Proteome Differences in Placenta and Endometrium between Normal and Intrauterine Growth Restricted Pig Fetuses.

Intrauterine growth restriction (IUGR) commonly occurs in both human and livestock species [ 1 ] and impairs postnatal metabolism, growth, development and health [ 2 ]. In swine industry, IUGR is a major problem contributing to high neonatal morbidity and mortality, low efficiency of feed utilization and poor meat quality [ 3 ] and [ 4 ]. Therefore, it is of importance to investigate the underlying mechanisms behind IUGR and to find a solution to prevent its occurrence. Placental transport of nutrients is a key determinant of fetal intrauterine growth [ 5 ]. The endometrium (a mucosal layer underlying placenta and a part of uterus) is also crucial for embryonic development, implantation, and placentation [ 6 ]. To provide an optimal environment for fetal growth, both placenta and endometrium undergo dramatic changes in morphology and function during pregnancy [ 7 ]. In pigs, placental development starts at approximately d 15 and reaches maximum by d 60–70 of gestation [ 8 ]. As a type of the epitheliochorial placenta, the pig trophoblast is attached to the uterine luminal epithelium without invasion during implantation, which is very different from implantation processes in humans. Over the mouths of uterine glands, pig allantochorion is not directly attached to the endometrial epithelium but forms areolae, where uterine gland secretions are absorbed by trophoblast cells. In the inter-areolar regions, substances pass from uterine circulation, via the uterine epithelium, into the trophoblast, and after the placenta is formed, via the capillary in fetal placenta into fetal circulation [ 9 ]. Although previous studies indicate that insufficiency in placental and endometrial development and function contributes to IUGR, little is known about changes of their proteomes at different gestational stages. The objective of this study was to identify differences in placental and endometrial proteins between normal body weight (NBW) and IUGR fetuses at d 60, 90, and 110 of gestation.

Both IUGR and NBW fetuses grew between D60 and D110 of gestation ( Table 1 ). Body weights of selected IUGR fetuses were 93.4, 460, and 858 g respectively and were significantly lower than that of NBW at the same gestational day, which were 143.3, 914.3, and 1528 g respectively ( P < 0.01, Table 1 ). a Values are means ± SEM, n = 6 per group. b **, P < 0.01 vs the NBW group. A total of 50 placental protein spots were differentially expressed in placentae between IUGR and NBW fetuses at D60, 90 and 110 of gestation. Their appearances in gel images were labeled in Fig 1A and biochemical information is summarized in Table 2 and Fig 1B . According to their biological function, these proteins were classified into the following groups: (1) energy metabolism; (2) nutrient transport; (3) stress response; (4) nutrient metabolism; (5) cell proliferation and apoptosis; (6) cell morphology and motility. (A) Abundance of differentially expressed proteins in the placenta of normal and IUGR pig fetuses at D 60, 90 and 110 during gestation. (B) Functional classification of differentially expressed proteins between placentae from normal and IUGR pig fetuses. a Spot number refer to protein spot numbers that correspond to the labels in Fig 1 . b Protein score generated by MS-MS identification platform, with a score higher than 71 being considered as statistical significance. c Ratio (I/N) refer to the ratio of protein levels in the endometrium of IUGR fetuses to these for normal-weight fetuses. Five proteins involved in glucose and energy metabolism were expressed at lower levels in the IUGR placenta, as compared with the NBW placenta. These proteins included triosephosphate isomerase 1 (TPI1, Spot F285), dihydrolipoamide dehydrogenase precursor (DLD, Spot F176), isocitrate dehydrogenase 1 (IDH1, Spot F48), aconitase 1 (ACO1, F179), and creatine kinase (CKB, Spot F23). IUGR negatively affected the expression of several proteins involved in transport of nutrients, including alpha-fetoprotein precursor (AFP, Spot F223, F189), potassium voltage-gated channel subfamily H, member 7 (KCNH, Spot F219), albumin (ALB, Spot F118), albumin precursor (ALB, F110), and apolipoprotein A-I (APOA1, Spot F196, F254) (p < 0.01). Thirteen differentially expressed proteins that participate in stress response include: protein disulfide isomerase-associated 3 isoform 1 (PDIA3, Spot F264), protein disulfide isomerase-associated 3 (PDIA3, Spot 276) and chain A, porcine ribonuclese inhibitor (RNH, Spot F241), 78 kDa glucose-regulated protein (GRP78, Spot F191), 75 kDa glucose-regulated protein (GRP75, Spot183), heat shock 90 kD protein 1, alpha (HSP90A, F58), heat shock 71 kDa protein (HSPA8, Spot F63), heat shock 70 kDa protein 5 (HSPA5, Spot F291), and parkinson disease protein 7 (PARK7, Spot F135). All of these proteins were up regulated in the placenta of IUGR fetuses, as compared with NBW fetuses. Spermine synthase isoform 3 (SPMSY, Spot F218), which is related to nutrient metabolism, was expressed at lower levels in the placenta of the IUGR placenta, compared with the NBW placenta. Eight spots of proteins related to cell proliferation and apoptosis were affected by IUGR. Abundances of eukaryotic initiation factor 5A isoform I variant A (EIF5A2, Spot F260), eukaryotic translation initiation factor 6 (EIF6, Spot F287), aspartyl-tRNA synthetase (AspRs, Spot F174, F246) and zinc finger protein 420 (ZFP420, Spot F177) were lower in the IUGR placenta compared with the NBW placenta. In contrast, the levels of cystatin B (CSTB, Spot F300) and cathepsin Z (CTSZ, Spot F304) were higher in the IUGR group. Fifteen protein spots related to cell morphology and motility were differentially expressed between IUGR and NBW placentae. Ezrin (EZR, Spot F172), myosin regulatory light chain (MRLC2, Spot F323), myosin regulatory light chain 12B isoform 2 (MLC-2A, Spot F85), beta-actin (ACTB, Spot F195), tublin alpha-3 (TUBA1B, Spot F164) and actin-related protein 3 (ARP3, Spot F19) were significantly up regulated in the IUGR placenta. In contrast, down regulation of annexin A4 (Anx4, Spot F327), annexin A8 (Anx8, Spot F139), moesin (MSN, Spot F169), keratin 7 (KRT7, Spot F21), keratin 8 (KRT8, Spot F78), gelsolin (GEL, Spot F70, F71) and tublin alpha 1c (TUBA1C) was detected in the IUGR fetuses. A total of 52 protein spots were differentially expressed in the IUGR endometrium compared with NBW fetuses at D 60, 90 and 110 of gestation. Their appearances in gel images were labeled in Fig 2A and biochemical information is summarized in Table 3 and Fig 2B . According to the biological function, these proteins were involved in: (1) energy metabolism; (2) transportation; (3) stress response; (4) nutrient metabolism; (5) cell proliferation and apoptosis; (6) cell morphology and motility. (A) Abundance of differentially expressed proteins in the endometrium of normal and IUGR pig fetuses at D 60, 90 and 110 of gestation. (B) Functional classification of differentially expressed proteins between endometrium from normal and IUGR pig fetuses. a Spot number refer to protein spot numbers that correspond to the labels in Fig 2 . b Protein score generated by MS-MS identification platform, with a score higher than 71 being considered as statistical significance. c Ratio (I/N) refer to the ratio of protein levels in the endometrium of IUGR fetuses to these for normal-weight fetuses. Three proteins involved in glucose and energy metabolism exhibited differential expression in the endometria of IUGR and NBW fetuses. These proteins included H + -ATPase B subunit (V-ATPase, Spot F65, D90 and D110) and chain A, aldose reductase complexed with Idd384 inhibitor (Spot F212). Both proteins were expressed at lower levels in IUGR fetuses than in NBW fetuses. IUGR affected the expression of 9 proteins involved in the transport of nutrients. These proteins included albumin (Spot F27, F69, F7, F232, F203), transferrin (TF, Spot F109), and copine-1 (CPN1, Spot F7). All of these proteins were down regulated in the IUGR group, as compared with NBW fetuses. Three cellular redox homeostasis proteins were expressed at lower levels in the IUGR group. They were thioredoxin (TXN, Spot F10) and peroxiredoxin 6 (PRDX6, Spot F218, F79). Nine differentially expressed proteins play important roles in stress response. Compared with NBW fetuses, expression of thioredoxin (TXN, Spot F10), peroxiredoxin 6 (PRDX6, Spot F218, F79), glucose-regulated protein (GRP94, Spot L144) and alpha-crystallin B chain (CRYAB, Spot L135) was higher, while expression of latexin (LXN, Spot F265), complement component 3 (C3, Spot F215), immunoglobulin gamma (IgG, Spot L323), and guanine nucleotide binding protein (GAQ, Spot F76) was lower in the IUGR endometrium, as compared with the NBW group. Nine protein spots related to nutrients metabolism were differentially expressed in the endometria between IUGR and NBW fetuses. These proteins included argininosuccinate synthase (ASS1, Spot F204), arginine aminopeptidase (RNPEP, Spot F87), aspartate aminotransferase (AST, Spots F224, F70, L101), carnosinase (CNDP1, L285), phosphoserine phosphatase (PSP, Spots F81), bifunctional 3'-phosphoadenosine 5'-phosphosulfate synthase 2-like (PAPSS2, Spot F13) and UDP-glucose dehydrogenase (UDPGD, Spot F90). The expression of these proteins was reduced in the IUGR endometrium, as compared with the NBW group. Eight spots of proteins involved in cell proliferation and apoptosis were differentially expressed in the endometria of normal and IUGR fetuses. Abundances of alpha-1-antichymotrypsin 2 (ACT2, Spot F58), alpha-1-antichymotrypsin 3 (ACT3, Spot F41, F104) and serpin A3-6 (Spot F104) were increased in the IUGR group, as compared with the NBW group. In contrast, expression of lamin-A/C (LMNA, Spot L48), elongation factor 1 (eEF1A-1, Spot F11) and heterogeneous nuclear ribonucleoprotein K (HNRNPK, Spot F61, F62) was reduced in the IUGR endometrium, compared with the NBW group. Twelve endometrial protein spots, which participate in cell structure and motility, were differentially expressed between IUGR and NBW fetuses. Actin-related protein 3 (Spot F26), actinin, alpha 4 isoform 12 (ACTN4, Spot F52), cytokeratin 19 (CK19, Spot L12), cytokeratin 8 (CK8, Spot F93), beta actin (ACTB, Spot F71), and microtubule-actin (Macf1, Spot L90), were down-regulated in the IUGR group, while T-complex protein 1 subunit (TCP1, Spot F9), tubulin, alpha, ubiquitous isoform 19 (TBLA, Spot F230), beta actin (ACTB, Spot L221), and gamma-actin (ACTG, Spot L220) were up-regulated in the IUGR group, as compared with NBW fetuses. Fig 3 shows the western blotting analysis of GADPH, HIF-1α, VEGF and VREFR1. The expression of HIF-1α was higher in the IUGR placenta, compared with the NBW group. In contrast, GADPH, VEGF and VEGFR were reduced significantly (p < 0.01) in the IUGR placenta, as compared with the NBW fetuses. (A) Western blot analysis of GAPDH. (B) Western blot analysis of HIF-α. (C) Western blot analysis of VEGF. (D) Western blot analysis of VEGFR1.

All the nutrients provided to fetuses during pregnancy are transported from maternal circulation through uteroplacenta tissue. Therefore, sufficient development of both placenta and endometrium (tissue size, vasculature, metabolism, secretory function) is very important for substance exchange between maternal and fetal circulation, and plays a key role in fetal growth and development. In order to fulfil substance transport between mother and fetus, the utero-placenta needs (1) energy and transporters; (2) an established vascular system. Results of the current work reveal that expression of several proteins related to energy metabolism, transportation, and vasculargenesis was reduced in IUGR placenta and endometrium, which could lead to inadequate energy provision and insufficient nutrient transport. Such utero-placental dysfunction could be a factor contributing to IUGR. Additionally, based on our proteomics data, we suggest that increased oxidative stress and apoptosis in IUGR placenta and endometrium, which can compromise utero-placental function, is another factor for the development of IUGR. Collectively, these findings have important implications for both human health and animal production to reduce the occurrence of IUGR fetuses.

This experiment was approved by China Agricultural University Animal Care and Use Committee (No. 20080106–1). All surgery was performed under anesthesia with halothane, and all efforts were made to minimize discomfort. Twenty-four gilts (Landrace × Large White) were used in this study. These gilts were mated, housed individually. They had free access to drinking water and were fed a corn-and soybean meal-based diet (2 kg.d -1 ) formulated to meet or exceed nutrient requirements according to the National Research Council (2012). At d 60, 90 and 110 of gestation, 8 gilts were randomly selected and exsanguinated after induction of anesthesia with halothane [ 10 ]. The maternal abdomen was opened and the pregnant uterus was extracted immediately from the body cavity. IUGR fetuses were identified as those whose weights were less than two standard deviation of the mean body weight for gestational age. One NBW fetus and one IUGR fetus, as well as their corresponding placenta and endometrium, were collected from each one of gilts. Placenta was easily separated from endometrium on d 60, 90 and 110 of gestation, and we did not observe any contamination between these two tissues. The tissues were rapidly transferred to liquid nitrogen and stored at -80°C. The placenta and endometrium from 18 IUGR and 18 NBW (6/stage) were used to extract protein, as we described previously [ 3 ]. Briefly, approximately 0.2 g frozen samples were crushed to powder in liquid nitrogen, then homogenized in a lysis buffer containing 7 M urea, 2 M thiourea, 4% 3-[3-(-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, and 50 mM dithiothreitol with protease inhibitors (GE Healthcare, Piscataway, NJ). An ultrasonicater (Sonics Model VC 750, Sonics and Materials, Newtown, CT) was set at 20% power output and used to break down the mixture for 10 min at 0°C. After the addition of 1% (vol/vol) nuclease mix (GE Healthcare), the mixture solution was kept at room temperature for 1 h to completely solubilize proteins, followed by re-sonification for 10 min as described above to thoroughly break down cell membranes. The homogenate was centrifuged for 10 min at 13,000 g at 4°C to settle down the insoluble components. The supernatant fluid was obtained and its protein concentration was determined using the Brandford method. Portions of the homogenate (1 mg of protein for both placenta and endometrium) were stored at -80°C. Two-dimensional gel electrophoresis was run in triplicate for both placental and endometrial samples at each of 3 time-points (D60, 90, 110) to compare protein expression between IUGR and NBW fetuses. First, isoelectric focusing was performed using immobile DryStrip gels (pH 3 to 10, nonlinear, 24 cm long; GE Healthcare), as we described previously [ 11 ]. One mg protein was loaded onto immobile Drystrip gels strips using an in-gel sample rehydration technique. The first-dimensional isoelectric focusing was carried out at 20°C for 100,000 voltage h (IPGphor system, GE Healthcare), followed by equilibration in 4 mL of equilibration buffer 1 (6 M urea, 1% dithiothreitol, 30% glycerol, and 50 mM Tris-Cl pH 8.8) for 15 min on a shaker and then in 4 mL of equilibration buffer 2 (6 M urea, 2.5% iodoacetamide, 30% glycerol, 50 mM Tris-Cl pH 8.8) for 15 min as described above. The second-dimensional Gel was performed using 12.5% SDS-PAGE gels. The gels were run at 30 mA/gel for 30 min and then at 50 mA/gel until the Bromophenol Blue came out of the gels. Analytical gels were stained with Coomassie Brilliant Blue G-250 (Amresco, Solon, OH), and high-resolution gel images (400 dpi) were obtained using a scanner (ImageScanner, model PowerLook 2100XL, UMAX Technologies, Atlanta, GA). Images were analyzed using commercial software (Image-Master 2D Platinum Version 6.01, GE Healthcare) and differentially expressed protein spots that deviated more than 1.3-fold in their relative volume (% vol) were selected for in-gel digestion and protein identification. Protein spots of interest were cut from the gels to be destained with 100 μL of 50% (v/v) acetonitrile (ACN) in 25 mM ammonium bicarbonate for 1 h. Then an aliquot (3 μL) of trypsin solution (10 μg/mL; Amresco Inc., Solon, OH) in 25 mM ammonium bicarbonate was added to each gel piece after it was completely dried by vacuum centrifugation (Eppendorf Concentrator 5301, Eppendorf, Hamburg, Germany) for 30 min. The mixture was incubated in a trypsin solution at 4°C for rehydration for 1 h, followed by incubation at 37°C for 12 h. Thereafter, the gel pieces were vacuum-dried to evaporate the solvent and 8 μL of 5% trifluoroacetic acid (TFA) was added onto the dry gel pieces. After incubation at 37°C for 1 h, the solution was transferred into a microcentrifuge tube. The gel pieces were extracted twice separately with 8 μL of 2.5% TFA and 50% acetonitrile, and 5% TFA. The third extraction was performed using 8 μL of 100% acetonitrile and the solution was combined with the previous two extracts in the microcentrifuge tube. Finally, the combined solution was dried and resolubilized in 2 μL of 0.5% TFA for protein identification using MALDI-TOF/TOF MS (Matrix Assisted Laser De sorption Ionization-Time of Flight/Time of Flight MS). The peptide mass fingerprint analysis was performed using a search engine (MASCOT, Matrix Science, Lon- don, UK) [ 12 ]. Searching parameters included: (1) “trypsin” as the enzyme of protein digestion; (2) “monoisotopic” as mass value; (3) “unrestricted” as peptide mass; (4) “0.3 Da” as peptide mass tolerance; (5) “oxidation (M) and carbamidomethyl (C)” as variable modifications; and (6) “1” as maximum missed cleavages. Protein match with a score was considered significant ( P < 0.05). Extracted protein samples were boiled for 5 min and separated by electrophoresis (Bio-Rad, Richmond, CA) in 12% SDS-PAGE gel before electroblotted (Bio-Rad) onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA) [ 11 ]. After polyvinylidene fluoride membranes were blotted with Tris buffer containing 5% fat-free dry milk and 0.05% Tween-20 (TBST; 0.05% Tween 20, 100 mmol/L of Tris-HCl, and 150 mmol/L of NaCl, pH 7.5) for 1 h at 25°C, they were rinsed in TBST for 4 times and incubated overnight at 4°C with primary antibodies, which were mouse polyclonal GAPDH (Abcom, ab22556), rabbit polyclonal HIF-1α (Abcam, ab114977), mouse monoclonal VEGF (Abcam, ab1316) and rabbit polyclonal VEGFR1 (Abcam, ab2350) with 1:3000, 1:2000, 1:2000 and 1:2000 dilutions, respectively. The membranes were washed in the same manner as described above and incubated with the horseradish peroxidase-conjugated secondary antibody (1:5,000; Sigma) for 40 min. Then, the membranes were washed 6 times for 5 min in TBST and the blots were detected using Lumi-light Western Blotting substrates (Biofuture, Beijing, China) and analyzed with the Imaging Analysis Software of National Institutes of Health (Bethesda, MD). Data are expressed as means ± SEM. Differences in fetal weights or western blot results in placenta or endometrium between IUGR and normal fetuses at a given day of gestation were statistically analyzed by the t-test (version 8.2; SAS Institute, Cary, NC, USA). Proteomic data for placenta or endometrium were analyzed by the t-test, a statistic program embedded in the commercial software (Image-Master 2D Platinum Version 6.01, GE Healthcare), to compare the difference between IUGR and normal fetuses at a given day of gestation. To avoid systemic error, we further calculated the ratio of protein levels between IUGR fetuses to those for normal weight fetuses, and only the proteins with a ratio higher than 1.3 or lower than -1.3 were chosen for further identification. P < 0.05 was taken to indicate statistical significance and data are expressed as means ± SEM.

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