Abstract
Polyunsaturated fatty acids derived from the diet are incorporated into cell membranes where they act as precursors for prostaglandin (PG) synthesis. Linoleic acid (LA; 18:2 n-6) is a major constituent of plant oils and its consumption in Westernized populations is increasing. This study investigated the influence of LA on PG production by the uterus and placenta. Pregnant ewes were fed a control or an LA-enriched diet. Oxytocin (OT) was injected on day 45 (early) or day 133 (late) of gestation to measure the release of 13,14-dihydro-15-keto PGF2α (PGFM). Ewes were killed on day 46 or day 138 for collection of uterine intercaruncular endometrium and fetal allantochorion. Basal and stimulated PG release from explant cultures was assessed before and after in vitro treatment with OT, lipopolysaccharide (LPS), dexamethasone (DEX) or calcium ionophore (CaI). Expression of cyclooxygenase (COX)-1 and COX-2 was determined by Western blot in endometrium of late-gestation ewes. Circulating PGFM levels in vivo did not differ according to diet but there were highly significant differences in the release of PGs in vitro. Basal production of PGF2αand PGE2 by the endometrium and of PGE2 by the allantochorion were all higher in tissues from LA-supplemented ewes. Endometrial tissues produced more PG following OT and CaI treatment, whereas DEX inhibited production of both PGs at both stages of gestation. In allantochorion collected at day 46 LPS did not significantly alter PGE2 release and DEX increased output, whereas at day 138 LPS was stimulatory but DEX was inhibitory. These data show that a high-LA diet can significantly increase the ability of both endometrium and placental tissues to produce PGs in vitro. This effect of diet may only become apparent after a sustained period of PG release, so was not seen following the brief pulse caused by OT treatment in vivo. As COX protein levels were unaltered, the main influence was likely to be via conversion of LA to arachidonic acid, providing an increased supply of precursor. These results support previous studies which suggest that alterations in dietary polyunsaturated fatty acids may influence the time of labour.
Introduction
Dietary polyunsaturated fatty acids (PUFAs) provide the body with energy, contribute structural components to cell membranes and also act as the precursors for prostaglandin (PG) synthesis. Linoleic acid (LA; 18:2 n-6) is the major fatty acid component in plant oils such as corn, sunflower, rapeseed and soybean oils (Sargent 1977) and is thus a common constituent of both human and animal diets. LA can be metabolized within the body to arachidonic acid (AA; 20:4 n-6; Kinsella et al. 1990, Bezard et al. 1994). Two series of PGs, including PGF2αand PGE2, are derived from AA following its release from phospholipid membrane stores by the action of the enzyme phospholipase A2 (PLA2). Free AA is then metabolized to PGF2α and PGE2 through the action of the cyclooxygenase (COX) enzymes COX-1 and COX-2 (Smith et al. 1991). COX-1 is generally considered to be a housekeeping gene and responsible for immediate responses whereas COX-2 is inducible by a variety of agents and is crucial for more-sustained PG generation (Smith et al. 1996, Murakami & Kudo 2004). There is, however, evidence showing that COX-1 can be upregulated in response to angiogenic factors (e.g. Sales et al. 2002) and during cellular differentiation (Murakami & Kudo 2004). Experiments using both gene-deletion and pharmacological treatments with specific COX-1 and COX-2 inhibitors have shown that parturition in mice is normally dependent on upregulation of COX-1 in the endometrium (Gross et al. 1998, Reese et al. 2000, Loftin et al. 2002). In the ewe, however, COX-2 expression in the endometrium is increased during both spontaneous and dexamethasone (DEX)-induced labour, whereas there is no change in COX-1 expression at this time (Zhang et al. 1996, Gibb et al. 2000, McClaren et al. 2000). In the uterus and placenta PG production and release can be modulated by a variety of physiological and pharmacological stimuli, for example oxytocin (OT; Lee and Silvia 1994), lipopolysaccharide (LPS; Flynn & Hoff 1995), calcium ionophore (CaI; Poyser 1987) and glucocorticoids (Whittle et al. 2001). Manipulation of the dietary PUFA content can also influence the production of PGs. Following increased human consumption of n-6 fatty acids, eicosanoid metabolic products from AA, specifically PGs and thromboxanes, are formed in larger quantities than when the diet is high in n-3 fatty acids (Simopoulos 2002).
However, not all reports on the effects of n-6 PUFA on PG synthesis are consistent. Some evidence suggests that LA enhances PG production by providing more precursor for conversion to AA (e.g. Connolly et al. 1996, Nakaya et al. 2001). Other studies have found an inhibitory effect. Both LA and n-3 PUFAs may suppress PG production at a number of different stages of the biosynthetic pathway. These include (i) inhibition of AA synthesis during the desaturation and elongation processes in the liver (Bezard et al. 1994), (ii) n-3 competing with n-6 PUFAs, especially AA, for incorporation into cell-membrane phospholipids, decreasing the available AA for COX (German et al. 1988) and (iii) direct blocking of COX activity (Staples et al. 1998, Marnett et al. 1999). In ruminants the effects of PUFAs have been investigated with a view to modulating uterine PG production during luteolysis or in early pregnancy (Staples et al. 1998, Abayasekara & Wathes 1999). PGs also play a critical role in many aspects of parturition (Thorburn & Challis 1979, Challis et al. 1997) and there is considerable evidence that dietary PUFAs can influence the timing of labour. For example, parturition is delayed in n-6 PUFA-deficient rats (Leaver et al. 1986), and women who delivered early were found to have a raised pool of n-6 PUFAs in plasma (Allen & Harris 2001). On the other hand, women with an increased n-3 PUFA intake had increased pregnancy duration (Olsen et al. 1992). The literature thus shows that manipulation of dietary PUFAs can influence PG production, but the end result is hard to predict. This study investigated the effect of increased n-6 PUFA supplementation on PG production during pregnancy by feeding ewes a diet enriched with LA. PG production in control and LA-supplemented ewes was compared: (a) in response to an in vivo OT challenge and (b) in vitro in cultured explants of endometrium and allantochorion. The experiment was repeated at two stages of gestation, early (day 45) and late (day 133).
Materials and Methods
Animals
All experimental work was performed under the Animals (Scientific Procedures) Act 1986. Mature Welsh mountain ewes had oestrous cycles synchronized by insertion of an intravaginal progestagen sponge (Upjohn, Crawley, West Sussex, UK) for 14 days. Immediately after sponge removal, 125 μg synthetic PG (Estrumate, Schering-Plough Animal Health, Welwyn Garden City, Herts, UK) was administered i.m. and the ewes were housed with a raddled ram for 48–96 h after sponge removal. Pregnancies were confirmed initially by measurement of plasma progesterone concentrations 16 days after mating. In the late-gestation group this was followed by an ultrasound scan at mid-gestation. The mean gestation length in a similar group of untreated ewes was 145 ± 0.4 days (n=7). Two experiments were performed. In both cases ewes were individually housed prior to the start of the experimental protocol and were gradually introduced to the experimental diet over a 7-day period. In experiment 1 (early gestation), ewes were placed on a control (n=5) or LA-supplemented (LA diet; n=6) diet from 7 days prior to the start of oestrous synchronization until euthanasia at 46 days gestation. In experiment 2 (late gestation) ewes were placed on a control (n=16) or LA-supplemented (n=16) diet from 96 days of gestation until euthanasia at 138 days of gestation.
Diets
Diets were calculated on an individual ewe basis (reviewed every week) to meet the maintenance requirement of the ewe according to her weight and stage of gestation and were mixed individually and fed each morning. The n-6 PUFA was fed as SoyPreme (Boregaard UK, Warrington, UK), a heat-treated product of xylose and cracked soyabean. This reaction reduces the degradability of the protein and protects the PUFAs from biohydrogenation in the rumen (Abel-Caines et al. 1998). Following such treatment approximately 55% of the dietary lipid bypasses the rumen, whereas for untreated feed this figure is only about 15%. Each diet was based on a mixture of maize silage, grass silage and concentrates with added SoyPreme on the LA-supplemented diet only. The diets were formulated to be isonitrogenous and isoenergetic. Details of dietary analyses and PUFA contents (ADAS Laboratories, Wolverhampton, UK) are given in Tables 1 and 2 respectively. Experiments 1 and 2 were performed in different years, so there were minor differences in dietary composition. Based on these figures it was estimated that, in experiment 1, 1.3 g/day n-6 PUFA would reach the small intestine in control-fed ewes compared with 8.5 g/day in the LA-supplemented ewes. In experiment 2, where dietary intake was higher later in gestation, the equivalent figures were 2.5 and 13.7 g/day. The LA-supplemented diet therefore increased the LA consumption approximately 6-fold.
OT challenge
When pregnant sheep reached day 44 (experiment 1) or 132 (experiment 2) of gestation a jugular-vein cannula was fitted. The following day, 5 ml blood samples were taken 60, 45, 30, 15 and 0 min before and 5, 10, 15, 30, 45, 60, 75, 90, 105 and 120 min after administration of 10 IU OT given via the jugular-vein cannula (Oxytocin-s; Intervet UK, Milton Keynes, Bucks, UK). The blood samples were put into heparinized tubes, placed on ice and centrifuged at 1600 g at 4 °C. The resulting plasma was frozen and stored at −20 °C. The stable metabolite of PGF2α, (PGFM) was then quanti-13,14-dihydro-15-keto PGF2 fied at each time point by RIA.
Explant culture
Cell-culture reagents were from Sigma-Aldrich (Poole, Dorset, UK) unless otherwise stated. Ewes were killed by captive bolt on day 46 (all ewes in experiment 1) or day 138 of gestation (eight ewes per diet from experiment 2). Reproductive tracts were placed in a laminar flow hood to maintain sterility. The uterus was opened and the fetus was removed. Maternal intercaruncular (IC) endometrium was separated from fetal allantochorion by manual dissection. Strips of each tissue were placed in basic Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 medium (Gibco Life Technologies, Paisley, UK) containing 1.125 g/l fatty acid-free BSA and antibiotics (50 000 units/l penicillin and 50 mg/l streptomycin). The method for explant culture has been described previously (Leung et al. 2001). Strips of IC and allantochorion tissue were chopped into 1 mm3 cubes. Tissue samples were blotted dry and weighed to provide 150 mg/dish. This was placed on a lens tissue on a stainless steel grid in a petri dish (30 mm×30 mm×0.5 mm) containing 6 ml basic medium supplemented with ITS media supplement to provide a final concentration of 0.5 μg/ml insulin, 0.5 μg/ml transferrin and 0.5 ng/ml selenium. The tissue explants were then cultured at 39 °C with 5% CO2 for 42 h in a humidified incubator with the medium changed after 24 h. At the end of the 42 h culture period the explants were challenged for 24 h with (i) control medium, (ii) OT (250 nM), (iii) DEX (5 μM), (iv) CaI (A23187; 10 μM) or (v) LPS (Escherichia coli 026.B6; 0.1 μg/ml). OT was only tested on maternal endometrial tissues whereas LPS was used on fetal allantochorion (which lacks OT receptors). Medium was collected at 2 and 24 h after the start of the challenge. The rationale behind the times chosen for medium collection for PG measurement was as follows. The first 24 h period was discarded as this represented release from damaged tissue following the chopping procedure. The next 18 h provided a period to assess basal PG production. The challenges were performed after 42 h as we have previously shown that this is the time needed to get upregulation of OT receptors in vitro, and we wished to challenge with OT (Leung & Wathes 2000). Following challenge, we investigated both the immediate (2 h) and longer-term (24 h) responses as the different ligands used were known to act over differing time courses in terms of influencing PG synthesis. Each treatment was performed in quadruplicate or triplicate. The culture medium was frozen and stored at −20 °C for subsequent measurement of PGF2α and PGE2 by RIA. Both PGs were measured for the IC explants but, as PGE2 is the main product of allantochorion, only this was measured in the allantochorion samples.
Treatment preparation
OT powder was dissolved in sterile distilled water containing 0.01% acetic acid. The reconstituted stock OT was diluted further with DMEM/Ham’s F12 basic medium to give a final concentration of 250 nM. DEX was dissolved in 100% ethanol at 20 mM and diluted with basic medium to a final concentration of 5 μM. The CaI A23187 was dissolved in 100% ethanol to a stock solution of 10 mM. This was diluted with basic medium to a final concentration of 10 μM. LPS was dissolved in distilled water at 500 μg/ml and diluted in basic culture medium to provide a final concentration of 0.1 μg/ml. The final ethanol concentration in the cultures did not exceed 0.1%.
PG RIAs
PGFM in the blood samples was measured as described previously (Robinson et al. 2002). The tracer (13,14-dihydro-15-keto-[5,6,8,9,11,12,14(n)-3H]PGF2α) was from Amersham International (Little Chalfont, Bucks, UK), the standards were supplied by Sigma-Aldrich and the antiserum was a kind gift from Dr H. Kindahl (Dept. of Obstetrics and Gynaecology, Swedish University of Agricultural Science, Uppsala, Sweden). The limit of detection was 1 pg/tube. The intra-assay and inter-assay coefficients of variation were 7.6 and 14.3% respectively.
PGE2 and PGF2α in the media samples were quantified as described previously following dilution in 0.05 M Tris buffer containing 0.1% gelatin and 0.01% sodium azide (Cheng et al. 2003). The tritiated tracers of PGE2 ([5,6,8,11,12,14,15(n)-3H]PGE2) and PGF2α ([5,6,8,9,11,12,14,15(n)-3H]PGF2α) were from Amersham International and the standards of PGE2 and PGF2α were from Sigma-Aldrich. The antisera to these two PGs were a kind gift from Dr N.L. Poyser (University of Edinburgh, Edinburgh, UK; Poyser 1987). Their cross-reactivities were as follows: PGF2α antiserum, 34% with PGF1α and 25% with PGF3α; PGE2 antiserum, 23% with PGE1 and 15% with PGE3. The limit of detection was 1 pg/tube for PGF2α and 2 pg/tube for PGE2. The intra-assay and inter-assay coefficients of variation were 3.5 and 6.3% for PGE2, and 4.1 and 9.6% for PGF2α respectively.
Preparation of tissue lysates
In experiment 2, additional strips of IC were collected at slaughter, placed in cryovials, frozen immediately on dry ice and stored at −80 °C. Tissues (1 g) were homogenized in 10 mM Tris/HCl buffer, pH 7.5, containing 1 mM EDTA, 250 mM sucrose and protease-inhibitor tablets (Protease inhibitor cocktail tablets, one dissolved per 50 ml buffer; Roche catalogue no. 1 836 145; F. Hoffman La Roche, Basel, Switzerland). The homogenate was then centrifuged at 800 g for 10 min at 4 °C, and the supernatant was collected and ultracentrifuged at 100 000 g for 60 min at 4 °C to obtain the microsomal fractions. Microsomal preparations were then solubilized in homogenization buffer containing 100 mM phenyl-methanesulfonyl flouride (PMSF) and stored at −80 °C. The protein content was determined by BCA protein assay kit (Sigma-Aldrich).
SDS/PAGE and Western immunoblotting for COX
Tissue lysate proteins were separated using 10% SDS/PAGE (Wheeler-Jones et al. 1996). Following electrophoresis, proteins were transferred to Immobilon polyvinilidine difluoride (PVDF) membrane (Millipore UK, Watford, UK) using the Bio-Rad semi-dry blotting system. After transfer of proteins to a PVDF membrane, non-specific sites were blocked using a solution of Tris-buffered saline with Tween (TBS-T; 50 mM Tris, 150 mM NaCl, 0.02% (v/v) Tween-20, pH 7.4) containing 10% (w/v) BSA, for 2 h at room temperature with gentle agitation. The membrane was then incubated with either anti-COX-1 antibody (Cayman Chemical Company, Ann Arbor, MI, USA) at 1:500 dilution or anti-COX-2 antibody (Santa Cruz Biotechnology, Calne, Wilts, UK) at 1:1000 dilution in TBS-T containing 10% (w/v) BSA overnight at 4 °C at the maximum agitation speed. The membrane was then washed for 6×10 min in TBS-T. Following the incubation of the membrane in a 1:10 000 dilution (in TBS-T plus 0.2% (w/v) BSA) of horseradish peroxidase-conjugated goat anti-rabbit IgG or rabbit anti-goat IgG (both from Pierce, Chester, UK) for 1 h, the membrane was washed a further eight times for 10 min in TBS-T. Immunoreactive proteins were visualized using enhanced chemiluminescence (ECL; Amersham).
Blots were also assessed for β-actin expression using anti-β-actin antibody (Abcam, Cambridge, UK) and horseradish peroxidase-conjugated goat anti-mouse IgG (Transduction Laboratories, Cowley, Oxford, UK). Densitometric quantification of immunoreactive bands was carried out using a Bio-Rad model GS-690 imaging densitometer with Bio-Rad Quantity One software, version 4.4.0 (Bio-Rad Laboratories, Hercules, CA, USA).
Data analysis
The values are quoted as means ± s.e.m. Analysis of experimental data in this study was performed on the basis of the two dietary groups, control and LA-supplemented. All data were checked for homogeneity and if not homogenous data were either log- or square-root-transformed to achieve a normal distribution. Statistical analysis for the PGFM concentrations following in vivo OT challenge was carried out using a repeated-measures analysis via Proc Mixed built in SAS 8.0 (SAS Institute, Cary, NC, USA). Comparisons of PGF2α and PGE2 production by placental tissue explants were carried out using analysis of variance via general linear and mixed linear models in SPSS 12.0 for Windows (SPSS, Chicago, IL, USA). Where significant difference was achieved (P<0.05), least-square means based on Fisher’s least significant difference (LSD) for multiple comparisons were calculated to examine the differences between the fixed effects. Optical density units from the Western blot analysis were analysed with an unpaired t-test using GraphPad Prism version 2.01 (GraphPad Software, San Diego, CA, USA).
Results
OT challenge
Basal and peak levels of plasma PGFM were measured in ewes on the two diets at 45 and 133 days gestation in plasma samples taken for 1 h before until 2 h following the OT challenge. Basal PGFM concentrations were higher at day 133 than at day 45, but peak values were similar (Table 3). There were no significant differences in either basal or peak levels of PGFM between the control and LA-supplemented diets at either time point and the pattern of release of PGFM into the maternal blood plasma in response to OT was not influenced by the experimental diet (Fig. 1).
Explant cultures: basal production of PGs
Basal concentrations of PGs were estimated by measurement in medium over the 18-h period from 24 to 42 h following establishment of the explant cultures (Table 4). In experiment 1 (early gestation) the PGF2α and PGE2 production by IC explants was increased in tissue obtained from ewes on the LA-supplemented diet. Synthesis of PGE2 by the fetal allantochorion was also significantly increased by dietary n-6 PUFA. Similar results were obtained for experiment 2 (late gestation), when all tested PGs were again significantly increased by the LA-supplemented diet. Whereas PG production by the endometrium was of a similar order of magnitude in tissues obtained at both stages of gestation, production of PGE2 by the allantochorion increased over 30-fold between 46 and 138 days.
Explant cultures: stimulated production of PGs
Following the initial 42 h culture, the tissue explants were challenged in vitro. The effects of treatment on PGF2α secretion by maternal IC endometrium are illustrated in Fig. 2. The OT and CaI treatments stimulated PGF2α output at both 2 and 24 h from tissue obtained in both early and late gestation. DEX had no effect at 2 h but caused a significant inhibition after 24 h in the early gestation tissue. Tissue obtained from ewes on the LA-supplemented diet produced consistently more PGF2α at 2 h than the tissue obtained from control-fed ewes and subsequently treated similarly in vitro. The effect of the LA diet was less profound after 24 h; significant increase in PGF2α production was observed only in the tissue challenged with DEX in the early-pregnancy ewes and following control-medium and CaI treatment in the late-pregnancy ewes.
PGE2 was only measured in the IC tissue collected in late gestation (Fig. 3). The effects of the challenge treatments were similar to those reported for PGF2α. OT stimulated PGE2 at 24 h, CaI treatment stimulated PGE2 output at both 2 and 24 h and DEX had no effect at 2 h but caused a significant inhibition after 24 h. Tissue from the LA-supplemented ewes produced more PGE2 following control-medium, OT and DEX treatments at 2 h and following CaI treatments at 24 h.
PGE2 production was assessed in explants of fetal allantochorion collected from both early- and late-gestation ewes (Fig. 4). In these cultures LPS rather than OT was used as a potential modulator of PG production. The effects of the challenges were somewhat different to those observed in the maternal IC tissue, possibly associated with the large overall increase in the ability of the tissue to produce PGE2 at the later time period. After 2 h treatment of the early-pregnancy tissue none of the in vitro treatments had any effect. After 24 h of treatment, PGE2 production was enhanced by DEX but inhibited by CaI. In the late-pregnancy tissue, none of the challenge treatments influenced PGE2 output after 2 h. After 24 h, PGE2 output was unaffected by DEX and increased by LPS and CaI. In relation to the diet, LA supplementation in vivo increased PGE2 output at both time points regardless of treatment in vitro for the tissue collected in early pregnancy. In the late-pregnancy tissue, there was again a significant increase following all challenges at 2 h and after the control challenge at 24 h.
Discussion
Few previous studies have investigated the effect of a high n-6 PUFA diet on PG production during pregnancy, yet PGs play central roles in both fetal maturation and parturition (Liggins & Thorburn 1994). Westernized populations are currently increasing their intakes of vegetable oils containing high concentrations of PUFAs, in particular LA (18:2 n-6; Department of Health 1995). Within the UK, 2.5% of the female population consume over 20 g n-6 PUFA daily, mostly as LA (Office of Population Censuses and Surveys 1990). We show here that placental tissues from ewes fed a diet containing a 6-fold increase in LA consistently produced significantly more PG in vitro than tissues from ewes on the control diet.
In general, diets with a high AA content will increase phospholipid levels of AA and also increase PG production (German et al. 1996, Sinclair & Mann 1996). When diets contain n-6 PUFAs from further up the n-6 pathway, results are more variable (see Introduction). LA is converted to AA by position-specific Δ6- and Δ5-desaturase and CoA-dependent chain elongases, located primarily in the liver (Bezard et al. 1994, Sprecher 2000). This will lead to an increased AA component of plasma lipids and greater incorporation of AA into the phospholipid membranes of tissues which can subsequently be released for PG synthesis. This is supported by in vitro studies of ovine endometrium (Cheng et al. 2004) and human decidual cells (Arntzen et al. 1998), which both showed enhanced PG production in reponse to the n-6 PUFAs AA and dihomo-γ-linolenic acid (DGLA) supplemented in vitro. In related studies we have shown that a similar LA-supplemented diet to that used here increased plasma AA concentrations (Elmes et al. 2004) and was associated with earlier delivery in DEX-induced late-gestation ewes (Elmes et al. 2003). Women giving birth to preterm infants were also reported to have a higher AA concentration in their blood than those delivering at normal term (Reece et al. 1997, Araya et al. 1998).
Our data suggest that the stimulatory effect of dietary LA on utero-placental PG production was more likely due to increased precursor availability than to upregulation of COX, as neither COX-1 nor COX-2 levels were increased in tissues obtained from the LA-supplemented ewes. However COX expression was not assessed following the explant culture period so it is possible that COX expression may have been further upregulated in vitro in tissues obtained from ewes on the high-LA diet. In support of this suggestion there is evidence that AA can itself upregulate COX-2 expression (Parent et al. 2003). PG production by particular tissues can also be influenced by the extent to which either COX-1 or COX-2 colocalize with particular terminal PG synthases in different sub-cellular compartments and the fact that COX-1 requires a greater supply of AA than COX-2 to be functional (Murakami & Kudo 2004). As the diet used in this study increased the supply of AA to the uterine tissues, activation of existing COX-1 could represent another potential mechanism whereby PG production could rise.
The in vitro study of stimulated production of PGs also produced interesting results, notably the different responses of the maternal and fetal tissues to DEX. The production of PGs by maternal endometrium was inhibited when challenged with this synthetic glucocorticoid, whereas in the fetal allantochorion PG secretion was stimulated in allantochorion tissues collected on day 46. In allantochorion obtained later in gestation (day 138) DEX caused slight, but non-significant, inhibition. An increase in fetal cortisol production is a key element in the signalling pathway which leads to parturition in sheep via induction of placental enzymes that redirect steroidogenic pathways to favour the production of oestrogen at the expense of progesterone (Anderson et al. 1975, Steele et al. 1976). This change in progesterone/oestrogen ratio activates COX-2, ultimately leading to an increase in PG production (McClaren et al. 1996, 2000). However, Whittle et al. (2000) showed that infusion of cortisol into the fetus beginning on day 125 of pregnancy increased both expression of COX-2 mRNA in placental tropho-blast tissue and levels of fetal plasma PGE2, even in the prescence of the aromatase inhibitor 4-OHA. This suggested that a preceding increased output of placental oestrogen was not essential in the DEX-induced upregulation of placental PG production. On the other hand, DEX suppresses PG production from many tissues via its anti-inflammatory action. Glucocorticoids can inhibit PLA2 activity by a variety of mechanisms, thus reducing precursor AA availability for the biosynthesis of PGs (Bailey 1991). Maternal endometrial COX-2 mRNA and protein concentrations only rose in the prescence of increases in both fetal plasma cortisol and placental oestradiol production (Whittle et al. 2000). Whittle et al. (2000, 2001) therefore proposed that there are two separate pathways of intrauterine PG production. The first is a cortisol-dependent/oestradiol-independent pathway within fetal tissue and the other an oestradiol-dependent pathway within maternal intrauterine tissues. Our data thus link well to this hypothesis, although they also suggest changes in responsiveness of the fetal allantochorion with stage of gestation.
Maternal explants challenged with OT significantly increased the production of PGF2α. This result was as expected based on many previous studies showing that OT can increase PG output via activation of cytosolic PLA2 and COX-2 (Flint et al. 1986, Lee & Silvia 1994, Asselin et al. 1997). CaI acts by increasing intracellular calcium concentrations, another step in the OT signalling pathway (LaFrance & Goff 1990, Danet-Desnoyers et al. 1995). In maternal tissues PGF2α was consistently higher at both 2 and 24 h following CaI treatment. In the fetal allantochorion the time course of the PGE2 response to CaI differed between early pregnancy (decrease at 24 h only) and late pregnancy (increase at 24 h only). There is no obvious reason for this discrepancy.
Fetal explants were challenged with LPS instead of OT as the allantochorion has no OT receptors (Wathes et al. 1996). LPS is a bacterial endotoxin that causes inflammation and fever and which increases PG production by stimulation of PLA2 release (Flynn & Hoff 1995). LPS also utilizes the Toll-like receptor 4 (Tlr4) to induce the transcription and expression of COX-2 (Poltorak et al. 1998, Rhee & Hwang 2000). Mitchell et al. (1991) reported a differential dose–response of human amnion and decidual cells to low-dose bacterial products, which stimulated PG production, and high doses, which were inhibitory. This upregulation of PG synthesis is widely attributed to being part of the mechanism by which uterine infection can cause preterm labour (Romero et al. 1989, Goldenberg et al. 2000). In our results LPS clearly stimulated PGE2 release in the allantochorion collected in late gestation but had no significant effect on PGE2 release in tissue collected in early pregnancy. This interesting result might perhaps be explained by alterations in Tlr4 signalling at different stages of gestation. In our experiments this effect of LPS was not yet apparent after 2 h, so the time course of response was slower than that for OT.
The clear in vitro effect of dietary LA supplementation on enhanced production of two-series PGs was not, however, apparent by measurement of PGFM following the OT challenge in vivo. Development of uterine OT receptors is inhibited by interferon τ production in early pregnancy (Roberts et al. 1992, Wathes and Lamming 1995), but OT receptors subsequently appear from day 22 until term (Wathes et al. 1996). There is a peak in OT receptor expression in the placentome capsule at day 45 of gestation (Leung et al. 1998), coincident with the onset of regular uterine contractility (Garcia-Villar et al. 1984). Pregnant ewes continue to release PGF2α in response to bolus OT administration during early pregnancy (Burgess et al. 1990), with a marked increase in response between days 25 and 65 and a further increase on day 145, near term (Meier et al. 1995). Our results showed that the PGFM production in response to a bolus injection of OT at either day 45 or 133 of gestation was not affected by feeding a diet high in LA. Previous work in the non-pregnant cow has produced conflicting results. Oldick et al. (1997) found that a fat source high in LA caused attenuation in OT-induced PGFM production in the peripheral plasma compared with control animals on day 15. Robinson et al.(2000) fed cows a similar SoyPreme diet to that used here and challenged with OT on days 15, 16 and 17 of the oestrous cycle. In this experiment the diet was found to have no effect on days 15 and 16, but induced a significant increase in PGFM release in SoyPreme fed cows on day 17.
With three studies on PGFM release in response to OT in LA-supplemented ruminants all having different outcomes, it is hard to interpret the results. The experimental diets used did have some differences in their PUFA contents, which may have been reflected in uterine phospholipid stores. Meier et al. (1997) also reported cyclical variation in ovine endometrial phospholipid concentrations, which increased as the cycle progressed from days 3 to 15 and then declined in early pregnancy. Further changes in the processing of phospholipids is likely to occur later in gestation. In a companion experiment conducted alongside that reported here, ewes were fed a similar LA-supplemented diet and monitored following DEX-induced labour, initiated on day 139 of pregnancy. In these ewes circulating maternal PGFM and fetal PGE2 concentrations were higher on the high-LA diet following but not preceding the DEX infusion (Elmes et al. 2003). Therefore the dietary effect of LA may only become apparent when PG production is upregulated over a sufficiently long period of time for precursor availability to become rate limiting.
In conclusion, these experiments provide evidence that a diet supplemented with the n-6 PUFA LA can increase PG production in both maternal and fetal tissues of the ovine placenta. These findings support ideas that a high n-6 diet may influence fetal and placental development, and may also play a role in determining the timing of birth.
Sheep diets. The amounts fed are based on wet weight for ewes in experiment 1 at day 45 of gestation (mean ewe weight, 47 kg) and in experiment 2 at day 135 of gestation (mean ewe weight, 60 kg). The actual amounts fed were adjusted according to ewe weight, which increased during pregnancy
| (a) | ||||
|---|---|---|---|---|
| Diet (g/sheep per day) | ||||
| Control diet | LA diet | |||
| Day 45 | Day 135 | Day 45 | Day 135 | |
| Ingredient | ||||
| Grass silage | 893 | 1390 | 893 | 1465 |
| Maize silage | 833 | 1390 | 833 | 1465 |
| Wheat | 95 | 160 | 85 | 96 |
| Sugar beet pulp | 50 | 106 | 60 | 48 |
| Rapeseed meal | 75 | 106 | 60 | 48 |
| Soypass | 65 | 160 | – | 0 |
| Megalac | 32.5 | 20 | 15 | 4 |
| SoyPreme | – | – | 140 | 217 |
| Maize gluten meal | – | – | – | 96 |
| Total | 2043 | 3332 | 2086 | 3439 |
| (b) | ||||
|---|---|---|---|---|
| Feed analysis (mixed ration) | ||||
| Dry matter | As fed | Dry matter | As fed | |
| DM, dry matter; NCGD, neutral cellulase gammanse digestibility; ME, metabolizable energy. | ||||
| Day 45 | ||||
| DM (%) | 43.4 | 46.7 | ||
| Total crude protein (%) | 14.5 | 6.3 | 14.1 | 6.6 |
| NCGD (%) | 62.0 | 65.9 | ||
| Oil (acid-hydrolysed) (%) | 4.6 | 2.0 | 5.8 | 2.7 |
| Total ash (%) | 7.1 | 3.1 | 6.5 | 3.0 |
| ME (MJ/kg DM) | 10.3 | 10.7 | ||
| Day 135 | ||||
| DM (%) | 29.7 | 28.9 | ||
| Total crude protein (%) | 13.3 | 4.0 | 13.6 | 3.9 |
| NCGD (%) | 62.6 | 62.9 | ||
| Oil (acid-hydrolysed) (%) | 3.8 | 1.9 | 4.5 | 2.6 |
| Total ash (%) | 7.6 | 2.2 | 7.6 | 2.2 |
| ME (MJ/kg DM) | 9.7 | 9.9 | ||
PUFA analysis of feed. Results are given as percentages of total fatty acids in the lipid component; all other fatty acids were not detectable (<1%)
| (a) | |||
|---|---|---|---|
| Proportion of total fatty acids (%) | |||
| Contol diet | LA diet | Neat SoyPreme | |
| Fatty acid | |||
| C16:0 | 13 | 12 | 11 |
| C18:0 | 3 | 3 | 4 |
| C18:1(n-7)cis | 2 | 2 | 1 |
| C18:1(n-9)cis | 13 | 16 | 21 |
| C18:2(n-6)cis | 29 | 41 | 53 |
| C18:3(n-3)cis | 22 | 16 | 8 |
Effects of the control or LA-supplemented diet on basal and peak concentrations of PGFM (mean ±s.e.m.) before and after an OT challenge. Basal values were obtained from five samples collected at 15 min intervals before the OT challenge. Peak concentration was the highest value reached in the 2 h sampling period after the OT challenge
| PGFM concentration (ng/ml) | ||||
|---|---|---|---|---|
| Day 45 | Day 133 | |||
| Control (n=5) | LA (n=6) | Control (n=14) | LA (n=14) | |
| No differences between diets were significant, but basal values were higher at day 133 than at day 45 (a>b, P<0.001). | ||||
| Basal | 0.06 ± 0.004b | 0.08 ± 0.007b | 0.18 ± 0.01a | 0.19 ± 0.01a |
| Peak | 0.77 ± 0.14 | 0.85 ± 0.14 | 0.75 ± 0.07 | 0.74 ± 0.06 |
Effects of a control or LA-supplemented diet on basal PGF2αand PGF2 release (mean ±s.e.m., measured in ng/ml per h) from cultured explants of maternal intercaruncular (IC) endometrium or fetal allantochorion (AC). Medium was analysed after an 18 h incubation period. There were 12 or 16 replicate dishes for each sheep
| PG release (ng/ml per h) | |||||
|---|---|---|---|---|---|
| IC | AC | ||||
| Diet | No. of ewes | PGF2α | PGE2 | PGE2 | |
| Differences between the Control and LA diets at each time point are indicated. *P<0.05 and **P<0.01. | |||||
| Gestation stage | |||||
| Day 46 | Control | 5 | 111 ± 4.2 | 92 ± 6.2 | 1.9 ± 0.11 |
| LA | 6 | 131 ± 5.8** | 121 ± 12.3* | 2.5 ± 0.16** | |
| Day 138 | Control | 8 | 71 ± 4.1 | 61 ± 3.6 | 69 ± 3.7 |
| LA | 8 | 144 ± 6.8** | 87 ± 5.8** | 89 ± 5.3** | |
Effect of a bolus treatment with 10 IU OT (given at time 0) on maternal plasma PGFM on (a) day 45 of gestation and (b) day 133 of gestation in ewes fed a control or LA-supplemented diet. Values are given as means ±s.e.m. for control (○) (n = 5) and LA-supplemented (▪) (n = 6) ewes at day 45 and for control (○) (n = 14) and LA-supplemented (▪) (n = 14) ewes at day 133.
Citation: Journal of Endocrinology 184, 1; 10.1677/joe.1.05910
PGF2α production by intercaruncular endometrium from ewes fed a control diet (open bars) or LA-supplemented diet (closed bars) following in vitro challenges with control medium (CM), 250 nM OT, 5 μM DEX or 10 μM CaI. The experiment was repeated at day 46 of gestation (5 control and 6 LA-supplemented ewes) and day 138 of gestation (8 control and 8 LA-supplemented ewes). The medium was collected at 2 and 24 h. Each treatment was replicated three or four times. Values are means ±s.e.m. Asterisks indicate significant differences between tissues from ewes on the control and LA-supplemented diets given the same treatment in vitro, *P<0.05, **P<0.01. Bars with letters indicate significant differences due to the in vitro treatment, a<b<c, P<0.01.
Citation: Journal of Endocrinology 184, 1; 10.1677/joe.1.05910
PGE2 production by intercaruncular endometrium obtained at day 138 of gestation from ewes fed a control (n = 8) or LA-supplemented (n = 8) diet following in vitro challenges with control medium (CM), 250 nM OT, 5 μM DEX or 10 μM CaI. The medium was collected at 2 and 24 h. Each treatment was replicated three or four times. Values are means ±s.e.m. Asterisks indicate significant differences between tissues from ewes on the control and LA-supplemented diets given the same treatment in vitro, *P<0.05, **P<0.01. Bars with letters indicate significant differences due to the in vitro treatment: in (a) a<b, P<0.05; in (b) a<b<c, P<0.01.
Citation: Journal of Endocrinology 184, 1; 10.1677/joe.1.05910
PGE2 production by fetal allantochorion from ewes fed a control or LA-supplemented diet following in vitro challenges with control medium (CM), 0.1 μg/ml LPS, 5 μM DEX or 10 μM CaI. The experiment was repeated at day 46 of gestation (5 control and 6 LA-supplemented ewes) and day 138 of gestation (8 control and 8 LA-supplemented ewes). The medium was collected at 2 and 24 h. Each treatment was replicated three or four times. Values are means ±s.e.m. Asterisks indicate significant differences between tissues from ewes on the control and LA-supplemented diets given the same treatment in vitro, *P<0.05, **P<0.01. Bars with letters indicate significant differences due to the in vitro treatment, a<b<c, P<0.01.
Citation: Journal of Endocrinology 184, 1; 10.1677/joe.1.05910
Western immunoblot data show COX expression in intercaruncular endometrium from ewes on day 138 of gestation. The data are representative of results from three (of five) animals for control and three (of seven) animals for LA-supplemented ewes. Densitometric analysis of each COX-1 or COX-2 band relative to the corresponding β-actin band has been pooled and is expressed as the mean ±s.e.m. in arbitrary optical density units. There was no difference in COX expression due to diet.
Citation: Journal of Endocrinology 184, 1; 10.1677/joe.1.05910
The project was funded by the BBSRC. We are grateful to David Manners and his staff for their excellent care of the experimental animals, to Dr Caroline Rymer for assisatance with the dietary formulations and to Dr NL Poyser and Dr H Kindahl for generous gifts of antiserum. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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