The Role of X/Y Linker Region and N-terminal EF-hand Domain in Nuclear Translocation and Ca2+ Oscillation-inducing Activities of Phospholipase Cζ, a Mammalian Egg-activating Factor
2006; Elsevier BV; Volume: 281; Issue: 38 Linguagem: Inglês
10.1074/jbc.m603473200
ISSN1083-351X
AutoresKeiji Kuroda, Masahiko Ito, Tomohide Shikano, Takeo Awaji, Ayako Yoda, Hiroyuki Takeuchi, Katsuyuki Kinoshita, Shunichi Miyazaki,
Tópico(s)Animal Genetics and Reproduction
ResumoSperm-specific phospholipase C-zeta (PLCζ) causes intracellular Ca2+ oscillations and thereby egg activation and is accumulated into the formed pronucleus (PN) when expressed in mouse eggs by injection of cRNA encoding PLCζ, which consists of four EF-hand domains (EF1-EF4) in the N terminus, X and Y catalytic domains, and C-terminal C2 domain. Those activities were analyzed by expressing PLCζ mutants tagged with fluorescent protein Venus by injection of cRNA into unfertilized eggs or 1-cell embryos after fertilization. Nuclear localization signal (NLS) existed at 374–381 in the X/Y linker region. Nuclear translocation was lost by replacement of Arg376, Lys377, Arg378, Lys379, or Lys381 with glutamate, whereas Ca2+ oscillations were conserved. Nuclear targeting was also absent for point mutation of Lys299 and/or Lys301 in the C terminus of X domain, or Trp13, Phe14, or Val18 in the N terminus of EF1. Ca2+ oscillation-inducing activity was lost by the former mutation and was remarkably inhibited by the latter. A short sequence 374–383 fused with Venus showed active translocation into the nucleus of COS-7 cells, but 296–309 or 1–19 did not. Despite the presence of these special regions, both activities were deprived by deletion of not only EF1 but also EF2–4 or C2 domain. Thus, PLCζ is driven into the nucleus primarily by the aid of NLS and putative regulatory sites, but coordinated three-dimensional structure, possibly formed by a folding in the X/Y linker and close EF/C2 contact as in PLCδ1, seems to be required not only for enzymatic activity but also for nuclear translocation ability. Sperm-specific phospholipase C-zeta (PLCζ) causes intracellular Ca2+ oscillations and thereby egg activation and is accumulated into the formed pronucleus (PN) when expressed in mouse eggs by injection of cRNA encoding PLCζ, which consists of four EF-hand domains (EF1-EF4) in the N terminus, X and Y catalytic domains, and C-terminal C2 domain. Those activities were analyzed by expressing PLCζ mutants tagged with fluorescent protein Venus by injection of cRNA into unfertilized eggs or 1-cell embryos after fertilization. Nuclear localization signal (NLS) existed at 374–381 in the X/Y linker region. Nuclear translocation was lost by replacement of Arg376, Lys377, Arg378, Lys379, or Lys381 with glutamate, whereas Ca2+ oscillations were conserved. Nuclear targeting was also absent for point mutation of Lys299 and/or Lys301 in the C terminus of X domain, or Trp13, Phe14, or Val18 in the N terminus of EF1. Ca2+ oscillation-inducing activity was lost by the former mutation and was remarkably inhibited by the latter. A short sequence 374–383 fused with Venus showed active translocation into the nucleus of COS-7 cells, but 296–309 or 1–19 did not. Despite the presence of these special regions, both activities were deprived by deletion of not only EF1 but also EF2–4 or C2 domain. Thus, PLCζ is driven into the nucleus primarily by the aid of NLS and putative regulatory sites, but coordinated three-dimensional structure, possibly formed by a folding in the X/Y linker and close EF/C2 contact as in PLCδ1, seems to be required not only for enzymatic activity but also for nuclear translocation ability. PLCζ 2The abbreviations used are: PLCζ, phospholipase C-ζ; [Ca2+]i, intracellular Ca2+ concentration; ΔEF1, deletion of the first EF-hand domain; ΔEF1-tr, truncation of the N terminus up to the end of EF1; F, fluorescence intensity; IP3, inositol 1,4,5-trisphosphate; IVF, in vitro fertilization; MII, metaphase of second meiosis; NLS, nuclear localization signal; NTR, nuclear transport receptor; PH domain, pleckstrin homology domain; PIP2, phosphatidylinositol 4,5-bisphosphate; PN, pronucleus or pronuclei; s-PLCζ, short form of PLCζ.2The abbreviations used are: PLCζ, phospholipase C-ζ; [Ca2+]i, intracellular Ca2+ concentration; ΔEF1, deletion of the first EF-hand domain; ΔEF1-tr, truncation of the N terminus up to the end of EF1; F, fluorescence intensity; IP3, inositol 1,4,5-trisphosphate; IVF, in vitro fertilization; MII, metaphase of second meiosis; NLS, nuclear localization signal; NTR, nuclear transport receptor; PH domain, pleckstrin homology domain; PIP2, phosphatidylinositol 4,5-bisphosphate; PN, pronucleus or pronuclei; s-PLCζ, short form of PLCζ. is a novel isozyme of PLC (the enzyme that hydrolyzes membrane PIP2 into IP3 and diacylglycerol) and a strong candidate of the mammalian sperm-derived egg-activating factor (1Swann K. Larman M.G. Saunders C.M. Lai F.A. Reproduction. 2004; 127: 431-439Crossref PubMed Scopus (141) Google Scholar). PLCζ is specifically expressed in the sperm (2Saunders 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 induces repetitive increase in [Ca2+]i called Ca2+ oscillations and subsequent early embryonic development when expressed in mouse eggs by injection of RNA encoding PLCζ (2Saunders 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, 3Yoda A. Oda S. Shikano T. Kouchi Z. Awaji T. Shirakawa H. Kinoshita K. Miyazaki S. Dev. Biol. 2004; 268: 245-257Crossref PubMed Scopus (120) Google Scholar). In mammalian fertilization, accumulated evidence indicates that a cytosolic sperm factor is driven into the ooplasm upon sperm egg fusion and induces Ca2+ oscillations (4Swann K. Rev. Reprod. 1996; 1: 33-39Crossref PubMed Scopus (76) Google Scholar, 5Jones K.T. Int. J. Dev. Biol. 1998; 42: 1-10PubMed Google Scholar), which are caused by Ca2+ release from the endoplasmic reticulum mainly through type 1 IP3 receptor (6Miyazaki S. Shirakawa H. Nakada K. Honda Y. Dev. Biol. 1993; 158: 62-78Crossref PubMed Scopus (501) Google Scholar) and are a pivotal signal for egg activation characterized by resumption of the second meiosis and formation of PN (5Jones K.T. Int. J. Dev. Biol. 1998; 42: 1-10PubMed Google Scholar). PLCζ is a strong candidate of the sperm factor, because 1) fertilization-like Ca2+ oscillations are produced by PLCζ expressed in a mouse egg at an estimated level comparable to the content in single mouse sperm (2Saunders 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, 3Yoda A. Oda S. Shikano T. Kouchi Z. Awaji T. Shirakawa H. Kinoshita K. Miyazaki S. Dev. Biol. 2004; 268: 245-257Crossref PubMed Scopus (120) Google Scholar). 2) Injection of recombinant PLCζ into mouse eggs induces Ca2+ oscillations as well (7Kouchi Z. Fukami K. Shikano T. Oda S. Nakamura Y. Takenawa T. Miyazaki S. J. Biol. Chem. 2004; 279: 10408-10412Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). 3) Ca2+ oscillation-inducing ability of sperm extract injected into eggs (4Swann K. Rev. Reprod. 1996; 1: 33-39Crossref PubMed Scopus (76) Google Scholar, 8Oda S. Deguchi R. Mohri T. Shikano T. Nakanishi S. Miyazaki S. Dev. Biol. 1999; 209: 172-185Crossref PubMed Scopus (65) Google Scholar) is lost when pretreated with an antibody against PLCζ (2Saunders 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). 4) PLCζ content in the mouse sperm and the number of Ca2+ spikes at fertilization are reduced by transgenic RNA interference of PLCζ (9Knott J.G. Kurokawa M. Fissore R.A. Schultz R.M. Williams C.J. Biol. Reprod. 2005; 72: 992-996Crossref PubMed Scopus (152) Google Scholar). 5) PLCζ has such a high Ca2+ sensitivity of PIP2-hydrolyzing activity that the enzyme can be active in the resting cells at ∼100 nm Ca2+ (7Kouchi Z. Fukami K. Shikano T. Oda S. Nakamura Y. Takenawa T. Miyazaki S. J. Biol. Chem. 2004; 279: 10408-10412Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 10Nomikos M. Blayney L.M. Larman M.G. Campbel K. Rossbach A. Saunders C.M. Swann K. Lai F.A. J. Biol. Chem. 2005; 280: 31011-31018Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), suitable for the sperm factor as the first stimulus in the egg cytoplasm at fertilization. Another important property of PLCζ is nuclear translocation ability. PLCζ expressed by RNA injection is accumulated into the formed PN (3Yoda A. Oda S. Shikano T. Kouchi Z. Awaji T. Shirakawa H. Kinoshita K. Miyazaki S. Dev. Biol. 2004; 268: 245-257Crossref PubMed Scopus (120) Google Scholar, 11Larman M.G. Saunders C.M. Carroll J. Lai F.A. Swann K. J. Cell Sci. 2004; 117: 2513-2521Crossref PubMed Scopus (117) Google Scholar, 12Sone Y. Ito M. Shirakawa H. Shikano T. Takeuchi H. Kinoshita K. Miyazaki S. Biochem. Biophys. Res. Commun. 2005; 330: 690-694Crossref PubMed Scopus (49) Google Scholar). This is consistent with earlier observation that sperm-derived Ca2+ oscillation-inducing activity is concentrated into PN formed several hours after fertilization, as examined by transfer of the ooplasm or PN into unfertilized eggs (13Kono T. Carroll J. Swann K. Whittingham D.G. Development. 1995; 121: 1123-1128Crossref PubMed Google Scholar). Ca2+ oscillations cease at about the time of PN formation (14Jones K.T. Carroll J. Merriman J.A. Whittingham D.G. Kono T. Development. 1995; 121: 3259-3266Crossref PubMed Google Scholar), but continue without stopping when PN formation was prevented by injection of a lectin, WGA (15Marangos P. FitzHarris G. Carroll J. Development. 2003; 130: 1461-1472Crossref PubMed Scopus (119) Google Scholar). Therefore, it is thought that translocation of the sperm factor or PLCζ into PN plays a key role in cessation of Ca2+ oscillations at the interphase of a cell cycle (11Larman M.G. Saunders C.M. Carroll J. Lai F.A. Swann K. J. Cell Sci. 2004; 117: 2513-2521Crossref PubMed Scopus (117) Google Scholar, 13Kono T. Carroll J. Swann K. Whittingham D.G. Development. 1995; 121: 1123-1128Crossref PubMed Google Scholar, 15Marangos P. FitzHarris G. Carroll J. Development. 2003; 130: 1461-1472Crossref PubMed Scopus (119) Google Scholar). Structure-function analysis of PLCζ is implicated, because PLCζ is a biologically important factor and can be practically utilized for artificial egg activation. PLCζ is composed of four EF-hand domains in the N terminus, X and Y catalytic domains, and C2 domain in the C terminus (2Saunders 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), common to other isozymes of PLC (16Williams R.L. Biochim. Biophys. Acta. 1999; 1441: 255-267Crossref PubMed Scopus (93) Google Scholar), but lacks N-terminal PH domain found in PLCβ, γ, δ, and ϵ (2Saunders 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). A short form of PLCζ, s-PLCζ, is thought to be expressed in the mouse sperm, because mRNA encoding a protein, which lacks three EF-hand domains but is identical to PLCζ in other region, has been found to exist in the mouse testis (AK006672 in EMBL). Both 74- and 65-kDa protein bands are detected by Western blotting of mouse sperm extract using anti-PLCζ antibody (17Kurokawa M. Sato K. Wu H. He C. Malcuit C. Black S.J. Fukami K. Fissore R.A. Dev. Biol. 2005; 285: 376-392Crossref PubMed Scopus (87) Google Scholar). We have found that s-PLCζ expressed in mouse eggs has much less Ca2+ oscillation-inducing activity and is hardly accumulated in PN (3Yoda A. Oda S. Shikano T. Kouchi Z. Awaji T. Shirakawa H. Kinoshita K. Miyazaki S. Dev. Biol. 2004; 268: 245-257Crossref PubMed Scopus (120) Google Scholar), suggesting that EF-hand domains are responsible for these important properties. Actually, deletion of N-terminal EF-hand domains results in the loss of Ca2+ oscillation-inducing activity (10Nomikos M. Blayney L.M. Larman M.G. Campbel K. Rossbach A. Saunders C.M. Swann K. Lai F.A. J. Biol. Chem. 2005; 280: 31011-31018Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 18Kouchi Z. Shikano T. Nakamura Y. Shirakawa H. Fukami K. Miyazaki S. J. Biol. Chem. 2005; 280: 21015-21021Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). In the present study, we addressed the molecular structure responsible for the nuclear translocation ability in a quantitative manner and in parallel with precise assay of Ca2+ oscillation-inducing activity, focusing on the putative NLS region and EF-hand domain region. PLCζ or its mutants fused with a fluorescent protein Venus (19Nagai T. Ibata K. Park E.S. Kubota M. Mikoshiba K. Miyawaki A. Nat. Biotechnol. 2002; 20: 87-90Crossref PubMed Scopus (2135) Google Scholar) were expressed by injection of respective cRNA into mouse eggs. Nuclear translocation was investigated in PN of 1-cell embryos after artificial activation by PLCζ or fertilization by the sperm. Translocation was also examined in the nucleus of cultured somatic cells after transfection of cDNA. Preparation of Gametes and Insemination—Mature eggs at MII were obtained from superovulated B6D2F1 mice (see Ref. 20Kumakiri J. Oda S. Kinoshita K. Miyazaki S. Dev. Biol. 2003; 260: 522-535Crossref PubMed Scopus (42) Google Scholar for details), and freed from cumulus cells by 0.05% hyaluronidase (Sigma). M2 medium was used during egg preparation, RNA injection, [Ca2+]i measurement, and observation of eggs or embryos. Twenty to thirty eggs were transferred to a 400-μl drop of M2 medium covered with paraffin oil in a glass-bottomed plastic dish, which was placed on the stage of an inverted fluorescence microscope (TMD, Nikon) and heated at 31–33 °C. Eggs were injected with cRNA (see below). In another experiments, cRNA was injected into 1-cell embryos after IVF. M16 medium was used for IVF and incubation of fertilized eggs. Spermatozoa were collected from the cauda epididymides and incubated at 37 °C (5% CO2 in air) for 1–1.5 h for capacitation (20Kumakiri J. Oda S. Kinoshita K. Miyazaki S. Dev. Biol. 2003; 260: 522-535Crossref PubMed Scopus (42) Google Scholar). A small amount of sperm suspension was added to a 200-μl drop of M16 medium containing M II eggs attached with cumulus cells. The eggs and spermatozoa were incubated for ∼5 h until the male and female PN were recognized. One-cell embryos were transferred to a 400-μl drop of M2 medium, treated with 0.05% hyaluronidase, and after washing, injected with cRNA. Construction of Plasmids—cDNA encoding full-length PLCζ (647 amino acid residues; see Fig. 1A) (GenBank™ accession number AF435950) or s-PLCζ lacking 110 amino acid residues from the N terminus (AK006672) was prepared using PCR techniques, fused with Venus (19Nagai T. Ibata K. Park E.S. Kubota M. Mikoshiba K. Miyawaki A. Nat. Biotechnol. 2002; 20: 87-90Crossref PubMed Scopus (2135) Google Scholar) in the C terminus, and subcloned into pBluescript II SK(+). The methods were the same as described previously (4Swann K. Rev. Reprod. 1996; 1: 33-39Crossref PubMed Scopus (76) Google Scholar). Point replacement of an amino acid or partial deletion of amino acid sequence in cDNA of PLCζ was constructed by GeneTailor™ site-directed mutagenesis system (Invitrogen), using PLCζ-Venus-pBluescript II-SK (+) as the template (see Fig. 1A for domain features and Tables for designation). To circumvent unwanted mutations, a region surrounding the targeted amino acid(s) and presenting unique restriction sites was subcloned in the parental vector and verified by DNA sequencing using Applied Biosystems ABI PRISM 310 DNA sequencer. Truncation was performed from the N terminus to a given number of amino acids between 4th to 39th residues or to the end of EF1 (D2–39), EF2 (D2–77), EF3 (D2–110), and EF4 (D2–167), leaving Met1. Truncated fragments were amplified by PCR using PLCζ-Venus-pBluescript II-KS (+) as the template. Amplified fragments were digested with KpnI and SpeI, and ligated to the KpnI and SpeI sites of the parental vector. All constructs were checked by sequence analysis. RNA and Polyadenylation—The constructed plasmids were digested with NotI, and resulting fragments were used as templates for in vitro transcription. RNA was synthesized by T3 or T7 polymerase using mMessage mMachine Kit (Ambion). To facilitate RNA translation in the egg, RNA was added with more than 200 poly(A) in the 3′-tail (see Ref. 21Aida T. Oda S. Awaji T. Yoshida K. Miyazaki S. Mol. Hum. Reprod. 2001; 7: 1039-1046Crossref PubMed Scopus (24) Google Scholar for details). Dried RNA was resolved in 150 mm KCl solution (final concentration, ∼1.5 μg/μl). RNA was diluted to the range between 10 and 1,000 ng/μl and injected into MII eggs or 1-cell embryos using a glass micropipette (injected amount, ∼4 pl per egg or embryo of which volume is 200 pl). To make the expression level of various PLCζ mutants comparable, the concentration of RNA for injection was adjusted in such way that fluorescence intensity (F) of Venus in the egg was in the range between 55 and 90 (arbitrary unit) at 3 h after RNA injection. A standard concentration of PLCζ-Venus RNA was 50 ng/μl. RNA concentration used was raised up to 1,000 ng/μl, when extreme overexpression was necessary. Measurement of Venus Fluorescence—Of 30–40 MII eggs injected with cRNA, 4–9 eggs were left in the same dish and subjected to continuous measurement of F. Fluorescent images of eggs were acquired every 3 min at 31–33 °C, using an EB-CCD camera (C7190–23; Hamamatsu Photonics) and an image processor (Argus 50; Hamamatsu Photonics). Excitation light was passed through a 470–490-nm bandpass filter and a 20× objective lens. Emitted light was passed through the objective lens, a 510-nm dichroic mirror (DM510; Nikon), and a 520–560-nm bandpass filter. Autofluorescence of the egg, probably derived from oxidized flavins (22Shirakawa H. Miyazaki S. Biophys. J. 2004; 86: 1739-1752Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), was subtracted from total fluorescence to obtain F. Other eggs were kept in another dish and subjected to precise observation at 3, 5, and 8 h after RNA injection using a confocal laser scanning microscope (LSM310, Carl Zeiss) with excitation light of 510 nm. Differential interference contrast images were recorded simultaneously by another sensor for transmitted laser light. All these procedures were also taken in the experiment in which cRNA was injected into 1-cell embryos about 5.5 h after insemination. Judgment of Nuclear Accumulation—The ratio of F in the PN to that in the cytoplasm (FPN/FC) at 6 h after RNA injection was taken as a parameter for nuclear accumulation. The evaluation criteria were tentatively defined, as indicated in the legend of Table 1. Values at 8 h were also presented to see the progression of nuclear accumulation.TABLE 1Point mutation in X domain or X/Y linker regionPLCζ mutantCa2+ oscillation-inducing abilityNuclear translocation abilityRNAaConcentration of RNA for injection.EggsF3hbF (mean value) of Venus at 3 h after RNA injection.Ca2+ cInduction of repetitive Ca2+ spikes. spikesDelaydThe time between RNA injection to 1st Ca2+ spike (mean value).RNA injeRNA injection into MII egg or 1-cell embryo.RNAaConcentration of RNA for injection.EggsF3hbF (mean value) of Venus at 3 h after RNA injection.FPN/FCfRatio of F (PN vs. cytoplasm) at 6 or 8 h after RNA injection (mean).NTgJudgment of nuclear translocation ability. Evaluation criteria (r = FPN/FC): R < 1.0, negative (-); 1.0 ≤ R ≤ 1.1, faint (±); 1.1 < R ≤ 1.5, positive (+); 1.5 < R ≤ 2.0, fair (++); 2.0 ≤ R, strong (+++).MII1-cell6 h8 hng/μlno.minng/μlno.Venus alone*1061750.980.98-PLCζ50770+28*506632.333.45+++*306561.802.08++D210R4007930-*504731.271.72+K299E10005360-*604470.820.83-K301E508120-*83430.710.78-K299A, K301A7505860-*255660.710.73-E296—V309103275-*33430.980.98-V373E50556+38*704781.431.73+K374E50475+42*5031151.141.29+K375E50836+46*1004701.412.08+R376E455170+31*254600.890.88-K377E505700+22*208950.870.87-K377A504380+25*3081202.573.94+++R378E50465+42*605801.021.08±K379E55946+41*903800.830.87-M380E504140+29*304751.201.47+K381E505355+28*153641.041.08±I 382E604250+26*2031002.193.77+++K374—A383109175-*1031750.980.98-a Concentration of RNA for injection.b F (mean value) of Venus at 3 h after RNA injection.c Induction of repetitive Ca2+ spikes.d The time between RNA injection to 1st Ca2+ spike (mean value).e RNA injection into MII egg or 1-cell embryo.f Ratio of F (PN vs. cytoplasm) at 6 or 8 h after RNA injection (mean).g Judgment of nuclear translocation ability. Evaluation criteria (r = FPN/FC): R < 1.0, negative (-); 1.0 ≤ R ≤ 1.1, faint (±); 1.1 < R ≤ 1.5, positive (+); 1.5 < R ≤ 2.0, fair (++); 2.0 ≤ R, strong (+++). Open table in a new tab [Ca2+]i Measurement—Ca2+ oscillations were recorded in another optical system by conventional Ca2+ imaging method using an image processor. Four to five MII eggs were injected with 50 μm solution of the Ca2+-sensitive fluorescent dye fura dextran (Molecular Probes Inc.) together with a cRNA and were subjected to [Ca2+]i measurement for 9 h after RNA injection. F of fura was measured without interference with that of Venus, by applying 340- and 380-nm UV lights alternatively and by leading emission light through a 400-nm dichroic mirror (DCLP; Omega) and a 500–520-nm bandpass filter. Fluorescence was detected by an EB-CCD camera (C7190–23; Hamamatsu Photonics). Ca2+ images were acquired at intervals of 20 s and processed to calculate F340/F380 later using NIH Image (a public domain image processing software for the Macintosh computer). Formation of the PN and nuclear translocation of a PLCζ mutant were examined 5 and 9 h after RNA injection, respectively. Nuclear Translocation in Cultured Somatic Cells—COS-7 cells cultured on glass coverslips were transfected with cDNA of Venus-tagged PLCζ mutants, using FuGENE6 (Roche Diagnostics) (23Awaji T. Hirasawa A. Shirakawa H. Tsujimoto G. Miyazaki S. Biochem. Biophys. Res. Commun. 2001; 289: 457-462Crossref PubMed Scopus (56) Google Scholar). Fluorescent cells were observed 9, 24, 48, or 72 h later by confocal microscopy (LSM510META, Carl Zeiss). For [Ca2+]i measurement, cells were loaded with fura-2 acetoxymethyl ester (fura-2 AM; Molecular Probes) by incubation in 4 μm fura-2 AM for 30 min at 24 °C. [Ca2+]i measurement was performed at 24 °C in Tyrode's solution 24 and 48 h after transfection. Ca2+ Oscillation-inducing Activity and Nuclear Translocation Ability of Wild-type PLCζ—The domain feature associated with amino acid number of PLCζ is illustrated in Fig. 1A. Under the present experimental conditions, expression of PLCζ in MII eggs was detected by Venus-derived F from 30 min after injection of 50 ng/μl RNA, increased up to 3–4 h, and attained a steady level (Fig. 2A). The magnitude of expression of PLCζ-Venus was compared in F at 3 h after RNA injection (Tables 1 and 2). The first Ca2+ transient was generated 30–40 min after injection of RNA of wild-type PLCζ (Fig. 1B). The delay time was a parameter that reflects Ca2+ oscillation-inducing activity of expressed PLCζ mutants (Tables 1 and 2); that is, the higher activity shortened the delay time. The second and third Ca2+ spikes occurred at an interval of ∼20 min. The interval was shortened up to 10 min for succeeding Ca2+ spikes (Fig. 1B). These Ca2+ oscillations, which are probably caused by continuously produced IP3 (24Jones K.T. Nixon V.L. Dev. Biol. 2000; 225: 1-12Crossref PubMed Scopus (65) Google Scholar), lasted for 3–4 h and suddenly ceased prior to the formation of (female) PN at about 5 h after RNA injection. The higher PLCζ activity resulted in earlier termination of Ca2+ oscillations, possibly because of a negative feedback via production of diacylglycerol and subsequent activation of protein kinase C (25Ali H. Richardson R.M. Haribabu B. Snyderman R. J. Biol. Chem. 1999; 274: 6027-6030Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), and/or down-regulation of IP3 receptor type 1 which develops as a result of Ca2+ oscillations, notably ∼4 h after fertilization or parthenogenetic activation (26Brind S. Swann K. Carroll J. Dev. Biol. 2000; 223: 251-265Crossref PubMed Scopus (108) Google Scholar, 27Jullerette T. He C.L. Wu H. Parys J.B. Fissore R.A. Dev. Biol. 2000; 223: 238-250Crossref PubMed Scopus (147) Google Scholar). It should be noted that expressed PLCζ was continuously accumulated into the formed PN (Fig. 2A) as described previously (4Swann K. Rev. Reprod. 1996; 1: 33-39Crossref PubMed Scopus (76) Google Scholar). F in the PN (FPN) became more than twice of F in the cytoplasm (FC) 6 h after RNA injection (Table 1). PLCζ that entered PN appeared to avoid the large nucleolus, which was identified as a round structure with a clear circumference in the bright field image (Fig. 2A, paired photographs at the right).TABLE 2Mutation in EF-hand domain region and C2 domainPLCζ mutantCa2+ oscillation-inducing abilityNuclear translocation abilityRNAaConcentration of RNA for injection.EggsF3hbF (mean value) of Venus at 3 h after RNA injection.Ca2+cInduction of repetitive Ca2+ spikes. spikesDelaydThe time between RNA injection to 1st Ca2+ spike (mean value).RNA injeRNA injection into MII egg or 1-cell embryo.RNAaConcentration of RNA for injection.EggsF3hbF (mean value) of Venus at 3 h after RNA injection.FPN/FCfRatio of F (PN vs. cytoplasm) at 6 or 8 h after RNA injection (mean).NTgJudgment of nuclear translocation ability. Evaluation criteria (r = FPN/FC): R < 1.0, negative (-); 1.0 ≤ R ≤ 1.1, faint (±); 1.1 < R ≤ 1.5, positive (+); 1.5 < R ≤ 2.0, fair (++); 2.0 ≤ R, strong (+++).MII1-cell6 h8 hng/μlno.minng/μlno.Venus alone*1061750.980.98-PLCζ50770+28*506632.333.45+++*306561.802.08++Δ2-460480+36*456752.153.55++Δ2-950855+50*504631.051.28(+)hDelayed nuclear translocation.*50055301.051.22(+)hDelayed nuclear translocation.Δ2-1410005325-*703550.69-Δ2-1910005890-*4071050.67-M1-Q192542750-*56930.98-E10A,R12A555150+42*356601.231.25+R12A,W13A,F14A10004345-*704820.75-R12E455170+29*203601.221.57+W13A1808195(+)iDelayed Ca2+ response (delay time >60 min).68*1204720.880.80-W13F505144+53*304721.021.07±F14A50480(+)iDelayed Ca2+ response (delay time >60 min).115*506650.870.79-F14W454275+32*124401.482.27+W13F,F14W455260(+)iDelayed Ca2+ response (delay time >60 min).72*154450.910.89-S16A555240+30*202881.171.25+K17A554120+34*4031351.131.48+K17E504230+32*203601.181.69+V18A30680+55*2041360.950.95-604220+40*300411550.820.85-Δ2-39 (ΔEF1-tr)jEF1-truncated PLCζ.508180-*407860.70-55041745-*400621200.73-Δ2-77 (ΔEF1-2-tr)5014115-*5061400.75-25014870(+)iDelayed Ca2+ response (delay time >60 min).173*500510500.84-Δ2-110 (s-PLCζ)50790-*509750.840.85-2505890(+)iDelayed Ca2+ response (delay time >60 min).187*50046350.85-Δ2-167 (ΔEF1-4-tr)100042160-*500428400.84-Δ10-19100051640-*205880.70-Δ45-110 (ΔEF2-3)100041270-*204800.85-Δ45-163 (ΔEF2-4)100061170-*409700.73-Δ522-610 (ΔC2)50092145-*508700.79-D542A709300+28*203921.412.73+D542R50455+36*7041541.111.54+a Concentration of RNA for injection.b F (mean value) of Venus at 3 h after RNA injection.c Induction of repetitive Ca2+ spikes.d The time between RNA injection to 1st Ca2+ spike (mean value).e RNA injection into MII egg or 1-cell embryo.f Ratio of F (PN vs. cytoplasm) at 6 or 8 h after RNA injection (mean).g Judgment of nuclear translocation ability. Evaluation criteria (r = FPN/FC): R < 1.0, negative (-); 1.0 ≤ R ≤ 1.1, faint (±); 1.1 < R ≤ 1.5, positive (+); 1.5 < R ≤ 2.0, fair (++); 2.0 ≤ R, strong (+++).h Delayed nuclear translocation.i Delayed Ca2+ response (delay time >60 min).j EF1-truncated PLCζ. Open table in a new tab Nuclear translocation of PLCζ was observed as well, when RNA was injected into the 1-cell embryo in which male and female PN were recognized 5 h after insemination. In the 1-cell embryo, Ca2+ oscillations induced by IVF had already ceased (14Jones K.T. Carroll J. Merriman J.A. Whittingham D.G. Kono T. Development. 1995; 121: 3259-3266Crossref PubMed Google Scholar, 28Deguchi R. Shirakawa H. Oda S. Mohri T. Miyazaki S. Dev. Biol. 2000; 218: 299-313Crossref PubMed Scopus (119) Google Scholar), and another series of Ca2+ spikes were induced by expressed PLCζ after a long delay of ∼80 min and at long intervals of 40–60 min (Fig. 1C). Phosphoinositide signaling pathway and/or IP3 receptor-mediated Ca2+ release seems to be suppressed in the 1-cell embryo, at the interphase of cell cycle (11Larman M.G. Saunders C.M. Carroll J. Lai F.A. Swann K. J. Cell Sci. 2004; 117: 2513-2521Crossref PubMed Scopus (117) Google Scholar). As shown in Fig. 2B (line 2), FPN was lower than FC at the early stage after RNA injection, but exceeded the latter at 3.5 h. Subsequently, FPN continuously increased and became twice of FC at about 8 h. Venus alone was more expressed than PLCζ-Venus (Fig. 2B, line 1) because of the smaller molecule. For Venus alone, FPN/FC was close to 1.0 (photographs of the inset in Fig. 2B; Table 1), indicating free diffusion through nuclear pores. The nuclear translocation of PLCζ expressed in the 1-cell embryo after IVF served as a control for mutants that had quite low or no Ca2+ oscillation-inducing activity and were incapable of activating the egg. For example, the mutant in which Asp210 in the X catalytic domain (see Fig. 1A) was replaced with arginine (D210R) was defective in Ca2+ oscillation-inducing activity even when overexpressed (Fig. 1D and Table 1), as shown previously (2Saunders 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). Nuclear accumulation of D210R took place (Fig. 2C), but it was substantially slower, compared with that of wild-type PLCζ (Table 1). F in the nucleolus was comparable to that in the nucleoplasm 12 h after RNA injection (Fig. 2C, paired photographs at the right). Some fraction of PLCζ may to be accumulated to the
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