expression system (13Nagano K. Fukami K. Minagawa T. Watanabe Y. Ozaki C. Takenawa T. J. Biol. Chem. 1999; 274: 2872-2879Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Using EcoRI and XbaI sites, the open reading frames of PLCζ, s-PLCζ, PLCδ1, and PLCζΔEFX were subcloned into a baculovirus expression vector, pFastBac HT (Invitrogen), which contains an N-terminal His tag sequence. The recombinant baculoviruses were generated by transformation into DH10Bac, and the transformants containing a PLC-inserted bacmid were selected by blue/white screening (see Instruction Manual by Invitrogen). Sf9 cells were grown at 28 °C in Sf900 serum-free medium (Invitrogen) containing 5% fetal bovine serum. The cells (1 × 106 cells/ml) were infected with baculoviruses and incubated for 3 days. The infected cells (3 liters for PLCζ and 300 ml for PLCδ1) were lysed with a French pressure cell in a lysis buffer (120 mm KCl, 10 mm imidazole, 0.2 mm phenylmethanesulfonyl fluoride, 2 μg/ml pepstatin, 5 μg/ml leupeptin, and 20 mm HEPES/KOH, pH 7.5). The lysate was centrifuged at 15,000 × g for 20 min, and the supernatant filtrated through a 0.22-μm filter was applied to nickel-chelating Sepharose (Amersham Biosciences). The column was washed with a wash buffer (400 mm KCl, 50 mm imidazole, and 20 mm HEPES/KOH, pH 7.5), and protein was eluted with an elution buffer (400 mm KCl, 300 mm imidazole, and 20 mm HEPES/KOH, pH 7.5). Purified PLC was dialyzed against an intracellular buffer (120 mm KCl and 20 mm HEPES/KOH, pH 7.5). The final solution was designated as PLC solution. Measurement of PLC Activity—The PLC activity was assayed by hydrolysis of PtdInsP2 (13Nagano K. Fukami K. Minagawa T. Watanabe Y. Ozaki C. Takenawa T. J. Biol. Chem. 1999; 274: 2872-2879Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) by mixing 10 μl of PLC solution with 30 μl of Ca2+ buffer and 10 μl of phospholipid micelle solution. The final concentration was 50 μm phosphatidylethanolamine (Sigma), 40 μm PtdInsP2 (Sigma), 1 μCi/ml (∼100 nm) [3H]PtdInsP2 (PerkinElmer Life Sciences), 50 mm HEPES (pH 7.0), 100 mm KCl, 2 mm EGTA, 1 nm to 100 μm Ca2+, and 0.5 mg/ml bovine serum albumin. Ca2+ buffers of various [Ca2+] were prepared by EGTA/CaCl2 mixture (14Fabiato A. Methods Enzymol. 1988; 157: 378-417Crossref PubMed Scopus (975) Google Scholar). The reaction mixture containing 25,000 dpm of [3H]PtdInsP2 was incubated at 37 °C for 5 min, and the reaction was stopped by adding 2 μm of chloroform:methanol (2:1, v/v). Radioactive InsP3 was extracted by adding 0.5 ml of 1 n HCl (13Nagano K. Fukami K. Minagawa T. Watanabe Y. Ozaki C. Takenawa T. J. Biol. Chem. 1999; 274: 2872-2879Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), and radioactivity in the upper aqueous phase was measured for 1 min in a liquid scintillation counter. PLC for assay was used at the concentration that produced a maximal [3H]InsP3 level of ∼4,000 dpm at a certain range of [Ca2+]. Under these conditions, [3H]InsP3 formation was linear as the function of time and enzyme concentration. Preparation of Eggs—Mature eggs were obtained from the oviducts of B6D2F1 female mice superovulated by intraperitoneal injection of gonadotropins (see Ref. 6Oda S. Deguchi R. Mohri T. Shikano T. Nakanishi S. Miyazaki S. Dev. Biol. 1999; 209: 172-185Crossref PubMed Scopus (67) Google Scholar for details). The eggs were collected into M2 medium supplement with bovine serum albumin (4 mg/ml) (6Oda S. Deguchi R. Mohri T. Shikano T. Nakanishi S. Miyazaki S. Dev. Biol. 1999; 209: 172-185Crossref PubMed Scopus (67) Google Scholar). The eggs were loaded with the Ca2+-sensitive fluorescent dye fura-2 acetoxymethyl ester (5 μm) (Molecular Probes Inc.) for 8 min at 37 °C. The eggs were then transferred to a plastic dish mounted on an inverted fluorescence microscope and heated at 30-32 °C. Injection of PLC and [Ca2+]i Measurement—PLC solution was injected into the eggs through a glass micropipette (15Kumakiri J. Oda S. Kinoshita K. Miyazaki S. Dev. Biol. 2003; 260: 522-535Crossref PubMed Scopus (43) Google Scholar). The injected amount was 5 pl (egg volume, ∼200 pl). [Ca2+]i was measured by a conventional Ca2+ imaging method using an image processor (Argus 200, Hamamatsu Photonics) (see Ref. 15Kumakiri J. Oda S. Kinoshita K. Miyazaki S. Dev. Biol. 2003; 260: 522-535Crossref PubMed Scopus (43) Google Scholar for details). Images were acquired every 20 s for 30-40 min by applying 340- and 380-nm lights and measuring fluorescence (F) of fura-2 at 510 nm. Data were processed to calculate the fluorescence ratio F340/F380. PLCζ and PLCδ1 with N-terminal His tags were purified up to a major single band shown by silver staining (Fig. 1B). The molecular mass including the His tag was ∼78 and ∼90 kDa, consistent with the full-length PLCζ (647 amino acids) (5Saunders C.M. Larman M.G. Parrington J. Cox L.J. Royse J. Blayney L.M. Swann K. Lai F.A. Development. 2002; 129: 3533-3544Crossref PubMed Google Scholar) and PLCδ1 amino acids) Rhee 1988; Full Text PDF PubMed Scopus Google Scholar), applied PLC was to InsP3 continuously in the eggs cause repetitive Ca2+ release from the endoplasmic Microinjection of recombinant PLCζ into the mouse eggs induce Ca2+ oscillations (Fig. n 10 The first Ca2+ transient for min was by Ca2+ spikes at of The was The is similar to that of Ca2+ oscillations induced by injection of sperm extract (2Jones K.T. Int. J. Dev. Biol. 1998; 42: 1-10PubMed Google Scholar, 6Oda S. Deguchi R. Mohri T. Shikano T. Nakanishi S. Miyazaki S. Dev. Biol. 1999; 209: 172-185Crossref PubMed Scopus (67) Google Scholar), Ca2+ oscillations of in eggs with a single R. Shirakawa H. Oda S. Mohri T. Miyazaki S. Dev. Biol. 2000; PubMed Scopus Google Scholar). The PLCζ concentration in the injection solution for of Ca2+ oscillations was and that found in the egg was to be The amount injected was as 300 It has been shown that low Ca2+ oscillations similar to those at are produced injection of PLCζ RNA by expressed PLCζ of estimated from using an antibody against PLCζ (5Saunders C.M. Larman M.G. Parrington J. Cox L.J. Royse J. Blayney L.M. Swann K. Lai F.A. Development. 2002; 129: 3533-3544Crossref PubMed Google Scholar). The in the amount of PLCζ for of Ca2+ oscillations be derived from that in of the PLCζ in the cell to a active Injection with a PLCζ μg/ml in the produced a single Ca2+ transient a time of min (Fig. n Ca2+ is usually upon injection of sperm extract The PLCζ concentration for of a single Ca2+ release was between and 30 μg/ml in the injection solution (Fig. and A protein similar to but than PLCζ is expressed in the mouse testis (GenBank™ accession number lacks amino from the to the and domains but is to PLCζ in and the (Fig. The has been found in the mouse and is probably a variant of PLCζ. Recombinant Ca2+ at the concentration higher than that of PLCζ (Fig. that the is significant in the of Ca2+ oscillations. Recombinant PLCδ1, which possesses similar domain to those of PLCζ except the PH domain (Fig. induced Ca2+ oscillations at the concentration of mg/ml in the injection solution μg/ml in the (Fig. n and a Ca2+ at mg/ml (Fig. n Thus, an higher concentration was required for of Ca2+ oscillations compared with PLCζ. PLCδ1 induced Ca2+ at mg/ml (Fig. or at the concentration range at which PLCζ induced Ca2+ oscillations The PLC activity hydrolyzing of PLCζ in vitro was measured at [Ca2+] between and in with that of PLCδ1 (Fig. PLC activity was for PLCζΔEFX at [Ca2+] (Fig. that the from Sf9 cells had PLC The assay was using the amount of PLC that was of maximal [3H]InsP3 of ∼4,000 which to of InsP3, the that the of InsP3 formation to the reaction time and the enzyme concentration for 5 The amount used was 50 for PLCζ and 1 for PLCδ1 in 50 μl of reaction mixture to 1 μg/ml and concentration of PLCζ was in the same range as the intracellular concentration of injected PLCζ that induced Ca2+ release (Fig. A and The activity of PLCζ was at 1 μm Ca2+ and that of PLCδ1 was at 30 μm Ca2+ (Fig. The activity was higher for PLCδ1. PLCζ induced Ca2+ oscillations at 20-fold concentrations than PLCδ1 and that PLCζ has a higher than PLCδ1 in of Ca2+ oscillations in the egg when on the of the PLC activity in vitro the of comparable on [Ca2+]. To the characteristics of PLCζ for of Ca2+ oscillations, the of PLC activity on [Ca2+] was in The PLC activity of recombinant PLCδ1 was at 1 μm Ca2+ (Fig. The activity increased between 1 and 30 μm Ca2+ and a level at 30 μm The [Ca2+] for PLC was obtained by a to the using the It was as 5.7 μm In the PLC activity of recombinant PLCζ was at [Ca2+] as low as 10 nm and a at 1 μm Ca2+ (Fig. EC50 was 52 nm that ∼100-fold than that for PLCδ1. It be that PLCζ had 70% activity of the maximal level at 100 nm Ca2+, which is the resting [Ca2+]i level in mouse eggs (6Oda S. Deguchi R. Mohri T. Shikano T. Nakanishi S. Miyazaki S. Dev. Biol. 1999; 209: 172-185Crossref PubMed Scopus (67) Google Scholar, Y. Shirakawa H. N. Y. Miyazaki S. Reprod. PubMed Scopus Google Scholar) as well as cells S. J. 1993; PubMed Scopus Google Scholar). that PLCζ can be active even at the resting state of cells. PLCζ has the Ca2+ sensitivity PLC isoforms identified and to R. S. Katan M. Biochem. J. PubMed Scopus Google Scholar, T. A. J. Biochem. 1998; PubMed Scopus Google Scholar, J. 2001; PubMed Scopus Google Scholar, K. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, 1998; PubMed Scopus Google Scholar). [Ca2+]i from 100 nm up to nm in Ca2+ Ca2+ oscillations in mouse eggs K.T. Dev. Biol. 2000; PubMed Scopus Google Scholar). In this [Ca2+] range a Ca2+ which be for a and of the PLC The Ca2+ of PLCζ as a a (Fig. which cause in the PLC activity with Ca2+ oscillations. this to be because the PLCζ activity at [Ca2+] as high as 10 μm. Ca2+ oscillations on the of Ca2+ of the InsP3 receptor (1Miyazaki S. Shirakawa H. Nakada K. Honda Y. Dev. Biol. 1993; 158: 62-78Crossref PubMed Scopus (506) Google Scholar), even of PLC activity is are produced by of InsP3 at a low level in the ooplasm Y. Miyazaki S. Shikano T. N. H. Mikoshiba K. Y. Biol. Reprod. 1998; PubMed Scopus Google Scholar). A single injection of a of InsP3, into the mouse egg produces Ca2+ oscillations Y. Shirakawa H. N. Y. Miyazaki S. Reprod. PubMed Scopus Google Scholar). The present study of PLCζ using purified recombinant PLCζ the high Ca2+ oscillation-inducing activity in the egg and the high Ca2+ sensitivity in the PtdInsP2 hydrolyzing on the N-terminal EF-hand domains of PLCζ, because lacking three EF-hand domains was of Ca2+ oscillations (Fig. and exhibited significant hydrolyzing activity when [Ca2+] was 1 μm The domains of PLCζ have a high to The higher Ca2+ sensitivity of PLCζ is to the ability to Ca2+ release in cells at the resting The of PLCζ are appropriate for a Ca2+ oscillation-inducing sperm factor, because the sperm factor is thought to be introduced into the ooplasm upon sperm-egg fusion, first Ca2+ release and then Ca2+ oscillations (2Jones K.T. Int. J. Dev. Biol. 1998; 42: 1-10PubMed Google Scholar, 4Swann K. Rev. Reprod. 1996; 1: 33-39Crossref PubMed Scopus (76) Google Scholar). It has been shown that sperm extract possesses of the maximal PtdInsP2 hydrolyzing activity at 100 nm Ca2+ A. Parrington J. K.T. Swann K. Dev. Biol. 2000; PubMed Scopus Google Scholar). Thus, PLCζ is a strong candidate of the mammalian egg-activating sperm factor. be an mechanism for PLCζ activity in the sperm it is introduced into the PLCδ1 the higher enzymatic activity in but had the Ca2+ oscillation-inducing activity in compared with PLCζ. PLCδ1 has been shown to be expressed in mouse but in and (10Lee K.L. Kim J.K. Seo M-K. Cha J.-H. Lee K.J. Rha H.K. Mi D.S. Jo Y.-H. Lee K-H. Biochem. Biophys. Res. Commun. 1999; 261: 393-399Crossref PubMed Scopus (31) Google Scholar). Thus, PLCδ1 is to be the sperm factor. The of PLCζ to PLCδ1 in the Ca2+ oscillation-inducing activity is thought to be derived from the higher Ca2+ sensitivity in the in the of Ca2+ oscillations is in of PLC activity in vitro and suggests an of PLCζ. be an egg factor that the activation of PLCζ or for PLCζ in the are necessary to whether PLCζ is the sperm factor at as well as to elucidate the activation and mechanism of PLCζ on the of the molecular T. H. and S. for and the present