§The current address of Xilong Li is Institute of Feed Research, The Chinese Academy of Agricultural Sciences, 100081 Beijing, China.
The current address of Huaijun Zhou is Department of Animal Science, University of California at Davis, Davis, CA 95616, USA.
Academic Editor: Graham Pawelec
Background: Increasing the dietary provision of L-arginine to pregnant swine beginning at Day 14 of gestation enhances embryonic survival, but the underlying mechanisms are largely unknown. Objective: This study determined the effects of dietary supplementation with 0.8% L-arginine to gilts between Days 14 and 25 of gestation on the global expression of genes in their placentae. Methods: Between Days 14 and 24 of gestation, gilts were fed 2 kg of a corn- and soybean meal-based diet (containing 12.0% crude protein and 0.70% Arg) supplemented with 0.8% L-arginine or without L-arginine (0.0%; with 1.64% L-alanine as the isonitrogenous control). On Day 25 of gestation, 30 min after the consumption of their top dressing containing 8 g L-arginine or 16.4 g L-alanine, gilts underwent hysterectomy to obtain placentae, which were snap-frozen in liquid nitrogen. Total RNAs were extracted from the frozen tissues and used for microarray analysis based on the 44-K Agilent porcine gene platform. Results: L-Arginine supplementation affected placental expression of 575 genes, with 146 genes being up-regulated and 429 genes being down-regulated. These differentially expressed genes play important roles in nutrient metabolism, polyamine production, protein synthesis, proteolysis, angiogenesis, immune development, anti-oxidative responses, and adhesion force between the chorioallantoic membrane and the endometrial epithelium, as well as functions of insulin, transforming growth factor beta, and Notch signaling pathways. Conclusion: Dietary supplementation with L-arginine plays an important role in regulating placental gene expression in gilts. Our findings help to elucidate mechanisms responsible for the beneficial effect of L-arginine in improving placental growth and embryonic/fetal survival in swine.
There is growing interest in the nutritional role of L-arginine (Arg) to enhance litter size in livestock species [1, 2, 3]. However, only a few studies have been conducted to explore the underlying mechanisms [4, 5, 6]. Thus, there is a limited understanding of regulatory functions of Arg in the placenta. Results of recent studies indicated that Arg is not only a building block for proteins, but also has multiple physiological roles in cell signaling and function [7, 8]. For example, Arg stimulates the production of nitric oxide (NO) and polyamines (key regulators of cell growth and development) by placental cells [9, 10], as well as the placental expression of aquaporins and the transport of water across the placentae [6]. In addition, Arg may influence the expression of genes related to amino acid transport, anti-oxidative responses, and protein synthesis in mammalian cells [9, 11]. As an approach to understanding how Arg acts on the placentae at the gene level, we used the 44-K Agilent porcine gene platform to determine changes in global gene expression in placentae at Day 25 of gestation from gilts receiving dietary Arg supplementation between Days 14 and 25 of gestation. This nutritional method is effective in enhancing placental growth and embryonic survival in swine [12].
The experimental design, including the diets of gilts before and after breeding,
has been described by Li et al. [12]. Briefly, gilts (F1 crosses of
Yorkshire
Each placenta was obtained from a live fetus. A portion of the placenta was
immediately snap-frozen in liquid nitrogen. All snap-frozen samples were stored
at –80
Total RNA was isolated from the frozen placenta (approximately 30 mg) according to the manual of the RNeasy Mini Kit (Qiagen Inc., Valencia, CA) [4]. The quantity of the total RNA was measured by NanoDrop 1000 Spectrophotometer (Thermo Scientific, USA). The quality of total RNA was determined by 1% agarose electrophoresis. In addition, we determined the ratio of absorbance at 260 nm and 280 nm, which was used to assess the purity of RNA, was approximately 2.0 for the total RNA isolated from porcine placentae. The total RNA from 3 placentae from each gilt was combined at equal quantity to represent one biological replicate, and there were 8 biological replicates for each treatment group in the following microarray analysis.
Total RNA (400 ng) was reverse-transcribed to cDNA. T7 RNA polymerase-driven RNA
synthesis was used for the preparation and labeling of cRNA with Cy3 or Cy5 dye.
In each treatment group, 4 samples were treated with the Cy3 (green) dye, and 4
samples were treated with the Cy5 (red) dye. The labeled cRNA probes were
purified with the RNeasy Mini Kit (Qiagen Inc., Valencia, CA). Purified cRNA was
quantified with the NanoDrop 1000, and 825 ng of each was hybridized on the 44-K
Agilent porcine gene expression microarray (Agilent, Santa Clara, CA). This array
included 43,803 probes that were prepared using gene sources from RefSeq,
UniGene, and TIGR. The slide format was printed using the Agilent’s 60-mer
SurePrint technology. The hybridized slides were washed according to the manual
of a commercial kit (Agilent Technology, Palo Alto, CA), followed by scanning
with a Genepix 4100A scanner (Molecular Devices Corporation, Sunnyvale, CA) with
the tolerance of saturation setting of 0.005%. A locally weighted linear
regression (LOWESS) method was applied to normalize the data by the median of the
signal intensity and local background values. SAS 9.1.3 program (SAS Institute
Inc. Cary, NC) for the mixed model was used to analyze the normalized data [13].
Statistical significance to detect differentially expressed genes was determined
by the approximate t-test for least-square means, where p
GO terms for biological processes (GO_TERM_BP) and KEGG pathways were
identified for differentially expressed genes (both up- and down-regulated genes)
using the database for DAVID version 6.8 [15, 16]. Ensembl gene IDs were
converted to official gene symbols for input into DAVID using Ensembl’s Biomart,
which is an open-source software and data service to the international scientific
community (https://m.ensembl.org/info/data/biomart/index.html). Significance
cutoff was p
Total RNA (1
Accession No. | Gene | Primer sequence | Product length (bp) | Annealing Temp. ( |
NM_001001861 | CXCL2 | Forward: 5′- CACTGTGACCAAACGGAA -3′ | 120 | 53 |
Reverse:5′- GTTGGCACTGCTCTTGTTT-3′ | ||||
NM_214003 | IGFBP2 | Forward: 5′- GTGGATGGGAACGTGAACTT-3′ | 111 | 56.8 |
Reverse:5′- GTGCTGCTCCGTGACTTTCT-3′ | ||||
TC267605 | PFKFB1 | Forward: 5′- GCCTAAGATGACTCAAGAGA-3′ | 187 | 53.3 |
Reverse:5′- CGTGGAGATGTAGGTCTTT-3′ | ||||
NM_213963 | PPARGC | Forward: 5′- AACCCACAGAGACCCGAAAC-3′ | 82 | 53 |
Reverse:5′- AAATGTTGCGACTGCGATTG-3′ | ||||
AK231515 | Presenilin 2 | Forward: 5′- AAGGAGCACAGCGGACTCT-3′ | 299 | 57 |
Reverse:5′- TGGGTACTGAACGGGTGTTT-3′ | ||||
TC275071 | RAG-2 | Forward: 5′- ATGCCAGATCCTTAACCCAC-3′ | 82 | 53 |
Reverse:5′- GCAGCAGAAATGAATCCAAC-3′ | ||||
BI341657 | RasGEF | Forward: 5′- CTCCCATCTACAGCGAGGAA-3′ | 104 | 56 |
Reverse: 5′- GAGCGTGGTCCTGAGGGTCT-3′ | ||||
TC243513 | RHBG | Forward: 5′- GTGCCTACTTTGGGTTGGTC-3′ | 103 | 56 |
Reverse:5′- ATGGCAAAGAGGTCCGAATG-3′ | ||||
TC257543 | RU2S | Forward: 5′- CACTTCTGGAACCCTGCACT-3′ | 103 | 53 |
Reverse:5′- TGATCCCACTGATTCAAGGC-3′ | ||||
NM_001001863 | TNNT3 | Forward: 5′- CCTGTACCARCTGGAGATTG-3′ | 78 | 51 |
Reverse: 5′- CTGAGGTTGATGATGTCGTA-3′ | ||||
DQ225365 | Tubulin |
Forward: 5′-GCAGTGTTTGTAGACCTG GA-3′ | 139 | 55 |
Reverse:5′-CAATGGTGTAGTGACCTCGG-3′ | ||||
EU288086 | MTOR | Forward: 5′- GTCTCTATCAAGTTGCTGGC-3′ | 126 | 53 |
Reverse: 5′- CTTTCGAGATGGCAATGGAA-3′ | ||||
NM_001012613 | SLC7A1 | Forward: 5′- ACTCGACTCTCGTGGACCTT-3′ | 134 | 54 |
Reverse:5′ GGTCAGTTGACTTTCTGCCT-3′ | ||||
Primers were prepared using the oligo6 program (www.oligo.net). |
All samples were run in triplicate and the average critical threshold cycle (Ct)
was used to calculate the relative mRNA levels of target genes by the
2
Data were analyzed by the unpaired t-test using the SPSS (Version 15.0,
Chicago, IL). Gilt was considered as the experimental unit. Probability values
One hundred and forty-six (146) expressed sequence tags (ESTs) were up-regulated (Supplementary Table 1) and 429 ESTs were down-regulated (Supplementary Table 2) in response to dietary supplementation with 0.8% Arg between Days 14 and 25 of gestation. Some of the up-regulated and down-regulated genes with known physiological functions are summarized in Tables 2 and 3, respectively. Among the up-regulated genes in the placentae of Arg-supplemented gilts, the mRNA level of troponin T type 3 (TNNT3) was the greatest, followed by leucine-rich repeat-containing protein 51-like, calcitonin receptor, presenilin 2, ceroid-lipofuscinosis, and leucine-rich repeat-containing protein 18-like in descending order. Among the down-regulated genes in the placentae of Arg-supplemented gilts, the reduction in the placental mRNA for cytochrome b was the greatest, followed by Ras GEF domain 1A-similar gene, probable dolichyl pyrophosphate GMGGT-like gene, doublecortin domain-containing protein 2 (RU2S), acetyl-coenzyme A carboxylase alpha, and pig endogenous retrovirus group beta3 polymerase in descending order.
Expressed sequence tag (EST; gene ID) | Accession No. | Gene name | Fold change | p-Value |
BX918610 | NM_001001863 | Troponin T type 3 (TNNT3) | 4.61 | 0.004 |
TC292911 |
XM_003129590 | Leucine-rich repeat-containing protein 51-like | 4.49 | 0.001 |
EW039857 | NM_001742 | Calcitonin receptor (CALCR) on chromosome 7 | 3.23 | 0.038 |
AK231515 | EU287432 | Presenilin 2 (PSEN2) | 2.31 | 0.006 |
TC278497 |
NM_018941 | Ceroid-lipofuscinosis, neuronal 8 | 2.23 | 0.010 |
TC289044 |
XM_001929300 | Sus scrofa leucine-rich repeat-containing protein 18-like | 2.10 | 0.020 |
PGM1 | NM_001076903 | Phosphoglucomutase 1 (PGM1) | 1.86 | 0.030 |
TC275071 |
NM_000536 | Recombination activating gene 2 (RAG-2) | 1.76 | 0.006 |
TC275071 |
AB091391 | Recombination activating gene 2 (RAG-2) | 1.76 | 0.006 |
TC274023 |
NM_001097446 | Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3F (APOBEC3F) | 1.70 | 0.005 |
BX666795 | XM_001924347 | Similar to solute carrier organic anion transporter family member 3A1 (SLCO3A1) | 1.67 | 0.003 |
TC278155 |
NM_214378 | Rh blood group polypeptide | 1.66 | 0.019 |
TC267605 | NM_001143721 | 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 (PFKFB1) | 1.55 | 0.025 |
EW660666 | NM_001045886 | Phenazine biosynthesis-like protein domain containing | 1.54 | 0.013 |
TC246855 |
AY208121 | Myostatin | 1.54 | 0.022 |
AY610045 | XM_001924474 | Similar to androgen-induced 1 | 1.42 | 0.018 |
DY428406 | NG_016762 | Pyruvate dehydrogenase kinase isozyme 3 (PDK3) | 1.40 | 0.042 |
CF361829 | A9YMB8 | NADH dehydrogenase subunit 2 | 1.34 | 0.012 |
AJ947745 | NG_007956 | Cytochrome P450 family 20 subfamily A polypeptide 1 (CYP20A1) | 1.33 | 0.022 |
TC261962 |
EW422073 | Hemoglobin subunit epsilon 1 (HBE1) | 1.31 | 0.035 |
AK234630 | XM_001927389 | FK506 (Tacrolimus)-binding protein | 1.28 | 0.009 |
AJ584674 | NM_213757 | Beta-Galactoside alpha-2,3-sialyltransferase 4 (ST3GAL4) | 1.27 | 0.000 |
BW980922 | XM_001113023 | dUTP pyrophosphatase isoform 2 transcript variant 4 (DUT) | 1.27 | 0.021 |
AK239509 | AB529869 | Peroxisomal trans-2-enoyl-CoA reductase (PECR) | 1.25 | 0.027 |
BX667232 | XM_001925672 | Similar to pecanex-like protein 1 | 1.23 | 0.030 |
CN155716 | EU617320 | Small calcium-binding mitochondrial carrier 1 | 1.23 | 0.038 |
EV880225 | DQ629170 | Ribosomal protein S6 (RPS6) | 1.22 | 0.017 |
CK467702 | NM_001035277 | Cadherin 13 (CDH13) | 1.22 | 0.013 |
CD572284 | AJ009912 | Proteolipid protein (PLP) | 1.21 | 0.006 |
TC258084 | NM_006690 | Matrix metallopeptidase 24 (MMP24) | 1.19 | 0.045 |
DN125568 | GQ184633 | Cell division cycle 2 (CDC2) | 1.18 | 0.048 |
EW299999 | XM_001498308 | Similar to eukaryotic translation elongation factor 1 beta 2 (EF1 β2) | 1.17 | 0.015 |
TC258796 | XM_001928025 | Calcineurin A protein transcript variant 2 | 1.14 | 0.049 |
SCYE1 | NM_001114283 | Aminoacyl tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1) | 1.12 | 0.026 |
*Sequence can be accessed on http://compbio.dfci.harvard.edu/cgi-bin/tgi. |
Expressed sequence tag (EST; gene ID) | Accession No. | Gene name | Fold change | p-Value |
AJ964783 | O48246 | Cytochrome b | 0.15 | 0.001 |
BI341657 | XM_001926447 | RasGEF domain 1A | 0.18 | 0.013 |
TC273367 |
XM_003129699 | Probable dolichyl pyrophosphate GMGGT-like | 0.20 | 0.010 |
TC257543 |
XM_001927988 | Doublecortin domain-containing protein 2 (RU2S) | 0.23 | 0.015 |
DN100844 | FJ263680 | Acetyl-coenzyme A carboxylase alpha | 0.27 | 0.003 |
NP321728 | AF274712 | Pig endogenous retrovirus group Beta3 polymerase | 0.29 | 0.014 |
BI360386 | XM_003133904 | Oncostatin-M-specific receptor subunit beta-like | 0.31 | 0.009 |
TC238637 |
NM_214376 | Amphiregulin | 0.31 | 0.045 |
CF178669 | AJ427478 | Agouti signaling protein | 0.33 | 0.023 |
CX061534 | XM_003130350 | Torsin-1A-interacting protein 1-like | 0.40 | 0.007 |
TC301037 |
XM_003357826 | Serine/threonine-protein kinase [doublecortin like kinase 1 (DCLK1)]-like | 0.42 | 0.012 |
TC243513 |
NM_213996 | Rh family, B glycoprotein (RHBG) | 0.45 | 0.006 |
DN106254 | NM_001098597 | Osteocrin (OSTN) | 0.47 | 0.025 |
AY577905 | NM_001001861 | Chemokine (C-X-C motif) ligand 2 (CXCL2) | 0.49 | 0.013 |
TC278652 |
NM_214003 | Insulin-like growth factor binding protein 2 | 0.49 | 0.002 |
BP443132 | XM_864245.3 | Cytochrome P450 family 2 subfamily C member 33 (CYP2C33) | 0.50 | 0.037 |
AY198323 | NM_214257 | Dipeptidyl peptidase 4 (DPP4) | 0.51 | 0.030 |
TC280345 |
XM_003122165 | Golgin A1 | 0.51 | 0.018 |
TC290654 |
NM_001105290 | Bone morphogenetic protein 7 (Bmp7) | 0.55 | 0.030 |
CO989438 | XM_001928917 | Potassium large conductance calcium-activated channel, subfamily M, beta member 4 | 0.56 | 0.017 |
DQ836054 | NM_001097442 | Disabled-1(DAB1) | 0.57 | 0.021 |
TC270858 |
AF228059 | Decay-accelerating factor CD55 | 0.58 | 0.026 |
CV878027 | XM_001926796 | Sterile alpha motif domain containing 4A (SAMD4A) | 0.58 | 0.018 |
TC290589 |
XM_003132094 | Upstream binding protein 1 | 0.58 | 0.005 |
CA513725 | XM_003129205 | Heat shock 70kDa protein 4-like | 0.58 | 0.016 |
EV881857 | XM_003132080 | Sodium bicarbonate cotransporter 3-like | 0.59 | 0.009 |
TC266622 |
XM_003127574 | Methylenetetrahydrofolate reductase (NAD(P)H), transcript variant 1 | 0.60 | 0.018 |
TC286353 |
NM_001243919 | Coupling of ubiquitin conjugation to ER degradation (CUE) domain containing 1 | 0.60 | 0.007 |
TC250322 |
NM_001037965 | Inhibitor of DNA binding 2 | 0.61 | 0.007 |
CN159399 | NM_001128506 | Charged multivesicular body protein 4b-like | 0.61 | 0.012 |
AK230591 | NM_001128488 | Antizyme inhibitor 1 | 0.62 | 0.016 |
AK234300 | XM_003125957 | RIB43A-like with coiled-coils protein 2-like | 0.63 | 0.005 |
TC247541 |
XM_003134192 | Pericentriolar material 1 | 0.64 | 0.015 |
CF181641 | XM_003128338 | Dystonin, transcript variant 2 | 0.64 | 0.015 |
AK233736 | XM_001927836 | Similar to Down syndrome critical region gene 1-like 1 protein | 0.65 | 0.033 |
DQ866834 | DQ279926 | Retinoid X receptor alpha (RXRalpha) | 0.65 | 0.047 |
AB271924 | NM_001099924 | Fibroblast growth factor receptor 2 (FGFR2) | 0.68 | 0.019 |
AY850382 | NM_001011505 | Kruppel-like factor 13 (KLF13) | 0.68 | 0.006 |
AB116561 | NM_213772 | Interferon alpha and beta receptor subunit 1 (IFNAR1) | 0.69 | 0.012 |
TC248589 |
NM_001077215 | Regulator of differentiation 1 (ROD1) | 0.70 | 0.025 |
AY610204 | NM_214296 | Rho family GTPase 3 (RND3) | 0.70 | 0.039 |
BP142559 | XM_001926474 | A-kinase anchoring protein 13 (AKAP13) | 0.70 | 0.016 |
TC257240 |
XM_001925375 | Similar to positive regulatory (PR) domain containing 1, with ZNF domain transcript variant 2 | 0.71 | 0.042 |
AY284842 | AY284842 | Glycerol-3-phosphate acyltransferase (GPAT) | 0.71 | 0.016 |
AK235700 | NM_001078670 | Interferon regulatory factor 9 | 0.71 | 0.024 |
AK235466 | DQ105589S2 | CDP-Diacylglycerol Synthase 2 (CDS2) | 0.71 | 0.013 |
EU095967 | NM_001105286 | TNF receptor associated factor 6 (TRAF6) | 0.71 | 0.023 |
BP444119 | NM_214224 | 4-Hydroxyphenylpyruvate dioxygenase (HPD) | 0.72 | 0.007 |
AY159788 | NM_214266 | 5’-AMP-activated protein kinase catalytic subunit alpha-2 (PRKAA2) | 0.72 | 0.025 |
AK235681 | NM_213963 | Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC-1) | 0.72 | 0.032 |
AK240475 | XM_001927539 | Similar to general transcription factor IIH | 0.73 | 0.006 |
BP446317 | NM_001097440 | Bridging Integrator 1 (BIN1) | 0.73 | 0.036 |
CK461960 | NM_001162401 | lysophosphatidic acid receptor 2 (LPAR2) | 0.73 | 0.048 |
BI184146 | XM_001927725 | Prostaglandin F2 receptor inhibitor (PTGFRN) | 0.74 | 0.002 |
CV875504 | XM_001926134 | Similar to chloride channel 3 | 0.74 | 0.040 |
EU009401 | NM_001098605 | Patatin-like phospholipase domain containing 2 (PNPLA2) | 0.74 | 0.014 |
TC261381 | NM_213973 | Heat-shock protein 90 (HSP90) | 0.75 | 0.036 |
AK233668 | NM_213830 | Folate-binding protein (FBP) | 0.75 | 0.029 |
AY609622 | AY609622 | Similar to small nuclear RNA activating complex | 0.76 | 0.037 |
TC299692 | NM_001025107 | Homo sapiens adenosine deaminase RNA-specific (ADAR) | 0.76 | 0.046 |
DY420532 | NM_017902 | Homo sapiens hypoxia inducible factor 1 alpha subunit inhibitor (HIF1AN) | 0.76 | 0.047 |
AB254406 | NM_001101814 | Nuclear receptor subfamily 1 group H member 3 (NR1H3) | 0.77 | 0.028 |
DN120475 | XM_001927228 | Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein | 0.77 | 0.013 |
AY644721 | NM_001009581 | Peripherial benzodiazepine receptor associated protein (PAP7) | 0.78 | 0.037 |
AJ955195 | XM_001929149 | Similar to transmembrane protein 77 | 0.79 | 0.036 |
AK237448 | XM_001928092 | Similar to Rab-1C | 0.79 | 0.033 |
AK234427 | XM_001928746 | Similar to adenosine deaminase-like protein | 0.79 | 0.046 |
TC278200 |
XM_001925656 | Similar to procollagen | 0.79 | 0.038 |
AK235686 | XM_001925381 | Similar to insulin-degrading enzyme | 0.80 | 0.016 |
AK237044 | XM_001499279 | Similar to ubiquitin-conjugating enzyme E2Z | 0.80 | 0.034 |
AK232486 | NM_001159481 | Pyruvate dehydrogenase kinase isozyme 2 (PDK2) | 0.81 | 0.042 |
DN100853 | AF339885 | Mannose-6-phosphate/insulin-like growth factor II receptor | 0.81 | 0.038 |
*Sequence can be accessed on http://compbio.dfci.harvard.edu/cgi-bin. CDP, cytosine diphosphate; RasGEF, Ras (rat sarcoma protein p21) guanine nucleotide exchange factor; GMGGT, Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase; RIB43A, ribbon protofilament protein 43A (43-kDa protein); TNF, tumor necrosis factor; ZNF, zinc finger. |
The placental expression of mRNAs for the following enzymes or proteins related
to amino acid metabolism did not differ (p
Dietary supplementation with 0.8% Arg did not affect (p
The functional analysis by the DAVID program revealed that the genes with altered expression are related to nutrient transport, protein synthesis, protein degradation, polyamine synthesis, ion transport, glucose metabolism, fatty acid biosynthesis, immune development, inflammation, and anti-oxidative responses, as well as insulin, transforming growth factor beta, and Notch signaling pathways (Table 4). Changes in metabolic pathways were associated with alterations in the expression of single genes or a group of related genes.
Gene name | Species | Database | Pathway |
5,10-methylenetetrahydrofolate reductase (NADPH) | Homo sapiens | KEGG_PATHWAY | hsa00670:One carbon pool by folate |
hsa00680:Methane metabolism | |||
Acetyl-coenzyme A carboxylase alpha | Homo sapiens | KEGG_PATHWAY | hsa00061:Fatty acid biosynthesis |
hsa00620:Pyruvate metabolism | |||
hsa00640:Propanoate metabolism | |||
hsa04910:Insulin signaling pathway | |||
Asparagine-linked glycosylation 8, alpha-1,3-glucosyltransferase homolog (S. cerevisiae) | Homo sapiens | KEGG_PATHWAY | hsa00510:N-Glycan biosynthesis |
Chemokine (C-X-C motif) ligand 2 | Sus scrofa | KEGG_PATHWAY | ssc04062:Chemokine signaling pathway |
Chromatin modifying protein 4B; similar to LOC616164 protein | Bos taurus | KEGG_PATHWAY | bta04144:Endocytosis |
Inhibitor of DNA binding 2 | Sus scrofa | KEGG_PATHWAY | ssc04350:TGF-beta signaling pathway |
Oncostatin M receptor | Homo sapiens | KEGG_PATHWAY | hsa04060:Cytokine-cytokine receptor interaction |
hsa04630:Jak-STAT signaling pathway | |||
Potassium large conductance calcium-activated channel, subfamily M, beta member 4 | Sus scrofa | KEGG_PATHWAY | ssc04270:Vascular smooth muscle contraction |
Presenilin 2 | Sus scrofa | KEGG_PATHWAY | ssc04330:Notch signaling pathway |
ssc05010:Alzheimer’s disease | |||
Recombination activating gene 2 | Sus scrofa | KEGG_PATHWAY | ssc05340:Primary immunodeficiency |
Ribosomal protein S6 (RPS6) | Sus scrofa | KEGG_PATHWAY | Protein synthesis |
Protein for ubiquitin conjugation | Sus scrofa | KEGG_PATHWAY | Protein degradation |
Antizyme inhibitor 1 | Sus scrofa | KEGG_PATHWAY | Polyamine synthesis |
Troponin T | Sus scrofa | KEGG_PATHWAY | Cell growth and development |
Cadherin 13 | Sus scrofa | KEGG_PATHWAY | Cell–cell adhesion in tissues |
Organic anion transporter | Sus scrofa | KEGG_PATHWAY | Transport of organic anions |
CYP20A1 | Sus scrofa | KEGG_PATHWAY | Removal of xenobiotics |
Heat shock 70kDa protein 4-like | Sus scrofa | KEGG_PATHWAY | Inflammation and oxidative stress |
Acetyl-coenzyme A carboxylase alpha | Homo sapiens | BIOCARTA | Leptin Pathway:Reversal of Insulin Resistance by Leptin |
5,10-Methylenetetrahydrofolate reductase (NADPH) | Homo sapiens | PANTHER_PATHWAY | P02743:Formyltetrahydroformate biosynthesis |
Doublecortin-like kinase 1 | Homo sapiens | PANTHER_PATHWAY | P00031:Inflammation mediated by chemokine and cytokine signaling pathway |
5,10-Methylenetetrahydrofolate reductase (NADPH) | Homo sapiens | REACTOME_PATHWAY | REACT_11193:Metabolism of vitamins and cofactors |
Acetyl-coenzyme A carboxylase alpha | Homo sapiens | REACTOME_PATHWAY | REACT_1505:Integration of energy metabolism |
REACT_602:Metabolism of lipids and lipoproteins | |||
Pericentriolar material 1 | Homo sapiens | REACTOME_PATHWAY | REACT_152:Cell cycle, mitotic |
KEGG, a database resource for understanding the metabolic network and functions of the biological system; STAT, signal transducer and activator of transcription; TGF, transforming growth factor. |
Table 5 summarizes the results of the GO terms and KEGG interaction pathways for selected genes that were differentially expressed in the placentae of arginine-supplemented gilts. We noted that supplementing Arg to the diet of gestating gilts influenced the following interaction pathways: phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway, regulation of circadian rhythm, glucagon signaling pathway, cell surface determinants, inflammation, osteoclast differentiation, Hippo signaling pathway, membranous septum morphogenesis, nitrogen utilization, mesenchymal cell differentiation, branching involved in salivary gland morphogenesis, ammonium transmembrane transport, organic cation transport, mesenchymal cell differentiation, beta-amyloid metabolic process, positive regulation of astrocyte differentiation, nutrient oxidation, extracellular space metabolism and remodeling, cell growth and development, regulation of gene transcription, and embryonic pattern specification.
Term | Count | % | p-Value | Genes |
ssc04151:PI3K-Akt signaling pathway | 7 | 13.46154 | 0.001015 | NM_213973, XM_001927228, NM_214266, NM_001099924 |
NM_001162401, NM_213772, XM_003133904 | ||||
GO:0042752 regulation of circadian rhythm | 3 | 5.769231 | 0.004521 | NM_213963, NM_214266, NM_001037965 |
ssc04922:glucagon signaling pathway | 4 | 7.692308 | 0.005019 | XM_001928025, NM_213963, NM_001143721, NM_214266 |
GO:0009986 cell surface determinants | 5 | 9.615385 | 0.008898 | XM_001925381, NM_001114283, NM_214376, NM_214257 |
NM_001162401 | ||||
ssc05160:inflammation | 4 | 7.692308 | 0.011177 | NM_001101814, NM_001078670, NM_001105286, NM_213772 |
ssc04380:osteoclast differentiation | 4 | 7.692308 | 0.012663 | XM_001928025, NM_001078670, NM_001105286, NM_213772 |
ssc04390:Hippo signaling pathway | 4 | 7.692308 | 0.013715 | NM_001105290, XM_001927228, NM_214376, NM_001037965 |
GO:0003149 membranous septum morphogenesis | 2 | 3.846154 | 0.01435 | NM_001099924, NM_001037965 |
GO:0019740 nitrogen utilization | 2 | 3.846154 | 0.01435 | NM_213996, NM_214378 |
GO:0060445 branching involved in salivary gland morphogenesis | 2 | 3.846154 | 0.017907 | NM_001105290, NM_001099924 |
GO:0072488 ammonium transmembrane transport | 2 | 3.846154 | 0.02145 | NM_213996, NM_214378 |
GO:0015695 organic cation transport | 3 | 3.846154 | 0.024981 | NM_213996, NM_214378, NM_214376 |
GO:0048762 mesenchymal cell differentiation | 2 | 3.846154 | 0.024981 | NM_001105290, NM_001099924 |
GO:0050435 beta-amyloid metabolic process | 2 | 3.846154 | 0.0285 | XM_001925381, NM_001078666 |
GO:0048711 positive regulation of astrocyte differentiation | 2 | 3.846154 | 0.0285 | NM_001097440, NM_001037965 |
GO:0014850 nutrient oxidation | 2 | 3.846154 | 0.0285 | NM_213963, NM_214266 |
GO:0005615 extracellular space metabolism and remodeling | 7 | 13.46154 | 0.032712 | XM_001925381, NM_001105290, NM_001114283, NM_001098597, |
– | – | – | – | NM_214376, NM_001001861, NM_214003 |
GO:0060749 cell growth and development | 2 | 3.846154 | 0.038982 | NM_214376, NM_001037965 |
GO:0048557 regulation of gene transcription | 2 | 3.846154 | 0.042451 | NM_001099924, NM_001037965 |
GO:0009880 embryonic pattern specification | 2 | 3.846154 | 0.042451 | NM_001105290, NM_001099924 |
PI3K, phosphoinositide 3-kinase. |
Data from the RT-PCR analysis of selected genes largely confirmed results from the microarray analysis (Table 6). These genes were chemokine (C-X-C motif) ligand 2 (CXCL2), MTOR, presenilin 2, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 (PFKFB1), recombination activating gene 2 (RAG-2), Ras (rat sarcoma protein p21) guanine nucleotide exchange factor (RasGEF), Rh family B glycoprotein (RHBG), RU2S, SLC7A1, and TNNT3.
Accession No. | Gene name | Microarray analysis | RT-PCR analysis | ||
Fold change | p-Value | Fold change | p-Value | ||
NM_001001863 | TNNT3 | 4.61 | 0.004 | 4.37 | 0.015 |
AK231515 | Presenilin 2 | 2.31 | 0.006 | 1.77 | 0.021 |
TC275071 | RAG-2 | 1.76 | 0.006 | 1.68 | 0.013 |
TC267605 | PFKFB1 | 1.55 | 0.025 | 1.51 | 0.036 |
XM_001926447 | RasGEF | 0.18 | 0.013 | 0.22 | 0.019 |
NM_001001861 | CXCL2 | 0.49 | 0.013 | 0.43 | 0.016 |
TC243513 | RHBG | 0.45 | 0.006 | 0.63 | 0.016 |
TC257543 | RU2S | 0.23 | 0.015 | 0.38 | 0.044 |
EU288086 | MTOR | 1.01 | 0.783 | 1.04 | 0.520 |
NM_001012613 | SLC7A1 | 0.97 | 0.602 | 0.94 | 0.550 |
CXCL2, chemokine (C-X-C motif) ligand 2; MTOR, mechanistic target of rapamycin; PFKFB1, 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 1; RAG-2, recombination activating gene 2; RasGEF, Ras (rat sarcoma protein p21) guanine nucleotide exchange factor; RHBG, Rh family, B glycoprotein; RU2S, doublecortin domain-containing protein 2; SLC7A1, Sus scrofa solute carrier family 7 member 1 (CAT-1); TNNT3, troponin T type 3. |
The placenta plays a critical role in transporting amino acids from mother to fetus, thereby having an enormous impact on fetal survival, growth, and development [18]. The pig has true epitheliochorial placentation, meaning that the placenta is only superficially attached to the uterine luminal epithelium. Such a placental structure increases the efficiency of gas and nutrient exchanges between fetus and mother [19]. Consistent with the increased availability of Arg in the conceptus of Arg-supplemented gilts [4], results of this microarray analysis revealed that dietary supplementation with 0.8% Arg to gilts between Days 14 and 25 of gestation altered the expression of 575 genes in their placentae. To our knowledge, this is the first study of effects of dietary Arg supplementation on in vivo expression of placental genes in any animal species. The microarray assay provides a powerful molecular technology to allow for the simultaneous determination of the expression of thousands of genes (particularly unexpected ones) in a tissue. The results can facilitate the elucidation of mechanisms responsible for the effects of nutrients or other substances.
Polyamines are crucial for cell growth, migration, and proliferation, as well
as angiogenesis [20]. We recently reported that dietary supplementation with Arg
to gilts increased the activity of ornithine decarboxylase (ODC) and the
synthesis of polyamines from ornithine in their placenta [4]. A novel and
unexpected finding of the present study is that Arg supplementation reduced the
placental expression of ODC antizyme inhibitor 1 (Table 3). This inhibitor
protein binds to and
destabilizes ODC, thereby suppressing ODC activity. Thus, a decrease in the
expression of the ODC antizyme inhibitor 1 alleviates the inhibitory effect on
ODC activity, leading to enhanced polyamine synthesis in placentae. This action
of Arg is associated with an increase in the transmembrane transport of Ca
Results of our previous in vitro studies revealed that, as compared with 10
Dietary Arg supplementation enhances placental angiogenesis (the growth of
new blood vessels from the existing vasculature) partly via the generation of
polyamines and NO [4, 5]. In addition, there is emerging evidence that glycans are
novel activators of angiogenesis under physiological conditions due to changes in
protein glycosylation [31]. Consistent with this notion, the expression of
beta-galactoside alpha 2–3 sialyltransferase (a glycosyltransferase), a key
enzyme that catalyzes protein glycosylation via the terminal sialylation of
glycoproteins and glycolipids, was enhanced in the placentae of Arg-supplemented
gilts as compared to the control group (Table 2). Likewise, calcitonin stimulated
all phases of angiogenesis through the calcitonin receptor [32], and matrix
metallopeptidases contributes to angiogenesis through the degradation of the
vascular basement membrane and remodeling of the extracellular matrix [33].
Furthermore, calcineurin (a calcium- and calmodulin-dependent serine/threonine
protein phosphatase) stimulates angiogenesis through Ca
Arg is known to alleviate inflammation [11, 36] and enhance immune responses [37] in animals but the underlying mechanisms are not fully understood. For example, dietary supplementation with Arg reduces risk for gastrointestinal infections and embryonic deaths in gestating gilts [38]. Interestingly, unexpected results of the present work revealed increases in the placental expression of the following key genes related to anti-oxidative and immune responses in gestating gilts supplemented with Arg (Table 3). These genes include: (a) the recombination-activating genes (RAGs), which encode part of a protein complex that plays important roles in the rearrangement and recombination of the genes for B-cell development and the production of immunoglobulins [39], as well as T-cell receptor molecules [40]; (b) leucine-rich repeat-containing proteins 51-like and 18-like, which promote the maturation of cells of the innate immune system [41, 42]; and (c) solute carrier organic anion transporter family member 3A1 (SLCO3A1), which encodes for a membrane protein in immune cells that mediates inflammatory processes in epithelial cells through the activation of the NF-kB cell signaling pathway [43]. Likewise, dietary supplementation with Arg to gestating gilts reduced the placental expression of mRNAs for heat shock protein 70, hypoxia inducible factor 1 alpha subunit inhibitor, decay-accelerating factor CD55 (that is involved in epithelial inflammation) [44], amphiregulin (a transmembrane glycoprotein that participates in cell inflammatory responses [45]), and CXCL2 (Table 3), indicating an improvement in cellular redox balance and a reduction in cellular inflammation.
There is much evidence that Arg regulates the metabolism of lipids and glucose
in mammalian liver, skeletal muscle, and white adipose tissue [8, 46], as well as
nutrient transport by the small intestine [47]. However, little is known about
the roles of Arg in these biochemical processes in placentae. Results of the
microarray analysis indicated, for the first time, that dietary supplementation
with Arg altered the expression of some key genes in porcine placentae that are
involved in: (a) glycolysis and glucose oxidation to CO
Another novel and important finding from the current work is that dietary Arg supplementation increased cadherin expression in porcine placentae (Table 2). Cadherin is a transmembrane protein that mediates cell–cell adhesion [48]. By regulating the stability of contacts between cells, cadherins play a crucial role in tissue morphogenesis and homeostasis. This is consistent with the analysis of interaction pathways for differentially expressed genes (Table 5) and the report that the apparent adhesion force between the chorioallantoic membrane and the endometrial epithelium was greater in Arg-supplemented gilts than control gilts [49]. Further analysis of the adhesion strength would require mechanical testing equipment.
Dietary supplementation with 0.8% Arg to gilts between Days 14 and 25 of gestation increased the expression of mRNAs for the syntheses of polyamines and protein, angiogenesis, cell-to-cell interactions, immune development, and antioxidative responses in placentae. Arginine supplementation reduced the placental expression of genes for protein degradation, inflammation, and cell injury. Furthermore, some of the key genes for glucose and fatty acid metabolism, ion transport, and cell signaling in placentae were differentially expressed between control and Arg-supplemented gilts to support placental growth and differentiation. Results from this microarray study will help to elucidate complex mechanisms responsible for the beneficial effects of Arg in improving conceptus growth, survival, and development in swine and possibly other mammals.
GW, FWB, and GAJ conceived and designed the study. XL, GW, FWB, GAJ, and RCB performed the experiment. XL and HZ analyzed the data. XL and GW summarized the results and wrote the manuscript. All authors contributed to data interpretation and manuscript revisions, and approved the final manuscript.
This study was approved by The Institutional Animal Care and Use Committee of Texas A&M University. No consent to participate was applicable.
Not applicable.
This work was supported by Agriculture and Food Research Initiative Competitive Grants (2015-67015-23276) from the USDA National Institute of Food and Agriculture.
The authors declare no conflict of interest. GW is serving as one of the Editorial Board members of this journal. We declare that GW had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to GP.