Skip to main content

Dietary supplementation with l-arginine between days 14 and 25 of gestation enhances NO and polyamine syntheses and the expression of angiogenic proteins in porcine placentae

Abstract

Dietary supplementation with 0.4 or 0.8% l-arginine (Arg) to gilts between days 14 and 25 of gestation enhances embryonic survival and vascular development in placentae; however, the underlying mechanisms are largely unknown. This study tested the hypothesis that Arg supplementation stimulated placental expression of mRNAs and proteins that enhance angiogenesis, including endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), placental growth factor (PGF), GTP cyclohydrolase-I (GTP-CH1), ornithine decarboxylase (ODC1), and vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and VEGFR2). Beginning on the day of breeding, gilts were fed daily 2 kg of a corn–soybean meal-based diet supplemented with 0.0 (control), 0.4, or 0.8% Arg. On day 25 of gestation, gilts were hysterectomized to obtain uteri and conceptuses for histochemical and biochemical analyses. eNOS and VEGFR1 proteins were localized to endothelial cells of maternal uterine blood vessels and to the uterine luminal epithelium, respectively. Compared with the control, dietary supplementation with 0.4 or 0.8% Arg increased (P < 0.05) the amounts of nitrite plus nitrate (NOx; oxidation products of NO) and polyamines in allantoic and amniotic fluids, concentrations of NOx, tetrahydrobiopterin (BH4, an essential cofactor for all NOS isoforms) and polyamines in placentae, as well as placental protein abundances of GTP-CH1 (the key enzyme for BH4 production) and ODC1 (the key enzyme for polyamine synthesis). Placental  mRNA levels for GTP-CH1, eNOS, PGF, VEGF, and VEGFR2 increased in response to both 0.4% and 0.8% Arg supplementation. Collectively, these results indicate that dietary Arg supplementation to gilts between days 14 and 25 of pregnancy promotes placental angiogenesis by increasing the expression of mRNAs and proteins for angiogenic factors as well as NO and polyamine syntheses.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3

Abbreviations

Arg:

l-arginine

BD:

Becton Dickinson Transduction Laboratories

BH4:

Tetrahydrobiopterin

EPC:

Endothelial precursor cells

GTP-CH1:

GTP cyclohydrolase-I

HPLC:

High-performance liquid chromatography

VEGF:

Vascular endothelial growth factor

NO:

Nitric oxide

cNOS:

Constitutive NO synthase

iNOS:

Inducible NO synthase

eNOS:

Endothelial NO synthase

NOx:

Oxidation end products of NO (nitrite plus nitrate)

ODC1:

Ornithine decarboxylase

PCR:

Polymerase chain reaction

PGF:

Placental growth factor

SPP1:

Secreted phosphoprotein 1

TBST:

Tris-buffered saline-Tween solution

VEGFR1:

Vascular endothelial growth factor receptors 1

VEGFR2:

Vascular endothelial growth factor receptors 2

References

  • Assaad HI, Zhou L, Carroll RJ, Wu G (2014) Rapid publication-ready MS-Word tables for one-way ANOVA. Springerplus 3:474

    PubMed  PubMed Central  Google Scholar 

  • Bazer FW, First NL (1983) Pregnancy and parturition. J Anim Sci 57:425–460

    CAS  PubMed  Google Scholar 

  • Bazer FW, Song G, Kim J, Dunlap KA, Satterfield MC, Johnson GA, Burghardt RC, Wu G (2012) Uterine biology in pigs and sheep. J Anim Sci Biotechnol 3:23

    PubMed  PubMed Central  Google Scholar 

  • Bazer FW, Johnson GA, Wu G (2015) Amino acids and conceptus development during the peri-implantation period of pregnancy. Adv Exp Med Biol 843:23–52

    CAS  PubMed  Google Scholar 

  • Bérard J, Bee G (2010) Effects of dietary L-arginine supplementation to gilts during early gestation on foetal survival, growth and myofiber formation. Animal 4:1680–1687

    PubMed  Google Scholar 

  • Bidarimath M, Khalaj K, Kridli RT, Kan FW, Koti M, Tayade C (2017) Extracellular vesicle mediated intercellular communication at the porcine maternal-fetal interface: a new paradigm for conceptus-endometrial cross-talk. Sci Rep 7:40476

    CAS  PubMed  PubMed Central  Google Scholar 

  • Borowicz PP, Arnold DR, Johnson ML, Grazul-Bilska AT, Redmer DA, Reynolds LP (2007) Placental growth throughout the last two thirds of pregnancy in sheep: vascular development and angiogenic factor expression. Biol Reprod 76:259–267

    CAS  PubMed  Google Scholar 

  • Carmeliet P, Moons L, Luttun A, Vincenti V, Compernolle V, De Mol M, Wu Y, Bono F, Devy L, Beck H, Scholz D, Acker T et al (2001) Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. Nat Med 7:575–583

    CAS  PubMed  Google Scholar 

  • Dantzer V, Leiser R (1994) Initial vascularisation in the pig placenta: I. Demonstration of nonglandular areas by histology and corrosion casts. Anat Rec 238:177–190

    CAS  PubMed  Google Scholar 

  • Dillon EL, Wu G (2021) Cortisol enhances ctrulline synthesis from proline in enterocytes of suckling piglets. Amino Acids. https://doi.org/10.1007/s00726-021-03039-y

    Article  PubMed  Google Scholar 

  • Douglas NC, Tang H, Gomez R, Pytowski B, Hicklin DJ, Sauer CM, Zimmermann RC (2009) Vascular endothelial growth factor receptor 2 (VEGFR-2) functions to promote uterine decidual angiogenesis during early pregnancy in the mouse. Endocrinology 150:3845–3854

    CAS  PubMed  PubMed Central  Google Scholar 

  • EFSA (European food Safety Authority) (2018) Safety and efficacy of l-arginine produced by fermentation with Escherichia coli NITE BP-02186 for all animal species. EFSA J 16:e05276

    Google Scholar 

  • Eis AL, Brockman DE, Pollock JS, Myatt L (1995) Immunohistochemical localization of endothelial nitric oxide synthase in human villous and extravillous trophoblast populations and expression during syncytiotrophoblast formation in vitro. Placenta 16:113–126

    CAS  PubMed  Google Scholar 

  • Fu WJ, Stromberg AJ, Viele K, Carroll RJ, Wu G (2010) Statistics and bioinformatics in nutritional sciences: analysis of complex data in the era of systems biology. J Nutr Biochem 21:561–572

    CAS  PubMed  PubMed Central  Google Scholar 

  • Furukawa K, He WL, Bailey CA, Bazer FW, Toyomizu M, Wu G (2021) Polyamine synthesis from arginine and proline in tissues of developing chickens. Amino Acids. https://doi.org/10.1007/s00726-021-03084-7

    Article  PubMed  PubMed Central  Google Scholar 

  • Gao H (2020) Amino acids in reproductive nutrition and health. Adv Exp Med Biol 1265:111–131

    CAS  PubMed  Google Scholar 

  • Gao H, Wu G, Spencer TE, Johnson GA, Bazer FW (2009) Select nutrients in the ovine uterine lumen. V. Nitric oxide synthase, GTP cyclohydrolase, and ornithine decarboxylase in ovine uteri and peri-implantation conceptuses. Biol Reprod 81:67–76

    CAS  PubMed  Google Scholar 

  • Garcia Fernandez RA, Sanchez Perez MA, Sanchez Maldonado B, Garcia-Palencia P, Naranjo Freixa C, Palomo Yague A et al (2015) Iberian pig early pregnancy: vascular endothelial growth factor receptor system expression in the maternofetal interface in healthy and arresting conceptuses. Theriogenology 83:334–343

    CAS  PubMed  Google Scholar 

  • Grazul-Bilska AT, Borowicz PP, Johnson ML, Minten MA, Bilski JJ, Wroblewski R, Redmer DA, Reynolds LP (2010) Placental development during early pregnancy in sheep: vascular growth and expression of angiogenic factors in maternal placenta. Reproduction 140:165–174

    CAS  PubMed  Google Scholar 

  • Halloran KM, Stenhouse C, Wu G, Bazer FW (2021) Arginine, agmatine and polyamines: key regulators of conceptus development in mammals. Adv Exp Med Biol 1332:85–105

    PubMed  Google Scholar 

  • Hood JD, Meininger CJ, Ziche M, Granger HJ (1998) VEGF upregulates ecNOS message, protein, and NO production in human endothelial cells. Am J Physiol 274:H1054–H1058

    CAS  PubMed  Google Scholar 

  • Jobgen WS, Jobgen SC, Li H, Meininger CJ, Wu G (2007) Analysis of nitrite and nitrate in biological samples using high-performance liquid chromatography. J Chromatogr B 851:71–82

    CAS  Google Scholar 

  • Johnson GA, Burghardt RC, Bazer FW (2014) Osteopontin: a leading candidate adhesion molecule for implantation in pigs and sheep. J Anim Sci Biotechnol 5:56

    PubMed  PubMed Central  Google Scholar 

  • Kong XF, Wang XQ, Yin YL, Li XL, Gao HJ, Bazer FW, Wu G (2014) Putrescine stimulates the mTOR signaling pathway and protein synthesis in porcine trophectoderm cells. Biol Reprod 91:106

    PubMed  Google Scholar 

  • Li H, Meininger CJ, Wu G (2000) Rapid determination of nitrite by reversed-phase high-performance liquid chromatography with fluorescence detection. J Chromatogr B Biomed Sci Appl 746:199–207

    CAS  PubMed  Google Scholar 

  • Li X, Bazer FW, Johnson GA, Burghardt RC, Frank JW, Dai Z, Wang J, Wu Z, Shinzato I, Wu G (2014) Dietary supplementation with L-arginine between days 14 and 25 of gestation enhances embryonic development and survival in gilts. Amino Acids 46:375–384

    CAS  PubMed  Google Scholar 

  • Liu XD, Wu X, Yin YL, Liu YQ, Geng MM, Yang HS, Blachier F, Wu GY (2012) Effects of dietary L-arginine or N-carbamylglutamate supplementation during late gestation of sows on the miR-15b/16, miR-221/222, VEGFA and eNOS expression in umbilical vein. Amino Acids 42:2111–2119

    CAS  PubMed  Google Scholar 

  • Martin D, Conrad KP (2000) Expression of endothelial nitric oxide synthase by extravillous trophoblast cells in the human placenta. Placenta 21:23–31

    CAS  PubMed  Google Scholar 

  • Meininger CJ, Wu G (2002) Regulation of endothelial cell proliferation by nitric oxide. Methods Enzymol 352:280–295

    CAS  PubMed  Google Scholar 

  • NRC (1998) Nutrient requirements of swine, 10th edn. National Academic Press, Washington

    Google Scholar 

  • Otrock ZK, Makarem JA, Shamseddine AI (2007) Vascular endothelial growth factor family of ligands and receptors: review. Blood Cells Mol Dis 38:258–268

    CAS  PubMed  Google Scholar 

  • Palencia JYP, Lemes MAG, Garbossa CAP, Abreu MLT, Pereira LJ, Zangeronimo MG (2018) Arginine for gestating sows and foetal development: a systematic review. J Anim Physiol Anim Nutr 102:204–213

    CAS  Google Scholar 

  • Regnault TR, Orbus RJ, de Vrijer B, Davidsen ML, Galan HL, Wilkening RB, Anthony RV (2002) Placental expression of VEGF, PlGF and their receptors in a model of placental insufficiency-intrauterine growth restriction (PI-IUGR). Placenta 23:132–144

    CAS  PubMed  Google Scholar 

  • Reynolds LP, McLean KJ, McCarthy KL, Diniz WJS, Menezes ACB, Forcherio JC, Scott RR, Ward AK, Dahlen CR, Caton JS (2022) Nutritional regulation of embryonic survival, growth and development. Adv Exp Med Biol 1354:63–76

    PubMed  Google Scholar 

  • Reynolds LP, Redmer DA (1992) Growth and microvascular development of the uterus during early pregnancy in ewes. Biol Reprod 47:698–708

    CAS  PubMed  Google Scholar 

  • Reynolds LP, Borowicz PP, Caton JS, Vonnahme KA, Luther JS, Buchanan DS, Hafez SA, Grazul-Bilska AT, Redmer DA (2010) Uteroplacental vascular development and placental function: an update. Int J Dev Biol 54:355–366

    CAS  PubMed  Google Scholar 

  • Risau W (1997) Mechanisms of angiogenesis. Nature 386:671–674

    CAS  PubMed  Google Scholar 

  • Seo H, Li X, Wu G, Bazer FW, Burghardt RC, Bayless KJ, Johnson GA (2020) Mechanotransduction drives morphogenesis to develop folding at the uterine-placental interface of pigs. Placenta 90:62–70

    CAS  PubMed  Google Scholar 

  • Shibuya M (2006) Differential roles of vascular endothelial growth factor receptor-1 and receptor-2 in angiogenesis. J Biochem Mol Biol 39:469–478

    CAS  PubMed  Google Scholar 

  • Steinhauser CB, Wing TT, Gao H, Li X, Burghardt RC, Wu G, Bazer FW, Johnson GA (2017) Identification of appropriate reference genes for qPCR analyses of placental expression of SLC7A3 and induction of SLC5A1 in porcine endometrium. Placenta 52:1–9

    CAS  PubMed  Google Scholar 

  • Tayade C, Black GP, Fang Y, Croy BA (2006) Differential gene expression in endometrium, endometrial lymphocytes, and trophoblasts during successful and abortive embryo implantation. J Immunol 176:148–156

    CAS  PubMed  Google Scholar 

  • Tayade C, Fang Y, Croy BA (2007) A review of gene expression in porcine endometrial lymphocytes, endothelium and trophoblast during pregnancy success and failure. J Reprod Dev 53:455–463

    CAS  PubMed  Google Scholar 

  • Tidwell SC, Ho HN, Chiu WH, Torry RJ, Torry DS (2001) Low maternal serum levels of placenta growth factor as an antecedent of clinical preeclampsia. Am J Obstet Gynecol 184:1267–1272

    CAS  PubMed  Google Scholar 

  • Vailhe B, Vittet D, Feige JJ (2001) In vitro models of vasculogenesis and angiogenesis. Lab Investig 81:439–452

    CAS  PubMed  Google Scholar 

  • Vonnahme KA, Wilson ME, Ford SP (2001) Relationship between placental vascular endothelial growth factor expression and placental/endometrial vascularity in the pig. Biol Reprod 64:1821–1825

    CAS  PubMed  Google Scholar 

  • Wang X, Frank JW, Xu J, Dunlap KA, Satterfield MC, Burghardt RC et al (2014a) Functional role of arginine during the peri-implantation period of pregnancy. II. Consequences of loss of function of nitric oxide synthase NOS3 mRNA in ovine conceptus trophectoderm. Biol Reprod 91:59

    PubMed  Google Scholar 

  • Wang X, Ying W, Dunlap KA, Lin G, Satterfield MC, Burghardt RC, Wu G, Bazer FW (2014b) Arginine decarboxylase and agmatinase: an alternative pathway for de novo biosynthesis of polyamines for development of mammalian conceptuses. Biol Reprod 90:84

    PubMed  Google Scholar 

  • Wing TT, Erikson DW, Burghardt RC, Bazer FW, Bayless KJ, Johnson GA (2020) OPN binds alpha v integrin to promote endothelial progenitor cell incorporation into vasculature. Reproduction 159:465–478

    CAS  PubMed  Google Scholar 

  • Winther H, Ahmed A, Dantzer V (1999) Immunohistochemical localization of vascular endothelial growth factor (VEGF) and its two specific receptors, Flt-1 and KDR, in the porcine placenta and non-pregnant uterus. Placenta 20:35–43

    CAS  PubMed  Google Scholar 

  • Wu G (2013) Functional amino acids in nutrition and health. Amino Acids 45:407–411

    CAS  PubMed  Google Scholar 

  • Wu G (2022) Nutrition and metabolism: Foundations for animal growth, development, reproduction, and health. Adv Exp Med Biol 1354:1–24

    PubMed  Google Scholar 

  • Wu G, Meininger CJ (2002) Regulation of nitric oxide synthesis by dietary factors. Annu Rev Nutr 22:61–86

    CAS  PubMed  Google Scholar 

  • Wu G, Morris SM (1998) Jr. Arginine metabolism: nitric oxide and beyond. Biochem J 336:1–17

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wu G, Bazer FW, Tuo W (1995) Developmental changes of free amino acid concentrations in fetal fluids of pigs. J Nutr 125:2859–2868

    CAS  PubMed  Google Scholar 

  • Wu G, Knabe DA, Flynn NE, Yan W, Flynn SP (1996a) Arginine degradation in developing porcine enterocytes. Am J Physiol 271:G913-919

    CAS  PubMed  Google Scholar 

  • Wu G, Bazer FW, Tuo W, Flynn SP (1996b) Unusual abundance of arginine and ornithine in porcine allantoic fluid. Biol Reprod 54:1261–1265

    CAS  PubMed  Google Scholar 

  • Wu G, Pond WG, Flynn SP, Ott TL, Bazer FW (1998) Maternal dietary protein deficiency decreases nitric oxide synthase and ornithine decarboxylase activities in placenta and endometrium of pigs during early gestation. J Nutr 128:2395–2402

    CAS  PubMed  Google Scholar 

  • Wu G, Bazer FW, Cudd TA, Meininger CJ, Spencer TE (2004) Maternal nutrition and fetal development. J Nutr 134:2169–2172

    CAS  PubMed  Google Scholar 

  • Wu G, Bazer FW, Hu J, Johnson GA, Spencer TE (2005) Polyamine synthesis from proline in the developing porcine placenta. Biol Reprod 72:842–850

    CAS  PubMed  Google Scholar 

  • Wu G, Bazer FW, Davis TA, Kim SW, Li P, Marc Rhoads J, Carey Satterfield M, Smith SB, Spencer TE, Yin Y (2009) Arginine metabolism and nutrition in growth, health and disease. Amino Acids 37:153–168

    CAS  PubMed  Google Scholar 

  • Wu G, Bazer FW, Dai Z, Li D, Wang J, Wu Z (2014) Amino acid nutrition in animals: protein synthesis and beyond. Annu Rev Anim Biosci 2:387–417

    CAS  PubMed  Google Scholar 

  • Wu G, Bazer FW, Johnson GA, Herring C, Seo H, Dai Z, Wang J, Wu Z, Wang X (2017) Functional amino acids in the development of the pig placenta. Mol Reprod Dev 84:870–882

    CAS  PubMed  Google Scholar 

  • Wu G, Bazer FW, Johnson GA, Hou Y (2018) Board-invited review: arginine nutrition and metabolism in growing, gestating, and lactating swine. J Anim Sci 96:5035–5051

    PubMed  PubMed Central  Google Scholar 

  • Wu G, Meininger CJ, McNeal CJ, Bazer FW, Rhoads JM (2021) Role of L-arginine in nitric oxide synthesis and health in humans. Adv Exp Med Biol 1332:167–187

    PubMed  Google Scholar 

  • Wu G, Self JT, Johnson GA, Bazer FW, Spencer TE (2003) Developmental changes in placental nitric oxide synthesis in pigs. Biol Reprod 68(Suppl 1):153

    Google Scholar 

  • Xiao XM, Li LP (2005) L-arginine treatment for asymmetric fetal growth restriction. Int J Gynecol Obstet 88:15–18

    CAS  Google Scholar 

  • Zhang Q, Hou YQ, Bazer FW, He WL, Posey EA, Wu G (2021) Amino acids in swine nutrition and production. Adv Exp Med Biol 1285:81–107

    PubMed  Google Scholar 

  • Zhu C, Li XL, Bazer FW, Johnson GA, Burghardt RC, Jiang ZY, Wu G (2021) Dietary L-arginine supplementation during days 14-25 of gestation enhances aquaporin expression in the placentae and endometria of gestating gilts. Amino Acids 53:1287–1295

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Agriculture and Food Research Initiative Competitive Grants (2015-67015-23276) from the USDA National Institute of Food and Agriculture supported this work. Data of this study have been presented previously as a poster in the 2018 Society of the Study of Reproduction (SSR)’s Annual Meeting (July 10-13, 2018; New Orleans, LA): Elmetwally MA, Li XL, Johnson GA, Burghardt RC, Herring C, Bazer FW, Wu G. “Dietary supplementation with l-arginine to gilts between days 14 and 25 of gestation enhances placental expression of angiogenic proteins”, P457. The present address of Mohammed A. Elmetwally is Department of Theriogenology, Faculty of Veterinary Medicine, Mansoura University, Mansoura, Egypt. The present address of Xilong Li is Institute of Feed Science, The Chinese Academy of Agricultural Sciences, Beijing, China 100081.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Guoyao Wu.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This study was approved by The Institutional Animal Care and Use Committee of Texas A&M University.

Informed consent

No informed consent is required for this study.

Additional information

Handling editor: E. I. Closs.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Elmetwally, M.A., Li, X., Johnson, G.A. et al. Dietary supplementation with l-arginine between days 14 and 25 of gestation enhances NO and polyamine syntheses and the expression of angiogenic proteins in porcine placentae. Amino Acids 54, 193–204 (2022). https://doi.org/10.1007/s00726-021-03097-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00726-021-03097-2

Keywords

  • Arginine
  • Polyamines
  • Placenta
  • Nitric oxide
  • Vascular endothelial growth factor
  • Angiogenesis