Molecular Human Reproduction, Vol. 7, No. 8, 723-729,
August 2001
© 2001 European Society of Human Reproduction and Embryology
Ovary and oogenesis |
Protein tyrosine kinase expression in the porcine ovary
1 Department of Pathology, Graduate School of Medicine and 2 Department of Animal Sciences, Graduate School of Agriculture Kyoto University, Sakyo-ku, Kyoto 606-8501, 3 Department of Obstetrics and Gynecology, Kumamoto University School of Medicine, Kumamoto 860-8556 and 4 Department of Pathology, Institute of Development, Aging and Cancer, Tohoku University, Aoba-ku, Sendai 980-8575, Japan
Abstract
Various growth factor receptors contain intrinsic tyrosine kinase activity, indicating that protein tyrosine kinases (PTK) play an important role in signal transduction pathways for cell proliferation and differentiation. To identify oocyte-derived factors which control follicle cells as well as oocyte-controlling factors produced by follicle cells, we examined the expression of genes which contain the PTK domain in the porcine ovary, using a polymerase chain reaction-based amplification technique with degenerate oligonucleotide primers that are specific to the PTK domain. Clones for the porcine homologues of platelet-derived growth factor receptor 
(PDGFR) and of insulin-like growth factor-I receptor (IGF-IR) were found during follicle growth both in oocytes and follicle cells. Clones for the porcine homologues of focal adhesion kinase (FAK), of c-kit and of fms-like tyrosine kinase (FLT)-3 were found only in oocytes. Moreover, after 24 h of in-vitro maturation of the cumulus–oocyte complexes, clones for the porcine homologues of FLT-1, of FLT-4, of Tie2 and of RYK in oocytes were observed. Immunohistochemical studies revealed the existence of PDGFR, platelet-derived growth factor A (PDGFA), FAK and FLT3 in oocytes at various stages of folliculogenesis. These results suggest that fluctuations in the expression of these PTK genes may be involved in follicle growth and maturation.
folliculogenesis/granulosa cell/oocyte/porcine/tyrosine kinase
Introduction
The molecular mechanisms of folliculogenesis and oocyte maturation are not fully elucidated. Early folliculogenesis is gonadotrophin independent, as primordial follicles reside in the ovarian cortex until a signal triggers their growth and development. Later, during the pre-antral stage, a selected group of follicles continue to grow while most are destined to undergo atresia (Gougeon, 1996
). In pre-ovulatory follicles, granulosa and theca cells respond to a gonadotrophic stimulus and ovulation is initiated, while oocytes resume meiosis. Many of the effects of LH/FSH are mediated directly via cell surface receptors or indirectly through the synthesis of growth factors. One family of growth factor receptors contain tyrosine kinase activity and their functions include cell proliferation, differentiation and signal transduction (Schlessinger and Ullrich, 1992). These receptor protein tyrosine kinases (PTK) transduce signals for diverse cell functions by receptor tyrosine autophosphorylation and tyrosine phosphorylation of other proteins in response to their ligands. To date, receptor PTK such as c-kit, c-fms, and insulin-like growth factor-I (IGF-I) receptor are known to be involved in ovarian function.
One of the receptor PTK, c-kit, and its ligand, Kit Ligand (KL), are involved in folliculogenesis through oocyte–follicle cell interaction, and mutations of either cause deficiencies in germ cell development. c-kit is expressed in oocytes and in theca cells (Manova et al., 1990; Orr-Urtreger et al., 1990; Horie et al., 1991) and is required at multiple steps of folliculogenesis (Yoshida et al., 1997). KL is expressed on granulosa cells (Keshet et al., 1991; Motro et al., 1991) and it promotes oocyte growth in vitro (Packer et al., 1994).
Colony-stimulating factor-1 (CSF-1/M-CSF) receptor is expressed on oocytes and intrafollicular macrophages (Arceci et al., 1992), and mutational inactivation of CSF-1 is responsible for the phenotype of op/op mice which includes osteopetrosis and low fertility (Yoshida et al., 1990). This ligand-receptor interaction is involved in oocyte growth, granulosa cell proliferation and ovulation (Nishimura et al., 1995; Araki et al., 1996; Cohen et al., 1997).
Expressions of the IGF-I gene and the IGF-I receptor (IGF-IR) gene have been observed in growing oocytes, of the infant and mature oocytes of the adult human ovaries (Zhou et al., 1991; Zhou and Bondy, 1993) and in rat oocytes (Zhang et al., 1994), suggesting that the IGF-I–IGF-IR system is involved in the process of oocyte growth and maturation. IGF-I and its receptor also participate in granulosa cell proliferation (Zhou et al., 1991). IGF-I may regulate the selection and meiotic maturation of oocytes during follicle development (Yoshimura et al., 1996), and the expansion of the cumulus cells (Singh and Armstrong, 1997).
Platelet-derived growth factor (PDGF) consists of dimers of either A, B or AB polypeptide chains linked by disulphide bonds. These chains are encoded by different genes and share >50% of amino acid identity. Although all possible isoform combinations are mitogenic for cultured cells (Cross and Dexter, 1991), the developmental and functional significance of these various isoforms remains unclear. PDGF receptor (PDGFR) gene expression in adult chicken ovary has been reported (Marcelle and Eichmann, 1992), but its distribution has not been determined.
In order to determine which PTK are involved in mammalian folliculogenesis and oocyte maturation, we used a polymerase chain reaction (PCR)-based amplification technique with degenerate oligonucleotide primers that are specific to the PTK domain. This methodology allowed us to determine known and unknown proteins with PTK domains that are expressed in ovary. Additionally, we confirmed the expression of genes by reverse transcription (RT)–PCR with sequence specific primers and the presence and distribution of the encoded proteins by immunohistochemistry.
Materials and methods
Collection of porcine oocytes and granulosa cells and in-vitro culture of the cumulus–oocyte complexes (COC)
Ovaries of prepubertal gilts (~6 months old) were collected at a local slaughterhouse and carried to the laboratory at room temperature. Ovaries were washed in phosphate-buffered saline (PBS) containing 0.1% polyvinylalcohol (PBS–PVA) three times and medium-sized follicles (4–6 mm in diameter) were dissected from ovaries. Follicles were opened in Roswell Park Memorial Institute (RPMI) medium 1640 (Nissui pharmaceutical Co., Tokyo, Japan) supplemented with 10% fetal calf serum (Life Technologies Inc., Grand Island, NY, USA), 100 µg/ml of penicillin and 0.1 mg/ml sodium pyruvate (Gibco BRL Life Technologies, Gaitherburg, MD, USA). Atretic follicles were excluded by carefully examining the granulosa cell layer and COC were isolated from healthy follicles under a dissecting microscope using forceps (Motlik et al., 1991). From a pool of 5–10 ovaries, 10–20 oocytes were then denuded with a fine-bore pipette in PBS–PVA. The granulosa cell layer was scraped away from a single healthy follicular shell with forceps and was immediately frozen and stored at –80°C until use. In the in-vitro maturation experiment, COC were cultured with a single follicular shell of a healthy follicle from which follicular fluid and granulosa cells had been removed (Kano et al., 1993). They were cultured for 24 h in RPMI medium with or without 0.1 IU/ml of human menopausal gonadotrophin (HMG; Nikken Kagaku, Tokyo, Japan) at 37°C in a humidified atmosphere of 5% CO2–95% air. After 24 h of culture, oocytes were denuded of surrounding cells. Some of the oocytes were mounted on a slide glass, fixed in ethanol and acetic acid (3:1) and stained with 1% aceto-orcein, and then they were examined for nuclear maturity using previously established methods (Kano et al., 1993). Others were immediately frozen and stored at –80°C.
RNA extraction and RT–PCR with degenerate primers
Total RNA was extracted from collected oocytes and granulosa cells of a single follicle with Trizol reagent (Life Technologies Inc., Gaithersburg, MD, USA) according to the manufacturer's protocol. Synthesis of cDNA was prepared from total RNA from 10–20 oocytes or the same number of granulosa cells by a First-strand cDNA Synthesis kit (Pharmacia LKB Biotechnology, Piscataway, NJ, USA) with random primers in a volume of 15 µl. For PCR, two sets of degenerate primers were designed from four highly conserved regions within the catalytic domain of the PTK (Kaneko et al., 1995). Primer 1 [5'-GGIC(G)A(C)IGGIC(G)A(C)ITTC(T)GG-3' (I: inosine)] encoded the amino acid sequence GXGXFG; primer 2 [5'-GAC(T)C(T)TIGCIGCIA(C)GIAA-3'] encoded DLAARN which is located downstream of GXGXFG; primer 3 [5'-C(G)A(T)C(T)TCIA (G)A(G)IC(G)ICATCCA-3'] was the comlementary sequence encoding WMXXES, and primer 4 [5'-CCA(G)A(T)AIC(G)IIA(C)AIAC (T)A(G)TC-3'] was the complementary sequence encoding DV(M) W(F)S(A)F(Y)G located downstream of WMXXES. An aliquot of synthesized cDNA (2 µl) was initially amplified with primers 1 and 4. PCR products were subjected to a second round of PCR, using primers 2 and 3. Each PCR mixture contained 2 µl of template solution, 1 nmol of each primer, 10 µl of 10x PCR buffer, 16 µl of 1.25 mmol/l dNTP, 0.5 units of TaqPlus Long PCR System (Stratagene, La Jolla, CA, USA), and distilled water to 100 µl. The first PCR was repeated for three cycles at 94°C, 37°C and 72°C for periods of 1, 2 and 3 min respectively followed by 27 cycles at 94°C, 37°C and 72°C for periods of 1 min each. The second PCR reaction was repeated for 30 cycles at 94°C, 37°C and 72°C for periods of 1 min each. The PCR products were subjected to 9% polyacrylamide gel electrophoresis (PAGE) and purified by cutting the ethidium bromide positive bands located at ~150 base pairs (bp) from the gel. We confirmed that bands were not observed at 150 bp when porcine genomic DNA was used as a template. The PCR products were then cloned into the pCRII or pCR2.1 vectors using the Original TA Cloning kit (Invitrogen, San Diego, CA, USA). About 30–40 colonies were isolated from each plating and were sequenced by an automatic sequencer (DSQ-1000; Shimadzu, Kyoto, Japan) using a ThermoSequenase kit (Amersham, Chalfont, Buckinghamshire, UK). Computer analyses of nucleotide and predicted amino acid sequences were performed using Blast Sit (Genomenet WWWserver, Kyoto Center).
RT–PCR with specific primers for PDGFR and IGF-IR
In order to measure expression levels of specific genes, we utilized known primers according to porcine genes that were sequenced in this study. The nucleotide sequence of the upstream primer for the PDGFR gene was [5'-AACGTCCTCCTGGCACAAGG-3'] corresponding to nucleotides 2606–2625 of the human PDGFR gene, and the downstream primer was [5'-ACTTCACCGGGAGGAAAGTA-3'] corresponding to complement sequence of nucleotides 2701–2720 of the human PDGFR gene. This primer pair predicts a PCR product of 115 bp. The sequence of the upstream primer for IGF-IR was [5'-TGGAGAGATTGCAGATGGC-3'] corresponding to the nucleotides 3376–3394 of the porcine IGF-IR gene, and the downstream primer was [5'-TGAAGACTCCGTCCTTGAGG-3'] corresponding to the complement sequences of nucleotides 3577–3596. This primer pair predicts a PCR product of 221 bps. Each PCR mixture contained 1 µl cDNA solution, 20 pmol of each primer, 2 µl of 10xPCR buffer, 0.6 µl of 50 mmol/l MgCl2, 3.2 µl of 1.25 mmol/l dNTP, 0.5 units of Taq DNA polymerase (Gibco), and distilled water to 20 µl. The PCR reaction was performed for 40 cycles at 94°C, 55°C and 72°C for periods of 1, 2 and 1 min respectively for each cycle. The PCR products were subjected to PAGE and stained with ethidium bromide. When porcine genomic DNA were used as templates, the predicted bands were not observed.
Immunohistochemistry
Anti-PDGFR (sc-338, x1000), anti-PDGF-A (sc-128, x2000), anti-focal adhesion kinase (FAK) (sc-557, x500) and anti-Flt-3/Flk-2 (sc-480, x300) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). They are affinity-purified rabbit polyclonal antibodies raised against peptides deduced from human nucleotide sequences.
Porcine ovaries were fixed in 4% paraformaldehyde and embedded in paraffin. Paraffin-embedded blocks were serially sectioned at 4 µm thickness and a series of sections which contained an oocyte of an antral follicle was picked up. After one of them had been stained with haematoxylin and eosin to determine stage and health status of the follicles, adjacent sections were used for immunohistochemistry. After the sections were deparaffinized and endogenous peroxidase blocked with 0.3% hydrogen peroxide in methanol, primary antibodies in buffer were applied and incubated overnight at 4°C. Sections were then washed, followed by addition of the second antibody. Further processing was carried out using the Vectastain ABC Elite kit (Vector Laboratories Inc., Burlingame, CA, USA) according to the manufacturer's protocol. The tyramide signal amplification system in combination with antigen retrieval was applied for immunohistochemical staining of PDGFR and its ligand PDGFA because they were not detectable by conventional immunohistochemistry (Toda et al., 1999). For negative controls, primary antibodies were preabsorbed with the corresponding oligopeptides (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA) used as immunogens at concentrations of 0.2–2 µg/ml. At least three different ovaries were examined to confirm consistency of immunohistochemical staining.
Results
Tyrosine kinase gene expression in oocytes and granulosa cells
A total of 276 clones containing PTK domains were obtained from oocytes and granulosa cells and were classified into six groups according to their most homologous PTK nucleotide sequences so far identified. Each clone consisted of 111–114 bp and its nucleic acid sequence showed 85–90% similarity to its most homologous human PTK gene or 100% similarity to their corresponding porcine PTK gene (Figure 1A).
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In oocytes, five of the six PTK clones were detected, the number of clones homologous to the PDGFR
gene was the greatest, with the second highest number of clones being for the IGF-IR gene, followed by the focal adhesion kinase (FAK), fms-like tyrosine kinase (FLT)-3 and c-kit genes, respectively (Table I). In granulosa cells, only three of the six PTK genes cloned were detected. The number of clones for the PDGFR gene predominated followed by those for the IGF-IR and epidermal growth factor receptor (EGFR) genes. Clones corresponding to the FAK, c-kit and FLT3 genes were not detected in granulosa cells (Table II). Amino acid sequences were deduced from PCR products. The fourth dominant sequence showed 95% amino acid similarity to human FLT3 and is likely to be porcine FLT3. Amino acid sequences encoded by all other PCR products were 100% identical to their corresponding human homologues (Figure 1B
).
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Tyrosine kinase gene expression in oocytes after in-vitro culture
We confirmed that >90% of the oocytes showed germinal vesicle breakdown (GVBD) 24 h after incubation with HMG and that no maturation occurred without HMG.
After 24 h of culture with or without HMG, degenerate PCR cloning was performed on the oocytes and 124 and 18 clones were sequenced respectively. These clones were classified into nine genes, four of which were observed only in the HMG-treated group (Table III). These were the FLT1, FLT4, TIE2 and RYK genes. Each clone consisted of 105–114 bp and showed 85–100% similarity to their most homologous PTK. The deduced amino acid sequences were 100% identical to their corresponding human homologues, except for FLT3 and RYK (Figure 1B).
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RT–PCR for PDGFR
and IGF-IR in oocytes and granulosa cellsExpression of PDGFR
and IGF-IR genes was clearly observed by RT–PCR both in oocytes and in granulosa cells (Figure 2). However, the intensity of the PCR products was not dramatically different between the two cell types. When porcine genomic DNA was used as a template with these primers, bands larger than predicted length were observed (data not shown), demonstrating that these PCR products were derived from mRNA.
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X174 DNA digested with Hinf I. The sizes (bp) of the predicted amplified products are indicated.Immunohistochemistry for PTK in the porcine ovary
In order to confirm that the polypeptides which were predicted from the deduced amino acid sequences of PCR products were correct and to investigate the distribution of these substances, immunohistochemical studies was performed on porcine ovaries (Figure 3
). Oocytes and theca cells were positive for PDGFR irrespective of stages of follicular maturation. Basal granulosa cells were weakly positive (Figure 3A, C
). Both oocytes and granulosa cells were intensely positive for the PDGFR ligand, PDGFA, at earlier stages of follicle growth whereas they were weakly positive in antral follicles. In antral follicles, a small number of cumulus cells and granulosa cells at the basal layer tended to be positive for PDGFA (Figure 3B, D
). Immunostaining for FAK was strongly positive in the oocytes from early antral to mature follicles. FAK was weakly positive in the granulosa cells of antral follicle (Figure 3G). Oocytes were positive and granulosa and theca cells were weakly positive for FLT3 in antral follicles (Figure 3H). No appreciable immunostaining was observed after the antibodies were preabsorbed with the PDGFR and the PDGFA peptides (Figure 3E, F respectively
).
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Discussion
This study showed PTK gene expression in porcine ovarian cells. Of the porcine PTK genes cloned in this study, sequences of only IGF-IR and KIT genes are available in the GenBank database. Other PTK gene sequences were 85–90% homologous to corresponding human gene sequences. However, it is highly possible that these PTK are the porcine homologues, because the deduced amino acid sequences showed 100% similarity to corresponding human PTK sequences. Here we have demonstrated that the PDGFR and IGF-IR genes were expressed both in oocytes and in follicle cells and that FAK, c-kit and FLT3 gene expression occurred only in oocytes. Both PDGFR and IGF-IR gene expression was uniform in each experiment. On the other hand, FAK gene expression was different between experiments, that is, the expression was observed in some experiments but not detected in others. This discrepancy may be due to the difference of the follicle sources for each experiment.
In the present study, expression of the PDGFR gene, whose product binds both PDGF-A and -B chains with high affinity, was the most abundantly observed among the PTK both in oocytes and in granulosa cells. In many instances including embryogenesis, PDGFR are found in mesenchymal tissues, whereas their ligands are present in adjacent epithelial layers (Orr-Urtreger and Lonai, 1992). In the present study, PDGFR was positive in theca cells and in the cell membrane of oocytes but weakly positive in basal granulosa cells. This staining pattern is consistent with the origin of these cell types. PDGFA gene expression has been observed in unfertilized oocytes of Xenopus (Mercola et al., 1988) and of the mouse (Rappolee et al., 1988). PDGFA was positive in both oocytes and granulosa cells of early stage follicles and partly in cumulus cells and granulosa cells of antral and mature follicles. These data suggest that the PDGFA–PDGFR system is involved in follicle growth in an autocrine and paracrine manner between oocytes and granulosa cells. It remains uncertain whether the number of clones indicates expression levels of individual genes due to possible biased efficiency of amplification during PCR. Although absolute expression levels of genes are difficult to determine, the immunohistochemical study revealed that all the stages of oocytes including atretic follicles were strongly positive for PDGFR whereas granulosa cells were weakly positive.
The second most predominant gene we observed was IGF-IR. This specific member of the PTK family has been extensively studied in folliculogenesis (Gougeon, 1996). The result of the current study, that IGF-IR gene expression was observed both in oocytes and granulosa cells, is consistent with a previous study by in-situ hybridization (Zhou et al., 1991; Zhou and Bondy, 1993).
FAK is a 125 kDa cytosolic PTK associated with focal adhesions and is involved in the interaction between integrins and the actin-based cytoskeleton. Overexpression and activation of FAK increases cell migration and promotes cell survival (Hanks and Polte, 1997). In the present study, degenerate PCR could detect FAK gene expression only in oocytes and not in granulosa cells. Immunohistochemical expression of FAK was detected in the oocytes of antral follicles and weakly in granulosa cells. Further studies are needed to compare expression at the transcriptional and translational levels. Considering that oocytes do not undergo apoptosis even in an atretic follicle (Manabe et al., 1996) and that an oocyte does not migrate within the follicle, the role of FAK in the oocyte may be to prevent oocytes from apoptosis during follicle growth. Growth factors including PDGF and IGF-I induce FAK activation by tyrosine phosphorylation (Baron et al., 1998). FAK can also be phosphorylated by integrin 6ß1. On the oocyte, integrin 6ß1 functions as a sperm receptor (Almeida et al., 1995), suggesting that FAK may be at the crossroads of multiple signalling pathways of oocyte maturation and fertilization.
FLT3 is a new member of the class III receptor PTK family to which PDGFR, c-kit and CSF-1 receptor (CSF-1R) also belong. As is the case with c-kit and CSF-1R, FLT3 is expressed mainly in haematopoietic stem cells (Lyman and Jacobsen, 1998). Although FLT3 gene expression in the ovary has been reported (Rosnet et al., 1991), its distribution has been unknown. In this study, we demonstrated that the FLT3 gene is expressed in oocytes and that the FLT3 protein is detected on the membrane of oocytes by immunohistochemistry, suggesting that FLT3 is involved in folliculogenesis, such as c-kit (Packer et al., 1994; Yoshida et al., 1997) and CSF-1R (Nishimura et al., 1995; Araki et al., 1996; Cohen et al., 1997). Although c-kit plays a crucial role in oocyte maturation, its expression levels as determined by clone frequency were much lower compared with those of PDGFR and IGF-IR in the current study. To clarify the variation in PTK gene expression observed between each experiment in this study, we need further studies of specific gene expression at the single oocyte level treated with various gonadotrophin concentrations.
In the present study, EGFR gene expression was detected in granulosa cells but not in oocytes. Using RT–PCR with specific primers, a much higher expression level of the EGFR gene in porcine granulosa cells compared with oocytes has been observed (Singh et al. 1995). Considering that the number of cloned EGFR in granulosa cells was less than 1/10 of cloned PDGFR, expression of the EGFR gene in oocytes must be under the levels detectable by RT–PCR with degenerate primers.
Expression of FLT1, FLT4 and TIE2 genes was induced in oocytes after in-vitro maturation. The FLT1 and FLT4 genes encode vascular endothelial growth factor (VEGF) receptor (VEGFR)-1 and VEGFR-3 respectively and the TIE2 gene encodes the receptor for angiopoietin (Ang)-1 and Ang-2 (Tallquist et al., 1999). Expression of the VEGF and the Ang genes in the rat ovary are well characterized in relation to vascular remodelling (Maisonpierre et al., 1997). Production of VEGF and Ang by primate granulosa cells is promoted in periovulatory follicles via direct stimulation of gonadotrophins (Christenson and Stouffer, 1997; Hazzard et al., 1999). Although receptors for angiogenic factors are considered to be specifically localized in the vascular endothelium, expression of TIE2 in haematopoietic stem cells and trophoblasts has been reported (Vuorela et al., 2000). This suggests that the angiogenic factor–receptor relationship plays an important role other than in vascular development, such as physiological cell invasion and implantation.
RYK is an atypical receptor tyrosine kinase which is catalytically inactive and its expression is detected ubiquitously in normal tissues. Although RYK overexpression is associated with progression of malignant epithelial ovarian tumours, its physiological role in the ovary is unknown (Hovens et al., 1992; Tamagnone et al., 1993; Wang et al., 1996).
In conclusion, the PDGFR and IGF-IR genes are expressed in both porcine oocytes and granulosa cells, and the c-kit, FAK and FLT3 genes are expressed exclusively in oocytes. The FLT1, FLT4 and TIE2 genes are expressed in mature oocytes. Immunohistochemical studies demonstrated PDGFR, PDGFA and FLT3 expression in oocytes and in follicle cells, while FAK expression was almost exclusively in oocytes. These findings suggest that paracrine/autocrine mechanisms between the oocyte and the granulosa cells via the PDGFA–PDGFR system may play an important role in folliculogenesis and that complex changes in signal transduction by fluctuation of receptor tyrosine kinases happen in the oocyte during folliculogenesis. Recently, molecular phenotyping of the human oocyte at germinal vesicle stage has been reported. Serial analysis of gene expression techniques revealed a relative abundance of transcripts for cytoskeletal proteins (Neilson et al., 2000). It will be interesting to elucidate how these molecules are linked and how maternal expression of these genes is involved in early embryogenesis.
Acknowledgements
We would like to thank Dr L.K.Christenson for critical reading of the manuscript, Dr T.Miyano for his advice on the treatment of porcine ovaries, and Y.Toda for his excellent assistance in immunohistochemistry. This work was supported by grants from the Ministry of Education, Science and Culture of Japan, Japanese Owners Association and HIROMI Medical Research Foundation.
Notes
5 To whom correspondence should be addressed. E-mail: fukumoto{at}idac.tohoku.ac.jp 
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Submitted on October 16, 2000; accepted on April 25, 2001.
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