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Reproduction (2004) 128 229-235
DOI: 10.1530/rep.1.00193
Copyright © 2004 Society for Reproduction and Fertility
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RESEARCH

Placental transport of leucine in a porcine model of low birth weight

Angela M Finch1, Li G Yang1, Margaret O Nwagwu1,2,3, Kenneth R Page3, Harry J McArdle1 and Cheryl J Ashworth1,2

1 Development, Growth and Function Division, Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK, 2 Sustainable Livestock Systems Group, Scottish Agricultural College, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, UK and 3 School of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK

Correspondence should be addressed to Cheryl J Ashworth at SAC; Email: c.ashworth{at}ab.sac.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Low birth weight is a major factor in neonatal morbidity and mortality in humans and domestic species and is a predictor of physiological disorders in adulthood. This study utilised the naturally occurring variation in pig fetal size within a uterus to test the hypothesis that placental amino acid transport capability is associated with fetal growth. Leucine uptake by trophoblast vesicles prepared from placentas supplying an average-sized fetus and the smallest fetus in the uterus was assessed. On days 45 and 65 of gestation, uptake of leucine by the porcine placenta was predominantly sodium independent and was inhibited by the non-metabolised leucine analogue 2-amino-2-norbornane-carboxylic acid, indicating that uptake occurs via system L. By day 100 the uptake of leucine by placentas supplying average-sized fetuses had changed from being predominantly sodium independent to involving both sodium-dependent (system B0) and -independent (system L) pathways. This change was not seen in placentas supplying the smallest fetus, which continued to display predominantly sodium-independent uptake. In conclusion, these data show gestational- and fetal size-dependent changes in the transport of leucine across the porcine placenta.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
In both humans and pigs, low birth weight is a major factor in neonatal morbidity and mortality. Furthermore, low birth weight has been linked to the development of endocrine and cardiac disease during adulthood in humans (Godfrey & Barker 2000), while in pigs birth weight is recognised as the major determinant of subsequent survival and total weaning weight of the litter (Winters et al. 1947). Attempts to alleviate the detrimental effects of low birth weight postnatally have achieved only limited success. Rather, data indicating that the in utero environment can programme fetal gene expression and metabolism suggest that strategies to promote post-natal well-being should focus on key stages of pre-natal development.

In order to gain understanding of the factors associated with differential fetal growth, this study compared the fetoplacental unit associated with the smallest pig fetus in the uterus with a normally sized littermate. This permits assessment of local factors associated with fetal size, free from the confounding effects of maternal husbandry, genetic background and nutrition. Such studies are important for both the pig industry, where low birth weight is associated with significant loss, and for the human condition, as the pig is recognised as a valuable model to study low birth weight infants (Cooper 1975).

The capacity of the placenta to transport nutrients has a direct effect on fetal growth. We have demonstrated that placentas supplying the smallest fetus in the uterus are disproportionally lighter than those supplying average-sized fetuses in the same litter (Ashworth et al. 2001). These data, combined with the positive relationship between placental blood flow and fetal weight in the pig (Wootton et al. 1977), imply that there is a generalised reduction in the ability of placentas supplying small fetuses to deliver nutrients. In addition, there is increasing evidence in a variety of species, including rat, guinea pig, sheep and human, of an association between placental amino acid transport and fetal growth (reviewed in Sibley et al. 1997). Direct evidence for a role of placental amino acid transport in fetal growth comes from in vivo studies in sheep, where 13C-labelled leucine was infused during growth-retarded pregnancies and a reduction in maternal leucine flux into the placenta was observed (Ross et al. 1996). Further, abnormalities in amino acid transporter function have been observed in vesicles prepared from the microvillous membrane of the human placental syncytiotrophoblast associated with low birth weight babies (Dicke & Henderson 1988, Mahendran et al. 1993, Glazier et al. 1997, Jansson et al. 1998, Norberg et al. 1998).

This study tested the hypothesis that the capacity of the placenta to transport amino acids differs between placentas supplying inadequately grown and normally grown fetuses. Leucine was selected as an exemplar amino acid for several reasons. First, it is taken up by both Na+-dependent and Na+-independent mechanisms (Christensen 1985). Secondly, it is an essential amino acid, and may be rate limiting for growth. Thirdly, day 100 fetal plasma leucine concentrations are lower in the smallest fetus of the litter compared with a normal-sized littermate (C J Ashworth and H J McArdle, unpublished observations).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Experimental animals
Eleven Large White x Landrace sows were used in these studies. On days 45 (n = 4), 65 (n = 4) or 100 (n = 3) following natural mating, sows were exsanguinated under deep anaesthesia which was induced and maintained by inhalation of 8% (v/v) halothane in oxygen (Halothane-M&B, Rhone Merieux Ltd, Harlow, Essex, UK) in a semi-closed system. The reproductive tract was removed, and all feto-placental units were excised in an aseptic manner. Fetal and placental weights were recorded. The lightest fetus and a littermate of average weight were identified and their placentas collected. All experimental procedures were approved by the appropriate ethical committee and conducted in accordance with the UK Animals (Scientific Procedures) Act 1986.

Preparation of placental membrane vesicles
Placental amino acid transporter function was investigated using an adaptation of a method used extensively to study uptake across the apical membrane of several species (McArdle et al. 1984, Dicke & Henderson 1988, Boyd 1991, Mahendran et al. 1993, Glazier et al. 1997, Jansson et al. 1998, Norberg et al. 1998). On homogenisation, the microvilli of the apical membrane form sealed vesicles that accumulate amino acids and other nutrients and reflect the transport characteristics of the placenta in situ.

Placental membrane vesicles were prepared from the freshly obtained fetal aspect of each placenta (consisting of trophoblast, connective tissue and allantoic layers) using an adaptation of previously published methods (McArdle et al. 1984). Placental tissue was placed in 150 mM NaCl, 5 mM Hepes (pH 7.4), chopped finely with scissors, washed twice in ice-cold 150 mM NaCl, 5 mM Hepes (pH 7.4), and then three times in 100 mM CaCl2, 5 mM Hepes (pH 7.4), followed by two washes in 250 mM sucrose, 20 mM Hepes (pH 7.4). The placental fragments were homogenised (13 000 r.p.m.) (30 s, 4 °C, x 3). The homogenate was centrifuged at 1500 g for 10 min, 15 000 g for 20 min and 105 000 g for 60 min. The pellet was discarded after the first two spins and the final pellet was resuspended in 10% sucrose and stored in aliquots at –70 °C. A sample of placenta was homogenised in 10% sucrose and kept for comparison with the vesicles. The purity of these membrane vesicle preparations was shown by enrichment of staining by the porcine trophoblast specific antibody SN1/38 (Whyte et al. 1984) on Western blots. Alkaline phosphatase is the standard measure of apical membrane enrichment. However, in early gestation it is not expressed in the pig placenta and in late pregnancy it only localises to discrete patches (Skolek-Winnisch et al. 1984). Therefore, vesicles were identified immunologically using the antibody SN1/38.

Immunoblotting
Placental vesicles were prepared as described above. Whole placental homogenate was prepared by homogenisation of placental tissue (consisting of trophoblast, connective tissue and allantoic layers) in 10% sucrose solution at a 1:5 w/v ratio. Protein concentration was determined by the Bradford assay (BioRad, Hemel Hempstead, Herts, UK) using BSA as a standard. Crude homogenate and placental vesicle preparations were resolved by SDS/PAGE (10% gel) in the presence of 5% v/v beta-mercaptoethanol as a reducing agent, and electroblotted to a nitrocellulose membrane. The monoclonal antibody SN1/38 (a gift from A Whyte, Babraham Institute, Cambridge, UK) was used (1:100). Immunoreactive species were detected with horseradish peroxidase-conjugated anti-mouse antibody (1:5000) using the enhanced chemiluminescence detection system (ECL; Amersham).

Immunohistochemistry
Fixed placentas were processed into wax blocks and sections (thickness, 5 µm) were cut for immunohistochemistry. Treatment with 3% hydrogen peroxide (Sigma, UK) in water (25 °C) quenched endogenous peroxidase activity. The samples were pretreated with a non-immune serum block (1.5% normal horse serum) for 20 min, and incubated with the SN1/38 antibody at a 1:400 dilution (made up in 1% BSA) overnight at 4 °C. Samples were again pre-treated with a non-immune serum block (1.5% normal horse serum; Vector Laboratories, Bretton, Peterborough, UK) and incubated for 30 min at 25 °C with 0.005% bio-tinylated mouse IgG diluted in 1.5% normal horse serum. Sections were labelled with the avidin-biotin-peroxidase detection system (Vector Laboratories). Thereafter, sections were counterstained with haematoxylin, dehydrated, and cleared in xylene. A matching concentration of mouse IgG was used as a negative control. Appropriate controls were prepared, omitting either primary or secondary antibody as necessary.

Vesicle transporter studies
L-Leucine uptake by microvillous membrane vesicles was determined by a modification of a rapid-filtration technique as described previously (McArdle et al. 1984). Briefly, 25 µg vesicle protein were incubated with 4.5 nM L-[4,5-3H] leucine (Amersham Life Science) in a balanced salt solution with or without Na+. The balanced salt solution consisted of 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 18 mM HEPES (pH 7.4) and 136 mM of either NaCl (Na+-buffer) or choline chloride (choline-buffer). In inhibition studies, unlabelled amino acids (2-amino-2-norbornane-carboxylic acid (BCH; Sigma), leucine or lysine) were added to the incubation buffer at the final concentrations indicated in the figure legends. BCH is a non-metabolised leucine analogue that is transported by system L (the transport system responsible for the sodium-independent transport of large neutral amino acids). The reaction was started by the addition of vesicles and stopped at 60 s by passing an aliquot (80 µl) through nitro-cellulose filters (Whatman, Brentford, Middlesex, UK) followed by two washes of 1 ml ice-cold incubation buffer. The filters were dissolved in 5 ml scintillation fluid (Ultima Gold, Packard Instrument Company, Meriden, CT, USA) and radioactivity was measured in a Packard beta counter. Appropriate blanks and standards were included as required.

Uptake, as opposed to binding, was measured by incubating vesicles in solutions of increasing osmolarity, achieved by the addition of mannitol, for 5 min by which time equilibrium was achieved (data not shown). Vesicle size (µl/mg protein) was calculated from the specific activity of the extra-vesicular medium.

Data analysis and expression of results
All uptake data were expressed as the difference between uptake at 39 °C and at 4 °C. The total and Na+-independent uptakes were defined as the uptake in the presence of sodium and choline respectively. Data were expressed as the uptake rate (fmol/s mg vesicle protein) or total placental uptake capacity. The total placental uptake capacity (fmol/s/placenta), an approximation of uptake capacity across the whole placenta, was derived from the uptake rate (fmol/s/mg vesicle protein) x placenta protein concentration (mg protein/placenta). No difference in protein concentration was seen between placentas (or the vesicles derived from them) supplying the low weight fetuses in comparison with those supplying the average-sized fetuses. Hence, total placental uptake capacity is a function of amino acid uptake and placental mass. Significance was determined via ANOVA and a post hoc Newman Keuls test, with the exception of the amino acid inhibition data for which significance was determined via ANOVA and Dunnet’s multiple comparison test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Fetal and placental size
At each gestational age studied, the smallest fetus from each litter was significantly lighter than that of the average-sized fetus to which it was compared (Table 1Go). By day 100 the smallest fetus was 52 ± 3% the size of its average-sized littermate. Interestingly, the placentas supplying the smallest fetuses were only significantly smaller than their normal counterparts at day 45 (Table 1Go). The small fetuses also displayed an increased fetal/placental weight ratio at day 45 (P < 0.05) (Table 1Go).


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  		Table 1 Fetal weight, placental weight and fetal/placental weight ratio in the smallest fetus and an average sized littermate. Data are means±S.E.
 
Vesicle validation
The monoclonal antibody SN1/38 shows specificity for the trophoblast brush border of the pig placenta (Fig. 1AGo). The purity of the membrane vesicle preparations was shown by enrichment of staining by the porcine trophoblast specific antibody SN1/38 on Western blots (Fig. 1BGo). The relationship between leucine uptake and the inverse of the osmolarity was linear, with no uptake when the line was extrapolated to zero (infinite osmolarity) (Fig. 2Go). This indicated that the leucine was transported into a vesicle space rather than binding to the vesicle membrane. From these data the vesicles have an apparent size of 10 µl/mg protein, similar to that described for rat microvillar vesicles, but larger than human (McArdle et al. 1984).



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  		Figure 1 (A) i: Localisation of SN1/38 on day 100 of gestation to the microvillous border of the fetal trophoblast. ii: Corresponding negative control. The bar represents 10 µM at a x10 magnification. MV, microvillous membrane of the fetal trophoblast; FC, fetal connective tissue; ME, maternal epithelium. (B) Vesicle preparations (V) and matching homogenate (H) at equal protein concentrations. Upper panel: Coomassie stained; lower panel: Western blot for SN1/38. The vesicle preparations show positive SN1/38 immunoreactivity; when the blot was exposed for longer the homogenate showed immunoreactivity (data not shown).

 


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  		Figure 2 Hyperosmolar behaviour of porcine trophoblast vesicles. Calculated vesicle volume of trophoblast vesicles prepared from day 45 (open circles) and day 100 (closed circles) placentas. Volume derived from 3H-leucine uptake in conditions of increasing osmolarity. Data represent means of 4 determinations ±S.E. on day 45 and of duplicate determinations on day 100.

 
Characterisation of leucine uptake in differential fetal growth
On day 45 of pregnancy, the calculated total uptake of leucine per placenta was decreased in placentas supplying the smallest fetus of each litter (Fig. 3Go). However on days 65 and 100 of pregnancy this difference in the total uptake capacity was no longer detected (Fig. 3Go).



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  		Figure 3 Total uptake capacity. The calculated total uptake capacity (pmols/s/placenta) for placentas supplying the smallest fetus (open bars) and an average-sized littermate (closed bars) at days 45, 65 and 100 of pregnancy. Data represent means±S.E., n = 3–4 litters. * P < 0.05.

 
To examine further the transport system/s involved in leucine transport by placentas supplying fetuses of differing sizes, we determined the sodium dependence of uptake and the inhibition of leucine uptake by other amino acids. At days 45 and 65 of pregnancy, in both the placentas supplying a normal-sized and the smallest fetus of each litter, the uptake of leucine was predominantly via a sodium-independent transport system (Fig. 4Go). The synthetic amino acid, BCH, inhibited this sodium-independent uptake of leucine (P < 0.05) (Fig. 5Go). In contrast, at day 100 of pregnancy leucine uptake by the placenta supplying the normal-sized fetuses was only 51 ± 12% sodium independent (Fig. 4Go). Both the sodium-dependent and -independent transport in the placentas supplying the average-sized fetus of each litter, at day 100, was inhibited by BCH (P < 0.05) (Fig. 5Go). At day 100, in the placentas supplying the average-sized fetus of each litter, leucine but not lysine inhibited the uptake of 3H-leucine in the presence of sodium in a dose-dependent manner (0.1 mM leucine P < 0.05, 1 mM leucine P < 0.01) (Fig. 6Go). This change in the sodium dependence of leucine uptake was not observed in the placentas supplying the smallest fetus of each litter, which continued to display predominantly sodium-independent uptake inhibited by BCH (P < 0.05) (Figs 4Go and 5Go).



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  		Figure 4 Sodium dependence of uptake of 3H-leucine into membrane vesicles. Uptake was performed in the presence (closed bars) and absence (open bars) of sodium using vesicles from placentas supplying both the smallest and an average-sized fetus of each litter at days 45 (A), 65 (B) and 100 (C) of pregnancy. Data represent means±S.E., n = 3–4 litters. *P < 0.05.

 


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  		Figure 5 Inhibition of uptake of 3H-leucine into membrane vesicles by 2-amino-2-norbornane-carboxylic acid (BCH). Uptake in the presence of 5 mM BCH was performed either in the presence (closed bars) or absence (open bars) of sodium using vesicles from placentas supplying both the smallest and an average-sized fetus of each litter at days 45 (A), 65 (B) and 100 (C) of pregnancy. Percent inhibition was calculated as percentage uptake in the presence of BCH versus uptake in the absence of BCH for each condition. Data represent means±S.E., n = 3–4 litters.

 


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  		Figure 6 Inhibition of 3H-leucine uptake into day 100 membrane vesicles from placentas supplying average-sized fetuses. Uptake was performed in the presence of sodium and 0.1 or 1 mM leucine (Leu; open bars) or lysine (Lys; closed bars). Percent of control was calculated as percentage uptake in the presence of unlabelled amino acid versus uptake in the absence (control) of unlabelled amino acid. Data represent means±S.E., n = 4 preparations. *P < 0.05, **P < 0.01 compared with control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
While there have been many studies of amino acid transport systems in the haemochorial placenta, those present in the epitheliochorial placenta of the pig remain uncharacterised. In this study we identify two of the amino acid transport systems present in the trophoblast brush border of the pig placenta and establish the trophoblast membrane vesicle as an experimental system to investigate amino acid transport in epitheliochorial placenta.

Leucine transport across the trophoblast brush border of the placentas supplying the average sized fetuses, during early to mid gestation (day 45/65), is predominantly via system L, as demonstrated by the sodium-independent nature of uptake and its inhibition by BCH (Christensen 1985, Christensen et al., 1968). The particular system L transporter involved resembles the recently described LAT3 transporter (Babu et al. 2003). LAT3 mediates facilitated diffusion and does not require 4f2 for its function, unlike the obligatory exchangers LAT1 and LAT2 (Yanagida et al. 2001).

During late gestation, leucine uptake by the placenta supplying the average-sized fetus occurs by both sodium-independent and -dependent systems. The inhibition by BCH indicates that the sodium-independent component continues to be via system L. The sodium-dependent component can be identified as system B0 also by its inhibition by BCH and its inability to be inhibited by lysine (Stevens et al. 1982). System B0 (ASCT2) is present in late gestational rat placenta and in human placenta (Carbo et al. 1997, Knerr et al. 2003). In humans, ASCT2 is a dual function protein also acting as the receptor for syncytin. The expression of syncytin and ASCT2 are essential for the formation of the syncytium trophoblast layer (Frendo et al. 2003). However, in the pig placenta, in which a cellular trophoblast is maintained, the physiological significance of its expression in late gestation remains to be elucidated.

On day 45 placentas supplying small fetuses had a reduced leucine uptake capacity compared with those supplying average-sized fetuses. However, by day 65 there was no difference in either the rate or the total uptake capacity between the placentas, and this trend was continued at day 100. There was, however, a difference in the proportion of the transport systems used. The placentas derived from the average-sized fetus utilised both sodium-independent and -dependent systems, while the placentas supplying the smallest fetuses continued to use the sodium-independent system L. It is not clear whether placentas supplying low weight fetuses are developmentally delayed and would show system B0 activity later in gestation or if this switch to system B0 transport never occurs in these placentas.

We have demonstrated that approximately one third of pig litters contain fetuses classified as intra-uterine growth retarded (IUGR) on the basis that they are outliers in an otherwise normally distributed population within the uterus (Finch et al. 2002). Although it was outside the scope of the present study, it will be important to determine how the observed differences in placental nutrient transport are manifest during IUGR. The full implications of the altered amino acid transport in placentas supplying small fetuses require further investigation.

The forces driving these changes in amino acid transporter expression need to be elucidated. It can be speculated that these changes in amino acid transporter expression are not driven by the placenta but are a consequence of the low fetal weight, as low weight fetuses can be identified in pig litters as early as day 30 of pregnancy (van der Lende et al. 1990, Finch et al. 2002). Low levels of epidermal growth factor (EGF) in amniotic fluid are associated with IUGR (Varner et al. 1996) and the expression of ASCT2 (system B0) has been shown to be stimulated by EGF (Torres-Zamorano et al. 1997). This may be the mechanism behind the lack of system B0 seen in the placentas supplying the low weight fetuses.

In conclusion, this study has demonstrated that the porcine placenta shows similar transport systems for neutral amino acids as those of other species (Carbo et al. 1997, Jansson et al. 1998) with the presence of system L, and later in gestation, system B0 transport across the brush border trophoblast membrane of the normal porcine placenta. In placentas associated with small fetuses, system L transport is present throughout pregnancy and system B0 is absent.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
We are grateful to SEERAD and the Royal Society for financial support. The authors express special appreciation to members of the Maternal-Fetal Physiology Group (Rowett Research Institute) and N Lokke (SAC) for technical assistance. We are grateful to Professor Colin Sibley (University of Manchester, UK) for discussion on the analysis of the total uptake capacity data and to Dr Tony Whyte (Babraham Institute, Cambridge, UK) for the gift of the SN1/38 antibody.


   Footnotes
 
Received 9 February 2004
First decision 31 March 2004
Revised manuscript received 12 May 2004
Accepted 19 May 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

Ashworth CJ, Finch AM, Page KR, Nwagwu MO & McArdle HJ 2001 Causes and consequences of fetal growth retardation in pigs. Reproduction Supplement 58 233–246.

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