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Departments of Animal Science3 and of
Veterinary Integrative Biosciences,4 Texas A&M University, College Station, Texas 77843-2471
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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conceptus, placenta, pregnancy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Polyamines (putrescine, spermidine, and spermine) play a crucial role in regulating gene expression, signal transduction, ion-channel function, DNA and protein synthesis, as well as cell proliferation and differentiation [9]. Polyamines also are scavengers of reactive oxygen species, thereby protecting DNA, proteins, and lipids from oxidative damage [10]. Available evidence shows that polyamines are key regulators of angiogenesis, early mammalian embryogenesis, placental trophoblast growth, and embryonic development [7]. Thus, knowledge about placental polyamine synthesis will aid in developing new means to enhance placental and fetal growth.
Ornithine decarboxylase (ODC) catalyzes the decarboxylation of ornithine to yield putrescine, which subsequently is converted to spermidine and spermine (Fig. 1). Arginine and proline are potentially major substrates for ornithine production in mammalian cells [11], whereas S-adenosylmethionine (a metabolite of methionine) provides the methyl group for spermidine and spermine synthesis [12]. Despite recent studies of polyamine synthesis in the ovine placenta [13] and ODC activity in the porcine placenta at Days 40 and 60 of gestation [14], little is known about changes in placental polyamine synthesis associated with porcine conceptus development. Of note, we recently reported substantial increases (up to 50-fold) in concentrations of the arginine-family amino acids (arginine, ornithine, glutamine, and glutamate) in porcine allantoic fluid (a reservoir for nutrients) between Days 20 and 40 of gestation [15]. Such changes coincide with the period of most rapid growth of the porcine placenta [3]. Based on these findings, we hypothesized that arginine and proline are the major amino acids for polyamine production in pig placentae and that placental polyamine synthesis is maximum during early gestation. This hypothesis was tested using gilts between Days 20 and 110 of gestation (term = 114 days). Because amniotic and allantoic compartments are integral parts of the porcine conceptus essential for fetal growth [16], we also determined concentrations of ornithine and polyamines in amniotic and allantoic fluids.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The L-[U-14C]proline, L-[U-14C]ornithine, and L-[U-14C]arginine were obtained from American Radiolabeled Chemicals (St. Louis, MO). High-performance liquid chromatography (HPLC)-grade water and methanol were purchased from Fisher Scientific (Fair Lawn, NJ). Soluene 350 and UltimaGold were purchased from PerkinElmer (Boston, MA), and NCS-II was obtained from Amersham Biosciences (Piscataway, NJ). All other chemicals, including putrescine, spermidine, spermine, amino acids, and dithiothreitol (DTT), were purchased from Sigma (St. Louis, MO).
Experimental Animals and Collection of Placentae and Fetal Fluids
Sexually mature crossbred gilts (Yorkshire x Landrace dams and Duroc x Hampshire sires) of approximately 8 mo of age and weighing 95– 100 kg were observed daily (0700 h) for estrous behavior through direct exposure to intact boars. Gilts exhibiting at least two estrous cycles of normal duration (18–21 days) were bred to crossbred boars (Yorkshire x Landrace dams and Duroc x Hampshire sires). A total of eight boars, ranging in age between 8 mo and 1.5 yr and in weight between 135 and 205 kg, were randomly distributed across all treatments for the present study. Throughout gestation, gilts had free access to drinking water and were individually fed once daily with 2.3 kg of a sorghum- and soybean meal-based diet (consisting of 72.35% milo, 10.0% wheat middlings, 7.55% soybean meal [47.5% grade], 5.0% soy hulls, 3.5% meat and bone, 0.58% limestone, 0.50% salt, 0.34% monocalcium phosphate, 0.08% trace mineral premix, 0.05% choline chloride, and 0.05% vitamin premix) that met the recommended National Research Council nutrient requirements [17]. The major protein source of the diet is soybean meal (47.5% grade). The diet provided the following nutrients: 89.8% dry matter, 14.2% crude protein, 13 372 kJ/kg of metabolizable energy, 0.56% lysine, 0.83% arginine, 1.26% proline, 0.71% calcium, and 0.61% phosphorus. The weight gain of pregnant gilts averaged 0.36 kg/day. Pregnant gilts were hysterectomized on either Day 20, 30, 35, 40, 45, 50, 60, 90, or 110 of gestation (n = 6 per day) [18]. Briefly, gilts received an i.m. injection of Telazol (1 mg/kg) to induce anesthesia, followed by administration of isofluorane (1– 5%) via inhalation during surgery. The uterus was removed by midventral laparotomy. Placentae (the chorioallantois) were exposed and isolated by dissection as recently described [18]. We collected the entire chorioallantois from each fetoplacental unit and took great care not to include the amnion or necrotic tips. The placenta of the first fetus located near the uterotubal junction of the left uterus horn was weighed and used for metabolic and enzymatic assays. A portion of placental tissue was used immediately for proline transport, proline degradation, and polyamine synthesis; the remaining placental tissue was stored at –80°C for analyses of metabolites and enzyme assays within 1 wk. Allantoic and amniotic fluids were obtained directly by aspiration into a syringe via an 18-gauge needle [15], and total volume of each fluid was measured in a graduated cylinder. No measurable amniotic fluid was present at Day 20 of gestation. The present study was approved by the Texas A&M University Institutional Agricultural Animal Care and Use Committee.
Determination of Amino Acids, Pyrroline-5-Carboxylate, and Polyamines in Pig Placentae and Fetal Fluids
Placentae (
200 mg) were homogenized at 4°C in 2 ml of 1.5 mM HClO4 using a glass homogenizer. The solution was transferred to a 12- x 75-mm polypropylene tube and neutralized with 1 ml of 2 M K2CO3. The homogenates were centrifuged at 3000 x g and 4°C for 15 min to obtain the supernatant fluid, which was stored at –80°C. Samples of fetal allantoic and amniotic fluids (10 ml) were centrifuged at 3000 x g at 4°C for 10 min; an aliquot of the supernatant fluid (0.5 ml) was deproteinized with 0.5 ml of 1.5 M HClO4, followed by neutralization with 0.25 ml of 2 M K2CO3. Polyamines and amino acids (including S-adenosylmethionine) were analyzed using HPLC methods involving precolumn derivatization with o-phthaldialdehyde as described previously [18, 19]. The retention times of S-adenosylmethionine, methionine, and ornithine were 21.4, 35.5, and 44.1 min, respectively. Pyrroline-5-carboxylate (P5C) was determined through its reduction to proline [20], followed by HPLC analysis of proline [19]. Briefly, 0.5 ml of neutralized samples were loaded into an AG 1-X8 resin (Bio-Rad Laboratories, Hercules, CA) column (0.6 x 6.5 cm), followed sequentially by elution with 8 ml of water and 6 ml of 25 mM acetic acid. The column was finally eluted with 4 ml of 25 mM acetic acid, and the effluent solution (containing 99% P5C but no proline) was mixed with 1 ml of 50 mM sodium borohydride. The resultant solution (containing proline) was dried in a Model RC10.10 centrifugal evaporator (Jouan, Inc., Winchester, MA), and the residue was suspended in 0.3 ml of HPLC-water for HPLC analysis of proline [19].
Proline Transport in Placentae
Proline transport in pig placentae was determined using L-[U-14C]proline as described previously for branched-chain amino acids [18]. Briefly, samples of placentae (200 mg) were washed three times in oxygenated (95% O2/5% CO2, v/v) Krebs-Henseleit bicarbonate (KHB) buffer containing 20 mM Hepes (pH 7.4) and 5 mM glucose. Samples were then incubated at 37°C for 5 min in 1 ml of oxygenated KHB buffer consisting of 20 mM Hepes, 2 mM glutamate, 5 mM glucose, 0.5 or 2 mM proline, 0.05 µCi L-[U-14C]proline, and 0.05 µCi [3H]inulin (an extracellular marker). After the 5-min incubation, the tissues were rinsed thoroughly with fresh KHB buffer and then solubilized in 0.5 ml of Soluene 350. The solution was measured for 14C and 3H radioactivities using a dual-channel counting program in a Packard 1900 liquid scintillation counter (Meriden, CT). The specific activity of [14C]proline in the medium was used to calculate proline uptake by placentae. Results from preliminary experiments established that proline uptake was linear over a 5-min period.
Proline and Arginine Degradation in Placentae
Proline degradation in placentae was quantified using L-[U-14C]proline as described previously for pig enterocytes [21]. Briefly, samples of placentae (200 mg) were preincubated at 37°C for 30 min in 2 ml of oxygenated (95% O2/5% CO2, v/v) KHB buffer and then incubated at 37°C for 2 h in 2 ml of oxygenated KHB containing 20 mM Hepes (pH 7.4), 5 mM glucose, 2 mM glutamate, 0.5 or 2 mM proline, and 0.5 µCi L-[U-14C]proline. After a 2-h incubation at 37°C, 0.2 ml of Soluene 350 was injected through the rubber cap into suspended center-wells, and 0.2 ml of 1.5 M HClO4 acid was injected into the incubation medium to liberate 14CO2. After a 1-h incubation period, suspended wells were transferred to scintillation vials containing 15 ml of cocktail for measurement of 14CO2 using a Packard liquid scintillation counter [21]. The neutralized medium was analyzed for [14C]ornithine and other amino acids using HPLC and liquid scintillation spectrometry [21]. Arginine degradation in placentae was determined as described above for proline degradation except that 0.5 and 2 mM arginine plus 0.5 µCi L-[U-14C]arginine were included instead of proline in the incubation medium.
Determination of Polyamine Synthesis in Placentae
Polyamine synthesis was determined in porcine placentae using L-[U-14C]proline, L-[U-14C]ornithine, and L-[U-14C]arginine as described previously for ovine placentae [13]. Briefly, placental tissues (500 mg) were rinsed twice with oxygenated (95% O2/5% CO2, v/v) Basal Eagle medium (BEM; Gibco RL, Grand island, NY) and then incubated at 37°C for 3 h in 2 ml of oxygenated (95% O2/5% CO2) BEM containing 5 mM glucose, 0.5 mM L-methionine, and one of the following: 0.5 or 2 mM L-proline plus 2 µCi L-[U-14C]proline and 2 mM glutamate, 0.5 or 2 mM L-ornithine plus 2 µCi L-[U-14C]ornithine, or 0.5 or 2 mM L-arginine plus 2 µCi L-[U-14C]arginine. Blank incubations were run using medium containing all the above components but no tissues. The 14C-labeled substrates were used to improve the sensitivity of detecting polyamine synthesis in placental tissue. Incubations were terminated by addition of 0.2 ml of 1.5 M HClO4. The acidified tissues plus medium were analyzed for [14C]putrescine, [14C]spermidine, and [14C]spermine by HPLC and liquid scintillation spectrometry [19]. Blank radioactivities were subtracted from sample values. Rates of production of putrescine, spermidine, and spermine were calculated on the basis of intracellular specific activities of [14C]ornithine, which were measured as described by Wu [21].
Measurement of Proline Oxidase Activity in Placentae and Fetal Fluids
Placentae (200 mg) were homogenized in 2 ml of homogenization buffer (pH 7.2) containing 250 mM sucrose, 1 mM EDTA, 2.5 mM DTT, protease inhibitors (5 µg/ml of phenylmethylsulfonyl fluoride, 5 µg/ml of aprotinin, 5 µg/ml of chymostatin, 5 µg/ml of pepstatin A), and 50 mM potassium phosphate buffer with the use of a glass pestle. The homogenate was centrifuged at 600 x g and 4°C for 10 min, and the supernatant fluid was centrifuged at 12 000 x g and 4°C for 10 min. The resultant mitochondrial pellets were suspended in 1.5 ml of 50 mM potassium phosphate buffer (pH 7.5), stored at –80°C, and used for enzyme assay within 3 days. Fetal allantoic and amniotic fluids were centrifuged at 3000 x g for 15 min, and the supernatant was used directly for enzyme assays. Proline oxidase activity was determined as described previously [19]. Briefly, the enzyme assay mixture (1.0 ml), which consisted of 15 mM proline, 20 µM ferricytochrome C, mitochondrial pellet (0.5 and 1 mg of protein), and 50 mM potassium phosphate buffer (pH 7.5), was incubated at 37°C for 0, 15, or 30 min. The reaction was terminated by addition of 0.5 ml of 10% TCA, followed by addition of 0.1 ml of 100 mM o-aminobenzaldehyde. The mixture was allowed to stand at room temperature for 30 min before centrifugation at 600 x g for 5 min. Absorbance of the supernatant was measured at 440 nm. Blanks (0-min incubation) were subtracted from sample values before calculating the formation of P5C from proline based on the molar extinction coefficient of P5C (2.7 x 103 M–1 cm–1).
Measurement of Arginase, Ornithine Aminotransferase, and P5C Dehydrogenase Activities in Placentae and Fetal Fluids
Placentae (500 mg) were homogenized at 4°C in 2 ml of homogenization buffer (pH 7.4) containing 300 mM D-mannitol, 5 mM Hepes, 0.2 mM EDTA, 3 mM DTT, and protease inhibitors (5 µg/ml of phenylmethylsulfonyl fluoride, 5 µg/ml of aprotinin, 5 µg/ml of chymostatin, and 5 µg/ml of pepstatin A) with the use of a glass pestle. The homogenate was centrifuged at 600 x g for 10 min at 4°C, and the supernatant fluid was centrifuged at 12 000 x g for 15 min at 4°C. The resultant supernatant (cytosol) was used for assays of arginase I. The pellet was resuspended in 1.5 ml of fractionation buffer (300 mM D-mannitol, 5 mM Hepes, 5 mM EDTA, and 3 mM DTT; pH 7.4) and centrifuged at 600 x g for 4 min at 4°C. The resulting supernatant was centrifuged at 4000 x g for 10 min at 4°C, and the pellet (the mitochondrial fraction) was suspended in 0.5 ml of the homogenization buffer containing 0.5% Triton X-100 for assays of arginase II, ornithine aminotransferase (OAT), and P5C dehydrogenase. Fetal allantoic and amniotic fluids were centrifuged at 3000 x g for 15 min, and the supernatant was used directly for enzyme assays.
Activities of arginase I and II, OAT, and P5C dehydrogenase were determined as described previously [22–24]. The assay mixture (0.15 ml) for arginase I (a cytosolic enzyme) consisted of 50 mM Tris-HCl buffer (pH 7.5), 3 mM MnCl2, 10 mM arginine, and cytosolic extracts (0.5 and 1 mg of protein). The enzyme and MnCl2 mixture was preheated at 55°C before addition of arginine. After cooling to room temperature, the solution was incubated at 37°C for 0, 10, and 20 min. At the end of the predetermined period of incubations, the reaction was terminated by addition of 50 µl of 1.5 M HClO4, and the neutralized solution was analyzed for ornithine by HPLC. The activity of arginase II (a mitochondrial enzyme) was determined as described for arginase I except that the enzyme and MnCl2 mixture was not preheated at 55°C before addition of arginine, because such a procedure resulted in a substantial reduction of arginase II activity. The assay mixture (2.0 ml) for OAT contained 75 mM potassium phosphate buffer (pH 7.5), 20 mM ornithine, 0.45 mM pyridoxal phosphate, 5 mM o-aminobenzaldedhyde, 0 (blank) or 3.75 mM 
-ketoglutarate, and mitochrondria (0.1 and 0.2 mg of protein). At the end of the 7.5- and 15-min incubation periods at 37°C, the colorimetric complex resulting from the reaction of P5C with o-aminobenzaldehyde was determined at 440 nm. The assay mixture (1.2 ml) for P5C dehydrogenase contained 4 mM NAD+, 2 mM DL-P5C, mitochondria (0.5 and 1 mg), and 100 mM potassium phosphate buffer (pH 7.5). The increase in NADH over a 5-min period after addition of 0 or 2 mM DL-P5C was measured at 37°C by a fluorometer (excitation wavelength, 340 nm; emission wavelength, 460 nm). Assay mixtures without P5C was used as blanks.
Determination of ODC Activity in Placentae
The ODC activity in porcine placentae was measured using L-[1-14C]ornithine as described for ovine placentae [13]. Briefly, tissues (200 mg) were homogenized, using a glass homogenizer, in 0.5 ml of 50 mM sodium phosphate buffer (pH 7.2) containing 0.2 mM pyridoxal-5-phosphate, 1 mM EDTA, 2.5 mM DTT, 150 mM sucrose, and protease inhibitors (5 µg/ml of phenylmethylsulfonyl fluoride, 5 µg/ml of aprotinin, 5 µg/ml of chymostatin, and 5 µg/ml of pepstatin A). The homogenizer was rinsed with 0.5 ml of the buffer, and the combined homogenates were centrifuged at 13 000 x g for 15 min at 4°C. The supernatants (free of mitochondria) were used for ODC assays; the assay mixture (0.5 ml) consisted of 2 mM L-[1-14C]ornithine (2500 dpm/nmol), 0.2 mM pyridoxal-5-phosphate, 0.2 mM EDTA, 0.5 mM DTT, enzyme preparations (equivalent to 10 and 20 mg of tissue), and 50 mM sodium phosphate buffer (pH 7.2). Radioactivity blanks containing [1-14C]ornithine but no enzyme preparations were included. After incubation at 37°C for 1 h, 14CO2 was collected in 0.2 ml of NCS-II, and its radioactivity was measured in a liquid scintillation counter. Rates of ornithine decarboxylation by ODC were calculated by dividing the radioactivity of collected 14CO2 (dpm) by the specific activity of [1-14C]ornithine (dpm/nmol) in the assay solution.
Calculations and Statistical Analyses
Concentrations of polyamines and amino acids in placentae, allantoic fluid, and amniotic fluid were calculated based on the recovery rates (93– 98%) of these substances from the samples as described previously [13]. Concentrations of total polyamines were a mathematical sum of putrescine, spermidine, and spermine. Data were subject to least-squares analysis of variance and correlation analysis [25] using the PROC GLM and PROC CORR procedures of the Statistical Analysis System software (SAS Institute, Cary, NC). Probability values of 0.05 or less were taken to indicate statistical significance. Data are presented as the mean ± pooled SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rate of placental growth (expressed as percentage increase) was highest (P < 0.01) between Days 20 and 60 and declined (P < 0.01) thereafter (Table 1). Placental weight of gilts increased (P < 0.01) 267-, 3.1-, and 1.3-fold between Days 20 and 40, Days 40 and 60, and Days 60 and 110 of gestation, respectively (Table 1). Placental growth was nearly completed by Day 60 of gestation. Fetal weights increased (P < 0.01) with gestational age (Table 1). The weight gain (130 g) of the fetal pig was small between Days 20 and 60 of gestation but increased rapidly thereafter (1045 g between Days 60 and 110 of gestation). Indeed, the weight gain (589 g) of the fetal pig between Days 90 and 110 of gestation was similar to that (596 g) between Days 20 and 90 of gestation. The volume of allantoic fluid increased (P < 0.01) 55-fold between Days 20 and 30 of gestation, declined (P < 0.01) progressively between Days 30 and 40 of gestation, and then increased (P < 0.01) fivefold between Days 40 and 60 of gestation. The volume of allantoic fluid decreased (P < 0.01) progressively between Days 60 and 110 of gestation. The volume of amniotic fluid increased (P < 0.01) progressively between Days 30 and 60 of gestation, remained at the elevated level between Days 60 and 90 of gestation, and then declined (P < 0.01) thereafter.
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Concentrations of Proline, Ornithine, and P5C in Placentae and Fetal Fluids
Concentrations of proline, ornithine, and P5C in placentae increased (P < 0.05) by 51%, 46%, and 79%, respectively, between Days 20 and 40 of gestation, remained constant between Days 40 and 60, and declined (P < 0.05) thereafter (Table 2). In allantoic fluid, concentrations of proline increased (P < 0.01) 130% between Days 20 and 30 of gestation, declined (P < 0.01) progressively between Days 30 and 60, and then increased (P < 0.01) progressively between Days 60 and 110. A very different pattern of developmental change was observed for ornithine and P5C in allantoic fluid (Table 2). Concentrations of ornithine and P5C in allantoic fluid increased (P < 0.01) 34- and 8-fold, respectively, between Days 20 and 40 of gestation, declined (P < 0.01) progressively between Days 40 and 90, and remained at the reduced level through Day 110. Allantoic fluid concentrations of proline were greater (P < 0.01) than those of ornithine during early (Days 20–30) and late (Days 90–110) gestation, whereas the opposite was determined between Days 35 and 60. In amniotic fluid, concentrations of proline increased (P < 0.01) twofold between Days 30 and 45 of gestation, remained constant between Days 45 and 60, and declined (P < 0.01) thereafter. In contrast, amniotic fluid concentrations of ornithine or P5C increased (P < 0.01) progressively between Days 30 and 40 of gestation and then decreased (P < 0.01) progressively between Days 40 and 90 of gestation.
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Concentrations of Methionine and S-Adenosylmethionine in Placentae
Concentrations of methionine were 10- to 14-fold greater than those of S-adenosylmethionine in porcine placentae throughout pregnancy (Table 3). Concentrations of methionine increased (P < 0.01) by 29% between Days 20 and 40 of gestation, remained at the elevated level between Days 40 and 60, and declined (P < 0.01) thereafter. Concentrations of S-adenosylmethionine doubled (P < 0.01) between Days 20 and 40 of gestation, declined (P < 0.01) progressively between Days 40 and 90, and remained at the reduced level through Day 110.
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Proline Transport and Degradation in Placentae
Increasing extracellular concentrations of proline from 0.5 to 2 mM increased (P < 0.01) the rate of protein transport by porcine placentae on all days of gestation (Table 4). Proline transport increased (P < 0.05) threefold between Days 20 and 40 of gestation, decreased (P < 0.01) progressively between Days 40 and 50, remained constant between Days 50 and 60, and declined (P < 0.01) thereafter. Relatively large amounts of P5C and ornithine were produced from proline in porcine placentae (Table 5). Formation of P5C and ornithine from proline increased (P < 0.05) approximately 2.5-fold between Days 20 and 40 of gestation and decreased (P < 0.01) thereafter. No production of [14C]glutamate, [14C]aspartate, [14C]alanine, or 14CO2 from L-[U-14C]proline was detectable in porcine placentae between Days 20 and 110 of gestation.
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Polyamine Synthesis in Placentae
Synthesis of polyamines from proline or ornithine was detected in porcine placentae between Days 20 and 110 of gestation (Tables 6 and 7). Rates of placental polyamine synthesis from [14C]proline or [14C]ornithine increased (P < 0.01) by 150–160% between Days 20 and 40 of gestation and declined (P < 0.05) thereafter. Spermidine and spermine were the major polyamines synthesized from proline or ornithine at all days of gestation. No production of [14C]ornithine, [14C]glutamate, [14C]putrescine, [14C]spermidine, or [14C]spermine from [U-14C]arginine was detectable in porcine placentae between Days 20 and 110 of gestation. Between Days 20 and 40 of gestation, the synthesis of total polyamines was correlated positively with placental weight (r = 0.916, P < 0.01). Maximum placental polyamine synthesis occurred in advance of major changes in fetal growth.
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Activities of Ornithine Metabolic Enzymes
Arginase I, arginase II, and P5C dehydrogenase activities were not detected in porcine placentae, allantoic fluid, or amniotic fluid between Days 20 and 110 of gestation. However, proline oxidase, OAT, and ODC activities were present in porcine placentae throughout pregnancy (Table 8). The activities of these three enzymes increased (P < 0.01) by 132%, 148% and 240%, respectively, between Days 20 and 40 of gestation and declined (P < 0.01) thereafter. At all days of gestation, OAT activity was higher (P < 0.01) than proline oxidase activity, whereas ODC activity was the lowest (P < 0.01). Both ODC and proline oxidase activities showed positive correlations with rates of polyamine synthesis from proline in porcine placentae (r = 0.938 and 0.925, respectively; P < 0.01). In allantoic fluid, proline oxidase and OAT activities increased (P < 0.01) 4- and 10-fold, respectively, between Days 20 and 40 of gestation and declined (P < 0.01) thereafter (Table 9). In amniotic fluid, the activities of both enzymes increased (P < 0.01) approximately threefold between Days 20 and 40 of gestation and declined (P < 0.01) thereafter (Table 9). Except at Days 40 and 45 of gestation, allantoic fluid activity of proline oxidase was similar to that of OAT at all other days of gestation (P > 0.05). In contrast, amniotic fluid activity of proline oxidase was higher (P < 0.01) than that of OAT at all days of gestation. No ODC activity was detected in porcine allantoic or amniotic fluid between Days 20 and 110 of gestation.
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Polyamine Concentrations in Placentae and Fetal Fluids
Porcine placentae contained high levels of putrescine, spermidine, and spermine at all days of gestation (Table 10). Placental concentrations of spermidine were similar to (P > 0.05) and lower than (P < 0.01) those of spermine between Days 20 and 50 and between Days 60 and 110 of gestation, respectively. Placental concentrations of total polyamines increased (P < 0.01) 160% between Days 20 and 40 of gestation and declined (P < 0.01) between Days 40 and 90. Positive correlations were found between concentrations of total polyamines and polyamine synthesis in porcine placentae (r = 0.946, P < 0.01). Concentrations of polyamines in allantoic fluid were higher than (P < 0.01) and similar to (P > 0.05) those in amniotic fluid at Days 30–60 and Days 90–110 of gestation, respectively (Table 10). In both fluids, concentrations of polyamines increased (P < 0.01) progressively between Days 30 and 40 of gestation, declined (P < 0.01) progressively between Days 40 and 60, and then remained at the reduced level through Day 110. During late gestation (Days 90–110), spermine was the most abundant polyamine in fetal fluids, followed in decreasing order by spermidine and putrescine. Concentrations of total polyamines were positively correlated with placental weights between Days 20 and 40 of gestation (r = 0.963, P < 0.01).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Despite a previous report that dealt with polyamine synthesis from arginine in ovine placentae [13], little is known about changes in placental polyamine synthesis associated with conceptus development in pigs. Arginine is considered to be a major substrate for ornithine production via arginase in animal tissues [11, 26]. However, the present results clearly indicate that neither arginase activity nor conversion of arginine into ornithine was detected in porcine placentae, allantoic fluid, or amniotic fluid. These results are in sharp contrast to those that we recently reported for ovine placentae and fetal fluids, in which a relatively high arginase activity plays an important role in the production of ornithine and, thus, polyamines from arginine [13]. The lack of arginine degradation via arginase in the porcine placenta and fetal fluids maximizes placental transfer of arginine from maternal and fetal blood. This finding aids in explaining the unusual abundance (4–5 mM) of arginine in porcine allantoic fluid during early gestation [15].
Proline oxidase is the only known enzyme for initiating proline catabolism in animal cells [27]. Results of the present study demonstrate that proline was extensively catabolized via proline oxidase and OAT to yield P5C and, subsequently, ornithine in the porcine placenta. In this tissue, concentrations of glutamate were three- to fivefold greater than those of ornithine [18], and concentrations of P5C were two- to threefold greater than those of -ketoglutarate (3.5–6 nmol/g tissue; unpublished data). Thus, the equilibrium of the placental OAT reaction favors the formation of ornithine. The proline-derived ornithine was readily used for the synthesis of putrescine, spermidine, and spermine via ODC, spermidine synthase, and spermine synthase, respectively (Table 6). These results suggest that proline oxidase is coupled efficiently with OAT in placental mitochondria and that the proline-derived ornithine readily enters the cytoplasm to serve as the substrate for ODC. A similar finding has been reported for the porcine small intestine [19]. Thus, either extracellular or mitochondrially generated ornithine is used for the synthesis of polyamines in the porcine tissues. Interestingly, P5C dehydrogenase was not detectable in porcine placentae between Days 20 and 110 of gestation in the present study. The lack of conversion of P5C to glutamate limits the irreversible loss of P5C while maximizing the formation of ornithine from P5C via OAT. This mechanism helps to conserve proline for polyamine production, because P5C can be recycled into proline by the cytosolic P5C reductase, a widespread enzyme in animals [27], including fetal pigs [28]. Notably, the rate of placental polyamine synthesis from proline increased with increasing extracellular concentrations of proline from 0.5 to 2 mM (Table 6) for the provision of P5C at all days of gestation, indicating a high capacity for this biosynthetic pathway. Additionally, our results suggest that proline oxidase, which catalyzes the first and irreversible reaction in proline catabolism, is a rate-controlling enzyme in placental synthesis of polyamines.
Another novel finding of the present study is that porcine allantoic and amniotic fluids contain both proline oxidase and OAT (Table 8). These two proteins may be secreted from allantoic and amniotic membranes of the placenta as well as the uterus. In the fetal fluids, concentrations of glutamate were approximately 50% lower than those of ornithine [15], but concentrations of P5C were 3- to 10-fold greater than those of -ketoglutarate (2.3–3.5 nmol/ml; unpublished data). Thus, as in the placenta, the equilibrium of the OAT reaction favors the generation of ornithine from proline. Importantly, the production of P5C and ornithine in the fetal fluids represents a hitherto-unrecognized pathway for extracellular metabolism of proline in mammals. The high activity of proline oxidase would actively catabolize proline to generate P5C in porcine allantoic and amniotic fluids, thereby explaining the relatively low concentrations of proline [15, 29]. Indeed, we have reported previously that proline is the second least abundant nonessential amino acid in porcine allantoic and amniotic fluids throughout pregnancy [29]. Interestingly, concentrations of P5C in porcine fetal fluids (Table 2) are approximately 10- to 20-fold higher than those in the plasma of fetal pigs (1.0–1.4 µM), pregnant gilts (0.6 µM; unpublished data), and adult humans (0.4 µM) [30], suggesting an important role for P5C in conceptus development. In this regard, it is noteworthy that P5C regulates cell redox state, proliferation, and differentiation in cultured cell lines [30, 31]. Whether P5C is a signaling molecule that regulates placental metabolism and function remains to be determined. Nonetheless, our discovery of proline oxidase in the conceptus opens a new avenue for future studies regarding the role of proline in placental and fetal development.
Although early anatomical studies suggested that the allantoic sac served as a reservoir for fetal wastes, it is now clear that allantoic fluid nutrients can be absorbed by the allantoic epithelium into the fetoplacental circulation and used by fetal tissues [16]. In addition, amniotic fluid is actively swallowed by the fetus and is a significant source of nutrients for the gut and other fetal tissues [32]. Allantoic and amniotic fluids derive, in part, from secretions and transport of water and solutes across the placenta and endometrium [16, 32]. Consistent with this notion, the pattern of changes in concentrations of total polyamines in allantoic and amniotic fluids between Days 20 and 110 of gestation (Table 10) closely matched the rates of placental polyamine synthesis from proline (Table 6) and ornithine (Table 7). With an increase in the transport capacity of fetal enterocytes [33], amniotic fluid taken up by the fetus provides a significant source of polyamines for supporting the proliferation and differentiation of intestinal epithelial cells. The nutritional significance of amniotic fluid is illustrated by the finding that esophageal ligation, which prevents the entry of this fluid into the small intestine, results in intrauterine growth retardation in fetal pigs [34].
Transport of proline across the plasma membrane represents the first step for its utilization by the placenta. Our results show that the rates of placental transport of proline (Table 4) were the highest at Day 40 of gestation. The active transport of proline ensures an adequate supply of intracellular proline for its metabolism by placentae. Several results are notable: First, the activities of proline oxidase, OAT, and ODC were maximal at Day 40 of gestation, which would maximize the production of ornithine and, subsequently, of all polyamines from proline in porcine placentae. Second, concentrations of ornithine in allantoic fluid were highest at Day 40 of gestation (Table 2), and the dynamic exchange of nutrients between this fluid and the placenta provides an additional source of ornithine for enhancing polyamine synthesis in the placenta. Third, placental production of glutamate (a major substrate for OAT) and glutamine (a stimulator of ODC activity) from branched-chain amino acid catabolism [18] as well as their concentrations in porcine placentae [18] and allantoic fluid [15, 29] were highest at Day 40 of gestation. Fourth, placental concentrations of methionine and its metabolite, S-adenosylmethionine, which provides a methyl group for the synthesis of spermidine and spermine from putrescine (Fig. 1), were highest during early pregnancy (Table 3). Collectively, our results indicate metabolic coordination among several integrated pathways that support maximal polyamine synthesis in the porcine placenta at Day 40 of gestation (Tables 6 and 7), when placental growth and morphological changes are most rapid [3]. Similarly, polyamine synthesis is highest in the ovine placenta during early gestation (Day 60) [13]. This common phenomenon from two divergent mammalian species supports the view that polyamines play a crucial role in promoting conceptus development [7]. Thus, modulation of the polyamine-synthetic pathways may provide a novel and useful method to regulate fetal growth and survival.
Based on the water content (82%) of porcine placentae between Days 20 and 110 of gestation [35], concentrations of total polyamines in this tissue were estimated to be 220– 550 µM. These values are substantially higher than concentrations of polyamines in maternal or fetal plasma (4–6 µM; unpublished data), indicating an abundance of polyamines in the porcine placenta, as we have reported previously for the ovine placenta [13]. Thus, polyamines are substantially concentrated in mammalian placentae, which is consistent with their crucial role in placental growth [7]. Indeed, in porcine placentae, polyamines are more abundant than most essential amino acids [14], and polyamine concentrations were highly correlated with placental growth in the first half of pregnancy (Tables 1 and 10).
The present findings raise important questions regarding the physiologic significance of polyamine synthesis in fetoplacental nutrition and development. In this regard, it is noteworthy that maternal dietary protein deficiency (0.5% protein) decreased the activities of proline oxidase and OAT in porcine placentae, allantoic fluid, and amniotic fluid by 70–80% at Day 40 of gestation (unpublished data); concentrations of proline and ornithine in porcine fetal plasma, placentae, and allantoic fluid [36]; as well as placental and fetal growth [37]. In addition, marked changes were found in placental tissue growth and vascularity during pregnancy [38]. In both pigs [8] and humans [39], low birth weight is a major factor contributing to high neonatal morbidity and mortality and is associated with the development of chronic disease (e.g., diabetes, hypertension, and coronary heart disease) later in life [40–42]. New knowledge about placental synthesis of polyamines may have important implications for preventing both intrauterine growth retardation and fetal origins of adult-onset diseases.
In conclusion, the present results indicate that polyamine synthesis and concentrations were highest in porcine placenta at Day 40 of gestation. Importantly, metabolic coordination occurs among the several integrated pathways that support high rates of polyamine synthesis in the placenta during early pregnancy. Our discovery of the novel pathway for the synthesis of polyamines from proline via proline oxidase in the porcine conceptus provides a new framework for future studies to define the roles of amino acids in fetoplacental growth and development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Guoyao Wu, Department of Animal Science, 212 Kleberg Center, 2471 TAMU, Texas A&M University, College Station, TX 77843-2471. FAX: 979 845 6057; g-wu{at}tamu.edu
Received: 15 September 2004.
First decision: 19 October 2004.
Accepted: 9 November 2004.
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