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Embryo; |
Department of Physiology,3 Tokyo Women's Medical University School of Medicine, Shinjuku-ku, Tokyo 162-8666, Japan
Department of Safety Research on Blood and Biological Products,4 National Institute of Infectious Diseases, Musashimurayama-shi, Tokyo 208-0011, Japan
Department of Integrated Biosciences,5 Graduate School of Frontier Sciences, University of Tokyo, Kashiwa City, Chiba 277-8562, Japan
Department of Biology, Faculty of Science,6 Toho University, Funabashi City, Chiba 274-8510, Japan
Department of Pharmacology,7 Saitama Medical University School of Medicine, Moro-machi, Saitama 350-0495, Japan
Tokyo Women's Medical University School of Medicine,8 Shinjuku-ku, Tokyo 162-8666, Japan
ABSTRACT
Mouse phospholipase C, zeta 1 (PLCZ1), a strong candidate of egg-activating sperm factor, induces Ca2+ oscillations and accumulates into formed pronucleus (PN) when expressed by cRNA injection. These activities were compared among mouse and human PLCZ1, newly cloned rat Plcz1, and medaka fish plcz1. The PLCZ1 proteins of the four species have an approximately homologous sequence of nuclear localization signal. However, the nuclear translocation ability was defective in rat, human, and medaka PLCZ1 expressed in mouse eggs. Rat PLCZ1 could not enter rat PN, whereas mouse PLCZ1 could. Mouse and human PLCZ1 translocated into the nucleus of COS-7 cells transfected with cDNA. There was little medaka PLCZ1 accumulated in the nucleus, and rat PLCZ1 was never located in the nucleus. All PLCZ1 proteins including fish could induce Ca2+ oscillations in mouse eggs, but the activity was variable in the order of human >> mouse > medaka >> rat, estimated from minimal RNA concentration to induce Ca2+ spikes. Ca2+ oscillations by human PLCZ1 continued far beyond the time of PN formation (TPN), whereas those by mouse PLCZ1 ceased slightly before TPN. High-frequency Ca2+ spikes by overexpressed rat PLCZ1 stopped far before TPN, possibly by feedback inhibition. Ca2+ oscillations by fertilization of rat eggs stopped at TPN, despite defective nuclear translocation of rat PLCZ1. Thus, PLCZ1 sequestration into PN participates in termination of Ca2+ oscillations at the interphase of mouse embryos but does not always operate in other mammals, notably in rat embryos.
calcium, Ca2+ oscillation-inducing activity, early development, egg-activating sperm factor, fertilization, nuclear translocation ability, phospholipase C zeta 1, species difference, sperm
Fertilized mammalian eggs show a repetitive transient increase in intracellular calcium ion concentration ([Ca2+]i) due to repeated Ca2+ release from the endoplasmic reticulum mainly through inositol 1,4,5-trisphosphate receptor 1 [1, 2]. The Ca2+ oscillations are a pivotal signal for egg activation and cause resumption of the second meiosis and subsequent formation of male and female pronuclei (PN) [3, 4]. Repetitive Ca2+ release is induced by a cytosolic sperm factor driven into the ooplasm upon sperm-egg fusion [5, 6]. Several lines of evidence indicate that a sperm-specific isozyme "zeta" of InsP3-producing enzyme phospholipase C (PLCZ1) is a strong candidate of the sperm factor [6–10]. Fertilization-like Ca2+ oscillations are produced by PLCZ1 expressed in mouse eggs by injection of cRNA encoding PLCZ1, at an estimated PLCZ1 level comparable to the level in single sperm [10, 11]. The Ca2+ oscillation-inducing ability of sperm extract injected into eggs is lost when the sperm extract was pretreated with an antibody against PLCZ1 [10].
A long-lasting series of Ca2+ spikes is the common Ca2+ response in fertilization of mammalian eggs, but there is some difference in the frequency and pattern of Ca2+ oscillations among hamster [12], pig [13], mouse [14], cow [15], and human [16]. PLCZ1 has been cloned in mouse, human, monkey, pig, and cow, but the Ca2+ oscillation-inducing activity of PLCZ1 seems to be different among species as well, when compared in the effective concentration of injected RNA [6, 11, 17, 18].
Besides its Ca2+ oscillation-inducing ability, mouse PLCZ1 possesses nuclear translocation ability. PLCZ1 expressed by injection of cRNA is accumulated into the formed PN of the mouse one-cell embryo [11, 19]. This ability is notable because the sperm-derived Ca2+ oscillation-inducing activity or egg-activating activity is concentrated into the PN, as assayed by transfer of the ooplasm or PN to an unfertilized mouse egg [20, 21]. Evidence has shown that sequestration of the sperm factor or PLCZ1 into the PN participates in the cessation of Ca2+ oscillations around the time of PN formation when the cell cycle enters the interphase [19, 22, 23]. The nuclear localization signal (NLS) sequence of mouse PLCZ1 exists in K374-K381 in the linker region of X and Y catalytic domains (Fig. 1A), involving a cluster of basic amino acids (Fig. 1B) [19, 24]. Ca2+ oscillations induced by a point mutant in the NLS sequence, K377E, do not stop at PN formation but continue over 10 h [19, 23]. The presumptive NLS region involving a cluster of basic amino acids is recognized in various mammalian species (Fig. 1B) in the C-terminal end of the X-Y linker region (Fig. 1A) [6].
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The nuclear translocation ability of PLCZ1 has not been examined except in mouse. In the present study, rat Plcz1 was cloned, and the ability of nuclear sequestration was addressed in the rat and human and compared among mammalian species. PLCZ1 tagged with a yellow fluorescent protein, Venus, was expressed in mouse eggs injected with cRNA or in COS-7 cells transfected with cDNA. The efficiency of PLCZ1 for inducing Ca2+ oscillations was also examined by changing the RNA concentration for injection. As to nonmammalian species, PLCZ1 of the chicken has been cloned and found to induce Ca2+ oscillations [25]. In the present study, plcz1 of the medaka fish, Oryzias patipes, was cloned and subjected to examination.
Preparation of Eggs and Sperm and Insemination
B6D2F1 female mice and Wistar female rats (Saitama Experimental Animals Supply Co. Ltd., Saitama, Japan) were superovulated by intraperitoneal injection of 5 IU or 30 IU eCG (Teikokuzoki, Tokyo, Japan) followed by 5 IU or 15 IU of hCG (Mochida Pharmaceutical Co., Tokyo, Japan) 48 h or 52 h later, respectively. Mature mouse and rat eggs at the metaphase of the second meiosis (M II) were obtained from the oviducts 16 h or 24 h after hCG injection, respectively, and freed from cumulus cells using 0.05% hyaluronidase (Sigma, St. Louis, MO). Hepes-buffered M2 medium [26] supplemented with BSA (4 mg/ml) was used for egg preparation, RNA injection, [Ca2+]i measurement, and observation of eggs or embryos. Eight to ten eggs were transferred to a 300-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, Tokyo, Japan) and heated at 31 to 33°C. Eggs were injected with cRNA (see the following).
Spermatozoa were collected from the cauda epididymides and incubated at 37°C (5% CO2 in air) for 1.5 to 2 h for capacitation. A small amount of mouse or rat sperm suspension was added to a 200-µl drop of M16 medium [27] or IVF-30 medium (Vitrolife, Kungsbacka, Sweden) containing M II eggs attached with cumulus cells, respectively. Inseminated eggs were incubated for 1.5 h, and [Ca2+]i measurement was started from 2 h after insemination.
All of the procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of Tokyo Women's Medical University School of Medicine and were performed in accordance with the guiding principles for the care and use of laboratory animals.
We prepared cDNA encoding mouse PLCZ1 (647 amino acid residues [aa]; GenBank accession number AF435950) using PCR techniques, fused with a yellow fluorescent protein, Venus [28], in the C terminus and subcloned into pBluescript II SK(+) (Stratagene, La Jolla, CA), as described previously [11]. We amplified cDNA encoding rat PLCZ1 (645 aa; AY885259) and human PLCZ1 (608 aa; AF532185) using Pfu Turbo DNA polymerase (Stratagene) from rat testis cDNA synthesized by superscript III (Invitrogen Corp., Carlsbad, CA) and human testis cDNA (PCR Ready First Strand cDNA; C1234260; BioChain Institute, Inc., Hayward, CA), respectively. The following primers were used for rat PLCZ1: 5'-GGGGTACCGCCACCATGGAGAGCCATTATGA GCTCGCAG-3' (forward primer involving a KpnI site) and 5'-CACACTAGTCTCTCT GTAGTACCAAACG-3' (reverse primer involving a SpeI site). Primers for human PLCZ1 were 5'-GGGGTACCGCCACCATGGAAATGAGATGGTTTTTGTC-3' (forward primer involving a KpnI site) and 5'-CACACTAGTTCTGACGTACCAAACATA-3' (reverse primer involving a SpeI site). Fragments of rat Plcz1 and human PLCZ1 cDNA were digested with KpnI and SpeI and ligated to the KpnI and SpeI sites of Venus-pBluescript II SK(+).
To obtain the cDNA encoding medaka PLCZ1, we first searched the medaka genome database at UTGB (http://medaka.utgenome.org/) by BLAST [29] and found a sequence that shows a strong similarity to the mammalian PLCZ1 gene and a conserved syntenic location in the medaka genome. CLUSTALW analysis (http://clustalw.ddbj.nig.ac.jp/top-e.html) [30] strongly suggested that the sequence is the medaka orthologue of mouse PLCZ1 (data not shown). We amplified cDNA fragment from testis RNA of the medaka (Hd-rR strain) using KOD DNA polymerase (derived from Thermococcus kodakaraensis; Toyobo, Osaka, Japan) and a pair of medaka plcz1-specific primers. Then, full length cDNA (567 aa; not registered in GenBank) was obtained by 5'- and 3'-RACEs (rapid amplification of cDNA ends) and cloned into pBluescript II SK(+).
For expression in COS-7 cells, Plcz1-Venus-pBluescript II SK(+) were digested with KpnI and NotI, and resulting fragments were ligated to the KpnI and NotI site of the mammalian expression vector pEF6/V5-HisA (Invitrogen Corp.). The sequences of all constructs were verified by DNA sequencing using ABI PRISM 310 DNA sequencer (Applied Biosystems, Foster City, CA).
cRNAs and Polyadenylation for Expression of PLCZ1
The constructed plasmids were digested with NotI, and resulting fragments were used as templates for in vitro transcription (for details, see [11]). Briefly, cRNA was synthesized by T7 polymerase using mMessage mMachine Kit (Ambion, Austin, TX). To facilitate RNA translation in the egg, RNA was added with more than 200 poly(A) in the 3' tail [31] by poly(A) polymerase (GE Healthcare Bio-Science Corp., Piscataway, NJ). Dried RNA was resolved in 150 mM KCl solution (final concentration, about 1.5 µg/µl) and concentrations of synthesized cRNA solution were measured by NanoDrop (ND-1000; NanoDrop Technologies, Wilmington, DE). We diluted cRNA to the range between 0.005 and 500 ng/µl and injected it into M II eggs using a glass micropipette (injected amount, about 4 pl per egg, of which volume is 200 pl).
Tissue Expression of PLCZ1 in Rat and Medaka
Northern blot analysis for rat tissues was performed by using Rat MTN Blot (Clontech Laboratories, Inc., Mountain View, CA) containing approximately 2 µg of polyadenylated RNA per lane. A 685-bp fragment from the 3' terminal of rat Plcz1 open reading frame (ORF) and Actb (supplied along with the MTN Blot) were labeled with biotin-N4-dCTP using North2South Biotin Random Prime Kit (Pierce, Rockford, IL) as a probe. Hybridization and chemiluminescent signal detection were done using the North2South Chemiluminescent Hybridization and Detection Kit (Pierce). The membrane was stripped with nuclease-free water containing 0.5% SDS for 10 min at 100°C.
For RT-PCR, cDNAs from rat tissues (Sprague-Dawley rats; 8 to 12 wk; heart, brain, spleen, lung, liver, smooth muscle, kidney, and testis) were purchased from Clontech (Rat MTC Panel I; Clontech Laboratories). We synthesized cDNAs from medaka tissues (brain, liver, skeletal muscle, spleen, ovary, and testis) from purified RNAs of each tissue of the Hd-rR strain. PCR amplification was performed at 30 cycles for rat Plcz1, medaka plcz1, and medaka actb, and 25 cycles for rat Gapdh using TaKaRa LA Taq Hot Start Version (Takara Bio Inc., Otsu, Japan). The following primers were used: 5'-AGAGAATCACTGCTCCCC-3' and 5'-CACCATCTGACAGCCCAC-3' for rat Plcz1 (a 0.6-kb region within rat Plcz1), 5'-TGAAGGTCGGTGTCAACGGATTTGGC-3' and 5'-CATGTAGGCCATGAGGTCCACCAC-3' for rat Gapdh, 5'-CAGGAGGAGAAA GTCTCTGTG-3' and 5'-CAGAGAAACTGGTTCTGTATG-3' for medaka plcz1 (full sequence), 5'-CACTCTGAGCGCCGTCACACACAG-3' and 5'-TGACACCCTGGTGC CTGGGGCGAC-3' for medaka actb. A reaction without cDNA (water control) was run to monitor reagent contamination.
Observation of Venus Fluorescence
To observe nuclear accumulation of PLCZ1, Venus-derived fluorescence images of eggs and one-cell embryos were acquired 6 h after RNA injection, using confocal laser scanning microscopy (LSM510META; Carl Zeiss, Oberkochen, Germany) with excitation light of 510 nm. Differential interference contrast images were simultaneously recorded by another sensor for transmitted light.
COS-7 cells cultured on glass coverslips were transfected with cDNA of Venus-tagged PLCZ1, using FuGene6 (Roche Diagnostics, Basel, Switzerland) [32]. Fluorescent cells were observed 48 h later by confocal microscopy.
Ca2+ oscillations were recorded by a conventional Ca2+ imaging method using an image processor (Argus 200; Hamamatsu Photonics, Hamamatsu, Japan). Of 10 to 20 M II eggs injected with 50 µM solution of the Ca2+-sensitive fluorescent dye fura dextran (Molecular Probes, Inc., Eugene, OR) together with cRNA, six to nine eggs were left in the same dish and subjected to continuous [Ca2+]i measurement. Ultraviolet lights of 340 nm and 380 nm were applied alternatively, and emission light was led through a 400-nm dichroic mirror (DCLP; Omega) and a 500-nm to 520-nm bandpass filter, and 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 developed by the National Institutes of Health). Other eggs were kept in another dish and subjected to the measurement of Venus fluorescence at the defined time, using an EB-CCD camera and an image processor (Argus 50; Hamamatsu Photonics). Excitation light was passed through a 470-nm to 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-nm to 560-nm bandpass filter. Formation of the PN was examined using bright-field optics every 10 min after extrusion of the second polar body. To avoid the possibility of RNA degradation, the patterns of Ca2+ oscillations and their Venus fluorescence were checked with 3, 4, 3, and 2 batches of mouse and rat Plcz1, human PLCZ1, and medaka plcz1 cRNAs, respectively.
Translocation Ability of PLCZ1 into the Pronucleus of One-Cell Embryos
Mouse Plcz1 and human PLCZ1 have been cloned and shown to be expressed specifically in the testis [10, 17]. In the present study, rat Plcz1 and medaka plcz1 were cloned, having 87% and 47% homology to mouse Plcz1 and 71% and 48% homology to human PLCZ1, respectively (Fig. 2). For precise functional analysis of PLCZ1, all cloned sequences and expression vectors containing Plcz1 cDNA were fully sequenced and verified to ensure that no extraneous mutations were introduced during PCR. Specific expression of rat Plcz1 in the rat testis is shown in Figure 3, A and Ba, using RT-PCR and Northern blotting. A similar result was obtained for medaka plcz1 (Fig. 3Bb). The domain feature of PLCZ1 is shown in Figure 1A, and the NLS sequence of mouse PLCZ1, together with the presumptive NLS region of various animal species, is presented in Figure 1B.
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The translocation ability of PLCZ1 into the PN was examined by expressing enough PLCZ1-Venus in mouse eggs after injection of 100 ng/µl cRNA. When RNA encoding mouse PLCZ1 was injected, complete formation of the PN was obviously recognized 5 h later, as indicated by the nucleolus having a clear circumference in the bright-field differential interference contrast image (Fig. 4Aa, bottom panel). It has been shown that expressed PLCZ1 begins to enter the PN as soon as the nuclear envelope is formed [23] and is accumulated in the developing PN [11, 23]. The accumulation in the nucleoplasm but not in the nucleolus is shown in Figure 4Aa, taken 6 h after RNA injection.
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When RNA encoding rat, human, or medaka PLCZ1-Venus was injected, the PN was completely formed 5 h later as well, and photographs were taken 6 h after RNA injection. Fluorescence of rat PLCZ1-Venus was observed in the cytoplasm of the mouse one-cell embryo, but it was absent in the PN, as indicated by a dark, round area (Fig. 4Ab). That was also the case for human and medaka PLCZ1 (Fig. 4, Ac and Ad). These results were also obtained by expressing PLCZ1-FLAG (addition of only 10 aa in the C terminus of PLCZ1) with Plcz1-FLAG RNA (100 ng/µl) and by immunohistochemical staining with anti-FLAG mAb 6 h later (not shown). Thus, rat, human, and medaka PLCZ1 are incapable of targeting into the mouse PN.
When mouse PLCZ1-Venus was expressed in rat eggs, it was accumulated into the PN formed 5 h after RNA injection (Fig. 4Ba), indicating that the nuclear transport receptor for mouse PLCZ1 is present in rat eggs as in mouse eggs. In contrast, rat PLCZ1 expressed in rat eggs did not enter the PN (Fig. 4Bb), unlike mouse PLCZ1. Thus, rat PLCZ1 has no ability to translocate even into the PN of the homologous species.
Translocation Ability of PLCZ1 into the Nucleus of Somatic Cells
Our previous experiments [24] have shown that mouse PLCZ1-Venus can translocate into the nucleus of cultured somatic cells (monkey kidney COS-7 cells), whereas a point mutant in the NLS sequence, K377E-Venus, cannot enter the nucleus. A fragment of the NSL sequence from K374 to A383 fused with Venus is accumulated into both the nucleoplasm and nucleoli of COS-7 cells [24]. Therefore, COS-7 cells can be used for examination of the nuclear translocation ability. Figure 5A shows nuclear distribution of mouse PLCZ1-Venus in COS-7 cells 48 h after transfection. Fluorescence of Venus was remarkable in the nucleoli, whereas fluorescence intensity in the nucleoplasm (FN) was rather lower than that in the cytoplasm (FC). At 72 h, mouse PLCZ1 is accumulated in the nucleoplasm as well as nucleoli [24].
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For rat PLCZ1, the nucleus was quite dark (Fig. 5B), indicating that rat PLCZ1 has primarily no nuclear translocation ability. For human PLCZ1, FN was substantially higher than FC, although PLCZ1 was not accumulated in the nucleoli as indicated by black spots in the nucleus (Fig. 5C). For medaka PLCZ1, FN was faintly detected but was lower than FC (Fig. 5D). No fluorescence was visible in the nucleoli. Thus, human PLCZ1 translocates into the nucleus of COS-7 cells to a lesser extent compared with mouse PLCZ1. Medaka PLCZ1 seems to be weakly accumulated in the nucleoplasm.
Temporal Pattern of Ca2+ Oscillations after Fertilization of Mouse and Rat Eggs
Ca2+ oscillations in fertilized mouse eggs cease around the time of PN formation [14, 33]. This phenomenon was examined in rat eggs, because rat PLCZ1 was not sequestered into the PN. We tried to record Ca2+ oscillations under conditions close to those of usual fertilization as far as possible. Mouse or rat eggs having the intact zona pellucida attached with cumulus cells were inseminated and kept in M16 medium or IVF-30 medium, respectively, in a CO2 incubator until [Ca2+]i recording was started 2 h later, to avoid any effect of long-term ultraviolet irradiation on Ca2+ dynamics at the early stage of fertilized eggs. The time of complete PN formation, TPN, was defined as the time when the nucleolus having a clear circumference was obviously recognized in the bright-field image taken every 10 min during [Ca2+]i measurement.
Ca2+ oscillations in monospermic mouse eggs under this experimental condition consist of low-frequency Ca2+ spikes with interspike intervals of 20 to 30 min [23], although higher-frequency Ca2+ oscillations have been recorded so far in zona-free eggs [14, 34–36] in which multiple sperm entry tends to occur. In the mouse egg shown in Figure 6A, the interval of the last two Ca2+ spikes was prolonged to about 40 min, and the last Ca2+ spike occurred 30 min prior to TPN. We have shown that the time of the initiation of nuclear envelope formation is 50 to 60 min before TPN [23]. Therefore, Ca2+ oscillations disappear during PN formation.
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Ca2+ oscillations in fertilized rat eggs consisted of Ca2+ spikes with the interval of about 6 min (Fig. 6B), a much higher frequency than that in mouse eggs. Because the egg was monospermic, the amount of the sperm factor driven into the egg may be not greatly different between mouse and rat. The high-frequency Ca2+ oscillations may be derived from the property of rat PLCZ1 (see Discussion). Ca2+ oscillations ceased close to TPN (Fig. 6B). Ca2+ spikes become absent at the interphase after fertilization, even if rat PLCZ1 is not sequestered into the PN.
Ca2+ Oscillation-Inducing Activity of Mouse and Rat PLCZ1 Expressed in Mouse Eggs
The Ca2+ oscillation-inducing activity of PLCZ1 expressed in mouse eggs was precisely analyzed by changing the RNA concentration in a wide range. For an RNA concentration less than 10 ng/µl, PLCZ1-Venus expression was so low that FC was indistinguishable from autofluorescence of the egg; that is, the amount of expressed PLCZ1 was unknown. However, comparison of PLCZ1 among species was possible, because FC measured 3 h after injection of 50 ng/µl RNA was 200 ± 12 (mean ± SD; n = 9 eggs) for mouse PLCZ1, 112 ± 5 for rat PLCZ1 (n = 5), 115 ± 15 (n = 8) for human PLCZ1, and 85 ± 6 (n = 8) for medaka PLCZ1. Thus, mouse Plcz1-Venus RNA was the most efficient in expression of the encoding protein, but the difference in the efficiency among four species was thought to be within 2.3-fold. For the following analysis, concentration of diluted RNAs (>10 ng/µl) were calculated by their absorbance. And, concentrations of 0.005 to 10 ng/µl of RNAs were prepared by the dilution from 50 ng/µl of the measured RNA solution and taken as the indicated ones.
Figure 7, A–D, shows Ca2+ oscillations after injection of mouse Plcz1. The time lag (Tlag) from RNA injection (the zero time) to the occurrence of the first Ca2+ spike depends on expressed PLCZ1 [11, 24]. Tlag was about 2 h for 0.1 ng/µl RNA, and it shortened to 30 min for increasing doses of RNA. For 0.1 ng/µl RNA, Ca2+ spikes occurred at 50 min intervals, and the last spike was generated 50 min prior to TPN (Fig. 7A). With increasing RNA concentration, the Ca2+ spikes increased in frequency and ceased around TPN (Fig. 7B) or 2 to 3 h after TPN (Fig. 7C). On the contrary, Ca2+ oscillations stopped far before TPN when PLCZ1 was overexpressed, probably because of feedback inhibition.
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Ca2+ oscillations induced by rat PLCZ1 expressed in mouse eggs differed from those induced by mouse PLCZ1 in several points. In Figure 7E, Tlag was as long as 3.5 h even for injection of 10 ng/µl RNA. The amplitude of each Ca2+ spike increased gradually up to the maximal spike, taking 3 h. TPN was much delayed (8 h after the first Ca2+ spike), suggesting that the early Ca2+ spikes might fail to trigger egg activation. Ca2+ oscillation as a whole had the duration of 5.5 h and ended 3 h prior to TPN (Fig. 7E). The RNA concentration of 10 ng/µl was nearly critical, as lower concentrations induced no Ca2+ spike. Considering that the efficiency for PLCZ1 expression was roughly half that of mouse PLCZ1 (see earlier), the Ca2+ oscillation-inducing activity of rat PLCZ1 is estimated to be 50-fold lower than that of mouse PLCZ1. With increasing RNA concentration, both Tlag and TPN were earlier, and Ca2+ spikes had the higher frequency and disappeared 2 to 2.5 h prior to TPN (Fig. 7, F and G). For RNA concentration as high as 500 ng/µl, Tlag was still 1 h, and Ca2+ oscillations consisting of a burst of small spikes followed by high-frequency spikes lasted for 3 h and ended 2 h before TPN (Fig. 7H). Feedback inhibition seemed to operate on these Ca2+ oscillations.
Ca2+ Oscillation-Inducing Activity of Human and Medaka PLCZ1 in Mouse Eggs
Surprisingly, for human PLCZ1, an RNA concentration as low as 0.005 ng/µl was able to induce Ca2+ oscillations (Fig. 8A). Considering that the efficiency for PLCZ1 expression was roughly half that of mouse Plcz1 RNA, the Ca2+ oscillation-inducing activity of human PLCZ1 is estimated to be 40-fold higher than that of mouse PLCZ1. Ca2+ oscillations shown in Figure 8A had a Tlag of 3 h and interspike intervals of 50 to 70 min and ceased 2 h after TPN. Increased RNA concentration caused Ca2+ spikes with the higher frequency, and Ca2+ oscillations lasted for several hours beyond TPN, although the interspike interval was prolonged (Fig. 8, B and C). Feedback inhibition of Ca2+ oscillations was observed after injection of 0.5 ng/µl RNA (Fig. 8D).
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Medaka PLCZ1 was revealed to induce Ca2+ oscillations in mouse eggs. Dose-dependent changes in the Ca2+ oscillation pattern induced by medaka PLCZ1 were basically similar to those induced by human PLCZ1, although the critical concentration was roughly two orders of magnitude higher than that of human PLCZ1 (Fig. 8, E–H). The activity was slightly lower than that of mouse PLCZ1.
Nuclear Translocation Ability of PLCZ1
The present study demonstrated that translocation into the mouse PN, a characteristic of mouse PLCZ1, is defective in rat, human, and medaka PLCZ1. These results were anticipated, because the NLS sequence of K374-K381 in mouse PLCZ1 (Fig. 1B) is so rigid that the nuclear translocation ability is lost by replacement of any one of the basic amino acids Arg376, Lys377, Arg378, Lys379, or Lys381 with glutamate [24]. Rat Plcz1 was cloned for the first time in the present study. Rat PLCZ1 was unable to translocate into the rat PN, whereas mouse PLCZ1 was accumulated into the rat PN. The same situation was observed in the translocation into the nucleus of cultured somatic cells, COS-7. It has been shown that addition of the sequence K374-A383 of mouse PLCZ1 to Venus causes active nuclear import of Venus in COS cells [24]; that is, the region functions as an NLS. Differences in the NLS region between mouse/rat PLCZ1 exist in Arg376/Lys376, Lys377/Ile377, and Lys381/Arg381 (Fig. 1B). Both Lys and Arg are basic amino acids. The defective nuclear translocation of rat PLCZ1 may be attributed to Ile377. The results of rat PLCZ1 provide information that the nuclear translocation ability is not always the common property of mammalian PLCZ1.
Mouse PLCZ1 was strongly accumulated in the nucleoli of COS cells at 48 h after transfection, whereas FN was rather lower than FC (Fig. 5A). It appears that the nuclear import of mouse PLCZ1 is relatively slow, whereas PLCZ1 that entered the nucleoplasm is concentrated to the nucleoli. One day later (72 h), mouse PLCZ1 was accumulated in the nucleoplasm as well as nucleoli [24]. On the other hand, mouse PLCZ1 was not concentrated to the large nucleolus of the mouse and rat (Fig. 4, Aa and Ba). K374-A383 involving a cluster of basic amino acids may serve as a nucleolar localization signal [37, 38], but the presumptive nucleolar localization signal receptor might not be expressed in the PN of the one-cell embryo.
The nuclear translocation was observed for human PLCZ1 in COS cells, whereas it was absent in the mouse PN. A difference in the NLS region between mouse/human PLCZ1 exists in Arg376/Lys376, and Thr378 is inserted between Lys and Arg in human PLCZ1 (Fig. 1B). The difference may be critical for translocation into the mouse PN but not for that into the nucleus of somatic cells. COS is monkey kidney cells. Nuclear translocation of human PLCZ1 may be possible, because PLCZ1 is identical in the NLS region (Fig. 1B). Translocation of human PLCZ1 into the human PN remains to be examined. Human PLCZ1 was not accumulated in the nucleoli of COS cells, unlike mouse PLCZ1, suggesting some variance in the nuclear distribution of PLCZ1 among animal species.
Medaka plcz1 was cloned for the first time in the present study as well. A basic amino-acid-rich region that appears to be homologous to the NLS region of mouse PLCZ1 exists in chicken and fish (medaka and tetradon) PLCZ1 (Fig. 1B). Medaka PLCZ1 appeared to be slightly accumulated in the nucleoplasm of COS cells when compared with rat PLCZ1, although medaka PLCZ1 is more different from mouse PLCZ1 in the NLS region than is rat PLCZ1. A very small fraction of overexpressed medaka PLCZ1 (65 kDa) might diffuse into the nucleus, because its size is less than rat PLCZ1 (Fig. 2).
Ca2+ Oscillation-Inducing Activity of PLCZ1
The present study showed a great variance in the Ca2+ oscillation-inducing activity of PLCZ1 among species. The Tlag to the first Ca2+ spike is an indicator of the activity of expressed PLCZ1 [11, 24]. The minimal Tlag for mouse PLCZ1 is 25 min, when enough PLCZ1 is expressed [24]. In the present study, the RNA concentration that induced Ca2+ oscillations in mouse eggs after the Tlag over 2 h was considered as the critical concentration. Although the comparison relies on rough estimation from the critical RNA concentration, the order of the Ca2+ oscillation-inducing activity of PLCZ1 was human (0.005 ng/µl) >> mouse (0.1 ng/µl) > medaka (0.5 ng/µl) >> rat (10 ng/µl). Taking the efficiency of PLCZ1 expression in mouse eggs into account, the activity of human PLCZ1 was roughly 40-fold higher, and the activity of rat PLCZ1 was roughly 50-fold lower, than that of mouse PLCZ1. This high Ca2+ oscillation-inducing activity of human PLCZ1 in mouse eggs is consistent with previous reports. Yu et al. [39] showed that about 1 fg of human PLCZ1 protein can effectively initiate Ca2+ signals in mouse oocytes with the direct quantification of luciferase. This threshold was prominently less than the estimated 10 to 50 fg of mouse PLCZ1 required to trigger Ca2+ oscillations [10, 11]. On the contrary, Cox et al. [17] have reported the high Ca2+ oscillation-inducing activity of human PLCZ1 in mouse eggs, presenting it by RNA concentration for injection (0.02 ng/µl). When injected into human eggs, the critical RNA concentration is 0.1 ng/µl [18], about 5-fold higher in homogenous species. It is unclear why human PLCZ1 possesses such higher activity in mouse eggs. PLCZ1 is thought to induce Ca2+ oscillations in mouse eggs by slightly elevating the cytoplasmic InsP3 level, as discrete repetitive Ca2+ spikes are generated in the presence of the nonmetabolizable agonist of InP3R, adenophostin B [40], or mimicked by release of InsP3 from caged form [36]. The Ca2+ oscillation-inducing activity of PLCZ1 in mouse eggs depends on not only the enzymatic activity in vitro but also on various factors such as accessibility to the target membranes of the egg, affinity to phosphoinositides, or egg-derived modulating factors.
Rat PLCZ1 exhibited unique characteristics. Tlag was as long as 2 h, even though 50 ng/µl RNA was injected, and expressed PLCZ1 was confirmed by fluorescence of Venus. The long Tlag led us to estimate the lowest Ca2+ oscillation-inducing activity among four species. Even for the critical RNA concentration, crescendo and high-frequency Ca2+ spikes were generated and terminated 3 h prior to TPN (Fig. 7E), contrasted with Ca2+ spikes induced by mouse PLCZ1 (Fig. 7A). The similar difference in the temporal pattern of Ca2+ oscillations was also seen in mouse and rat eggs fertilized by a single spermatozoon (Fig. 6). The threshold for inducing Ca2+ oscillations might be relatively high for rat PLCZ1 in the beginning and then lowered by repetitive [Ca2+]i rises, resulting in Ca2+ oscillations similar to those usually induced by higher levels of expressed PLCZ1 of other species. Actually, all of those high-frequency Ca2+ oscillations suddenly ceased far before TPN, possibly by feedback inhibition (see the following).
Medaka fish PLCZ1 was found to induce Ca2+ oscillations in mouse eggs, as chicken PLCZ1 does [25]. The response pattern was similar to that for human PLCZ1, and the activity was only slightly lower than that in mouse PLCZ1. It should be examined whether PLCZ1 operates as the sperm factor at fertilization of nonmammalian vertebrates.
Termination of Ca2+ Oscillations
Ca2+ oscillations after fertilization of mouse eggs cause degradation of metaphase promoting factor via activation of calcium/calmodulin-dependent protein kinase II alpha, and thereby lead to resumption of second meiosis [3, 4, 8, 41]. Disappearance of [Ca2+]i rises at the PN stage may be required for preparing metaphase promotion by synthesizing cyclin and producing metaphase promoting factor again [41], thereby leading to the first mitosis of the one-cell embryo.
Ca2+ oscillations induced by mouse PLCZ1 using relatively low RNA concentrations are terminated several tens of min before or slightly after TPN (Fig. 7, A and B) [19, 23]. There are at least three mechanisms to terminate Ca2+ oscillations: decrease of cytoplasmic PLCZ1 due to sequestration of PLCZ1 into the PN [6, 19, 23], decline in the ability of IP3 to cause repetitive Ca2+ transients due to down-regulation of inositol 1,4,5-trisphosphate receptor 1 resulting from Ca2+ oscillations [42–44], and feedback inhibition by overexpressed PLCZ1 [11, 23], possibly via production of diacylglycerol and subsequent activation of protein kinase C [45]. As to feedback inhibition, we previously reported [11] that Ca2+ oscillations once ceased within 90 min and reappeared approximately 180 min after the onset of the first Ca2+ transient with higher RNA concentration (200 ng/µl). The second Ca2+ oscillations consisted of a relatively long-lasting Ca2+ transient and subsequent high-frequency Ca2+ spikes (interspike intervals, approximately 3 min; data not shown). Halet et al. [46] showed that overstimulation of protein kinase C (PKC) by the addition of the PKC agonist phorbol-12-myristate-13-acetate induced the high frequency of Ca2+ oscillation in the fertilized eggs, which had interspike intervals of approximately 2 min. It is likely that this state might correspond to the second Ca2+ oscillations induced by overexpressed PLCZ1. Further, the moderate activation of PKC might inhibit the generation of Ca2+ transients after the first Ca2+ oscillations like inhibition of the PLCB isoform [45, 47]. The first two mechanisms are likely to operate on sperm-induced and PLCZ1-induced Ca2+ oscillations [19, 22, 23]. Ca2+ oscillations induced by human and medaka PLCZ1 lasted beyond TPN at intervals of 30 to 60 min (Fig. 8, B, C, F, and G). This phenomenon occurs presumably because human and medaka PLCZ1s do not move into the PN, supporting the view of nuclear sequestration-dependent termination of Ca2+ oscillations. On the other hand, Ca2+ oscillations in fertilized rat eggs ceased around TPN (Fig. 6B), although rat PLCZ1 lacked the nuclear translocation ability. The result suggests that Ca2+ oscillations can stop at the PN stage without nuclear sequestration of PLCZ1. Disappearance of [Ca2+]i rises at the interphase of a cell cycle is thought to be guaranteed after fertilization of rat eggs by the second and third mechanisms described earlier. It is necessary to address how the role of the nuclear sequestration mechanism in the cessation of Ca2+ oscillations is common in mammals.
ACKNOWLEDGMENTS
We thank Drs. H. Shirakawa and S. Mitani for discussion and valuable advice and Mr. Y. Konuma for technical assistance.
FOOTNOTES
1Supported by grants-in-aid for general scientific research (B) to S.M. and young scientists (B) to M.I. from the Japanese Ministry of Education, Science, Sports, and Culture. 
Correspondence: 2Masahiko Ito, Department of Physiology, Tokyo Women's Medical University School of Medicine, 8–1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. FAX: 81 3 5269 7414; e-mail: mito557{at}nih.go.jp
Received: 17 January 2008.
First decision: 5 February 2008.
Accepted: 21 February 2008.
REFERENCES
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  P. Grasa, K. Coward, C. Young, and J. Parrington The pattern of localization of the putative oocyte activation factor, phospholipase C{zeta}, in uncapacitated, capacitated, and ionophore-treated human spermatozoa Hum. Reprod., November 1, 2008; 23(11): 2513 - 2522. [Abstract] [Full Text] [PDF]
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