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Protein restriction in early pregnancy alters fetal and placental growth and allantoic fluid proteins in swine
Theriogenology
42:2 17-226,1994
PROTEIN RESTRICTION IN EARLY PREGNANCY ALTERS FETAL AND PLACENTAL GROWTH AND ALLANTOIC PLUID PROTEINS IN SWINE P.A. Schoknecht”,
G.R. Newton*, D.E. Weise* and W.G. Pond’
1 USPAIARS Children’s Nutrition Research Center, Houston, TX Prairie View A&M University, Prairie View, TK Received for publication: Accepted:
December 31, 1993 June 10, 1994
ABSTRACT This study was designed to determine the impact of protein malnutrition during early pregnancy on fetal and placental growth and on the protein synthesis capacity of placental and endometrial tissues. Twelve crossbred sows received 1.8 kg/d of a control (13% protein) or protein-restricted (0.5% protein) diet from the day of breeding to Day 63 of pregnancy, when dissections were performed on each conceptus unit. ‘Ihe de novo protein synthetic rate of placental and endometrial explants was measumd using %-methionine. ‘l&se proteins and tbe proteins from amniotic and allantoic fluids were separated by polyacrylamide gel electrophoresis. Placental weight was significantly reduced in the sows fed the restricted diet, with a tendency for decreased fetal wei$tt as well. No difterences were found due to dietary treatment in de novo protein synthesis or in the electrophoretic patterns of secreted proteins of the placenta or endometrium. The. apparent quantity of 3 proteins in the allantoic fluid of the restricted diet fetuses decreased, while 1 protein iucreased in comparison with that of the control fetuses. These data suggest that protein malnutrition in early pregnancy decreases placental growth, thereby decreasing both fetal growth and the opportunity for compensatory growth upon nutritional rehabilitation. Key words: mahmtrition, fetal growth, protein synthesis, placenta INTRODUCTION Malnutrition has a detrimental impact on fetal growth, with the timing and severity of nu’uitional insult determining the degree of fetal growth retardation (8). In the early stages of pregnancy, conceptus growth is characterixed by rapid expansion of the placenta, with maximum sixe beii achieved by mid-pregnancy (12). At the mid-point of gestation, all major fetal organ systems have developed (11); however, the fetus weighs only one-tenth of its eventual birth weight (21). Maternal nutritional deficiencies during early pregnancy would, therefore, be expected to alter primarily placental size. This would likely compromise the ability of the placenta to transport nutrients at a time critical to the fetus and fetal organ development.
Acknowledgements This work is a publication of the USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX ‘Ihe project was funded in part under federal funds from the US Department of Agriculture, Agricultural Research Service under Cooperative Agreement Number 58-625@1~3. The contents of this publication do not necessarily reflect the views or policies of the US Department of Agriculture, nor does the mention of trade names, commercial products or organizations imply endorsement from the US Government.’ We thank Larry Solomon and associates of Praiie View A&M University for animal care and Kathy McKee, Lynn Walton and Gary Cook for technical assistance. $resent address: Department of Animal Sciences, Rutgers University, New Brunswick, NJ C@O3.
Copyright
0 1994 Buttetworth-Heinemann
Theriogenology
218
A reduction in fetal size would be a secondary effect of the maternal malnutrition. Sows receiving a protein-restricted diet from breeding to Day 63 of pregnancy (13) did produce smaller fetuses; however, these fetuses had larger placentae than fetuses of sows which received a normal protein diet. This apparently anomalous tinding raises the possibiity that local paracrine and/or autocriac factors may have contributed to compensatory growth of the placenta to insure that an adequate flow of nutrients was available for fetal development. Therefore., the objectives of this study were to reevaluate the effects of maternal protein restriction during the first 63 days of pregnancy on 1) indices of fetal and placental growth and development, 2) de novo protein synthesis by endometrial and placental tissues, and 3) the proteins contained in fetal fluids. MATERIALS AND METHODS Materials
Tissue culture supplies were obtained from GLBCO Laboratories (Grand Island, NY) and Sigma Chemical Co. (St. Louis, MO). Eagle’s minimal essential medium with Earl’s salts (MHM) for tissue culture was modified in the following manner. Medium was supplemented with penicillin (100 U/ml), amphotericin B (250 ng/ml), streptomycin (100 ~glml), insulin (0.2 U/ml), nonessential amino acids (1%) v/v), glucose (5 mg/ml), chloride (100 @ml), folk acid (100 &ml), pyridoxal-HC! (100 &nl), riboflavin (10 &ml), and thiamine (100 pg/ml). Acrylamide and N,N’-methylene-bis-acryhunide (electrophoresis grade) were obtained from Boekiqer Mannheim Biochemicals (Mdianapolis, IN). Ampholytes were obtained from Serva Bkhetnicals (Heidelberg, Germany). X-Gmat AR film was purchased from Eastman-Xodak (Rochester, NY). I$Qnethionine (specific activity = 1140 Ciimmol) was purchased from Dupont Company (Boston, MA). All other supplies and reagents were obtained from either Fisher Scientific (Houston, TX) or Sigma Chemical Co., ad were of reagentgradeor better. Tissue and Fluid Collection Twelve first or second parity crossbred swine (Chester White X Landrace X Large white X Yorkshire) were bred upon standing eshus (Day 0). Each sow received 1.8 kg/d of either control (13% protein) or protein-restricted (0.5% protein) diet from Day 1 to Day 63 of pregnancy (Table 1). On Day 63, sows were hilled by exsanguination after stunniq with a captive bolt pistol, and the entire pregnant uterus was excised. Each conceptus unit was separated and dissected individually. ‘lhe placenta was carefully stripped away from the endometrium, separated from the fetal membranes, and weighed. Fetal weight, crownrump length and heart girth were measured on each fetus, and the following tissues were removed and weighed: liver, heart, lungs, spleen, kidneys, gut (stomach + inkstines), brain, and the plantarls and gastrocnemius muscles. The first 4 conceptuses separated were those in the second and third positions (Position 1 beii nearest to the ovary) in both the right and left uterine horns. Protein (6) and DNA (4) concentrations were measured on the tissues from these 4 units. Samples of chorioallantois, endometrium, and allantoic and amniotic fluids were also collected from these tirst 4 units. Conceptus units in the fifth position in both the right and left uterine horns were separated next, and the placental and fetal tissues were used for dry matter determinations. All remaining conceptus units were separated, dissected as described above, and the tissues were weighed.
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Table
1. Composition of control and protein-restricted diets fed to sows from Day 1 to Day 63 of pregnancy
Ingredient
Corn Sugar Soybean Oil Alfalfa Meal Corn Soybean Meal Dicalcium Phosphate Limestone Iodized salt Choline chloride Vitamin Premixa Trace Element Premixb Protein Concentration Digestible Energy (kcalflig)
Control diet (%)
1.0 84.1 11.0 2.4 0.5 0.4 0.2 0.2 Q.2 100 13 3350
Protein-restricted
diet
(%) 92.1 3.0 1.0
2.4 0.5 0.4 0.2 0.2 Q.2 100 0.5 3350
?jupplies the following nutrients (per kg of mixed diet): retinyl pahnitate, 5280 IU; cholecalciferol, 704 IU; vitamin E (a-tocopheryl acetate), 35.2 IU; vitamin K (menadione), 3.52 mg; vitamin B-12,26.4 mg; riboflavin, 5.28 mg; niacin, 28.16 mg; D-pantothenic acid, 21.12 mg; biotin, 88 mg; thiamin, 2.2 mg. bSupplies the following nutrients (mgkg diet): Cu (as CuSo,) 10; Fe (as FeS0,.7H,O) MnSO,) 20; Zn (as ZnO) 100. Ground limestone was used as a carrier.
160; Mn (as
Tissue Culture Chorioallantoic and endometrial explants (500 mg; 2 to 3 mm’) were cultured in an atmosphere of 5% CO, and air at 37°C in sterile plastic lOO-mm Petri dishes containing 15 ml of MEM. The content of methionine in radiolabeled cultures was reduced to one-tenth of normal to facilitate incorporation of 50 FCi of SsS-methionine. Cultures were terminated after 30 h by centrifugation at 12,tXlO x g for 10 mitt at 4°C. Supernatant fractions were harvested, dialyzed against deionized water and nondialyzable radioactivity was determined using scintillation spectroscopy. All samples were stored froxen at -30°C until analysis. Polyacrylamide Gel Electrophoresis (PAGE) Samples (50 pg) of proteins in fetal fluids and radiolabeled proteins (50,000 dpm) from chorioallantoic and endometrial cultures were lyophilized and solubilii in 62.5 mM Tris-HCI buffer @H 6.8) containing 5% (w/v) sodium dodecyl sulfate (SDS), 10% (w/v) sucrose, and 5% (v/v) 2mercaptoethanol. Proteins were separated by SDS-PAGE using 12.5% (w/v) polyacrylamide gels and the buffer system of Laemmli (5). Lyophilixed samples (200 pg) of proteins in fetal fluids and radiolabeled proteins (200,WO dpm) from chorioallantoic and endometrial cultures were solubilixed in 5 mM potassium carbonate @H 10.5) containing 9 M urea, 2% (v/v) NP-40, and 5.1% (v/v) ampholytes.The ampholytes employed were 3-10, 5-7, and 9-11 (49:35: 16 by volume, respectively). All gels were stained with
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y
Coomassie Blue R-250 (0.125%. w/v, dissolved in acetic acid:ethanol:water; 7:40:53 v/v/v), and then destained. Gels containing radiolabeled proteins were then soaked in water, impregnated with sodium salicylate and dried. Fhrorographs were prepared by exposing dried gels next to Kodak X-AR t?bn at 8OC. statistical Ardysea Statistical analyses on all weights and measurements were performed, with sow being the experimental unit. Placental weight, fetal weight and measurements, and tissue weights from all fetuses within a titter were used to calculate a mean for each sow, and the 6 values for each diet treatment were averaged. Differences due to diet were determined using the two-sample r-test (9). Nondialyrable radioactivity in endometrial explant culture supernatants was standardized to represent dpm/ml/mg of wet tissue. These data were analyzed by analysis of variance (19) to evaluate differences resulting from maternal diet, tissue type (chorioallantois or endometrium), sow-within-treatment, and all two-way interactions. RESULTS Litter size restricted, 12.2 (control, 155 f restricted, 15.4
was not different between the. sows of the 2 maternal diet groups (control, 10.5 f 0.3; f 0.3; P>O. 1). Fetal body weight tended to be reduced in the restricted diet sows 5 g; restricted, 145 f 6 g; P=O.O74). Crown-rump length (control, 15.5 f 0.3 cm; f 0.3cm;P>O.1)andheartgirth(control, 11.4 f O.lcm;restricted, 11.1 f O.lcm; P>o.1)werenotdifferentbehveenthe 2groups. The absolute and relative weight (g/100 g body wt) and percentage of dry matter of all fetal tissues were compared. The percentage of dry matter was not different in any tissue and weight was different in only 3 tissues: liver, kidneys, aml brain (Table 2). The relative (P=O.O7) weight of the liver tended to be decreased in the restricted diet fbtutma; weights of the ki&ys were also significantly decreased in the restricted diet &sea, and brain tight tended to be heavier in the restricted diet fetuses then the control diet fetuses on a relative basis (P=O.Cn; Table 2). Protein and DNA concentrations were not different in the liver, kidneys, or brain of the control and restricted diet fetuses; however, the total DNA content was significantly decreased in the liver of the restricted diet fetuses compared with the control diet fetuses (Table 2). Placental weight was significantly reduced in the restricted diet fetuses, although there was M significant difference in the fUal:placentitl weight ratio between the control and restricted diet groups (Table 3). Placental protein and DNA concentration and content, and percentage of dry matter were also not different between the placentae of the 2 groups (Table 3). The concentrations of endometrial protein (control, 82.3 f 3.3; restricted, 79.0 f 5.2 mg/g) and DNA (control, 4.2 f 0.5; restricted, 4.7 f 0.8 mg/g) were not affected by maternal dietary treatment. However, there was a tendency for endometrial percentage of dry matter to be greater in the control sows (14.6 f 0.5%) than in the restricted diet sows (13.3 f 0.1%; P = 0.086). De novo protein secretion was greater in endometrial than placental tissue (P
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Amniotic and allantoic fluid samples from control and restricted diet fetuses were also subjected to SDS PAGE. Maternal dietary treatment bad no effect upon the electrophoretic pattern observed in amniotic fluid (F’igure 3). A trend was observed, however, in the allantoic fluid samples (Figure 3). Three proteins (Mr = 75,69 and 55 kDa) were reduced snd one protein (Mt = 32 kDa) was enbanced in alla&c fluids obtained from restricted diet fetuses. l%is trend was consistent wbetber tite gels were staid with Coomassie blue or silver stain. The 55 and 69 kDa proteins were subsequently identified by amino acid sequencing as alpha-1-antitrypsin and alpha-f&protein, respectively (G. Cook, Department of Pediatrics, Baylor College of Medicine, personal ~~~~tion).
Table
2. Weight (g and g/kg body wt) and protein and DNA concentration (mg/g) and content (mg) of tbe fetal liver, kidneys and brain at Day 63 of gestation after tbe sows bad been &d a control or pro~~-r~c~ diet from breeding (mean f SEi%; n=6) ContIol diet
Protein-restricted diet
P-value
Liver Weight %g)
8.1 f 0.5 5.2 f 0.1
6.9 f 0.3 4.8 f 0.1
0.11 0.07
108 i8 732 f 103
NS NS
prOtftiEi (m&t) (mg)
112 f3 900 f71
DNA (mg@ tmg)
11.1 f 0.9 87 i3
9.3 f 0.5 61 i2 Kidneys
0.12 0,001
WeigM 0 (glkg) Protein
2.5 f 0.2 1.6 f 0.1
2.0 f 0.2 1.3 f 0.1
0.05 0.05
59 It4 140 f 10
56 *4 110 f 12
NS 0.11
3.3 f 0.3 8fl
3.6 f 0.2 7fl Brain
NS NS
4.2 f 0.3 2.8 f 0.1
4.4 f 0.4 3.1 f 0.1
0.07
41 *l 174 i7
38 il 176 i 10
NS NS
4.2 f 0.4 18 rt2
4.3 f 0.5 20 It 3
NS
(mg@ (mg) DNA (mg&) (mg) Weight (g) @kg) Protein tmglg) (mg) DNA
NS
NS
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Theriogenology
C
R
6957-
Figure 1. Representative fluorograph of ‘Hleucine labelled eudometrial proteins from sows fed a control (C) or protein-restricted (R) diet. No apparent differeuces in de nova protein secretion were detected.
Figure 2. Representative fluorograph of 3Hleucine labelled proteins secreted by chorioallantoic me&mnesfromsows fed a control (C) or protein-restricted (RI diet. No apparent difkences in de nova protein secretion were detected.
DISCUSSION A positive, aud usually high, mrrelation exists between placental weigh and fetal weight (14). However, Pond et al. (13) reported &at feeding a diet severely restricted in proteh durihg early pregnancy resulted in smaller fetuses with larger placentae. The present study was performed to amfirm those findings and to determiue if protein restriction iu early pregnancy altered the protein synthetic properties of the endometrium or fetal placenta. We also wished to determine if the proteins found in the amniotic and allantoic fluids were affected by maternal protein restriction. Fetal weight was reduced in the present study, as was expected due to maternal malm~trition. The decrease in fetal weight must be considered part of a continuum, with the reduction in weight greater the
Theriogenology
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Theriogenology
Table 3. Placental weight (g), fetahplacemal weight ratio (F/P), dry matter (%), and protein and DNA concentration (mg/g) and content (mg) at Day 63 of gestation for fetuses whose dams were fed either a control or protein-restricted diet from breeding (mean f SEhf; n=6) Control Weight (g) F/P Dry Matter (96)
Protein-restricted
P-value
194 f20 0.93 f 0.12 5.0 f 0.5
155 f 12 0.97 f 0.07 5.0 f 0.7
0.025 NS NS
14.4 f 0.9 2347 f 212
15.6 f 1.0 2415 f 168
NS NS
1.0 f 0.1 166 f 15
1.0 f 0.1 135 f 10
NS 0.15
PfOtein
DNAm ?F (mgig) (mg)
longer the period of protein restriction. When sows were restricted in dietary protein to Day 44 of pregnancy, no change in fetal weight was recorded (14); to Day 63 of pregnancy, a 13% reduction (13); throughout pregnancy, a 27% reduction in birth weight (15). Fetal bone, liver, lddney, and brain growtb were also affected by this continuum, with no differences seen by Day 44 (14); however, significant reductions in fetal weight were seen by Day 63 (13). In tbe present study of maternal dietary protein restriction to Day 63 of pregnancy, the sows seemed better capable of buffering the nutritional insult because fetal body weight was reduced only 7% and bone and brain growth were not at&ted. Kidney weight was significantly reduced, with the reduction in liver weight tending to be significant. Placental weight was also reduced in the present study, in contrast to the report of Pond et al. (13). The fetakplacental weight ratio was not different between the control and protein-restricted fetuses, implying that the fetus was growing appropriately for its placenta. We believe that the difference in the 2 studies is the manner in which the placenta was stripped from the endometrium. The matern 1prcl’tein restriction seemed to alter the attachment of these 2 tissues, such that there was a greater tendency for the endometrium to remain attached to the placenta in the protein-restricted conceptuses. In-the present study, we took great care to ensure that the separation was complete. It is possible that the protein restriction altered some component of the attachment matrix, such as the glycosaminoglycam (2,M). This would explain the increased placental weight in only the protein-restricted group. Placental growth is more rapid in early pregnancy than is fetal growth (12), so malnutrition in early pregnancy would be expected to have a greater impact on the size of the placenta. This was true in the present study with placental weight reduced 21% compared with a 7% reduction in fetal weight. The reduction in placental weight may also be permanent, since the content of DNA was slightly reduced in the placentae of the protein-restricted sows. A permanent reduction in placental size would be expected to decrease the nutrient transport capacity of that placenta (1, IO), even if the nutritional restriction was removed. Reduced birth weight was recorded for piglets whose dams had received a protein-restricted diet only until Day 44 of pregnancy (14). It is interesting to note that there were no measurable differences in the placentae of control and protein-restricted sows at Day 44. There were no differences due to dietary treatment in the total protein and dry matter content of the placenta, the & novo protein synthesis of placental tissue explants, or the identity of these secreted proteins as measured by gel electrophoresis. This
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suggests that, at this stage of gestation, protein accretion by the placenta has not been altered by protein restriction. Endometrial protein concentration, de novo protein synthesis, and the identity of the secreted proteins in the 2 groups were similar, yet the dry matter content was reduced in the restricted sows. This suggests that the secretory activity of the endometrium may have been increased in the restricted sows. We hypothesize that some of tbat increased activity may be in the local production of peptides which interact with the placenta to control nutrient flux. The protein composition of the amniotic fluid was not altered by maternal protein restriction; that of the allantoic fluid, however, was slightly different, with 3 proteins reduced in concentration and 1 increased. Since allantoic fluid is generally considered to contain fetal metabolic products (7), these differences suggest that fetal protein metabolism was altered by maternal protein restriction. Alpha-lantitrypsin is known to be reduced in humans during periods of protein malnutrition (3), as it was in the allantoic fluid of the restricted fetuses. Alpha-fetoprotein is the predominant protein in fetal circulation (20), so its reduction in the restricted fetuses may well be an adaptation by the fetus to spare amino acids. Proteins of uterine origin also gain access to fetal fluids, with the classic example beiig uteroferrin (17). The differences in allantoic fluid proteins could , therefore, also reflect differential transport and/or metabolism of uterine proteins. We believe these results suggest that maternal protein restriction in early pregnancy permanently reduces fetal growth by decreasing placental size in swine. The fetus is able to alter its protein metabolism to spare some amino acids, but the longer the period of deficiency, the greater the impact on fetal grow& The effect of returning the sow to a protein-sufficient diet on both placental size and function is not known, however it appears that a permanent decrease in placental nutrient transport would occur. REFERENCES 1. BeII AW, Wiiening RB, Meschia G. Some aspects of placental function in chronically heat-stressed ewes. I Devel Physiol. 1987;9:17-29. 2. Burton A, Lockhart F, Bosnjak S, Yong S. Stimulation by 17 alpha-hydroxyprogesterone of glycoprotein and glycosaminoglycan synthesis in human placenta in vitro. Biol Neonate 1989;55:151-155. 3. Grant GH, Kachmar JF. The proteins of body fluids. In: Tietz NW (ed), Textbook of Clinical Chemistry. Philadelphia, W.B.Saunders Co., 1976;345-346. 4. J..abarca C, Paigen K. A simple, rapid, and sensitive DNA assay procedure. Anal B&hem. 1980;102:344-352. 5. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 1970;227:680-685. 6. Lowry OH, Rosebrougb NJ, Farr AL, Rundall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275. 7. McCance RA, Dickerson IWT. The composition and origin of the foetal fluids of the pig. J Embryo1 Exptal Morphol. 1957;5:43-50. 8. McCance P-4, Widdowson EM. Nutrition and growth. Proc R Sot London 1962;356:326-335. 9. Minitab. Release 7. Minitab Incorporated, State College, PA, 1989. 10. Noblet I, Close WH, Heavens RP. Studies on the energy metabolism of the pregnant sow. 1. Uterus and mammary tissue development. Brit J Nutr. 1985;53:251-265. Il. Patten BM. The early development of the body form and the establishment of the organ systims. In: Embryology of the Pig. New York, McGraw-Hill Book Company, Inc., 1959;60-93. 12. Pond WG, Houpt KA. Prenatal development. In: The Biology of the Pig. Ith?c :, NY, Cornell University Press. 1978;82-105.
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13. Pond WG, Maurer RR, Klindt J. Fetal organ response to maternal protein deprivation during pregnancy in swine. J Nutr 1991;121:504-509. 14. Pond WG, Maurer RR, Mersmann HI, Commins S. Response of fetal and newborn piglets to maternal protein restriction during early or late pregnancy. Growth, Develop, Aging 1992;56: 115-127. 15. Pond WG, Yen J-T, Mersmann HJ. Effect of severe dietary protein, nonprotein calories or feed restriction during gestation on postnatal growth of progeny in swine. Growth 1987$1:3X-371. 16. Roberts RM, Baombach GA, Bohi WC, Denny JB, Fitzgerald LA, Babelyn SF, Horst MN. Analysis of membrane polypeptides by two dimensional polyactylamide gel electrophoresis. In: Vnter CJ, Harris EC (eds), Molecular and Chemical Characterization of Membrane Receptors. New York, Alan R. Liss, 1984$6-l 13. 17. Roberta RM, Bazer FW. Uterofetin: A protein in search of a function. Bioessays 1984;1:8-11. 18. Rohde LH, Carson DD. Heparin-like glycosaminoglycans participate in binding of a human trophoblastic cell lii (JAR) to a homan uterine epithelial cell line (RL95). J Cell Physiol. 1993;155:185-1%. 19. SAS. SAS User’s Guide. Statistical Analysis Systems, Inc., Gary, NC, 1988. 20. Stone RT, Christenson RK. The relationship of fetal weight to serum albumin and alpha-fetoprotein in swine. J Anim Sci. 1982;55:818-825. 21. Ullrey DE, Sprague JI, Becker DE, Miller ER. Growth of the swine fetus. J Anim Sci 1%5;24:711-717.
42:2 17-226,1994
PROTEIN RESTRICTION IN EARLY PREGNANCY ALTERS FETAL AND PLACENTAL GROWTH AND ALLANTOIC PLUID PROTEINS IN SWINE P.A. Schoknecht”,
G.R. Newton*, D.E. Weise* and W.G. Pond’
1 USPAIARS Children’s Nutrition Research Center, Houston, TX Prairie View A&M University, Prairie View, TK Received for publication: Accepted:
December 31, 1993 June 10, 1994
ABSTRACT This study was designed to determine the impact of protein malnutrition during early pregnancy on fetal and placental growth and on the protein synthesis capacity of placental and endometrial tissues. Twelve crossbred sows received 1.8 kg/d of a control (13% protein) or protein-restricted (0.5% protein) diet from the day of breeding to Day 63 of pregnancy, when dissections were performed on each conceptus unit. ‘Ihe de novo protein synthetic rate of placental and endometrial explants was measumd using %-methionine. ‘l&se proteins and tbe proteins from amniotic and allantoic fluids were separated by polyacrylamide gel electrophoresis. Placental weight was significantly reduced in the sows fed the restricted diet, with a tendency for decreased fetal wei$tt as well. No difterences were found due to dietary treatment in de novo protein synthesis or in the electrophoretic patterns of secreted proteins of the placenta or endometrium. The. apparent quantity of 3 proteins in the allantoic fluid of the restricted diet fetuses decreased, while 1 protein iucreased in comparison with that of the control fetuses. These data suggest that protein malnutrition in early pregnancy decreases placental growth, thereby decreasing both fetal growth and the opportunity for compensatory growth upon nutritional rehabilitation. Key words: mahmtrition, fetal growth, protein synthesis, placenta INTRODUCTION Malnutrition has a detrimental impact on fetal growth, with the timing and severity of nu’uitional insult determining the degree of fetal growth retardation (8). In the early stages of pregnancy, conceptus growth is characterixed by rapid expansion of the placenta, with maximum sixe beii achieved by mid-pregnancy (12). At the mid-point of gestation, all major fetal organ systems have developed (11); however, the fetus weighs only one-tenth of its eventual birth weight (21). Maternal nutritional deficiencies during early pregnancy would, therefore, be expected to alter primarily placental size. This would likely compromise the ability of the placenta to transport nutrients at a time critical to the fetus and fetal organ development.
Acknowledgements This work is a publication of the USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX ‘Ihe project was funded in part under federal funds from the US Department of Agriculture, Agricultural Research Service under Cooperative Agreement Number 58-625@1~3. The contents of this publication do not necessarily reflect the views or policies of the US Department of Agriculture, nor does the mention of trade names, commercial products or organizations imply endorsement from the US Government.’ We thank Larry Solomon and associates of Praiie View A&M University for animal care and Kathy McKee, Lynn Walton and Gary Cook for technical assistance. $resent address: Department of Animal Sciences, Rutgers University, New Brunswick, NJ C@O3.
Copyright
0 1994 Buttetworth-Heinemann
Theriogenology
218
A reduction in fetal size would be a secondary effect of the maternal malnutrition. Sows receiving a protein-restricted diet from breeding to Day 63 of pregnancy (13) did produce smaller fetuses; however, these fetuses had larger placentae than fetuses of sows which received a normal protein diet. This apparently anomalous tinding raises the possibiity that local paracrine and/or autocriac factors may have contributed to compensatory growth of the placenta to insure that an adequate flow of nutrients was available for fetal development. Therefore., the objectives of this study were to reevaluate the effects of maternal protein restriction during the first 63 days of pregnancy on 1) indices of fetal and placental growth and development, 2) de novo protein synthesis by endometrial and placental tissues, and 3) the proteins contained in fetal fluids. MATERIALS AND METHODS Materials
Tissue culture supplies were obtained from GLBCO Laboratories (Grand Island, NY) and Sigma Chemical Co. (St. Louis, MO). Eagle’s minimal essential medium with Earl’s salts (MHM) for tissue culture was modified in the following manner. Medium was supplemented with penicillin (100 U/ml), amphotericin B (250 ng/ml), streptomycin (100 ~glml), insulin (0.2 U/ml), nonessential amino acids (1%) v/v), glucose (5 mg/ml), chloride (100 @ml), folk acid (100 &ml), pyridoxal-HC! (100 &nl), riboflavin (10 &ml), and thiamine (100 pg/ml). Acrylamide and N,N’-methylene-bis-acryhunide (electrophoresis grade) were obtained from Boekiqer Mannheim Biochemicals (Mdianapolis, IN). Ampholytes were obtained from Serva Bkhetnicals (Heidelberg, Germany). X-Gmat AR film was purchased from Eastman-Xodak (Rochester, NY). I$Qnethionine (specific activity = 1140 Ciimmol) was purchased from Dupont Company (Boston, MA). All other supplies and reagents were obtained from either Fisher Scientific (Houston, TX) or Sigma Chemical Co., ad were of reagentgradeor better. Tissue and Fluid Collection Twelve first or second parity crossbred swine (Chester White X Landrace X Large white X Yorkshire) were bred upon standing eshus (Day 0). Each sow received 1.8 kg/d of either control (13% protein) or protein-restricted (0.5% protein) diet from Day 1 to Day 63 of pregnancy (Table 1). On Day 63, sows were hilled by exsanguination after stunniq with a captive bolt pistol, and the entire pregnant uterus was excised. Each conceptus unit was separated and dissected individually. ‘lhe placenta was carefully stripped away from the endometrium, separated from the fetal membranes, and weighed. Fetal weight, crownrump length and heart girth were measured on each fetus, and the following tissues were removed and weighed: liver, heart, lungs, spleen, kidneys, gut (stomach + inkstines), brain, and the plantarls and gastrocnemius muscles. The first 4 conceptuses separated were those in the second and third positions (Position 1 beii nearest to the ovary) in both the right and left uterine horns. Protein (6) and DNA (4) concentrations were measured on the tissues from these 4 units. Samples of chorioallantois, endometrium, and allantoic and amniotic fluids were also collected from these tirst 4 units. Conceptus units in the fifth position in both the right and left uterine horns were separated next, and the placental and fetal tissues were used for dry matter determinations. All remaining conceptus units were separated, dissected as described above, and the tissues were weighed.
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Table
1. Composition of control and protein-restricted diets fed to sows from Day 1 to Day 63 of pregnancy
Ingredient
Corn Sugar Soybean Oil Alfalfa Meal Corn Soybean Meal Dicalcium Phosphate Limestone Iodized salt Choline chloride Vitamin Premixa Trace Element Premixb Protein Concentration Digestible Energy (kcalflig)
Control diet (%)
1.0 84.1 11.0 2.4 0.5 0.4 0.2 0.2 Q.2 100 13 3350
Protein-restricted
diet
(%) 92.1 3.0 1.0
2.4 0.5 0.4 0.2 0.2 Q.2 100 0.5 3350
?jupplies the following nutrients (per kg of mixed diet): retinyl pahnitate, 5280 IU; cholecalciferol, 704 IU; vitamin E (a-tocopheryl acetate), 35.2 IU; vitamin K (menadione), 3.52 mg; vitamin B-12,26.4 mg; riboflavin, 5.28 mg; niacin, 28.16 mg; D-pantothenic acid, 21.12 mg; biotin, 88 mg; thiamin, 2.2 mg. bSupplies the following nutrients (mgkg diet): Cu (as CuSo,) 10; Fe (as FeS0,.7H,O) MnSO,) 20; Zn (as ZnO) 100. Ground limestone was used as a carrier.
160; Mn (as
Tissue Culture Chorioallantoic and endometrial explants (500 mg; 2 to 3 mm’) were cultured in an atmosphere of 5% CO, and air at 37°C in sterile plastic lOO-mm Petri dishes containing 15 ml of MEM. The content of methionine in radiolabeled cultures was reduced to one-tenth of normal to facilitate incorporation of 50 FCi of SsS-methionine. Cultures were terminated after 30 h by centrifugation at 12,tXlO x g for 10 mitt at 4°C. Supernatant fractions were harvested, dialyzed against deionized water and nondialyzable radioactivity was determined using scintillation spectroscopy. All samples were stored froxen at -30°C until analysis. Polyacrylamide Gel Electrophoresis (PAGE) Samples (50 pg) of proteins in fetal fluids and radiolabeled proteins (50,000 dpm) from chorioallantoic and endometrial cultures were lyophilized and solubilii in 62.5 mM Tris-HCI buffer @H 6.8) containing 5% (w/v) sodium dodecyl sulfate (SDS), 10% (w/v) sucrose, and 5% (v/v) 2mercaptoethanol. Proteins were separated by SDS-PAGE using 12.5% (w/v) polyacrylamide gels and the buffer system of Laemmli (5). Lyophilixed samples (200 pg) of proteins in fetal fluids and radiolabeled proteins (200,WO dpm) from chorioallantoic and endometrial cultures were solubilixed in 5 mM potassium carbonate @H 10.5) containing 9 M urea, 2% (v/v) NP-40, and 5.1% (v/v) ampholytes.The ampholytes employed were 3-10, 5-7, and 9-11 (49:35: 16 by volume, respectively). All gels were stained with
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Coomassie Blue R-250 (0.125%. w/v, dissolved in acetic acid:ethanol:water; 7:40:53 v/v/v), and then destained. Gels containing radiolabeled proteins were then soaked in water, impregnated with sodium salicylate and dried. Fhrorographs were prepared by exposing dried gels next to Kodak X-AR t?bn at 8OC. statistical Ardysea Statistical analyses on all weights and measurements were performed, with sow being the experimental unit. Placental weight, fetal weight and measurements, and tissue weights from all fetuses within a titter were used to calculate a mean for each sow, and the 6 values for each diet treatment were averaged. Differences due to diet were determined using the two-sample r-test (9). Nondialyrable radioactivity in endometrial explant culture supernatants was standardized to represent dpm/ml/mg of wet tissue. These data were analyzed by analysis of variance (19) to evaluate differences resulting from maternal diet, tissue type (chorioallantois or endometrium), sow-within-treatment, and all two-way interactions. RESULTS Litter size restricted, 12.2 (control, 155 f restricted, 15.4
was not different between the. sows of the 2 maternal diet groups (control, 10.5 f 0.3; f 0.3; P>O. 1). Fetal body weight tended to be reduced in the restricted diet sows 5 g; restricted, 145 f 6 g; P=O.O74). Crown-rump length (control, 15.5 f 0.3 cm; f 0.3cm;P>O.1)andheartgirth(control, 11.4 f O.lcm;restricted, 11.1 f O.lcm; P>o.1)werenotdifferentbehveenthe 2groups. The absolute and relative weight (g/100 g body wt) and percentage of dry matter of all fetal tissues were compared. The percentage of dry matter was not different in any tissue and weight was different in only 3 tissues: liver, kidneys, aml brain (Table 2). The relative (P=O.O7) weight of the liver tended to be decreased in the restricted diet fbtutma; weights of the ki&ys were also significantly decreased in the restricted diet &sea, and brain tight tended to be heavier in the restricted diet fetuses then the control diet fetuses on a relative basis (P=O.Cn; Table 2). Protein and DNA concentrations were not different in the liver, kidneys, or brain of the control and restricted diet fetuses; however, the total DNA content was significantly decreased in the liver of the restricted diet fetuses compared with the control diet fetuses (Table 2). Placental weight was significantly reduced in the restricted diet fetuses, although there was M significant difference in the fUal:placentitl weight ratio between the control and restricted diet groups (Table 3). Placental protein and DNA concentration and content, and percentage of dry matter were also not different between the placentae of the 2 groups (Table 3). The concentrations of endometrial protein (control, 82.3 f 3.3; restricted, 79.0 f 5.2 mg/g) and DNA (control, 4.2 f 0.5; restricted, 4.7 f 0.8 mg/g) were not affected by maternal dietary treatment. However, there was a tendency for endometrial percentage of dry matter to be greater in the control sows (14.6 f 0.5%) than in the restricted diet sows (13.3 f 0.1%; P = 0.086). De novo protein secretion was greater in endometrial than placental tissue (P
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Amniotic and allantoic fluid samples from control and restricted diet fetuses were also subjected to SDS PAGE. Maternal dietary treatment bad no effect upon the electrophoretic pattern observed in amniotic fluid (F’igure 3). A trend was observed, however, in the allantoic fluid samples (Figure 3). Three proteins (Mr = 75,69 and 55 kDa) were reduced snd one protein (Mt = 32 kDa) was enbanced in alla&c fluids obtained from restricted diet fetuses. l%is trend was consistent wbetber tite gels were staid with Coomassie blue or silver stain. The 55 and 69 kDa proteins were subsequently identified by amino acid sequencing as alpha-1-antitrypsin and alpha-f&protein, respectively (G. Cook, Department of Pediatrics, Baylor College of Medicine, personal ~~~~tion).
Table
2. Weight (g and g/kg body wt) and protein and DNA concentration (mg/g) and content (mg) of tbe fetal liver, kidneys and brain at Day 63 of gestation after tbe sows bad been &d a control or pro~~-r~c~ diet from breeding (mean f SEi%; n=6) ContIol diet
Protein-restricted diet
P-value
Liver Weight %g)
8.1 f 0.5 5.2 f 0.1
6.9 f 0.3 4.8 f 0.1
0.11 0.07
108 i8 732 f 103
NS NS
prOtftiEi (m&t) (mg)
112 f3 900 f71
DNA (mg@ tmg)
11.1 f 0.9 87 i3
9.3 f 0.5 61 i2 Kidneys
0.12 0,001
WeigM 0 (glkg) Protein
2.5 f 0.2 1.6 f 0.1
2.0 f 0.2 1.3 f 0.1
0.05 0.05
59 It4 140 f 10
56 *4 110 f 12
NS 0.11
3.3 f 0.3 8fl
3.6 f 0.2 7fl Brain
NS NS
4.2 f 0.3 2.8 f 0.1
4.4 f 0.4 3.1 f 0.1
0.07
41 *l 174 i7
38 il 176 i 10
NS NS
4.2 f 0.4 18 rt2
4.3 f 0.5 20 It 3
NS
(mg@ (mg) DNA (mg&) (mg) Weight (g) @kg) Protein tmglg) (mg) DNA
NS
NS
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C
R
6957-
Figure 1. Representative fluorograph of ‘Hleucine labelled eudometrial proteins from sows fed a control (C) or protein-restricted (R) diet. No apparent differeuces in de nova protein secretion were detected.
Figure 2. Representative fluorograph of 3Hleucine labelled proteins secreted by chorioallantoic me&mnesfromsows fed a control (C) or protein-restricted (RI diet. No apparent difkences in de nova protein secretion were detected.
DISCUSSION A positive, aud usually high, mrrelation exists between placental weigh and fetal weight (14). However, Pond et al. (13) reported &at feeding a diet severely restricted in proteh durihg early pregnancy resulted in smaller fetuses with larger placentae. The present study was performed to amfirm those findings and to determiue if protein restriction iu early pregnancy altered the protein synthetic properties of the endometrium or fetal placenta. We also wished to determine if the proteins found in the amniotic and allantoic fluids were affected by maternal protein restriction. Fetal weight was reduced in the present study, as was expected due to maternal malm~trition. The decrease in fetal weight must be considered part of a continuum, with the reduction in weight greater the
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Table 3. Placental weight (g), fetahplacemal weight ratio (F/P), dry matter (%), and protein and DNA concentration (mg/g) and content (mg) at Day 63 of gestation for fetuses whose dams were fed either a control or protein-restricted diet from breeding (mean f SEhf; n=6) Control Weight (g) F/P Dry Matter (96)
Protein-restricted
P-value
194 f20 0.93 f 0.12 5.0 f 0.5
155 f 12 0.97 f 0.07 5.0 f 0.7
0.025 NS NS
14.4 f 0.9 2347 f 212
15.6 f 1.0 2415 f 168
NS NS
1.0 f 0.1 166 f 15
1.0 f 0.1 135 f 10
NS 0.15
PfOtein
DNAm ?F (mgig) (mg)
longer the period of protein restriction. When sows were restricted in dietary protein to Day 44 of pregnancy, no change in fetal weight was recorded (14); to Day 63 of pregnancy, a 13% reduction (13); throughout pregnancy, a 27% reduction in birth weight (15). Fetal bone, liver, lddney, and brain growtb were also affected by this continuum, with no differences seen by Day 44 (14); however, significant reductions in fetal weight were seen by Day 63 (13). In tbe present study of maternal dietary protein restriction to Day 63 of pregnancy, the sows seemed better capable of buffering the nutritional insult because fetal body weight was reduced only 7% and bone and brain growth were not at&ted. Kidney weight was significantly reduced, with the reduction in liver weight tending to be significant. Placental weight was also reduced in the present study, in contrast to the report of Pond et al. (13). The fetakplacental weight ratio was not different between the control and protein-restricted fetuses, implying that the fetus was growing appropriately for its placenta. We believe that the difference in the 2 studies is the manner in which the placenta was stripped from the endometrium. The matern 1prcl’tein restriction seemed to alter the attachment of these 2 tissues, such that there was a greater tendency for the endometrium to remain attached to the placenta in the protein-restricted conceptuses. In-the present study, we took great care to ensure that the separation was complete. It is possible that the protein restriction altered some component of the attachment matrix, such as the glycosaminoglycam (2,M). This would explain the increased placental weight in only the protein-restricted group. Placental growth is more rapid in early pregnancy than is fetal growth (12), so malnutrition in early pregnancy would be expected to have a greater impact on the size of the placenta. This was true in the present study with placental weight reduced 21% compared with a 7% reduction in fetal weight. The reduction in placental weight may also be permanent, since the content of DNA was slightly reduced in the placentae of the protein-restricted sows. A permanent reduction in placental size would be expected to decrease the nutrient transport capacity of that placenta (1, IO), even if the nutritional restriction was removed. Reduced birth weight was recorded for piglets whose dams had received a protein-restricted diet only until Day 44 of pregnancy (14). It is interesting to note that there were no measurable differences in the placentae of control and protein-restricted sows at Day 44. There were no differences due to dietary treatment in the total protein and dry matter content of the placenta, the & novo protein synthesis of placental tissue explants, or the identity of these secreted proteins as measured by gel electrophoresis. This
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suggests that, at this stage of gestation, protein accretion by the placenta has not been altered by protein restriction. Endometrial protein concentration, de novo protein synthesis, and the identity of the secreted proteins in the 2 groups were similar, yet the dry matter content was reduced in the restricted sows. This suggests that the secretory activity of the endometrium may have been increased in the restricted sows. We hypothesize that some of tbat increased activity may be in the local production of peptides which interact with the placenta to control nutrient flux. The protein composition of the amniotic fluid was not altered by maternal protein restriction; that of the allantoic fluid, however, was slightly different, with 3 proteins reduced in concentration and 1 increased. Since allantoic fluid is generally considered to contain fetal metabolic products (7), these differences suggest that fetal protein metabolism was altered by maternal protein restriction. Alpha-lantitrypsin is known to be reduced in humans during periods of protein malnutrition (3), as it was in the allantoic fluid of the restricted fetuses. Alpha-fetoprotein is the predominant protein in fetal circulation (20), so its reduction in the restricted fetuses may well be an adaptation by the fetus to spare amino acids. Proteins of uterine origin also gain access to fetal fluids, with the classic example beiig uteroferrin (17). The differences in allantoic fluid proteins could , therefore, also reflect differential transport and/or metabolism of uterine proteins. We believe these results suggest that maternal protein restriction in early pregnancy permanently reduces fetal growth by decreasing placental size in swine. The fetus is able to alter its protein metabolism to spare some amino acids, but the longer the period of deficiency, the greater the impact on fetal grow& The effect of returning the sow to a protein-sufficient diet on both placental size and function is not known, however it appears that a permanent decrease in placental nutrient transport would occur. REFERENCES 1. BeII AW, Wiiening RB, Meschia G. Some aspects of placental function in chronically heat-stressed ewes. I Devel Physiol. 1987;9:17-29. 2. Burton A, Lockhart F, Bosnjak S, Yong S. Stimulation by 17 alpha-hydroxyprogesterone of glycoprotein and glycosaminoglycan synthesis in human placenta in vitro. Biol Neonate 1989;55:151-155. 3. Grant GH, Kachmar JF. The proteins of body fluids. In: Tietz NW (ed), Textbook of Clinical Chemistry. Philadelphia, W.B.Saunders Co., 1976;345-346. 4. J..abarca C, Paigen K. A simple, rapid, and sensitive DNA assay procedure. Anal B&hem. 1980;102:344-352. 5. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 1970;227:680-685. 6. Lowry OH, Rosebrougb NJ, Farr AL, Rundall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275. 7. McCance RA, Dickerson IWT. The composition and origin of the foetal fluids of the pig. J Embryo1 Exptal Morphol. 1957;5:43-50. 8. McCance P-4, Widdowson EM. Nutrition and growth. Proc R Sot London 1962;356:326-335. 9. Minitab. Release 7. Minitab Incorporated, State College, PA, 1989. 10. Noblet I, Close WH, Heavens RP. Studies on the energy metabolism of the pregnant sow. 1. Uterus and mammary tissue development. Brit J Nutr. 1985;53:251-265. Il. Patten BM. The early development of the body form and the establishment of the organ systims. In: Embryology of the Pig. New York, McGraw-Hill Book Company, Inc., 1959;60-93. 12. Pond WG, Houpt KA. Prenatal development. In: The Biology of the Pig. Ith?c :, NY, Cornell University Press. 1978;82-105.
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13. Pond WG, Maurer RR, Klindt J. Fetal organ response to maternal protein deprivation during pregnancy in swine. J Nutr 1991;121:504-509. 14. Pond WG, Maurer RR, Mersmann HI, Commins S. Response of fetal and newborn piglets to maternal protein restriction during early or late pregnancy. Growth, Develop, Aging 1992;56: 115-127. 15. Pond WG, Yen J-T, Mersmann HJ. Effect of severe dietary protein, nonprotein calories or feed restriction during gestation on postnatal growth of progeny in swine. Growth 1987$1:3X-371. 16. Roberts RM, Baombach GA, Bohi WC, Denny JB, Fitzgerald LA, Babelyn SF, Horst MN. Analysis of membrane polypeptides by two dimensional polyactylamide gel electrophoresis. In: Vnter CJ, Harris EC (eds), Molecular and Chemical Characterization of Membrane Receptors. New York, Alan R. Liss, 1984$6-l 13. 17. Roberta RM, Bazer FW. Uterofetin: A protein in search of a function. Bioessays 1984;1:8-11. 18. Rohde LH, Carson DD. Heparin-like glycosaminoglycans participate in binding of a human trophoblastic cell lii (JAR) to a homan uterine epithelial cell line (RL95). J Cell Physiol. 1993;155:185-1%. 19. SAS. SAS User’s Guide. Statistical Analysis Systems, Inc., Gary, NC, 1988. 20. Stone RT, Christenson RK. The relationship of fetal weight to serum albumin and alpha-fetoprotein in swine. J Anim Sci. 1982;55:818-825. 21. Ullrey DE, Sprague JI, Becker DE, Miller ER. Growth of the swine fetus. J Anim Sci 1%5;24:711-717.