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BOR - Papers in Press, published online ahead of print March 3, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.026344
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BIOLOGY OF REPRODUCTION 71, 163–169 (2004)
DOI: 10.1095/biolreprod.103.026344
© 2004 by the Society for the Study of Reproduction, Inc.

Pregnancy

Differential Expression of the Vascular Endothelial Growth Factor-Receptor System in the Gravid Uterus of Yorkshire and Meishan Pigs

Kimberly A. Vonnahme, and Stephen P. Ford1

Department of Animal Science, University of Wyoming, Laramie, Wyoming 82071


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Litter size in the pig is limited by uterine capacity, which is dependent on uterine size, placental size, and vascularity. Placentae of U.S. pig breeds, such as the Yorkshire, exhibit marked growth from mid to late gestation, increasing their surface area of endometrial attachment. In contrast, placentae of the prolific Chinese Meishan pig exhibit little growth from mid to late gestation; instead, they exhibit a marked and progressive increase in the density of placental blood vessels. Vascular endothelial growth factor (VEGF) is a potent angiogenic and permeability-enhancing factor that is produced and secreted by placentae of several species, including the pig. The activity of VEGF is mediated through two specific receptors (VEGF-R1 and VEGF-R2), both of which are expressed by placental and endometrial tissues in pigs and are thought to play a role in mediating increased vascularization and/or permeability at the fetal-maternal interface. The objectives of the present study were to determine concentrations of VEGF in fetal blood and placental fluids as well as placental and adjacent endometrial mRNA expression of VEGF, VEGF-R1, and VEGF-R2 on Days 30, 50, 70, 90, and 110 of gestation in Yorkshire and Meishan pigs. Day 90 Meishan conceptuses exhibited marked increases (P < 0.05) in placental VEGF mRNA expression as well as fetal blood and allantoic fluid concentrations of VEGF, which remained elevated through Day 110. In contrast, Yorkshire conceptuses failed to exhibit increases in placental VEGF mRNA expression or concentrations of VEGF in fetal blood or allantoic fluid until Day 110. Receptor mRNA expression patterns differed between Meishan and Yorkshire conceptuses, but no difference was found in their expression levels. Placental efficiency (fetal weight/placental weight) was higher (P < 0.05) on Days 90 and 110 in Meishan than in Yorkshire conceptuses. The earlier increase in VEGF protein and mRNA expression in the Meishan versus the Yorkshire conceptus may explain the previously reported increased vascularity and increased placental efficiency of this breed compared the Yorkshire breed.

conceptus, developmental biology, placenta, pregnancy, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Conceptus losses after Day 30 are thought to result from intrauterine crowding in swine caused by limitations in uterine capacity [1]. In the pig, which has an epithelial-chorial and diffuse placental type [2], uterine capacity can also be considered as the endometrial surface area available for placental attachment [3]. The prolific Chinese Meishan pig can farrow three to five more live pigs per litter than the less prolific Yorkshire breed [4, 5], but it exhibits the same ovulation rate and uterine size [4, 5]. In contrast to their similarity in uterine size, Meishan conceptuses exhibit a markedly smaller placenta than the Yorkshire breed throughout gestation [6], which would allow greater numbers of Meishan conceptuses to occupy a given amount of limited uterine space. Furthermore, whereas Meishan placentae grow little from mid to late gestation, Yorkshire placentae grow markedly over the same interval [6]. To compensate for this lack of growth, the Meishan placenta exhibits a marked and progressive increase in the density of blood vessels on the placental surface in close contact with the associated endometrial vasculature [6, 7]. The increased vascularity of the Meishan placental membranes would be expected to increase maternal-fetal nutrient and waste product exchange per unit placental membrane, potentially eliminating the need for increases in placental size to accommodate the needs of an exponentially growing fetus. This conclusion was supported by Vonnahme et al. [8], who crushed alternate conceptuses in the uterine horns of Meishan and Yorkshire females on Day 40 of gestation. Those researchers found that Yorkshire placentae grew to occupy the adjacent unoccupied spaces after Day 40 but that placentae of Meishan conceptuses did not.

Vascular endothelial growth factor (VEGF) is a secreted, 45-kDa, dimeric, ligand-binding glycoprotein that increases endothelial cell proliferation, migration, and capillary permeability through its binding to two specific membrane receptors (VEGF-R1 and VEGF-R2) [9, 10]. Vascular endothelial growth factor and its receptors are present on chorionic and adjacent uterine epithelial cells as well as blood vessels of the pig from early to late gestation [11, 12], and placental VEGF mRNA levels are positively correlated with placental and adjacent endometrial vascularity [12]. As the profiles of placental growth and vascular development have been shown to be markedly different in the Meishan versus Yorkshire females [6], it was the objective of the present study to compare placental and adjacent endometrial VEGF, VEGF-R1, and VEGF-R2 mRNA expression as well as VEGF concentrations in fetal blood and placental fluid from Day 30 to Day 110 of gestation in both breeds.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Tissue Collection

Meishan (n = 29) and Yorkshire (n = 30) gilts (4–8 postpubertal estrous cycles) were bred to boars of the same breed at the onset of estrus (Day 0) and 12 h later. Females were humanely killed via stunning and exsanguination on Days 30 (n = 12), 50 (n = 10), 70 (n = 12), 90 (n = 13), and 110 (n = 11) of gestation in a federally accredited facility in accordance with procedures approved by the Institutional Animal Care and Use Committee of the University of Wyoming. The gravid uterus was immediately transported back to the laboratory for further processing. At the laboratory, corpora lutea (CL) were dissected from the ovary and counted as an index of the ovulation rate, and the length of the uterine horn was determined using a cloth tape measure. Each uterine horn then was opened over each conceptus along the antimesometrial side. The location of each conceptus was noted within the horn; then, a 10-ml sample of allantoic fluid was collected, placed on ice until centrifugation, and stored at –80°C. Thereafter, as each fetus was removed from the horn, a sample of cord blood was collected and placed on ice for 4 h until serum was removed after centrifugation to be stored at –80°C until assayed. The fetus was then weighed and the crown-rump length recorded. A small portion (~4 g) of both placental and adjacent endometrial tissue was collected from each conceptus and snap-frozen on liquid nitrogen for later RNA extraction. The ends of each placenta were marked with dissection pins placed in the uterine wall, and placentae were peeled from the endometrium and weighed. The implantation site length for each conceptus was then measured using a cloth tape measure. To obtain a measure of placental efficiency, the weight of each fetus was divided by the weight of its placenta.

Assays

A radioimmunoassay for VEGF was developed for pig serum and allantoic fluid according to the protocol described by Anthony et al. [13] with the following modifications: Human recombinant VEGF165 (cold hormone; G143AB; Genentech, Inc., Los Angeles, CA), primary antibody (polyclonal rabbit antiserum to VEGF165; no. 27906-17; Genentech), and human recombinant [125I]VEGF165 (tracer; NEX328; NEN Life Science Products, Inc., Boston, MA) were used in all assays. The 165-isoform of VEGF is the predominate soluble form of VEGF in mammals [14]. Sensitivity averaged 25 pg/ml, defined as the VEGF standard yielding 95% of the counts in the buffer control tube. Within-assay variability for VEGF was determined by assaying replicate samples from a pool of systemic serum from a pregnant gilt to which known quantities of VEGF had been added (0.0, 0.5, and 5.0 ng/ml). For serum, the resulting concentrations (mean±SEM) after subtraction of the serum blank (1.51 ± 0.14 ng/ml; n = 4) averaged 0.51 ± 0.04 (n = 4) and 4.77 ± 0.51 (n = 4) ng/ml, respectively. Coefficients of variation averaged 6.0%, 8.2%, and 5.7% for the serum blank and the 0.5- and 5.0-ng/ml VEGF additions, respectively. For allantoic fluid, the resulting concentrations (mean±SEM), after subtraction of the allantoic fluid blank (0.31 ± 0.02 ng/ml; n = 4) averaged 0.58 ± 0.08 (n = 4) and 5.07 ± 0.40 ng/ml, respectively. Coefficients of variation averaged 8.7%, 7.4%, and 5.1% for the allantoic fluid blank and the 0.5- and 5.0-ng/ml VEGF additions, respectively. Parallelism was obtained between a double-diluted pregnant serum pool and the standard curve. No cross-reactivity was found with basic fibroblast growth factor or {alpha}2-macroglobulin (Sigma, St. Louis, MO) at concentrations as high as 100 mg/L. The interassay coefficient of variation was 13.6% for fetal serum samples and 11.2% for allantoic fluid.

Placental VEGF, VEGF-R1, and VEGF-R2 mRNA expression

To determine the levels of VEGF, VEGF-R1, and VEGF-R2 mRNA in the pig placenta, a pig-specific probe was needed for use in the RNase protection assay. Details for the porcine VEGF probe generation have been previously published [15]. Primers for VEGF-R1 and VEGF-R2 were designed from the full-length porcine sequence established from ARS-USDA MARC (Clay Center, NE; VEGF-R1: sense, 5'-GCAGGATCCGGCTCTGGCCCAACAATCAGAG-3' antisense, 5'-GTAGAATTCCCTCGCACAAAGGGACACAT-3'; VEGF-R2: sense, 5'-GCAGGATCCGACGAGCAACAGCGGCTTCACG-3'; antisense, 5'-GTAGAATTCAGCTGGAGTGGCAGAAAGT-3'). Porcine placental RNA was reverse transcribed using Moloney murine leukemia virus reverse transcriptase. Conditions were optimized, and the polymerase chain reaction (PCR) was performed. A 5-min extension time was added to the cycling program to insure ample addition of dATP to the 3' end of the PCR amplicon for ligation in to the pCR2.1 plasmid (Invitrogen, Carlsbad, CA). The VEGF-R1 and VEGF-R2 inserts were then subcloned into Bluescript SK +/– plasmid (Stratagene, La Jolla, CA).

RNase Protection Assay

The in vitro transcription assay and RNase protection assay were performed as previously described in our laboratory [15, 16] with the following exceptions: Detection of placental VEGF mRNA and the housekeeping gene, pyruvate dehydrogenase (PD), was performed using HybSpeed RNase Protection Assay kit as previously described [12]. Plasmids containing either VEGF-R1 or VEGF-R2 were linearized with SacI restriction enzyme. Radiolabeled RNAs were synthesized from these linear DNA templates with 10 U of T7 RNA polymerase according to the manufacturer's protocol (Ambion, Austin, TX). To quantitate mRNA expression levels, counts from VEGF, VEGF-R1, or VEGF-R2 bands were divided by the counts in the PD band to correct for slight differences in sample loading. To correct for between-gel variation, a pool of placental RNA was included in each assay; therefore, data are presented relative to the mRNA levels in the control sample.

Statistics

Data were analyzed using the general linear model procedure of SAS (Version 8.0; SAS Institute, Inc., Cary, NC). Day of gestation and breed were included in the class statement. The effects of day of gestation and breed were tested for the variables ovulation rate, litter size, percentage survival (litter size/CL number), uterine horn length, fetal weight, placental weight, placental efficiency, crown-rump length, implantation site length, percentage occupied uterine horn (total implantation site length/ total uterine horn length), fetal blood and allantoic fluid VEGF concentrations, as well as placental and endometrial VEGF, VEGF-R1, and VEGF-R2 mRNA expression levels. Separation of means was accomplished using the least-squared means procedure. A P value of less than 0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
No differences were found in ovulation rate, litter size, or percentage survival by day or by breed, which averaged (across day and breed) 14.84 ± 0.43 CL, 10.29 ± 0.46 viable fetuses, and 70.61% ± 2.40%, respectively. However, an effect of breed was found on the uterine horn length (Fig. 1). Whereas no difference was observed in uterine horn length on Day 30 of gestation between Meishan and Yorkshire gilts, uterine horns of Yorkshire females were longer (P < 0.05) on all other days of gestation. This breed difference in uterine horn length after Day 30 resulted from the observed, progressive increase in horn length in Yorkshire females through Day 110, with no corresponding increase in Meishan females.



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  		FIG. 1. Uterine horn length in Meishan and Yorkshire gilts on Days 30, 50, 70, 90, and 110 of gestation. Asterisks indicate significant differences (means ± SEM) between breeds within a day (P < 0.05). Different subscripts indicate significant differences (means ± SEM) within a breed (P < 0.05)

Meishan and Yorkshire fetuses and placentae were similar in size and weight on Day 30 of gestation (Table 1). However, beginning on Day 50 and continuing through the rest of gestation, Yorkshire fetuses and placentae were both heavier and longer (P < 0.05) than those of Meishan conceptuses. Implantation sites for Meishan conceptuses were shorter than Yorkshire conceptuses on Days 50, 70, and 110 of gestation (Table 1), whereas this difference did not reach significance (P > 0.05) on Days 30 and 90. The percentage of uterine space occupied by Meishan conceptuses was greater (P < 0.05) on Day 30 of gestation compared to Yorkshire conceptuses (Table 1). No breed difference was found in the percentage of uterine horn space occupied on Days 50, 70, 90, or 100 of gestation. The placental efficiencies (fetal weight/placental weight) of the Meishan and the Yorkshire conceptuses were similar through Day 70, before diverging on Day 90, of gestation, with Meishan conceptuses having a greater (P < 0.05) placental efficiency than Yorkshire conceptuses on Days 90 and 110 of gestation (Fig. 2).


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  		TABLE 1. Characteristics of Meishan and Yorkshire conceptuses throughout gestation



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  		FIG. 2. Placental efficiency (fetal weight/placental weight) in Meishan and Yorkshire conceptuses on Days 30, 50, 70, 90, and 110 of gestation. Asterisks indicate significant differences (means ± SEM) between breeds within a day (P < 0.05)

Concentrations of VEGF in Meishan fetal blood remained constant from Day 50 to Day 70, then increased (P < 0.05) (Fig. 3a) progressively from Day 70 to Day 110 of gestation. In contrast, Yorkshire fetal blood VEGF concentrations increased slightly from Day 50 to Day 70, remained relatively constant from Day 50 to Day 90, then increased (P < 0.05) abruptly on Day 110 of gestation (Fig. 3a). Furthermore, VEGF concentrations in fetal blood were greater (P < 0.05) in Meishan fetuses compared to Yorkshire fetuses on Days 50, 90, and 110 of gestation (Fig. 3a). The overall concentrations of VEGF in allantoic fluid were lower than those in fetal blood (Fig. 3, a vs. b). No differences were found between Meishan and Yorkshire allantoic fluid concentrations of VEGF on Day 50 or 70 of gestation (Fig. 3b). Meishan allantoic fluid concentrations of VEGF increased markedly from Day 70 to Day 90 and remained elevated through Day 110 of gestation, but VEGF concentrations in Yorkshire allantoic fluid remained constant from Day 50 to Day 90 before increasing (P < 0.05) abruptly on Day 110 of gestation. As a result, VEGF concentrations in allantoic fluid of Meishan gilts were markedly greater (P < 0.05) than those of Yorkshire gilts on Day 90 of gestation.



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  		FIG. 3. VEGF concentrations from Meishan and Yorkshire fetal serum (a) and allantoic fluid (b) on Days 50, 70, 90, and 110 of gestation. Asterisks indicate significant differences (means ± SEM) between breeds within a day (P < 0.05). Different letters indicate significant differences (means ± SEM) within a breed (P < 0.05)

Figure 4 depicts the VEGF (Fig. 4, a and d), VEGF-R1(Fig. 4, b and e), and VEGF-R2 (Fig. 4, c and f) profiles for the Meishan and Yorkshire placental tissues. Unlike VEGF protein levels in allantoic fluid, which were greater in the Meishan compared to the Yorkshire, no breed differences in the mRNA levels were detected, only day effects within a breed. In Meishan placental tissue, VEGF mRNA levels (Fig. 4a) remained relatively steady from Day 30 to Day 70 of gestation, then increased (P < 0.05) from Day 70 to Day 90 and remained high on Day 110 of gestation. In the Yorkshire placenta, however, VEGF mRNA levels (Fig. 4d) increased from Day 30 to Day 50, remained steady through Day 90, and then increased abruptly on Day 110 of gestation. The patterns for VEGF-R1 and VEGF-R2 were similar within a breed across gestation (Fig. 4, b, c, e, and f). Expression of both receptors increased progressively from Day 50 to Day 110 in the Meishan placenta, but Yorkshire placental VEGF-receptor expression was relatively constant from Day 30 to Day 70, attaining greatest expression on Day 90 and on Day 110 of gestation for VEGF-R1 and VEGF-R2, respectively.



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  		FIG. 4. Meishan (a–c) and Yorkshire (d–f) placental mRNA expression of VEGF (a and d), VEGF-R1 (b and e), and VEGF-R2 (c and f). Different letters indicate significant differences (means ± SEM) within a gene (P < 0.05)

In the endometrium (Fig. 5), mRNA expression levels of VEGF, VEGF-R1, and VEGF-R2 appeared to follow similar trends within a breed. In the Meishan, endometrial expression of VEGF (Fig. 5a), VEGF-R1 (Fig. 5b), and VEGF-R2 (Fig. 5c) remained relatively constant from Day 30 through Day 70 of gestation before increasing (P < 0.05) abruptly from Day 70 and Day 90 and remaining elevated and constant through Day 110 of gestation. In contrast, the Yorkshire endometrium exhibited a progressive increase in the expression of VEGF (Fig. 5d), VEGF-R1 (Fig. 5e), and VEGF-R2 (Fig. 5f) from Day 30 to Day 110 of gestation.



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  		FIG. 5. Meishan (a–c) and Yorkshire (d–f) endometrial mRNA expression of VEGF (a and d), VEGF-R1 (b and e), and VEGF-R2 (c and f). Asterisks indicate significant differences between breeds on Day 90 of gestation (P < 0.05). Different letters indicate significant differences (means ± SEM) within a gene (P < 0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have demonstrated, to our knowledge for the first time, the occurrence of earlier (Day 90 vs. Day 110 of gestation) increases in placental VEGF mRNA expression as well as VEGF concentrations in fetal serum and allantoic fluid Meishan conceptus than in the Yorkshire conceptuses at a time of diverging placental efficiencies. Furthermore, whereas VEGF mRNA expression levels did not differ between Meishan and Yorkshire conceptuses, VEGF protein in fetal blood and allantoic fluid was increased in Meishan conceptuses compared to Yorkshire fetuses. This increased amount of VEGF protein was temporally associated with the increased placental vascularity of the Meishan conceptuses compared to Yorkshire conceptuses, as reported previously [6–8] and also observed in the present study (unpublished results). These data are consistent with previously reported data demonstrating that Meishan, but not Yorkshire, conceptuses exhibit a marked increase in placental vascularity from Day 70 to Day 110 of gestation [6]. These data lend support to the concept that the VEGF-receptor system may exert significant effects on increasing placental vascularity and efficiency in the pig; however,, initial increases in vascularity may also arise from other angiogenic factors. Platelet-derived growth factor, a mitogen with angiogenic effects, has also been localized to the porcine placenta [17]. We have demonstrated that a significant amount of the variation in placental efficiency in the pig is associated with the variation in both placental and endometrial vascular densities [18]. Furthermore, Vonnahme and Ford [16] recently demonstrated that Yorkshire piglets resulting from a multigenerational selection for increased placental efficiency exhibited an increased placental mRNA expression of VEGF, VEGF-R1, and VEGF-R2 during late gestation in association with decreased placental weights and increased litter sizes. Therefore, it appears that the VEGF-receptor system may play a significant role in mediating placental efficiency in the pig.

Many researchers have demonstrated that uterine horn length in the Meishan and Yorkshire do not differ during the estrous cycle or during early gestation [4, 19, 20], but to our knowledge, no reports have characterized uterine growth throughout gestation in these two breeds. In the present study, using gilts of similar reproductive age (i.e., 4–8 postpubertal estrous cycles), we demonstrate that Meishan uteri were significantly shorter than Yorkshire uteri after Day 30 of gestation. This difference resulted from breed-specific differences in uterine horn growth. Whereas a marked and progressive increase in uterine horn length was observed in the Yorkshire females with the advancement of gestation, little uterine growth was observed in the Meishan breed. The overall increase in uterine horn length observed in the Yorkshire gilts may result from growth factors emanating from their rapidly growing placentae [21, 22]. In contrast, the limited uterine horn growth seen in the Meishan breed may result from the failure of the Meishan placenta to grow and, thus, possibly secrete these growth factors after Day 50. This hypothesis is supported by the observation that as a percentage of uterine horn length, the amount of space occupied by Meishan and Yorkshire conceptuses in the present study did not differ after Day 30 of gestation. Thus, placental growth appears to be tied to equivalent increases in uterine horn length in both breeds. The reason for our present failure to observe breed differences in litter size, as we have reported in the past, is unknown. In the present study, because fetal and placental weights, placental efficiency, and placental vascularity (data not shown) are similar to previously published data [6–8], we feel that the biological differences between this group of Meishan and Yorkshire pigs remain, even though no breed differences in litter size were observed. Although we tried to maintain consistent environments for both our Meishan and Yorkshire herds, the possibility always exists for litter sizes to be skewed from the normal average, because reproductive efficiency can be altered by slight variations in environmental factors affecting herd health [23]. Whereas the capacity for producing larger litters is certainly greater in the Meishan than in the Yorkshire breed [4, 5, 19], this potential is not always realized [23].

One of the most potent stimulators of VEGF expression is local hypoxic conditions [24]. It would logically follow that as nutrient and, particularly, oxygen delivery becomes limiting at the endometrial-placental interface in association with accelerating fetal growth, both placental and adjacent endometrial tissues would upregulate the synthesis and secretion of VEGF to compensate via increases in angiogenesis and vascular permeability. Additionally, evidence suggests that estrogens upregulate VEGF expression in uterine tissues from rats [25], sheep [26], and humans [27]. This is likely a primary estrogen receptor-mediated effect, because VEGF induction is very rapid, is blocked by pure antiestrogens [28], and is inhibited by actinomycin D but not by puromycin or cycloheximide [25, 29]. This regulation by estrogens is consistent with the results of most studies concerning the expression of VEGF in the uterus throughout the estrous cycle in rodents [30, 31] and the menstrual cycle in humans [32]. Both fetal demand for oxygen and placental secretion of estrogen increase dramatically throughout late gestation in the pig [21].

Besides playing an role in increasing vascular proliferation and permeability in the endometrium and placenta, VEGF may be acting to decrease vascular resistance in the uteroplacental vasculature. Recently, it was shown that VEGF can induce vasodilatation in uterine vasculature of the pregnant rat [33]. Vasodilatation can be induced by VEGF acting on its receptors that, in turn, activate endothelial nitric oxide synthase (eNOS), which causes an increase in the vasodilator, nitric oxide [34–36]. Winther et al. [11] demonstrate an increase in the expression of VEGF-R1 and VEGF-R2 in the porcine uterine artery as pregnancy advances. To our knowledge, no report of eNOS expression in the gravid pig uterus or placenta has appeared, but eNOS expression in the sheep [37] and the rat [38] increase in the uterine artery as well as the placenta during late gestation [39, 40]. Our laboratory has demonstrated that uterine blood flow per gram of placenta is markedly greater in Meishan conceptuses compared to Yorkshire conceptuses [41]. It is possible that the increased concentration of VEGF in the Meishan pig may be not only driving the increased vascularity but also enhancing vasodilatation in the uteroplacental vascular bed via the nitric oxide system.

The differential expression of the VEGF-receptor system between the Meishan and the Yorkshire breeds demonstrates its potential role in the increased placental efficiency and increased placental vascularity that is demonstrated by the Meishan conceptus. Factors that may influence these unique patterns of VEGF, VEGF-R1, and VEGF-R2 expression in the pig are still unknown.


   FOOTNOTES
 
1 Correspondence: Stephen P. Ford, Department of Animal Science, University of Wyoming, 113 Animal Science/Molecular Biology Complex, Laramie, WY 82071. FAX: 307 766 2355; spford{at}uwyo.edu Back

Received: 9 December 2003.

First decision: 27 December 2003.

Accepted: 25 February 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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