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Biology of Reproduction 61, 900-905 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

A Protein Tyrosine Phosphatase Inhibitor, Sodium Orthovanadate, Causes Parthenogenetic Activation of Pig Oocytes via an Increase in Protein Tyrosine Kinase Activity1

Jae-Hwan Kima, Hyun-Jin Doa, Wei-Hua Wang3,a, Zoltan Machátya, Yong-Mahn Han4,a, Billy N. Daya, and Randall S. Prather2,a

a Department of Animal Science, University of Missouri, Columbia, Missouri 65211


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study was conducted to determine whether a protein tyrosine kinase (PTK) activity is involved in the initiation of the events that occur at fertilization in pig oocytes. After maturation for 47 h, a 7-h treatment of oocytes with 1 mM sodium orthovanadate, which is an inhibitor of protein tyrosine phosphatase, caused more than 90% pronuclear formation, cortical granule exocytosis, and a decrease in mitogen-activated protein kinase activity. Immunoblotting with an antibody specific for phosphotyrosine showed at least three proteins whose phosphotyrosine contents were significantly increased upon treatment of oocytes with 1 mM sodium orthovanadate. Preincubation of pig oocytes with 50 µM tyrphostin 47, a specific PTK inhibitor, completely blocked the ability of sodium orthovanadate to trigger activation events. In addition, when oocytes were pretreated with the calcium-chelating agent BAPTA-AM, sodium orthovanadate-stimulated pronuclear formation was significantly (P < 0.01) reduced (94.0% vs. 43.1%). These results suggest that PTK may be involved in pig oocyte activation in a calcium-dependent manner and that the stimulation of tyrosine kinase is able to signal a series of intracellular changes that lead to the activation events associated with fertilization.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oocyte activation refers to global changes in the oocyte after the interaction of sperm and oocyte plasma membranes. Oocyte activation can be classified into early and late events [1]. Early events include repetitive calcium transients and cortical granule exocytosis. Late events include recruitment of maternal mRNAs, pronuclear formation, and initiation of DNA synthesis.

There are two major theories concerning the mechanism by which sperm activate oocytes. One theory states that a certain diffusible substance that sperm carry in their cytoplasm is involved in oocyte activation [2–4]. According to the other theory, oocyte activation is mediated by a signal transduction pathway connected to a G protein- or a protein tyrosine kinase-coupled signal transduction. Several studies have provided evidence that a G protein-coupled signal transduction pathway is involved in mammalian oocyte activation by introducing G protein-linked receptors into oocytes [5–8].

In sea urchin, the possible role of protein tyrosine kinase (PTK) in oocyte activation has been supported by changes in the tyrosine phosphorylation of a variety of oocyte proteins after fertilization [9, 10]; an Abl-related kinase was identified as one of the PTKs involved in the oocyte activation process [11]. In support of the involvement of PTK in oocyte activation, frog [12] and starfish [13] oocytes were microinjected with mRNA encoding platelet-derived growth factor receptor and epidermal growth factor receptor, respectively, to express the receptor in the oocyte plasma membrane. After agonist application, the oocytes showed the several responses that normally occur at fertilization. These studies suggested that PTK activation may be sufficient to mimic activation events. To date, two groups have reported that changes in phosphotyrosine proteins of mammalian oocyte take place after fertilization [14, 15]. To examine the possibility that pig oocyte activation may occur via a PTK signal transduction pathway, we studied the stimulatory effects of sodium orthovanadate (Na3VO4) on endogenous pig oocyte PTKs and investigated whether these PTK activities play a role in pig oocyte activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

Unless otherwise stated, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Tyrphostin 47 (3,4-dihydroxy-a-cyanothiocinnamamid; RG-50864) and BAPTA-AM (1,2-bis[o-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester; Molecular Probes, Eugene, OR) were dissolved in dimethylsulfoxide (DMSO).

Experiments were conducted according to institutional Animal Care and Use Committee guidelines.

In Vitro Maturation

Porcine oocytes were aspirated from prepubertal pig ovaries collected at a slaughterhouse. Cumulus-oocyte complexes (COC) with uniform ooplasm and a compact cumulus cell mass were prepared in Hepes-buffered Tyrode's medium [16] containing 0.01% polyvinylalcohol (HbTP). After the COCs were washed in HbTP medium, they were matured in BSA-free NCSU-23 medium [17] for 44 or 47 h at 39°C in a humidified atmosphere of 5% CO2 in air. During the first 22 h of maturation, the medium contained 10 IU/ml eCG, 10 IU/ml hCG, 10 ng/ml epidermal growth factor, 0.1 mg/ml cysteine, and 10% porcine follicular fluid; the last 22 or 25 h of maturation was carried out in the same medium without hormonal supplementation [18]. After maturation, oocytes were denuded by vortexing in 0.3 M mannitol solution supplemented with 0.3 mg/ml hyaluronidase. After a rinse in HbTP, the oocytes with homogenous cytoplasm were used for experiments.

In Vitro Fertilization

After 44 h of maturation, oocytes were washed three times with modified Tris buffer medium (mTBM) consisting of 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl2·2H2O, 20 mM Tris (Fisher Scientific, Fair Lawn, NJ), 11 mM glucose, 5 mM sodium pyruvate, 0.1% BSA, and 1 mM caffeine. Thereafter, oocytes were transferred into a 50-µl drop of mTBM covered with mineral oil (30 each). A semen pellet was thawed and washed three times at 39°C in PBS supplemented with 1 mg/ml BSA, 75 µg/ml potassium penicillin G, and 50 µg/ml streptomycin sulfate. After the last washing step, sperm were resuspended with mTBM to give a concentration of 1 x 106 cells/ml, and 50 µl of the sperm suspension was added to the fertilization drop containing the oocytes. Six hours after insemination, oocytes were washed three times and cultured in NCSU-23 medium containing 0.4% BSA for an additional 4 h [19]. They were used for evaluation of pronuclei and assays as described below.

Treatments of Oocytes

All treatments were carried out in HbTP medium at 39°C in air. Since sperm penetration takes place about 3 h after insemination [20], after 47 h of maturation the oocytes were treated with sodium orthovanadate for 7 h. Then the oocytes were evaluated for pronuclear formation or used for assays as described below. In some experiments, oocytes matured for 46 h were preincubated for 1 h with tyrphostin 47, a specific inhibitor of PTK, and then incubated for an additional 7 h in the presence of sodium orthovanadate and tyrphostin 47. To induce artificial activation by the sulfhydryl reagent thimerosal [21] or protein kinase inhibitor HA100 [22], oocytes were treated as follows: 1) oocytes were exposed to 200 µM thimerosal for 10 min, with subsequent incubation in 8 mM dithiothreitol (Fisher Scientific, Pittsburgh, PA) for 30 min, and were cultured for an additional 12 h; 2) oocytes were treated with 350 µM protein kinase inhibitor HA100 (Calbiochem, La Jolla, CA) for 12 h. To chelate intracellular calcium, oocytes were preincubated with 10 µM BAPTA-AM for 30 min. After the treatment, oocytes were washed with HbTP and treated with sodium orthovanadate, HA100, or thimerosal, as described above, and incubated for 12 h.

Immunoblotting

After a 7-h treatment with sodium orthovanadate or 5 h of in vitro fertilization (IVF), oocytes were washed with ice-cold PBS-PVA. Each group of oocytes was lysed by freezing and thawing in 10 µl lysis buffer (50 mM Hepes [pH 7.6], 100 mM NaCl, 10 mM EDTA·2H2O, 1% Triton X-100, 4 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 mM sodium fluoride) and stored at -80°C until analysis. The crude lysates were mixed with 4-strength Laemmli buffer and boiled for 5 min. Samples were resolved on a 4–15% gradient SDS polyacrylamide gel (Bio-Rad, Hercules, CA) with prestained molecular weight markers in parallel (Rainbow Recombinant Protein Molecular Weight Markers; Amersham, Arlington Heights, IL; now Amersham Pharmacia Biotech, Piscataway, NJ), and the membrane transfer was carried out for 2 h at 100 V in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol, and 0.02% SDS) using a Mini Trans-blot Transfer cell (Bio-Rad). The polyvinylidene fluoride microporous membrane (Millipore Corp., Bedford, MA) blots were blocked in Tris-buffered saline (TBS; 20 mM Tris [pH 7.4], 150 mM NaCl, and 0.05% Tween 20) containing 3% BSA for 1 h at room temperature and then incubated overnight with antiphosphotyrosine antibody (1:200; PY20, Santa Cruz, CA) in TBS containing 3% BSA and 0.02% sodium azide at 4°C. After washing in TBS, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:2000; Amersham) for 1 h and washed with TBS. The bands were visualized by an enhanced chemiluminescence (ECL) system (Amersham).

Assessment of Cortical Granule (CG) Exocytosis

After 7 h of sodium orthovanadate treatment, or 10 h after IVF, CG exocytosis was quantitated with confocal microscopy as previously described [23]. Zonae pellucidae were removed with acidic Tyrode solution (pH 2.5). After being washed three times in PBS, oocytes were fixed with 3.7% paraformaldehyde in PBS for 30 min at room temperature, followed by three 15-min washes in PBS containing 3 mg/ml BSA and 100 mM glycine. The oocytes were then treated for 5 min in PBS containing 0.1% Triton X-100, washed two additional times, and cultured in 100 µg/ml fluorescein isothiocyanate-labeled peanut agglutinin in PBS for 30 min. Nuclear status of oocytes was determined after oocytes were stained with 10 µg/ml propidium iodide in PBS for 1 h at 39°C. Finally, these oocytes were mounted on slides and examined using a laser scanning confocal microscope. Confocal microscopy was performed by using a Bio-Rad MRC-600 (Bio-Rad Laboratories, Richmond, CA) equipped with a krypton argon ion laser and mounted on an Optiphoto II Nikon (Garden City, NY) microscope. The number of CGs at the cortex was counted at four different areas in a 100-µm2 area, and the average number per 100 µm2 of cortex in each oocyte was then calculated.

Measurement of Mitogen-Activated Protein Kinase (MAPK) Activity

The MAPK activity was measured with a MAPK assay kit (Upstate Biotechnology, Lake Placid, NY) according to the manufacturer's instructions. Briefly, 7 h after treatments or 10 h after sperm insemination, zonae pellucidae were removed with acidic Tyrode solution (pH 2.5), and oocytes were washed with PBS containing 0.01% polyvinylalcohol (PBS-PVA). Twenty-five oocytes of each group were lysed by freezing and thawing in 10 µl of assay dilution buffer (20 mM 3-[N-morpholino]propanesulfonic acid [pH 7.2], 25 mM ß-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, and 1 mM dithiothreitol). The kinase reactions were performed for 10 min at 30°C with agitation in a total volume of 50 µl assay dilution buffer containing the crude oocyte lysates with 0.4 mg/ml myelin basic protein, 4 µM protein kinase C inhibitor peptide, 0.4 µM protein kinase A inhibitor peptide, 4 µM compound R24571, and 10 µCi [{gamma}-32P]ATP (3000 Ci/mmol; DuPont NEN, Boston, MA). The reactions were terminated by spotting 25-µl aliquots onto P81 phosphocellulose papers, followed by washing three times with 0.75% phosphoric acid and once in acetone. Total incorporation of 32P was estimated by Beckman LS 1701 liquid scintillation counter (Beckman Instruments, Fullerton, CA). MAPK activity was determined by subtracting from each determination the value for the blank tube containing all materials except for oocytes. Experiments were replicated three times, and the MAPK activity was presented as the fold increase of control oocytes (metaphase II-arrested oocytes cultured for 7 h).

Evaluation of Pronuclei Formation

Oocytes were mounted under posted coverslips and fixed in ethanol : glacial acetic acid (3:1) for 48 h. After staining with 1% (w:v) aceto-orcein, the oocytes were evaluated for the presence of pronuclei by using Hoffman modulation contrast optics (Hoffman Modulation Optics, Greenvale, NY).

Statistical Analysis

All data were analyzed by ANOVA using General Linear Models procedures of the Statistical Analysis System (SAS Institute, Gary, NC). For multiple comparisons, MAPK activities were compared by Duncan's multiple range procedure, and the rest of the data were analyzed by Tukey test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Sodium Orthovanadate on Pronuclear Formation

Oocytes were treated with different levels of sodium orthovanadate for 7 h in order to determine the optimal concentration. Treatment of matured oocytes with sodium orthovanadate stimulated pronuclear formation in a dose-dependent manner (Table 1). Concentrations of 1 or 2 mM sodium orthovanadate induced pronuclear formation in 94.2% or 98.3% of the oocytes (P < 0.01), respectively. In subsequent experiments, sodium orthovanadate was used at the concentration of 1 mM.


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  		TABLE 1. Pronucleus formation after addition of various concentrations of sodium orthovanadate

Inhibition of Sodium Orthovanadate-Stimulated Pronuclear Formation by Tyrphostin 47

In order to determine whether sodium orthovanadate could stimulate PTK activity in oocyte activation, tyrphostin 47, which is a specific PTK inhibitor, was employed to examine the role of PTK in pig oocyte activation. As shown in Table 2, preincubation in 50 µM or 100 µM tyrphostin 47 inhibited sodium orthovanadate-stimulated pronuclear formation compared to sodium orthovanadate treatment alone. In this experiment, tyrphostin 47 itself or preincubation in DMSO did not cause or impair pronuclear formation.


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  		TABLE 2. Inhibition of sodium orthovanadate-stimulated pronuclear formation by tyrphostin 47

Effect of Sodium Orthovanadate on the Phosphorylation of Oocyte Proteins

Since PTK activity can be examined by analysis of phosphotyrosine-containing proteins, immunoblot analysis using specific anti-phosphotyrosine antibody PY20 was performed (Fig. 1). Sodium orthovanadate treatment of matured oocytes greatly stimulated the tyrosyl phosphorylation of three oocyte proteins, whose molecular masses were approximately 55, 60, and 120 kDa. In fertilized oocytes, however, there was no significant increase in phosphorylation of these proteins compared to that in nontreated oocytes.



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  		FIG. 1. Protein tyrosine phosphorylation in sodium vanadate-stimulated or fertilized pig oocytes. Proteins of 150 oocytes per lane were analyzed by 4–15% gradient SDS-PAGE and transferred to polyvinylidene fluoride microporous membrane. The blotted papers were immunoblotted with antiphosphotyrosine antibody, followed by detection with an ECL system. The position of the prestained molecular weight markers in lane M is shown on the left; the major proteins containing phosphotyrosine are marked by arrows on the right

Inhibition of Sodium Orthovanadate-Stimulated Pronuclear Formation by BAPTA-AM

In order to determine whether calcium release within the oocyte is necessary for sodium orthovanadate-stimulated oocyte activation, oocytes were treated with 10 µM BAPTA-AM, a cell-permeable calcium chelator, before sodium orthovanadate treatment (Table 3). This concentration was found to inhibit the electrostimulated pronuclear formation (data not shown). In this experiment, we included two controls: one was thimerosal treatment (calcium-dependent; [21]), and the other was the protein kinase inhibitor HA100 (calcium-independent; [22]). When oocytes were pretreated with 10 µM BAPTA-AM before the addition of 1 mM sodium orthovanadate or 200 µM thimerosal, the rate of pronuclear formation was decreased (P < 0.01: ~94% vs. ~43%). In contrast, the same concentration of BAPTA-AM pretreatment did not affect the pronuclear formation in 350 µM HA100 treatment (99.4% vs. 100%).


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  		TABLE 3. Effect of BAPTA-AM on sodium orthovanadate-stimulated pronuclear formation

CG Exocytosis

To ascertain the involvement of calcium in sodium orthovanadate-stimulated oocyte activation, we investigated CG exocytosis. After treatment, CGs distributed in the cortex of oocytes were examined in 100-µm2 areas (Fig. 2 and Table 4). The densities of CGs in the oocytes treated with 1 mM sodium orthovanadate (Fig. 2C) or DMSO preincubation and sodium orthovanadate (Fig. 2E), or in fertilized oocytes (Fig. 2D), were significantly decreased compared to densities in nontreated oocytes cultured for 7 h (Fig. 2A). However, pretreatment with 50 µM or 100 µM tyrphostin 47 before the addition of sodium orthovanadate completely blocked the sodium orthovanadate-stimulated CG exocytosis (Fig. 2F). Treatment with tyrphostin 47 alone did not affect CG exocytosis (Table 4 and Fig. 2B).



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  		FIG. 2. Confocal micrographs of CGs in pig oocytes. A) Matured oocyte cultured for 7 h; B) 100 µM tyrphostin-treated oocyte; C) 1 mM sodium orthovanadate-treated oocyte; D) fertilized oocyte; E) ooctye treated with 0.1% DMSO+1 mM sodium orthovanadate; F) oocyte treated with 100 µM tyrphostin 47+1 mM sodium orthovanadate. The insets in A, B, and F indicate the metaphase II chromosome; C, D, and E indicate pronucleus in treated oocytes. Arrowhead: sperm head. Bar = 16.25 µm


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  		TABLE 4. Cortical granule (CG) exocytosis of pig oocytes following various treatments

MAPK Activity

In order to determine whether a decrease in MAPK activity occurs in sodium orthovanadate-stimulated oocytes at the time of pronuclear formation, kinase activities were measured in the treated oocytes. As shown in Figure 3, MAPK activities remained steady in matured oocytes cultured for 7 h as well as in oocytes treated with 50 µM tyrphostin 47 alone. The oocytes treated with 1 mM sodium orthovanadate showed a dramatic decrease in MAPK activity. This decline in MAPK activity induced by sodium orthovanadate stimulation was similar to that measured in fertilized oocytes. However, 50 µM tyrphostin 47 treatment before treatment with sodium orthovanadate completely inhibited the decrease in MAPK activity induced by sodium orthovanadate stimulation.



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  		FIG. 3. MAPK activities at the time of pronuclear formation following various treatments. Cont 1: Matured oocytes; Cont 2: matured oocytes cultured for 7 h; Tyr: 50 µM tyrphostin 47-treated oocytes; Tyr+NaV: 50 µM tyrphostin 47+1 mM sodium orthovanadate-treated oocytes; NaV: 1 mM sodium orthovanadate-treated oocytes; IVF: fertilized oocytes. Bars indicate the MAPK activity presented as the fold increase of control oocytes (matured oocytes cultured for 7 h); vertical lines indicate the SEM. a,bDifferent letters indicate significant difference (P < 0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present studies have demonstrated that PTK activities stimulated by sodium orthovanadate initiate some of the events associated with oocyte activation: calcium release, CG exocytosis, decrease in MAPK activity, and progression to first interphase. Several studies have been undertaken to investigate the involvement of PTK during fertilization of mammalian oocytes. However, it is known that only a small proportion of all phosphoproteins are phosphorylated on tyrosine residues [24], and tyrosine phosphorylation of a potential substrate is transient during the first few minutes after fertilization [9]. Thus it is hard to detect PTK activities or phosphotyrosine contents of their putative substrates in mammalian oocytes in response to fertilization. This led us to hypothesize that the detection of intracellular phosphotyrosine contents could be facilitated if PTK activities were stimulated artificially under physiological conditions. We investigated this hypothesis by using sodium orthovanadate, known as an activator of certain PTKs—either directly or by virtue of its known inhibitory effect on phosphoprotein tyrosine phosphatase [25, 26]. Recently, Lee et al. [27] have demonstrated that treatment with sodium metavanadate, one of the vanadium salts, induces pronuclear formation by maturation-promoting factor inactivation via increase in tyrosine phosphorylation of p34cdc2 in pig oocytes. This suggests that vanadate indeed activates PTKs in oocytes. Our study provides several lines of evidence to indicate that sodium orthovanadate stimulates PTKs and that an increase in PTK activities is involved in pig oocyte activation. First, at least three polypeptides in oocytes underwent enhanced tyrosine phosphorylation in response to treatment with sodium orthovanadate (Fig. 1). Although it is difficult to relate any of these to a specific biological effect on oocyte activation, their presence suggests an increase in PTK in response to sodium orthovanadate actions. No difference in phosphotyrosine contents between fertilized and nontreated oocytes suggests that PTK activities may be transiently augmented during the first few minutes in response to fertilization; alternatively, the elevation in the phosphotyrosine contents may not be sufficient to be detected with present methods. Second, pretreatment with a specific PTK inhibitor, tyrphostin 47, completely inhibited activation events induced by sodium orthovanadate treatment (Tables 2 and 4 and Fig. 3). These results indicate that PTKs play important roles in pig oocyte activation.

During oocyte activation, an increase in intracellular calcium concentration occurs as an early event. It is well accepted that calcium release from intracellular stores is essential for CG exocytosis [28]. The observations (Tables 3 and 4) that sodium orthovanadate treatment causes CG exocytosis, and that preincubation with 10 µM BAPTA-AM significantly blocks the sodium orthovanadate-stimulated pronuclear formation, strongly support the hypothesis that the treatment with sodium orthovanadate provokes mobilization of calcium from intracellular stores. Since the same concentration of BAPTA-AM did not affect the pronuclear formation induced by the protein kinase inhibitor HA100, known to activate pig oocytes in a calcium-independent manner [22], it can be concluded that the blocking effect of BAPTA-AM on sodium orthovanadate-stimulated pronuclear formation originates from the calcium buffering action of BAPTA-AM.

The mechanisms responsible for release from the second meiotic arrest depend on a decrease in a specific kinase activity [29]. MAPK, a serine/threonine kinase, is activated by Mos protein in maturing and matured oocytes of several species. In mouse oocytes, MAPK activity is maintained at high levels throughout oocyte maturation and is responsible for maintaining condensed chromatin and suppressing S phase during the second meiotic division to induce a metaphase II arrest. This activity decreases in a time-dependent manner after fertilization and is lowest during the first mitotic cycle as the pronucleus forms [30, 31]. It has been suggested that a decrease in MAPK activity is required for chromosome decondensation and pronuclear envelope development. At pronuclear formation (Fig. 3), a significant decline in MAPK activity after sperm insemination or sodium orthovanadate treatment as compared to that in matured oocytes suggests that stimulation with sodium orthovanadate may follow a normal physiological pathway to the release of the metaphase II arrest and induce pronuclear formation.

In summary, in the present study we observed that treatment of matured pig oocytes with sodium orthovanadate resulted in an increase in PTK activities and that the stimulation of PTKs led to both early and late events of pig oocyte activation. Our results strongly suggest that pig oocyte activation might be mediated by a PTK signal transduction pathway. In future research, the tyrosine-phosphorylated substrates as well as PTKs stimulated by sodium orthovanadate need to be identified and characterized to further unravel the mechanism of oocyte activation.


   FOOTNOTES
 
1 This material is based upon work supported by the Food for the 21st Century and the Cooperative State Research, Education and Extension Service, U.S. Department of Agriculture, under agreement No. 95-37203-2073. The manuscript is a contribution from the Missouri Agriculture Experiment Station Journal Series No. 12,918. Back

2 Correspondence: Randall S. Prather, 162 Animal Science Research Center, University of Missouri-Columbia, Columbia, MO 65211. FAX: 573 882 6827; pratherr{at}missouri.edu Back

3 Current address: IVF Lab., Women & Infant's Hospital of Rhode Island, Brown University School of Medicine, Providence, RI 02905. Back

4 Current address: Korean Research Institute of Bioscience & Biotechnology, Taejon 305-600, Korea. Back

Accepted: May 10, 1999.

Received: March 23, 1999.


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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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