Molecular Human Reproduction, Vol. 7, No. 11, 1039-1046,
November 2001
© 2001 European Society of Human Reproduction and Embryology
Embryology |
Expression of a green fluorescent protein variant in mouse oocytes by injection of RNA with an added long poly(A) tail
1 Department of Physiology, Tokyo Women's Medical University School of Medicine, 8-1 Kawada-cho, Shinjuku-ku, Tokyo,162-8666, 2 Department of Obstetrics and Gynecology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, 113-8241 and 3 Laboratory of Intracellular Metabolism, Department of Molecular Physiology, National Institute for Physiological Sciences, Okazaki, 444-8585, Japan
Abstract
In oocytes, cytoplasmic 3' polyadenylation regulates translational activation of dormant mRNA during meiotic maturation. Thus exogenous proteins are hardly expressed after injection of conventional RNA. To circumvent this, we synthesized a long polyadenylated (~250 A) tail to encode RNA with an enhanced yellow fluorescent protein targeted to mitochondria (EYFP-mito), and injected it into mouse oocytes at the germinal vesicle (GV) stage. From this transcript, EYFP-mito was clearly expressed in ~80% of oocytes, while scarce expression from a transcript with only 30 A was observed. In strongly expressing oocytes, fluorescence was detected within 1–3 h after RNA injection, increased linearly up to 12 h, and reached a maximum at 12–15 h. The distribution of EYFP-mito matched the staining of mitochondria in these oocytes. About 80% of these oocytes underwent GV breakdown and 60% matured in vitro, comparable to non-expressing or non-RNA-injected oocytes. Some of the oocytes which strongly expressed EYFP-mito remained at the GV stage. Thus, the expression was not always accompanied by meiotic maturation, nor did it suppress the maturation process. Mature oocytes expressing EYFP-mito possessed normal fertilizability associated with intracellular Ca2+ oscillations, and developed into 2-cell embryos. Thus, polyadenylated RNA is a useful tool applicable to the expression of EYFP-fused functional proteins or of indicator protein probes for studies of mammalian fertilization.
gene expression/GFP variant/mouse oocyte/polyadenylated RNA/mitochondria-targeted protein
Introduction
The artificial expression of a defined protein fused with green fluorescent protein (GFP) is a powerful technique that enables us to visualize the localization and trafficking of the protein in living cells or to monitor concentration changes of second messengers that interact with the protein probe. The technique has been applied in a wide variety of cells. However, this strategy is often difficult in oocytes, particularly in mammalian oocytes, at meiotic maturation, since the translation of mRNA is suppressed for the majority of proteins (Davidson, 1986
) other than those necessary during maturation such as cyclin B1 (Polanski et al., 1998; Tay et al., 2000) and Mos (Gebauer et al., 1994). It has recently been found that, for the proteins that are expressed in maturing mouse oocytes, the 3' poly(A) tail of their mRNA is extended to 100–150 nucleotides following induction of meiotic maturation (Richter, 1999). It has also been revealed that the cytoplasmic polyadenylation is necessary for translational activation of dormant mRNA and is conferred by the cytoplasmic polyadenylation element and polyadenylation hexanucleotide AAUAAA, both of which are located in the distal portion of the 3' untranslated regions of responding mRNAs (Vassalli et al., 1989; Richter, 1999). Vassalli et al. (1989) have shown that exogenous plasminogen activator can be expressed in mouse oocytes, when its RNA is polyadenylated in vitro by adding 200–400 A and then injected into oocytes at the germinal vesicle (GV) stage.
In the present study, we applied this in-vitro polyadenylation method for expression of a GFP variant, enhanced yellow fluorescent protein (EYFP), in mouse oocytes, as a step towards further application of EYFP-fused functional proteins or indicator protein probes. RNA of mitochondria-targeted EYFP was used, because EYFP expression could be clearly determined by the characteristic distribution of mitochondria. Fluorescence of EYFP is stronger than that of GFP at a pH near 8 in the mitochondrial matrix (Llopis et al., 1998). We show here that the method was successful, without disturbing oocyte maturation in vitro, fertilizabilty, and Ca2+ responses at fertilization.
Materials and methods
Plasmid construction
A plasmid encoding EYFP fused with the mitochondria-targeting sequence from subunit VIII of human cytochrome c oxidase, `pEYFP-Mito' was used (Clontech, Palo Alto, CA, USA). The cDNA encoding mitochondria-targeted EYFP (EYFP-mito) in this plasmid was amplified by polymerase chain reaction (PCR) using the following primers; 5'-CGC GGT ACC GCC ACC ATG TCC GTC CTG ACG CCG CTG C-3' and 5'-TCG AGA TCG CGG CCG CTA CTT GTA CAG CTC GTC CAT GC-3'. Fragments of EYFP-mito cDNA were digested with KpnI and NotI and ligated to the KpnI and NotI sites of pBluescript KS+ (Stratagene, La Jolla, CA, USA). For non-mitochondria-targeted EYFP, pEYFP-Mito was digested with BamHI and NotI, to remove the mitochondria-targetting sequence, and the released fragments were introduced into the BamHI and NotI sites of pBluescript KS+.
RNA and polyadenylation
The constructed plasmids, containing cDNA encoding EYFP-mito or EYFP, were digested with SacI and NotI and the resulting fragments were used as templates for in-vitro transcription. RNA were synthesized by means of T3 polymerase using T3 MessageMachine Kit (Ambion, Austin, TX, USA). Synthesized RNA were treated with phenol–chloroform before precipitation with ethanol, and dried RNA were resolved in TE. RNA was polyadenylated by incubation for 30 min at 37°C in the presence of 100 µmol/l ATP and 30 IU/ml poly(A) polymerase (Gibco BRL, Rockville, MD, USA) in buffer A containing (mmol/l) 250 NaCl, 50 Tris–Cl (pH 8.1), 10 MgCl2, 2 dithiothreitol, 1000 IU/ml RNasin (Promega, Madison, WI, USA), and 100 µg/ml BSA (Vassalli et al., 1989). The RNA was resolved in 150 mmol/l KCl solution (final concentration, 
1 µg RNA/µl). To avoid folding of the RNA, the sample was heated at 90°C for 1 min and then cooled on ice. The polyadenylated RNA was designated as `EYFP-mito-polyA' or `EYFP-polyA'. The numbers of nucleotides in EYFP-mito-polyA and EYFP-mito (no polyA) RNA fragments were ~1150 and 900 respectively, examined by polyacrylamide gel electrophoresis. The length of added poly(A) tract was therefore estimated to be ~250 A. RNA of EYFP-mito added with 30 A (`EYFP-mito-short polyA') was also injected into some oocytes. To obtain the template for transcription, a fragment containing T3 promoter and EYFP-mito cDNA was amplified by PCR using the following primers: 5'-GGA AAC AGC TAT GAC CAT G-3' and 5'-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT CTA TCT AGA CTT GTA CAG CTC G-3'. In-vitro transcription was performed with the T3 polymerase and the synthesized RNA was designated as `EYFP-mito-short poly A'.
Oocytes and injection of RNA
B6D2F1 female mice (6–10 weeks old) were injected intraperitioneally with 5 IU pregnant mare's serum gonadotrophin (Teikoku Hormone Mfg., Tokyo, Japan), and immature oocytes at the GV stage were collected from mature follicles 48–52 h later. M2 medium (Fulton and Whittingham, 1978) supplemented with BSA (4 mg/ml) was used for cell manipulation. Oocytes with cumulus cells only partially attached were selected and inserted with a micropipette for injection with 5–10 pl of EYFP-mito-polyA- or EYFP-polyA-containing solution (1 µg/µl), to produce a concentration of 25–50 ng/µl RNA in the oocyte (assuming an oocyte volume of 200 pl). In each experiment, 5–15 oocytes were injected within 45 min after oocyte collection. After washing, the RNA-injected oocytes were transferred to a glass-bottomed dish and cultured in M16 medium (Whittingham, 1971) at 37°C under 5% CO2 in air for 18–20 h, during which time 60% of the oocytes spontaneously matured. Cumulus cells were detached from the oocytes.
Measurement of EYFP fluorescence
Fluorescence of EYFP expressed in oocytes was investigated under an inverted fluorescence microscope (TMD; Nikon, Tokyo, Japan) at a given time after RNA injection, using excitation light passed through a 470–490 nm bandpass filter. Emission was passed through a 510 nm dichroic mirror, a 520–560 nm bandpass filter, and a silicon intensifier target camera (C2400-08; Hamamatsu Photonics, Hamamatsu, Japan). Images were acquired by an image processor (Arugas 50; Hamamatsu Photonics). The fluorescence intensity (F) was averaged in an oocyte and presented as an arbitrary unit of values between 0 and 255. The relationship between F and the arbitrary unit was calibrated using fluorescein (Sigma, St Louis, MO, USA). The optical system and detector were set at the same conditions throughout the present study.
Mitochondria staining and confocal microscopy
Some RNA-injected oocytes were stained with the mitochondria-specific fluorescent dye MitoTracker Orange CM-H2TMRos (Molecular Probes Inc., Eugene, OR, USA) by incubation in the presence of 0.5 µmol/l MitoTracker for 30 min at 37°C. The distribution patterns of EYFP-mito and MitoTracker were investigated in the same cells using a confocal laser scanning microscope (CLSM; LSM310, Carl Zeiss, Oberkochen, Germany) equipped with an Achroplan x40 objective water immersion lens (NA 0.75, Carl Zeiss), as described previously (Shiraishi et al., 1995). EYFP and MitoTracker were excited by 488 nm and 543 nm laser, respectively, and each emitted fluorescence was collected through a 510–525 nm bandpass filter or a 570 nm longpass filter, respectively.
Insemination of zona-free oocytes and Ca2+ measurement
RNA-injected oocytes were inseminated after maturation. The oocytes were freed from the zona pellucida, since the cumulus cells had detached and cumulus-free zona-intact mouse oocytes are known to be less fertilizable (Bleil, 1993). To prevent hardening of the zona and make it removable, 5% fetal bovine serum was added to M16 medium for in-vitro maturation (Downs et al., 1986). The zonae of mature oocytes were partially dissolved by applying 0.001% chymotrypsin for several minutes (Boldt and Wolf, 1986) and removed mechanically by pipetting in acidic Tyrode solution (Nakano et al., 1997). Spermatozoa obtained from the caudae epididymides of B6D2F1 male mice (11–15 weeks old) were incubated in M16 medium for more than 2 h at 37°C under 5% CO2 in air. Oocytes were inseminated by adding a small amount of the sperm suspension to the oocyte-containing medium.
Prior to insemination, some oocytes were loaded with the Ca2+-sensitive fluorescent dye fura-2 by incubation in the presence of 5 µmol/l fura-2 AM (acetoxymethyl ester; Molecular Probes) for 8 min at 37°C. Increases in intracellular Ca2+ concentration ([Ca2+]i during fertilization were recorded by a conventional Ca2+ imaging method at 20 s intervals using an image processor (Arugas 50; Hamamatsu Photonics), for details of the method and calibration of [Ca2+]i), (Nakano et al., 1997). Fluorescence of fura-2 was obtained without interference with that of EYFP, by applying 340 and 380 nm UV lights alternatively and by passing emission light through a 500–520 nm bandpass filter.
Fertilization of oocytes was identified after 6–8 h culture in M16 medium by extrusion of the second polar body and formation of the male and female pronuclei, using differential interference contrast optics (LSM310, Carl Zeiss). Two-cell embryos were observed 24–28 h after fertilization.
Results
Expression of EYFP-mito in oocytes
Table I shows the rate of expression of EYFP-mito examined 20 h after RNA injection. Oocytes that shrank within 1 h after injection were discarded (30%). The fluorescence intensity (F) of all oocytes injected with non-polyadenylated RNA was the same level as autofluorescence seen in non-RNA-injected oocytes (Figure 1), indicating that EYFP-mito was not expressed. In contrast, EYFP-mito was clearly expressed in 83% of oocytes (79 of 95) injected with EYFP-mito-polyA (Table I), as judged by F > 10 (mean ± SD, 63 ± 35; Figure 1). Another 12 oocytes in which F was between 2 and 10 were considered as `weakly' expressing oocytes, since F did not increase after the initial 3 h (see later section for details). With EYFP-mito-short polyA (30 A), fluorescence was detected in some oocytes, but F was 5 ± 3 (n = 18; Figure 1) in the range of weakly expressing oocytes. Thus, the addition of a long poly(A) tail to RNA is much more efficient for the expression of EYFP-mito.
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Distribution of EYFP-mito in oocytes
Figure 2A
shows that the distribution of EYFP-mito fluorescence (green image at 488 nm excitation) in oocytes was almost consistent with MitoTraker-stained structures (red image at 543 nm excitation) and was composed of many granular spots of various sizes at the equatorial section of the oocyte examined using confocal microscopy ~20 h after RNA injection. No interference by the two excitation lights was involved in these images, since no fluorescence was detected in the oocyte injected with EYFP-mito-polyA but not treated with MitoTracker when examined with 543 nm light (Figure 2B, right) nor in a non-injected oocyte stained with MitoTracker and examined under 488 nm excitation (Figure 2C, left). In the oocytes injected with polyadenylated RNA of non-mitochondria-targeted EYFP, fluorescence was homogeneous in the entire oocyte without granular structures (Figure 2D) as the produced EYFP were located in the cytosol. These results indicate that EYFP-mito was properly targeted to mitochondria in the oocytes.
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The distribution of EYFP-mito appears to be different between the green images of Figure 2A and B
, despite of the same conditions for image acquisition and presentation. This may result from a non-homogeneous distribution of mitochondria in mouse oocytes (Van Blerkom and Runner, 1984; Calarco, 1995), observation at different optical sections and/or differences in expression. When Figure 2A is precisely inspected, small faint spots of MitoTracker are visible over the oocyte in the red image, whereas corresponding small spots are hardly recognized in the green image. Production of EYFP-mito is likely to be lower in this oocyte, compared with the oocyte of Figure 2B which exhibits more abundant granular structures and brighter background fluorescence. The oocyte of Figure 2B seems to contain excessively expressed EYFP-mito molecules that are not incorporated into mitochondria.
Time course of EYFP-mito expression
Fluorescence intensity was measured successively for 20 h after RNA injection in individual oocytes under the same conditions (Figure 3A). F was proportional to fluorescein concentration, up to the arbitrary unit of 230 (Figure 3C). F was unaltered for non-polyA EYFP-mito or only slightly increased for EYFP-mito-short polyA (dotted or broken line in Figure 3B, respectively). Figure 3A is an example showing fluorescence changes in individual oocytes injected with EYFP-mito-polyA (n = 19; three experiments on the same day). Except in two cases, substantial fluorescence was detected as early as 1 h after RNA injection, and a significant increase in F (>10) was recognized at 3 h. The succeeding increase in F was variable among oocytes. In four oocytes, F stayed at relatively low levels between 20 and 30 after 6 h and up to 20 h. In other oocytes, F increased to >50 in a linear fashion up to 12 h, and then showed variation of a slight decrease or increase among oocytes during the next 8 h. Figure 3B is an average profile of changes in F of these `strongly' expressing oocytes, showing that F linearly increased during 12 h and reached an almost maximum level at 12 or 15 h.
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Figure 4
shows the distribution of EYFP-mito at four stages of meiotic maturation. The oocyte in which the GV was still visible (2 h after RNA injection) showed small patches of EYFP-mito surrounding the GV and small spots in the peripheral region (Figure 4A), consistent with the distribution of mitochondria (Van Blerkom and Runner, 1984; Calarco, 1995). Thus, the trafficking of EYFP-mito into mitochondria occurred before GV breakdown (GVBD) was completed. After GVBD (6.5 h), a dense mass of EYFP-mito was localized in the perinuclear region (Figure 4B). In the oocyte immediately before formation of the first polar body (PB1) (12 h), EYFP-mito was aggregated around a dark region away from the extruding PB1 (Figure 4C), as with mitochondria which surround the second metaphase spindle region (Van Blerkom and Runner, 1984; Calarco, 1995). Mitochondria disperse over the cytoplasm in mature oocytes, but have a denser distribution in the central region (Van Blerkom and Runner, 1984; Calarco, 1995). In the mature oocyte, EYFP-mito was densely located in the central region of the mature oocyte (Figure 4D). Thus, EYFP-mito molecules were precisely localized in accordance with the distribution changes of mitochondria during oocyte maturation.
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In-vitro maturation of oocytes
About 80% of fluorescent oocytes (75 of 95) injected with EYFP-mito-polyA resumed first meiosis indicated by GVBD, and 60% underwent maturation, judged by extrusion of the PB1 (Table II
). These percentages were consistent with those in non-EYFP-expressing oocytes injected with non-polyA EYFP or not injected at all (Table II).
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To examine any correlation between the magnitude of EYFP-mito expression and the advance of oocyte maturation, F and the maturation stage of clearly expressing oocytes examined at 20 h after RNA injection were plotted in Figure 5
. Oocytes were divided into three groups: oocytes that were arrested at the GV stage even at 20 h, oocytes that underwent GVBD but did not form the PB1, and oocytes that were matured. The values of F were scattered in a wide range in each group of oocytes, and there was no correlation between F and oocyte maturation. EYFP-mito was well expressed even in some oocytes arrested at the GV stage. Oocytes were matured even when EYFP-mito was strongly expressed.
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Fertilization, Ca2+ oscillations and first cleavage
The fertilizability of EYFP-mito expressing oocytes was also examined. Mammalian oocytes exhibit repetitive increases in [Ca2+]i called `Ca2+ oscillations' at fertilization (Miyazaki et al., 1993
), and these are responsible for cortical granule exocytosis and resumption of the second meiosis (Kline and Kline, 1992). Ca2+ oscillations were recorded in strongly expressing oocytes (Figure 6), having the temporal pattern similar to that seen at normal fertilization of oocytes matured in vivo (Deguchi et al., 2000).
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The resumption of the second meiosis and completion of fertilization were determined by formation of the second polar body (PB2) and male and female pronuclei (PN + PB2) 6–8 h after insemination. About 90% of injected and inseminated oocytes (20 of 22) formed PN + PB2, and this was comparable to oocytes matured in vitro without RNA injection (Table III
). The first cleavage was examined in another series of experiments. The rate of formation of normal 2-cell embryos was 75% in control oocytes (Table III), probably because polyspermy tended to occur in zona-free oocytes, although diluted sperm suspension was used. The rate of cleavage was 67% in EYFP-expressing oocytes, almost comparable to that in the control oocytes.
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The first cleavage in these oocytes occurred 26–28 h after insemination, whereas it was completed within 24 h in control experiments. The period tended to be more delayed in the oocytes with brighter fluorescence. Thus, expression of EYFP-mito did not affect the fertilizability of oocytes, although excessive expression might slightly affect the efficiency of development into the 2-cell embryo.
Discussion
The expression of GFP variant-fused proteins in mouse oocytes is a potential tool for analysing mechanisms involved in oocyte maturation, fertilization, and early embryonic development. The present study demonstrated that mitochondria-targeted EYFP is efficiently expressed by injection of RNA with an added long poly(A) tail. The number of added A (~250) was in the range (200–400) reported for experiments with plasminogen activator RNA (Vassalli et al., 1989). The dormant mRNA in oocytes have short poly(A) tails fewer than 20 A (Richter, 1999). We confirmed that RNA having 30 A induced only weak expression. The expression of exogenous proteins in mouse oocytes by injecting RNA has been performed to introduce the human m1 muscarinic receptor (Williams et al., 1992, 1998; Moore et al., 1993) and human platelet-derived growth factor ß receptor (Mehlmann et al., 1998), but polyadenylation of RNA was not described. In these cases, a small amount of expressed receptor proteins might be sufficient to examine the involvement of G proteins in agonist-induced Ca2+ release. Polyadenylated RNA have, however, been used for expression of cyclin B1 (Polanski et al., 1998) or luciferase/cyclin B1 (Barkoff et al., 2000) in oocytes. For visualization of GFP variants, a higher expression level is thought to be required, and this was provided by using polyadenylated RNA.
The majority of isolated immature mouse oocytes undergo spontaneous maturation. In previous experiments, RNA have been injected into oocytes arrested at the GV stage in the presence of dibutyryl cAMP (Vassalli et al., 1989) or 3-isobutyl-1-methylxanthine (IBMX) (Williams et al., 1992). RNA-injected oocytes have also been kept in IBMX-containing medium for several hours (Moore et al., 1993) to give a longer time before maturation for RNA translation. The present method provided maximal expression of EYFP-mito without such treatments. EYFP-mito was strongly expressed both in maturing oocytes and in some oocytes that failed to undergo GVBD. It is conceivable therefore that RNA translation advances independently of meiotic maturation, when polyadenylated RNA is injected.
EYFP-mito became detectable as early as 1–3 h after RNA injection, and its trafficking into mitochondria advanced soon after production of the protein. Thus, the targeting sequence from subunit VIII of human cytochrome c oxidase (Rizzuto et al., 1989) was effective in the mouse oocyte. The expression of EYFP-mito increased with time, associated with incorporation into mitochondria. In strongly expressing oocytes, the fluorescence intensity increased in an approximately linear manner and attained a maximal level in 12 or 15 h. The time course is favourable for studies using expressed GFP variants in mature oocytes.
EYFP-mito was clearly expressed in 80% of oocytes. However, the magnitude of expression indicated by the fluorescence intensity was widely variable among individual oocytes. Possible factors that affect the magnitude of expression due to RNA activity include the length of poly(A) tail, the amount of RNA injected, the cellular activity, which could depend partly on the grade of cell growth of the oocytes collected from ovaries, and the injection technique which is likely to cause cell damage. In our imaging system, the brightness of EYFP fluorescence corresponding to F values of 20–50 would probably be sufficient for identifying the localization of EYFP-fused proteins or for monitoring concentration changes of second messengers using EYFP-fused probes. A fluorescence intensity above that level may indicate excessive expression with EYFP-mito remaining in the cytosol, not incorporated into mitochondria, as seen in Figure 2B. Regulated expression, by controlling the experimental conditions described above, might therefore be required. It should be noted that the present method provides such high levels of EYFP expression that it could be adjustable to various purposes.
The biological inspection of the RNA-injected oocytes showed that percentages of GVBD and maturation in vitro were about 80 and 60%, respectively, comparable to that in non-RNA-injected oocytes. The culture condition could be improved in future experiments, to obtain higher yields. Mature oocytes had normal fertilizability associated with Ca2+ oscillations leading to oocyte activation. The PB2 and male and female pronuclei were formed normally in 90% of inseminated oocytes. Fertilized oocytes developed into 2-cell embryos, although the first cleavage was a little delayed, compared with control oocytes. Since development to the 2-cell stage takes 1 day after activation of the oocyte at fertilization, excessive expression of EYFP in mitochondria could affect the process via some effects on cellular metabolism.
The present method could be applicable to the expression, in mouse oocytes, of GFP variant-fused functional proteins, for example: cell cycle-related proteins such as cyclin (Polanski et al., 1998; Tay et al., 2000) and Mos (Gebauer et al., 1994); fertilization-related proteins such as 
6ß1 integrin (Almeida et al., 1995), Cd9 (Kaji et al., 2000), and Ca2+ oscillation-inducing factor derived from spermatozoa (if cloned in the future) (Parrington et al., 2000); proteins involved in the meiotic and mitotic apparatus (Wianny et al., 1998), or signal proteins such as membrane receptors (Mehlmann et al., 1998; Williams et al., 1998), G proteins, and phospholipase C. It is also applicable to indicator probes for second messengers such as Ca2+ (Miyawaki et al., 1999), cyclic AMP (Zaccolo et al., 2000), or inositol 1,4,5-trisphosphate (Hirose et al., 1999), as have been developed in somatic cells. The present study provides a step forward for such technology in mammalian oocytes.
Acknowledgements
We thank Dr H.Shirakawa and Dr Z.Kohchi for discussion and comments and Mr T.Shikano, and Mr Y.Konuma for their technical assistance. This work was supported by Grant-in-Aid for General Scientific Research (B) to S.M. and for Encouragement of Young Scientists to S.O. from the Japan Ministry of Education, Science, Sports, and Culture.
Notes
4 To whom correspondence must be addressed. shunm{at}research.twmu.ac.jp 
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Submitted on March 30, 2001; accepted on August 23, 2001.
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  M. Ito, T. Shikano, S. Oda, T. Horiguchi, S. Tanimoto, T. Awaji, H. Mitani, and S. Miyazaki Difference in Ca2+ Oscillation-Inducing Activity and Nuclear Translocation Ability of PLCZ1, an Egg-Activating Sperm Factor Candidate, Between Mouse, Rat, Human, and Medaka Fish Biol Reprod, June 1, 2008; 78(6): 1081 - 1090. [Abstract] [Full Text] [PDF]
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 K. Kuroda, M. Ito, T. Shikano, T. Awaji, A. Yoda, H. Takeuchi, K. Kinoshita, and S. Miyazaki The Role of X/Y Linker Region and N-terminal EF-hand Domain in Nuclear Translocation and Ca2+ Oscillation-inducing Activities of Phospholipase C{zeta}, a Mammalian Egg-activating Factor J. Biol. Chem., September 22, 2006; 281(38): 27794 - 27805. [Abstract] [Full Text] [PDF]
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 L. Freudzon, R. P. Norris, A. R. Hand, S. Tanaka, Y. Saeki, T. L.Z. Jones, M. M. Rasenick, C. H. Berlot, L. M. Mehlmann, and L. A. Jaffe Regulation of meiotic prophase arrest in mouse oocytes by GPR3, a constitutive activator of the Gs G protein J. Cell Biol., October 24, 2005; 171(2): 255 - 265. [Abstract] [Full Text] [PDF]
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Z. Kouchi, T. Shikano, Y. Nakamura, H. Shirakawa, K. Fukami, and S. Miyazaki The Role of EF-hand Domains and C2 Domain in Regulation of Enzymatic Activity of Phospholipase C{zeta} J. Biol. Chem., June 3, 2005; 280(22): 21015 - 21021. [Abstract] [Full Text] [PDF]
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 C. M. Saunders, M. G. Larman, J. Parrington, L. J. Cox, J. Royse, L. M. Blayney, K. Swann, and F. A. Lai PLC{zeta}: a sperm-specific trigger of Ca2+ oscillations in eggs and embryo development Development, August 1, 2002; 129(15): 3533 - 3544. [Abstract] [Full Text] [PDF]
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