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Bioactive Constituents, Metabolites, and Functions
Maternal N-Carbamylglutamate supply during early pregnancy enhanced pregnancy outcomes in sows through modulations of targeted genes and metabolism pathways Shuang Cai, Jinlong Zhu, Xiangzhou Zeng, Qianhong Ye, Changchuan Ye, Xiangbing Mao, Shihai Zhang, Shiyan Qiao, and Xiangfang Zeng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01637 • Publication Date (Web): 27 May 2018 Downloaded from http://pubs.acs.org on May 27, 2018
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Maternal N-Carbamylglutamate supply during early pregnancy enhanced
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pregnancy outcomes in sows through modulations of targeted genes and
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metabolism pathways
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Shuang Cai,†,§ Jinlong Zhu,† Xiangzhou Zeng,† Qianhong Ye,† Changchuan Ye,†
5
Xiangbing Mao,‡ Shihai Zhang,|| Shiyan Qiao,† and Xiangfang Zeng†,*
6 7
†
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Centre, China Agricultural University, Beijing 100193, China;
9
‡
State Key Laboratory of Animal Nutrition, Ministry of Agriculture Feed Industry
Animal
Nutrition
Institute,
Sichuan
Agricultural
University,
No.
10
Gongpinghuimin Road, Wenjiang District, Chengdu 611130, China;
11
||
12
Animal Science, South China Agricultural University, Guangzhou 510642, China
211,
Guangdong Provincial Key Laboratory of Animal Nutrition Control, College of
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ABSTRACT: Reducing pregnancy loss is important for improving reproductive
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efficiency for both human and mammalian animals. Our previous study demonstrates
15
that maternal N-Carbamylglutamate (NCG) supply during early pregnancy enhances
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embryonic survival in gilts. However whether maternal NCG supply improves the
17
pregnancy outcomes is still not known. Here, we found maternal NCG supply during
18
early pregnancy in sows significantly increased the numbers of total piglets born alive
19
per litter (P < 0.05), and significantly changed the levels of metabolites in amniotic
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fluid and serum involved in metabolism of energy, lipid, and glutathione, and
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immunogical regulation. The expression of endometrial progesterone receptor
22
membrane component 1 (PGRMC1) were significantly increased by NCG
23
supplementation (P < 0.05), as well as the expression of PGRMC1, endothelial nitric
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oxide synthesases (eNOS), and lamin A/C in fetuses and placentae (P < 0.05). Among
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the NCG associated amino acids, arginine and glutamine markedly increased
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PGRMC1 and eNOS expression in porcine trophectoderm cells (P < 0.05). While
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glutamate could stimulate the expression of vimentin and lamin A/C in porcine
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trophectoderm (pTr) cells (P < 0.05), and proline stimulated lamin A/C expression (P
29
< 0.05). Collecetively, these data reveal mechanisms of NCG in reducing early
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embryo loss. These findings have important implications that NCG has great potential
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to improve pregnancy outcomes in human and mammalian animals.
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KEYWORDS: embryo, metabolome, N-carbamylglutamate, pregnancy outcome,
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sows
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INTRODUCTION
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Embryo implantation is a critically important biological process in pregnancy in
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mammals, which, to a large degree, has a direct impact on the pregnancy outcome.1
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Early pregnancy loss, accounting for 30% - 50% in mammals, profoundly restricts
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reproductive performance.2 Embryo implantation failure is the major cause of these
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early pregnancy losses.1 In women, about 15% of all pregnancies end due to early
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pregnancy loss.3 Even though in vitro fertilization (IVF) has become a successful
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technology in treating infertility, the pregnancy rate of IVF is still relatively low at
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around 25% - 30%, because of the failure of the IVF embryo to implant in the uterus.4
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Therefore, reducing early embryo implantation failure is a crucially important issue in
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improvement of the reproductive efficiency for both humans and mammalian animals.
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Although numerous studies have demonstrated that nutrition plays important roles
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in fetal development and subsequent pregnancy outcomes, only limited studies have
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focused on the impacts and roles of specific nutrients or general nutritional status on
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embryo development and embryo implantation.5-7 Arginine is a conditionally essential
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amino acid for most mammals. Many studies have shown that arginine regulates
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embryo development and improves embryo quality subsequently enhancing embryo
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implantation.8-10 Arginine supplementation during early pregnancy is demonstrated to
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improve embryo implantation through the PI3K/PKB/mTOR/NO signaling pathway,
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ultimately enhancing the pregnancy outcome in rats.11,12 As an activator of
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endogenous arginine synthesis, NCG has been reported to improve early embryonic
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implantation and pregnancy outcome in rats.13,14 In gilts, maternal NCG supply during
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early pregnancy also improves embryonic survival and development through
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modulation of endometrial proteomes.15,16 However, whether maternal NCG
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supplementation during early pregnancy improves pregnancy outcomes in sows is still 3
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unknown, as well as the specific roles of NCG in regulation of targeted genes’
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expression in fetuses, placentae and endometriums, and the metabolic changes in
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serum and amniotic fluid. Additionally, pig, which shares high similarity with humans
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in anatomic and physiologic characteristics, becomes one of the best models for
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human reproduction studie.17 Therefore, approaches to enhance sows’ reproductive
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performance have important implication for improving human reproduction. The
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objective of this study was to investigate whether maternal NCG supplementation
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during early pregnancy improved pergancy outcomes in sows and to examine the
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targeted genes’ expression in fetuses, placentae andendometriums, as well as the
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metabolic changes in serum and amniotic fluid.
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MATERIALS AND METHODS
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Ethics statement
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This study was carried out in strict accordance with the Chinese guidelines for animal
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welfare. Animal handling procedures reported herein were approved by the
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Institutional Animal Care and Use Committee of China Agricultural University.
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Animals and protocols
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In order to examine whether maternal NCG supplementation during early pregnancy
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improved pergancy outcomes in sows, a total of 80 sows (parity 3 or 4; Landrace ×
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Yorkshire) with an initial body weight of 210.00 ± 10.91 kg were used in this study.
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Sows were housed individually in gestation stalls (2.2 m × 0.65 m) with concrete
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floors. Sows were checked daily for estrus using direct boar exposure in the morning
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and artificially inseminated three times with fresh semen during estrus (10-12 h apart).
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Immediately after breeding, sows were allocated based on body weight, back fat, and
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parity into 4 groups. Sows in the control group were fed the basal diet for the entire
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gestation period. Sows in the three N-carbamylglutamate groups were fed the basal 4
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diet supplemented with 0.05% NCG (wt:wt) during d 1-8, d 9-28, or d 1-28.
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Thereafter sows were fed the basal diet until delivery (n = 20). Here, we designed
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three NCG supply phases to determine the optimal one. All sows were fed 1.25 kg
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diets during d 1-28 of pregnancy and 1.75 kg diets from d 29 of pregnancy to delivery
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twice daily at 06:00 h and 16:00 h. Sows had free access to water throughout the
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experiment. Diets were formulated to meet the recommended nutrient requirements of
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NRC (2012) for gestating sows (Table 1). NCG (purity, 97%) was obtained from
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National Feed Engineering Technology Research Center (Beijing, China) and 0.05%
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NCG level was based on results from our previous study.15 Litter size, live litter size,
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litter weight, number of stillborn and mummified piglets, and birth weight were
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recorded soon after birth.
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To investigate whether NCG improving pregnancy outcome was associated with
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the role of NCG in regulation of targeted genes’ expression in fetuses, placentae and
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endometriums, as well as the metabolic changes in serum and amniotic fluid, sixteen
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gilts (Duroc × Yorkshire × Landrace) with average initial body weight of 132.0 kg
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were randomly assigned to the the basal diet (control) or fed the basal diet
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supplemented with 0.05% NCG between d 1 and d 28 of pregnancy (0.05% NCG, n =
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8). This NCG supply phase was chosen based on the results of the first animal study.
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Diets were the same as in Exp. 1. On d 28 of gestation, blood samples were collected
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via jugular venepuncture into 5 mL tubes after overnight starvation before being
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centrifuged at 3,000 × g for 10 min at 4°C to obtain serum samples. Gilts were killed
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by electrocution to obtain uteri, which were transported on ice to the lab. A volume of
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5 mL amniotic fluid from each fetus was harvested with syringes. Placentae and
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endometriums were carefully isolated from the individual fetus. All the samples were
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immediately snap frozen in liquid nitrogen and stored at -80°C until further analysis. 5
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Chemicals and reagents
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HPLC-grade methanol and ACN were obtained from Fisher Scientific International
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(Hampton, NH, USA). HPLC-grade formic acid was purchased from Dikma
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Technology (RichmondHill, Ontario, Canada). Arginine, glutamine, glutamate, and
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proline were purchased from Sigma Aldrich (St. Louis, Missouri, USA). Ultrapure
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water (Milli-Q, Millipore Corporation, Bedford, MA, USA) was used to prepare all
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aqueous solutions.
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Cell culture and treatment
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To further examine the contributions of arginine, glutamine, glutamate, and proline,
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the concentrations of which are changed in maternal serum in response to maternal
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NCG supply during early pregnancy, pTr cells were used as the in vitro model. Cells
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were isolated originally from d 12 conceptuses and was kindly provided by Dr.
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Zhenlong Wu from China Agricultural University. Cells were cultured in 6-well plates
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in DMEM/F-12 medium with 10% FBS, 1% insulin–transferrin–selenium (ITS,
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Sigma–Aldrich, St. Louis, Missouri, USA) until reaching 80% confluence. Cells were
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serum starved overnight in customized medium, deprived of either arginine, proline,
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glutamate or glutamine, and then treated with different doses (0, 0.25, 0.50, 1.00 mM)
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of Arg, Pro, Glu or Gln for 24 h. All experiments were repeated at least 2-3 times.
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Non-targeted metabolic fingerprinting analysis
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Metabolite extraction
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Methanol was used to extract both intracellular and extracellular metabolites. Serum
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and amniotic fluid samples (200 µL) were centrifuged at 14,000 × g for 20 min at 4°C.
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The supernatant were transferred and mixed with 4 volumes of methanol. The samples
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were incubated at -80°C for 2 h and then centrifuged at 14,000 × g for 10 min at 4°C.
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The metabolites containing supernatant were transferred to new tubes on dry ice and 6
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then dried to pellets using speedvax/lyophilizer with no heat. The dried metabolite
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samples were stored at -80°C until further processing.
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HPLC methods for polar metabolites
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Ultimate 3000 UHPLC (Dionex) coupled with Q Exactive was used to perform LC
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separation. In positive mode, Waters Atlantis HILIC Silica column (2.1 × 100 nm)
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was used. Mobile phase A was prepared by dissolving 0.63 g of ammonium formate in
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50 mL of HPLC-grade water, then adding 950 mL of HPLC-grade acetonitrile and 1
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µL of formic acid. Mobile phase B was prepared by dissolving 0.63 g of ammonium
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formate in 500 mL of HPLC-grade water, followed by 500 mL of HPLC-grade
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acetonitrile and 1 uL of formic acid. Linear gradient was as follows: 0 min, 1% B; 2
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min, 1% B; 3.5 min, 20% B; 17 min, 80% B; 17.5 min, 99% B; 19 min, 99% B; 19.1
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min, 1% B; 22 min, 1% B. In negative mode, Waters BEH Amide column was applied
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(2.1 × 100 mm). Mobile phase A was prepared by dissolving 0.77 of ammonium
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acetate in 50 mL of HPLC-grade water, then 950 mL of HPLC-grade acetonitrile was
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added. pH was adjusted to 9.0 with ammonium hydroxide solution. Mobile phase B
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was prepared by dissolving 0.77 g of ammonium acetate in 500 mL of HPLC-grade
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water, subsequently, adding 500 mL of HPLC-grade acetonitrile and adjusting pH to
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9.0 with ammonium hydroxide solution. Linear gradient was as follows: 0 min, 5% B;
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2 min, 5% B; 4 min, 20% B; 18 min,85% B;19 min, 95% B; 21 min, 95% B; 21.1 min,
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5% B; 25 min, 5% B.
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MS methods for polar metabolites
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Polar metabolite analysis was performed on Q Exactive Orbitrap mass spectrometer.
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The detailed mass spectrometer parameters are as follows: spray voltage, 3.5 kV for
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positive and 2.5 kV for negative; capillary temperature, 275 °C for positive and 320°C 7
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for negative; sheath gas flow rate (arb), 35; aux gas flow rate (arb), 8; mass range
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(m/z), 70-1,050 for positive and 80-1,200 for negative; full MS resolution, 70,000;
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MS/MS resolution, 17,5000; topN, 10; NCE, 15/30/45; duty cycle, 1.2s.
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Data processing
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Two levels of identification were performed simultaneously using Tracefinder.
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Metabolites were first potentially assigned according to endogenous MS database by
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accurate masses. At the same time, those that can match with the spectra in fragment
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database were confirmed and potentially assigned metabolites were displayed in the
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results. 10 ppm and 15 ppm mass tolerance was applied for precursor and fragment
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matching. Still, 0.25 min retention time shift was allowed for quantitation.
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Western blot analysis
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The pTr cells, frozen placenta, endometrium and fetuses were separately homogenized
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in RIPA lysis buffer (150 mM NaCl, 1 % Triton X-100, 0.5 % sodium deoxycholate,
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0.1 % SDS, 50 mM Tris–HCl at pH 7.4) supplemented with a protease inhibitor
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cocktail (Apply gene, Beijing, China). After centrifugation at 1,4000 × g for 15 min at
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4oC, the protein concentration in the supernatant was determined using the BCA
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protein assay kit (Pierce, Rockford, IL, USA). Equal amount of proteins was
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electrophoresed (Bio-Rad, Richmond, CA, USA) on SDS polyacrylamide gels. Then
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proteins were electrotransferred to polyvinylidene difluoride membrane (Millipore,
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Bedford, MA, USA) and blocked with 5% Bovine Serum Albumin (BSA) overnight at
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4 °C or 1 h at room temperature. Prestained protein markers (Fermentas, Waltham,
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MA, USA) were used in each gel. Samples were incubated with primary antibodies
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(1:1,000 dilution) against Membrane-associated PGRMC1 (Santa Cruz Biotechnology,
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Santa Cruz, CA, USA), lamin A/C (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 8
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eNOS (Cell Signaling Technology, Beverly, MA, USA), and anti-β-actin antibody
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(Santa Cruz Biotechnology, Santa Cruz, CA, USA). After being washed with
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Tris-Tween 20 buffer (pH 7.4) for three times, membranes were incubated with the
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HRP-conjugated secondary antibody (Zhongshan Gold Bridge, Beijing, China) for 1 h
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at room temperature. The membrane was exposed by AlphaImager 2200 (Alpha
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Innotech, San Leandro, CA, USA) automatically. Band densities were detected with
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the Western Blot Luminance Reagent (Applygene, Beijing, China) and quantified
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using AlphaImager 2200 (Alpha Innotech, San Leandro, CA, USA).
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Statistical analysis
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For results of reproductive performance, PGRMC1, eNOS, vimentin, and lamin A/C
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expression of gilts and pTr cells, data were analyzed using MIXED procedures of
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SAS (version 9.0; SAS Institute Inc., Cary, NC) following a randomized complete
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block design. Sow was considered the experimental unit. Data for the number of
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stillborn and mummified, and embryonic mortality were analyzed using the chi-square
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test of SAS. A value of P < 0.05 was considered statistically significant. Statistical
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analysis for metabolics results were performed with identified polar metabolites. The
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student’s t test was evaluated by SAS. Principal component analysis (PCA) and
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pathway analysis was completed using Metaboanalyst. Compared with the control
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group, metabolites with a threshold of > 1.50- or < 0.55-fold, values of P < 0.1, and
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VIP > 1.35 were considered differentially metabolites.
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RESULTS
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The reproductive performance of sows and serum concentration of amino acids
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As shown in Table 2, dietary NCG supplementation during d 1-28 of pregnancy in
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sows increased the number of total piglets born per litter and total piglets born alive 9
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per litter, compared with the control diet and NCG supplementation during d 1-8 or d
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9-28 of pregnancy (P < 0.05). However, dietary NCG supplementation during three
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different stages of early pregnancy had no effect on sows’ weight gain, number of
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stillborn and mummified piglets or birth weight of piglets.
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The metabolomic profiles in serum and amniotic fluid
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To identify possible relationships within classes of data, PCA was employed.
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Performing PCA on the statistics-filtered data set, distinct differences was observed
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between the NCG group and control group, showing that NCG highly influences the
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serum and amniotic fluid metabolome. Data were visualized by constructing PC
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scores, where each point on the plot represented a single sample. In serum, PC1 and
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PC2 represented 19.4% and 13.1% of the variation, respectively. The first two PCs
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explained 32.5% of the total variances within the data (Fig. 1A). And in amniotic fluid,
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PC1 and PC2 represented 8.2% and 8% of variation, respectively (Fig. 1B).
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Metabolomes in serum from NCG and control groups were differentially
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expressed (Table 3). To identify which variables account for such a significant
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separation, we calculated the P value, VIP score (variable in project, the higher the
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score, the more important it is) and fold change (FC), defined as the ratio of the NCG
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group to the control group for a given expression profile. Taking the three aspects into
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account, 19 metabolites are selectively shown in Table 3. Fifteen of the metabolites
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detected were found to be up-regulated in NCG group, while 4 were down-regulated.
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The detailed analysis of the most relevant pathways was performed by
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MetaboAnalysis. The results showed that maternal NCG supplementation
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up-regulated metabolites mainly involved in fatty acid metabolism, energy
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metabolism and immunological regulation. 10
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For amniotic fluid, 14 metabolites are selectively shown in Table 4. Ten of the
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metabolites detected were found to be up-regulated in NCG group, while 4 were
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down-regulated. The up-regulated metabolites are mainly involved in fatty acid
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metabolism, energy metabolism and glutathione metabolism while down-regulated
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metabolites are involved in cysteine metabolism and absorption of vitamins.
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The protein abundance of PGRMC1, lamin A/C, and eNOS in the endometrium,
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fetuses, and placentaeplacentae of gilts on d 28 of pregnancy
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The protein abundance of PGRMC1, eNOS, and lamin A/C in placentae were
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significantly increased (P < 0.05) in gilts fed the NCG supplemented diet on d 28 of
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pregnancy, compared with those in gilts fed the control diet (Fig. 2A). Similarly, in
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fetuses, dietary NCG supplementation significantly increased (P < 0.05) the
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expression of PGRMC1, eNOS, and lamin A/C on d 28 of pregnancy, compared with
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the control diet (Fig. 2B). In endometrium, NCG supplementation also dramatically
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enhanced (P < 0.05) the expression of PGRMC1 and eNOS on d 28 of pregnancy,
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compared with the control diet (Fig. 2C), while the expression of lamin A/C was not
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affected.
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The expression levels of PGRMC1, lamin A/C, eNOS, and vimentin in pTr cells
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in the presence of arginine, proline, glutamine, or glutamate
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Western blot analysis revealed that compared to the control treatment, a 24-h arginine
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treatment markedly increased PGRMC1 expression in a dose dependent manner in
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pTr cells, which peaked at 0.5 mM. Similarly, the expression levels of eNOS were
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also dose-dependently increased in pTr cells, with the peak at 0.5 mM (Fig. 3A).
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However, arginine had no effect on vimentin and lamin A/C expression level in pTr
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cells (Fig. 3A). 11
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Glutamine also dose-dependently stimulated the expression levels of PGRMC1
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and eNOS in pTr cells, in comparison with the control treatment, with the peak at 0.5
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mM for PGRMC1, and 1.0 mM for eNOS (Fig. 3B). Glutamine treatment had no
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obvious effect on the expression of lamin A/C and Vimentin (Fig. 3B).
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Compared with the control treatment, glutamate stimulated the expression of
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vimentin and lamin A/C in pTr cells, peaking at 0.25 mM and 0.50 mM, respectively
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(Fig. 3C). However, glutamate had no effects on the expression of PGRMC1 and
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eNOS in pTr cells (Fig. 3C).
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A dose-dependent augmentation of lamin A/C was also observed in pTr cells
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treated with proline (Fig. 3D). Proline markedly increased the expression levels of
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lamin A/C at 0.25 mM treatment in pTr cells, compared to the control treatment (Fig.
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3D). Proline treatment had no effects on the expression of PGRMC1, Vimentin, and
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eNOS in pTr cells (Fig. 3D).
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DISCUSSION
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Our results showed that NCG supply during early pregnancy improved the pregnancy
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outcome in sows, which indicated that the beneficial effects of NCG on embryonic
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survival during early pregnancy subsequently improved pregnancy outcome in sows.
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NCG supplementation had a similar response compared with dietary arginine
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supplementation during early pregnancy in sows.18 However, NCG has unique
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advantages over arginine because: (1) there is no impact on intestinal absorption of
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dietary tryptophan, histidine, or lysine; (2) a low dose is highly effectivein stimulating
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endogenous arginine synthesis; (3) it has a relatively long half-life in vivo (perhaps
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8-10 h); and (4) substantially reduced cost.19-21 Therefore, dietary NCG
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supplementation perhaps is a better choice than arginine to improve pregnancy
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outcomes in mammals. 12
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To further explore the mechanism of NCG in improving pregnancy outcomes,
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metabolomic analysis in amniotic fluid and serum was conducted. The results
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indicated that maternal NCG supplementation increased levels of metabolites in
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amniotic fluids and serum primarily involved in energy metabolism, lipid metabolism,
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glutathione metabolism, and immunological regulation. This is consistent with our
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previous proteome results, which indicates that maternal NCG supplementation
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modulates endometrial proteins associated with energy metabolism, lipid metabolism,
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protein metabolism, antioxidative stress, and immune response.15 Energy metabolism
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is crucially important to embryo growth, differentiation, and viability.22 The
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reinforcement in energy metabolism probably provides more energy for embryonic
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and fetal development. During pregnancy, oxidative stress has negative impacts on
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embryo development, such as a decrease in the percentage of two-cell and blastocyst
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stage embryos. Reactive oxygen species are generated from embryo metabolism
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and/or exist in embryo surroundings, which cause embryonic growth retardation and
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embryonic dysmorphogenesis.23,24 Glutathione status reflects the antioxidant capacity
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of developing embryos. GSH reduces the formation of free oxygen radical species,
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ultimately protecting embryos against oxidative stress.24,25 Our data indicated that
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beta-citryl-L-glutamic acid level in amniotic fluids was increased which indicated that
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glutathione metabolism was enhanced in NCG supplemented sows. This was
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beneficial for embryo growth and protecting embryos against damage from free
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radical oxygen species. Another notable finding of the metabolomics data in amniotic
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fluids and serum was that immunological regulation pathways were up-regulated in
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NCG supplemented sows during early pregnancy. During pregnancy, the maternal
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uterus has tolerance to the semiallogenic fetus, and many immune cells (such as
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macrophages, dendritic cells, and regulatory T cells) and molecules (such as 13
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Leukemia Inhibitory Factor and heme oxygenase-1) are involved in this process.26
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Platelet-activating factor (PAF) may be used as an indicator of embryo viability and
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pregnancy outcome prediction. It has been reported that PAF level is highly correlated
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with pregnancy outcomes in human.27 PAF initiates early pregnancy factor synthesis,
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which is recognized as an immunosuppressive factor and whose amount is highest on
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d 30 of pregnancy in gilts.28,29 In this study, the improvement in pregnancy outcome
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induced by maternal NCG supply could be associated with the increase in serum PAF
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level. Collectively, alternations in levels of amniotic fluid and serum metabolites
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involved in energy metabolism, lipid metabolism, glutathione metabolism, and
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immunological regulation by maternal NCG supply were beneficial to embryonic
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development, which ultimately enhances pregnancy outcomes in sows.
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Our previous proteome data indicates that maternal NCG supplementation during
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early pregnancy up-regulates factors related to embryo implantation and development.
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15
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targeted genes to validate the modulation of NCG on the expression of these genes in
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vivo and in vitro. A recent study demonstrates that PGRMC1 is localized in oocytes,
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embryos/fetuses, and uteri.30 PGRMC1 is different from classical nuclear PGRs
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receptors. PGRMC1 and serpine 1 mRNA binding protein (SERBP1) form a complex
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to bind progesterone, leading to the increase in cAMP levels and activation of protein
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kinase G,31 to mediate anti-apoptosis and to modulate steroidogenesis and cellular
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homeostasis.32,33 In bovine endometrium, PGRMC1 is co-expressed with SERBP1
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and nuclear progesterone receptor (PGR) during the first trimester of pregnancy,
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indicating PGRMC1 plays an important role in early pregnancy possesses.34
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PGRMC1 is also expressed in uterus and placenta in mice and humans indicating its
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potentially important role in cellular differentiation, modulation of apoptosis and
In the present study, PGRMC1, NOS, vimentin, and lamin A/C were chosen as
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steroidogenesis during early pregnancy.35 In the current study, the enhanced
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expression of PGRMC1 observed in endometrium, fetuses, and placentae in NCG
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supplemented gilts on d 28 of pregnancy indicates that maternal NCG supply might
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improve the development of endometrium, fetuses, and placentae through modulation
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of local PGRMC1 expression. The in vitro results showed only arginine and
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glutamine but not glutamate and proline remarkably induced PGRMC1 expression in
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pTr cells which indicates that arginine and glutamine are the key metabolites related
336
to PGRMC1 stimulation by maternal NCG supply.
337
Three nitric oxide synthesases (NOS), including neuronal nitric oxide
338
synthesases (nNOS), eNOS, and inducible nitric oxide synthesases (iNOS) are
339
involved in nitro oxide synthesis from arginine in the body.36 NO modulates embryo
340
implantation through remodeling of the extracellular matrix,37 and plays critical roles
341
in placental vascular development.38 NOS expression in the uterus is beneficial for
342
endometrial decidualization and embryo implantation.39 Low expression of eNOS
343
increases embryo loss and decreases embryo implantation sites in the eNOS-knockout
344
mouse model.40 In contrast, high expression of eNOS in fetuses and placentae is
345
benefical for nutrient transfer from mother to fetus and efficient utilization of these
346
nutrients.41 Our results showed that arginine and glutamine supplementation increased
347
eNOS expression in pTr cells. We speculate that dietary NCG supplementation during
348
early pregnancy might improve embryo implantation through up-regulation of eNOS
349
expression induced by arginine and glutamine in pTr cells. NOSs, also highly
350
expressed in placenta, influence the placental vasculogenesis and angiogenesis
351
through modulation of VEGF and angiopoietin signaling pathways.38 We found that
352
eNOS expression was differentially modulated in uterus, fetus, and placenta in
353
response to maternal NCG supply in early pregnancy, with relatively stronger 15
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stimulation in the placenta and fetus than that in the uterus. These data indicate that
355
maternal NCG supply exerted a stronger influence on the development of fetuses and
356
placentae, compared with the uterus.
357
Vimentin, one of the intermediate filament proteins, plays pivotal roles in cell
358
migration.42 Trophoblastic giant cells (TGC) exert critical roles in embryo and fetal
359
development through interaction with decidual cells and the extracellular matrix.43-45
360
Vimentin is found to be expressed in mice trophoblastic giant cells, which is benefical
361
for development of the chorioallantoic placenta.46,47 Our data indicated that glutamate
362
enhanced vimentin expression in pTr cells. Serum glutamate concentration was
363
reported to increase by NCG supplementation.15 Therefore, maternal NCG supply
364
appears to benefit embryo and fetal development partially through modulation of
365
vimentin expression in pTr cells. Lamin A/C is expressed in mouse embryonic stem
366
cells and developing blastocyst.48 Lamin A/C mediates cell mechanics, polarization,
367
and migration 49 and is important for determination of cell fate.50 The increased lamin
368
A/C expression in fetuses and placentae could be benefical for the early development
369
of embryos in response to maternal NCG supply.
370
In conclusion, maternal NCG supply during early pregnancy enchanced
371
pregnancy outcomes in sows through differential modulation of factors related to
372
embryo implantation and development in endometrium, fetuses, and placentae, as well
373
as the regulation of metabolites in amniotic fluids and serum, which are primarily
374
involved in energy metabolism, lipid metabolism, glutathione metabolism, and
375
immunological regulation. These findings imply that NCG has great potential to
376
improve pregnancy outcomes in humans and mammalian animals.
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AUTHOR INFORMATION
378
Corresponding Authors
379
*(X.Z.)
380
[email protected].
381
Finding
382
This work was supported by National Natural Science Foundation of the P. R of China
383
(NO. 31301980 and NO. 31772614).
384
Notes
385
The authors declare no competing financial interest.
Phone:
+86
10-62733588.
Fax:
+86
10-62733688.
E-mail:
386 387
ABBREVIATIONS USED
388
eNOS, endothelial nitric oxide synthesases; FC, fold change; iNOS, inducible nitric
389
oxide synthesases; ITS, insulin–transferrin–selenium; IVF, in vitro fertilization; NCG,
390
N-carbamylglutamate; nNOS, neuronal nitric oxide synthesases; NOS, nitric oxide
391
synthesases; PAF, Platelet-activating factor; PCA, Principal component analysis; PGR,
392
progesterone receptor; PGRMC1, progesterone receptor membrane component 1; pTr,
393
porcine trophectoderm; SERBP1, serpine 1 mRNA binding protein; TGC,
394
Trophoblastic giant cells; VIP, variable in project
17
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395
Refercences
396
(1) Koot, Y. E. M.; Teklenburg, G.; Salker, M. S.; Brosens, J. J.; Macklon, N. S.
397
Molecular aspects of implantation failure. Biochim. Biophys. Acta. 2010, 1822,
398
1943-1950.
399
(2) Goff, A.K. Embryonic signals and survival. Reprod. Domest. Anim. 2002, 37,
400
133-139.
401
(3) McNamee, K. F.; Dawood, F.; Farquharson, R. G. Thrombophilia and early
402
pregnancy loss. Best. Pract. Res. Clin. Obstet. Gynaecol. 2012, 26, 91-102.
403
(4) Bazer, F.W.; Wu, G.; Johnson, G.A.; Kim, J.; Song, G. Uterine histotroph and
404
conceptus development: select nutrients and secreted phosphoprotein 1 affect
405
mechanistic target of rapamycin cell signaling in ewes. Biol. Reprod. 2011,
406
85,1094-1107.
407
(5) Cross, J.C.; Mickelson, L. Nutritional influences on implantation and placental
408
development. Nutr. Rev. 2006, 64, S12-S18.
409
(6) Ren, W. K.; Luo, W.; Wu, M. M; Liu, G.; Yu, X. L.; Fang, J.; Li, T. J.; Yin, Y. L.;
410
Wu G. Y. Dietary L-glutamine supplementation improves pregnancy outcome in mice
411
infected with type-2 porcine circovirus. Amino. Acids. 2013, 45, 479–488.
412
(7) Wu, X.; Xie, C. Y.; Zhang, Y. Z.; Fan, Z. Y.; Yin, Y. L.; Blachier, F. Glutamate–
413
glutamine cycle and exchange in the placenta–fetus unit during late pregnancy. Amino.
414
Acids. 2015, 47,45-53.
415
(8) Santana, P. D.; Silva, T. V.; Da, C. N.; Da, S. B.; Carter, T. F.; Cordeiro, M. S. Da,
416
S. B.; Santos, S. S.; Herculano, A. M.; Adona, P. R.; Ohashi, O. M.; Miranda, M. S.
417
Supplementation of bovine embryo culture medium with L-arginine improves embryo
418
quality via nitric oxide production. Mol. Reprod. Dev. 2014, 81, 918-927. 18
ACS Paragon Plus Environment
Page 18 of 36
Page 19 of 36
Journal of Agricultural and Food Chemistry
419
(9) Wu, G. Y.; Bazer, F. W.; Satterfield, M. C.; Li, X. L.; Wang, X. Q.; Johnson, G.
420
A.; Burghardt, R. C.; Dai, Z. L.; Wang, J. J.; Wu, Z. L. Impacts of arginine nutrition
421
on embryonic and fetal development in mammals. Amino. Acids. 2013, 45, 241-256.
422
(10) Wu, X.; Yin, Y. L.; Liu, Y. Q.; Liu, X. D.; Liu, Z. D.; Li, T. J.; Huang, R. L.;
423
Ruan, Z.; Deng, Z. Y. Effect of dietary arginine and N-carbamoylglutamate
424
supplementation reproduction and gene expression of eNOS, VEGFA and PlGF1 in s
425
placenta in late pregnancy of sows. Anim. Reprod. Sci. 2012, 132, 187-192.
426
(11) Zeng, X. F.; Mao, X. B.; Huang, Z. M.; Wang, F. L.; Wu, G. Y.; Qiao, S. Y.
427
Arginine enhances embryo implantation in rats through PI3K/PKB/mTOR/NO
428
signaling pathway during early pregnancy. Reproduction. 2013, 145, 1-7.
429
(12) Zeng, X. F.; Wang, F. L.; Fan, X; Yang, W. J.; Zhou, B.; Li, P. F.; Yin, Y. L.;
430
Wu, G. Y.; Wang, J. J. Dietary arginine supplementation during early pregnancy
431
enhances embryonic survival in rats. J. Nutr. 2008, 138, 1421-1425.
432
(13) Zeng, X. F.; Huang, Z. M.; Mao, X. B.; Wang, J. J.; Wu, G. Y.; Qiao, S. Y.
433
N-carbamylglutamate enhances pregnancy outcome in rats through activation of the
434
PI3K/PKB/mTOR signaling pathway. PLos. One. 2012, 7, e41192.
435
(14) Liu, X. D.; Wu, X.; Yin, Y. L.; Liu, Y. Q.; Geng, M. M.; Yang, H. S.; Blachier,
436
F., Wu, G. Y. Effects of dietary L-arginine or N-carbamylglutamate supplementation
437
during late gestation of sows on the miR-15b/16, miR-221/222, VEGFA and Enos
438
expression in umbilical vein. Amino. Acids. 2012, 42, 2111-2119.
439
(15) Zhu, J. L.; Zeng, X. F.; Peng, Q.; Zeng, S. M.; Zhao, H. Y.; Shen, H. X.; Qiao, S.
440
Y. Maternal N-carbamylglutamate supplementation during early pregnancy enhances
441
embryonic survival and development through modulation of the endometrial proteome
442
in gilts. J. Nutr. 2015, 145, 2212-2220. 19
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
443
(16) Zhang, B.; Che, L.Q.; Lin, Y.; Zhuo, Y.; Fang, Z. F.; Xu, S. Y.; Song, J.; Wang,
444
Y. S.; Liu, Y.; Wang, P. Wu, D. Effect of dietary N-carbamylglutamate levels on
445
reproductive performance of gilts. Reprod. Domest. Anim. 2014, 49, 740-745.
446
(17) Bazer, F. W.; Song, G.; Kim, J.; Dunlap, K. A.; Satterfield, M. C.; Johnson, G.
447
A.; Burghardt, R. C.; Wu, G. Y. Uterine biology in pigs and sheep. J. Anim. Sci.
448
Biotechnol. 2012, 3, 23.
449
(18) Li, J.; Xia, H.; Yao, W.; Wang, T. T.; Li, J. L.; Piao, X. S.; Thacker, P.; Wu, G.
450
Y.; Wang, F. L. Effects of arginine supplementation during early gestation (day 1 to
451
30) on litter size and plasma metabolites in gilts and sows. J. Anim. Sci. 2015, 93,
452
5291-303.
453
(19) Wu, G. Y.; Knabe, D. A.; Kim, S. W. Arginine nutrition in neonatal pigs. J. Nutr.
454
2004, 134, 2783S-2790S.
455
(20) Geng, M. M.; Li, T. J.; Kong, X. F.; Song, X. Y.; Chu, W. Y.; Huang, R. L.; Wu,
456
G. Y. Reduced expression of intestinal N-acetylglutamate synthase in suckling piglets:
457
a novel molecular mechanism for arginine as a nutritionally essential amino acid for
458
neonates. Amino. Acids. 2011, 40, 1513- 1522.
459
(21) Liu, Z. Q.; Geng, M. M.; Shu, X. G.; Wang, W. C.; Yin, Y. L. Dietary NCG
460
supplementation enhances the expression of N-acetylglutamate synthase in intestine
461
of weaning pig. J. Food. Agric. Enviro. 2012, 10, 408-412.
462
(22) Gardner, D. K.; Pool, T. B.; Lane, M. Embryo nutrition and energy metabolism
463
and its relationship to embryo growth, differentiation, and viability. Semin. Reprod.
464
Med. 2000, 18, 205-18.
465
(23) Guerin, P.; El, M. S.; Menezo, Y. Oxidative stress and protection against reactive
466
oxygen species in the pre-implantation embryo and its surroundings. Hum. Reprod.
467
Update. 2001, 7, 175-189. 20
ACS Paragon Plus Environment
Page 20 of 36
Page 21 of 36
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468
(24) Trocino, R. A.; Akazawa, S.; Ishibashi, M.; Matsumoto, K.; Matsuo, H.;
469
Yamamoto, H.; Goto, S.; Urata, Y.; Kondo, T.; Nagataki, S. Significance of
470
glutathione depletion and oxidative stress in early embryogenesis in glucose-induced
471
rat embryo culture. Diabetes. 1995, 44, 992-998.
472
(25) Yin, J.; Ren, W.; Yang, G.; Duan, J.; Huan, X.; Fang, R.; Li, C.; Yin, Y.; Hou, Y.;
473
Kim, S. W.; Wu, G. L-Cysteine metabolism and its nutritional implications. Mol. Nutr.
474
Food. Res. 2016, 60, 134-146.
475
(26) Leber, A.; Zenclussen, M. L.; Teles, A.; Brachwitz, N.; Casalis, P.; El, M. T.;
476
Jensen, F.; Woidacki, K.; Zenclussen, A. C. Pregnancy: tolerance and suppression of
477
immune responses. Methods. Mol. Biol. 2011, 677, 397-417.
478
(27) Roudebush, W. E.; Wininger, J. D.; Jones, A. E.; Wright, G.; Toledo, A. A.; Kort,
479
H. I.; Massey, J. B.; Shapiro, D. B. Embryonic platelet-activating factor: an indicator
480
of embryo viability. Hum. Reprod. 2002, 17, 1306-1310.
481
(28) Grosso, M. C.; Bellingeri, R. V.; Motta, C. E.; Alustiza, F. E.; Picco, N. Y.;
482
Vivas, A. B. Immunohistochemical distribution of early pregnancy factor in ovary,
483
oviduct and placenta of pregnant gilts. Biotech. Histochem. 2015, 90, 14-24.
484
(29) Lash, G. E.; Legge, M. Induction of early pregnancy factor activity in vitro by
485
platelet-activating factor in mice. J. Reprod. Fertil. 1997, 109, 187-191.
486
(30) Luciano, A. M.; Lodde, V.; Franciosi, F.; Ceciliani, F.; Peluso, J. J. Progesterone
487
receptor membrane component 1 expression and putative function in bovine oocyte
488
maturation, fertilization, and early embryonic development. Reproduction. 2010, 140,
489
663-672.
490
(31) Peluso, J. J. Multiplicity of progesterone's actions and receptors in the
491
mammalian ovary. Biol. Reprod. 2006, 75, 2-8.
492
(32) Rohe, H. J.; Ahmed, I. S.; Twist, K. E.; Craven, R. J. PGRMC1 (progesterone 21
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
493
receptor membrane component 1): a targetable protein with multiple functions in
494
steroid signaling, P450 activation and drug binding. Pharmacol. Ther. 2009, 121,
495
14-9.
496
(33) Peluso, J. J.; Romak, J.; Liu, X. Progesterone receptor membrane component-1
497
(PGRMC1) is the mediator of progesterone’s antiapoptotic action in spontaneously
498
immortalized granulosa cells as revealed by PGRMC1 small interfering ribonucleic
499
acid treatment and functional analysis of PGRMC1 mutations. Endocrinology. 2008,
500
149, 534-54.
501
(34) Kowalik, M. K.; Slonina, D.; Rekawiecki, R.; Kotwica, J. Expression of
502
progesterone receptor membrane component (PGRMC) 1 and 2, serpine mRNA
503
binding protein 1 (SERBP1) and nuclear progesterone receptor (PGR) in the bovine
504
endometrium during the estrous cycle and the first trimester of pregnancy. Reprod.
505
Biol. 2013, 13, 5-23.
506
(35) Zhang, L.; Kanda, Y.; Roberts, D. J.; Ecker, J. L.; Losel, R.; Wehling, M.; Peluso,
507
J. J.; Pru, J. K. Expression of progesterone receptor membrane component 1 and its
508
partner serpine 1 mRNA binding protein in uterine and placental tissues of the mouse
509
and human. Mol. Cell. Endocrinol. 2008, 287, 81-89.
510
(36) Rosselli, M.; Keller, R. J.; Dubey, R. K. Role of nitric oxide in the biology,
511
physiology and pathophysiology of reproduction. Hum. Reprod. Update. 1998, 4,
512
3-24.
513
(37) Hansson, S. R.; Nääv, Å.; Erlandsson, L. Oxidative stress in preeclampsia and
514
the role of free fetal hemoglobin. Front. Physiol. 2015, 5, 516.
515
(38) Krause, B. J.; Hanson, M. A.; Casanello, P. Role of nitric oxide in placental
516
vascular development and function. Placenta. 2011, 32, 797-805.
517
(39) Biswas, S.; Kabir, S. N.; Pal, A. K. The role of nitric oxide in the process of 22
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Page 23 of 36
Journal of Agricultural and Food Chemistry
518
implantation in rats. J. Reprod. fertil. 1998, 114, 157-161.
519
(40) Pallares, P.; Garcia, F. R.; Criado, L. M.; Letelier, C. A.; Esteban, D.; Fernandez,
520
T. J.; Flores, J. M.; Gonzalez, B. A. Disruption of the endothelial nitric oxide synthase
521
gene affects ovulation, fertilization and early embryo survival in a knockout mouse
522
model. Reproduction. 2008, 136, 573-579.
523
(41) Wu, G.; Bazer, F. W.; Wallace, J. M.; Spencer, T. E. Board-invited review:
524
intrauterine growth retardation: implications for the animal sciences. J. Anim. Sci.
525
2006, 84, 2316-2337
526
(42) Chernoivanenko, I. S.; Minin, A. A.; Minin, A. A. Role of vimentin in cell
527
migration. Ontogenez. 2013, 44, 186-202.
528
(43) Katz, S. G. Extracellular breakdown of collagen by mice decidual cells. A
529
cytochemical and ultrastructural study. Biocell. 2005, 29, 261-270.
530
(44) Katz, S.G. Extracellular and intracellular degradation of collagen by trophoblast
531
giant cells in acute fasted mice examined by electron microscopy. Tissue. Cell. 1995,
532
27, 713-721.
533
(45) Welsh, A. O.; Enders, A. C. Chorioallantoic placenta formation in the rat: II.
534
Angiogenesis and maternal blood circulation in the mesometrial region of the
535
implantation chamber prior to placenta formation. Am. J. Anat. 1991, 192, 347-365.
536
(46) Scherholz, P. L.; Souza, D. P.; Spadacci, M. D.; Katz, S. G. Vimentin is
537
synthesized by mouse vascular trophoblast giant cells from embryonic day 7.5
538
onwards and is a characteristic factor of these cells. Placenta. 2013, 34, 518-525.
539
(47) De, S. P.; Katz, S. G. Coexpression of cytokeratin and vimentin in mice
540
trophoblastic giant cells. Tissue. Cell. 2001, 33, 40-45.
541
(48) Eckersley, M. M.; Bergmann, J. H.; Lazar, Z.; Spector, D. L. Lamin A/C is
542
expressed in pluripotent mouse embryonic stem cells. Nucleus. 2013, 4, 53-60. 23
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
543
(49) Lee, J. S.; Hale, C. M.; Panorchan, P.; Khatau, S. B.; George, J. P.; Tseng, Y.;
544
Stewart, C. L.; Hodzic, D.; Wirtz, D. Nuclear lamin A/C deficiency induces defects in
545
cell mechanics, polarization, and migration. Biophys. J. 2007, 93, 2542-2552
546
(50) Rober, R. A.; Weber, K.; Osborn, M. Differential timing of nuclear lamin A/C
547
expression in the various organs of the mouse embryo and the young animal: a
548
developmental study. Development. 1989, 105, 365-378.
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Table 1 Composition and nutrient composition of the basal diet for gestating sows
550
(as-fed basis) Item
Content
Ingredients, % Corn grain
66.10
Wheat bran
19.50
Soybean meal, 43%
10.00
Soybean oil
0.50
Limestone
0.60
Dicalcium phosphate
2.10
Salt
0.40
L-Lysine hydrochloride
0.10
DL-Methionine
0.05
Choline chloride
0.15
Vitamin mineral premix1
0.50
Nutrient levels, % Dry matter2
89.60
Digestible energy3, Mcal/kg
3.07
Metabolizable energy3, Mcal/kg
2.93
Crude protein2
13.10
Calcium2
0.79
Available phosphorus3
0.49
Total phosphorus3
0.69
Lysine3
0.64
Arginine3
0.66
Methionine + cystine3
0.50
551
1
552
cholecalciferol, 0.04; d-α-tocopheryl acetate, 10.10; menadione sodium bisulfate, 1.60;
553
thiamine, 1.50; riboflavin, 3.00; vitamin B-6, 1.50; vitamin B-12, 0.01; niacin, 22.50;
Provided the following (mg/kg of the basal diet): retinyl acetate, 3.47;
25
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554
calcium pantothenate, 15.00; folic acid, 2.50; biotin, 0.20; manganese, 40.00; iron,
555
85.00; zinc, 75.00; copper, 1.50; iodine, 0.09; and selenium, 0.03.
556
2
Analyzed values.
557
3
Calculated values.
26
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Table 2 Effects of N-Carbamylglutamate (NCG) supplementation on reproductive
559
performance in sows Treatment1
SEM2 P value
Items Weight at breeding (kg)
Control
NCG 1 NCG 2 NCG 3
216.78
215.00
204.72
207.88
10.91
0.85
225.17
222.81
213.11
217.24
10.93
0.87
8.39
7.81
8.39
9.35
0.62
0.40
4.00
3.88
4.00
3.83
0.46
0.99
11.31ab 11.67ab 12.18a
0.46
0.04
10.70a
0.27
0.02
Weight at day 28 of gestation (kg) Weight gain through d 28 of gestaion(kg) Parity Total piglets born per litter Total piglets born alive per
11.11b 9.61b
9.81ab 10.39a
1.50
1.50
1.33
1.53
0.20
0.89
13.32
13.13
11.30
12.43
1.58
0.80
13.73
14.38
15.15
15.43
0.62
0.23
1.44
1.45
1.43
1.40
0.05
0.94
litter Stillborn and mummified (n) Fetal mortality (%)3 Litter birth weight of all piglets born alive (kg) Weight of piglets (kg) 560
Note: 1the numbers of sows for Control group, NCG 1, NCG 2 and NCG 3 were 18,
561
18, 16, and 17, respectively. a, b within a row, significant difference at P < 0.05.
562
2
SEM, Standard error of the mean.
563
3
Fetal mortality = number of stillborn and mummified piglets/number of total
564
piglets born per litter.
27
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565
Table 3 Serum metabolites that differed between control and N-Carbamylglutamate
566
groups Metabolite
Fold
VIP
P
change1
score
value
3.31
2.90
0.02
Related pathway Cysteine and methionine
Indoleacrylic acid
metabolism Isohyodeoxycholic acid
3.23
2.42
0.00
Platelet activating factor
2.75
1.72
0.05
Lipid metabolism Immunological regulation
Isofucosterol 3-O-6-O-9,12-octadecadi 2.69
2.27
0.01
Lipid metabolism
2.64
1.94
0.01
Hydrolysis of xenobiotic
enoyl -b-d-glucopyranoside Diisobutyl phthalate
Energy metabolism and Retapamulin
2.42
2.77
0.02
immunological regulation
Polysorbate 60
2.25
1.99
0.01
Fatty acid metabolism
Oleamide
2.20
1.65
0.02
Energy metabolism
Benzyl isothiocyanate
2.05
1.74
0.04
Glucosinolate breakdown
Pyridostigmine
2.09
1.70
0.03
Cholinesterase Inhibitors
1.89
1.35
0.04
Energy metabolism
1.88
1.56
0.02
3-cis-hydroxy-b,e-carote n-3'-one 3-hydroxyphenylpyruvic
Tyrosine catabolism
acid
pathway Phenylalanine
Tiropramide
1.84
1.73
0.00 metabolism
Cholesteryl acetate
1.53
1.47
0.04
Steroid biosynthesis
3alpha-acetomethoxy-11
1.60
1.52
0.04
Energy metabolism
28
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alpha-oxo-12-ursen-24-o ic acid Dilauryl 0.41
1.89
0.04
Redox reaction
0.32
1.76
0.04
Redox reaction
0.43
2.85
0.02
Purine metabolism
3,3'-thiodipropionate O-tyrosine Guanosine hexaphosphate adenosine 567
Note: 1Fold change, which was based on the normalized data, was defined as the fold
568
difference in the observed concentrations between NCG group and control group.
569
2
570
markers for distinguishing the two groups. The higher the score is, the more
571
important the metabolite is.
VIP, variable in project, was used to select distinct variables as potential
29
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572
Table 4 Metabolites that differed in amniotic fluid between control and
573
N-Carbamylglutamate groups Fold
VIP
P
change1
score
value
12- ketodeoxycholic acid
6.15
3.67
0.00
Fatty acid metabolism
Diisobutyl phthalate
4.30
2.70
0.00
Hydrolysis of xenobiotic
Beta-citryl-l-glutamic acid
3.14
2.57
0.03
Glutathione metabolism
Cladribine
2.92
2.00
0.02
Metabolite name
Related pathway
Immunological suppression Ethyl aconitate
2.41
2.20
0.01
Pristanal
2.15
2.05
0.01
Tricarboxylic acid cycle Oxidation of branched chain fatty acids
Lysopc100
1.94
1.94
0.03
Energy metabolism
9-hexadecenoylcholine
1.77
1.60
0.03
Fatty acid metabolism
1.75
2.76
0.04
1-Pyrroline-4-hydroxy-2-car
Arginine and proline
boxylate
metabolism
Polyoxyethylene 600 1.54
1.50
0.02
Energy metabolism
Benzyl thiocyanate
0.49
1.88
0.02
Cysteine metabolism
Pyroglutamic acid
0.49
2.26
0.02
Glutathione metabolism
Umbelliferone
0.55
1.89
0.03
monoricinoleate
Biosynthesis of phenylpropanoids Metabolism of 2-Nonadecanone
0.45
2.12
0.05 carbohydrates
1
574
Note: Fold change, which was based on the normalized data, was defined as the fold
575
difference in the observed concentrations between NCG group and control group.
576
2
VIP, variable in project, was used to select distinct variables as potential
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markers for distinguishing the two groups. The higher the score is, the more
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important the metabolite is.
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Figure legends
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Figure1. Principal component analysis (PCA). Each point represents an individual
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sample. (A) PCA of the profiling data from the serum metabolome. (B) PCA of the
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profiling data from amniotic fluid metabolome. Gilts were fed the control or 0.05%
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N-Carbamylglutamate supplemented diets during d 1-28 of pregnancy.
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Figure 2. The protein abundance of PGRMC1, eNOS, and lamin A/C expression in
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placentae (A), fetuses (B), and endometriums (C) of gilts on d 28 of pregnancy. Gilts
586
were fed the control or 0.05% N-Carbamylglutamate supplemented diets during d
587
1-28 of pregnancy. On day 28 of gestation, the placenta and endometrium were
588
carefully isolated from the uterus of individual fetuses. All tissue samples were
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immediately snap frozen in liquid nitrogen and stored at -80℃.
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Figure 3. The protein abundance of PGRMC1, eNOS, lamin A/C, and vimentin in
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poecine Tr cells treated with arginine (A), glutamine (B), glutamate (C), and proline
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(D). Cells were serum starved overnight in customized medium, deprived of either
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arginine, proline, glutamate or glutamine, and then treated with different doses (0,
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0.25, 0.50, 1.00 mM) of Arg, Pro, Glu or Gln for 24 h. All experiments were repeated
595
at least 2-3 times.
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Figure 1
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Figure 2
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Figure 3
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TOC Graphic
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