Molecular and Cellular Endocrinology 398 (2014) 53–68

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Molecular and Cellular Endocrinology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m c e

Review

Environmental factors affecting pregnancy: Endocrine disrupters, nutrients and metabolic pathways Fuller W. Bazer a,*, Guoyao Wu a, Gregory A. Johnson b, Xiaoqiu Wang a a b

Department of Animal Science, Texas A&M University, College Station, Texas 77843, United States Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, Texas 77843, United States

A R T I C L E

I N F O

Article history: Available online 16 September 2014 Keywords:: Arginine Glucose Secreted phosphoprotein 1 Pregnancy Endocrine Disrupter

A B S T R A C T

Uterine adenogenesis, a unique post-natal event in mammals, is vulnerable to endocrine disruption by estrogens and progestins resulting in infertility or reduced prolificacy. The absence of uterine glands results in insufficient transport of nutrients into the uterine lumen to support conceptus development. Arginine, a component of histotroph, is substrate for production of nitric oxide, polyamines and agmatine and, with secreted phosphoprotein 1, it affects cytoskeletal organization of trophectoderm. Arginine is critical for development of the conceptus, pregnancy recognition signaling, implantation and placentation. Conceptuses of ungulates and cetaceans convert glucose to fructose which is metabolized via multiple pathways to support growth and development. However, high fructose corn syrup in soft drinks and foods may increase risks for metabolic disorders and increase insulin resistance in adults. Understanding endocrine disrupters and dietary substances, and novel pathways for nutrient metabolism during pregnancy can improve survival and growth, and prevent chronic metabolic diseases in offspring. © 2014 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5.

6. 7.

Introduction ........................................................................................................................................................................................................................................................... Endocrine disrupters of uterine adenogenesis and fertility ................................................................................................................................................................. Pregnancy in sheep and pigs ........................................................................................................................................................................................................................... 3.1. Role of progestamedins ....................................................................................................................................................................................................................... Nutrients and fertility ........................................................................................................................................................................................................................................ Amino acids and pregnancy ............................................................................................................................................................................................................................ 5.1. Arginine (see Wu et al., 2009) ........................................................................................................................................................................................................... 5.2. Solute carrier family 7 (cationic amino acid transporter, Y+ system), member 1 (SLC7A1) ........................................................................................ 5.3. Nitric oxide synthase (NOS3) ............................................................................................................................................................................................................ 5.4. Ornithine decarboxylase (ODC1) ...................................................................................................................................................................................................... 5.5. Polyamines and trophoblast motility ............................................................................................................................................................................................. 5.6. Arginine and steroidogenesis ............................................................................................................................................................................................................ Glucose and fructose .......................................................................................................................................................................................................................................... Summary ................................................................................................................................................................................................................................................................ Acknowledgements ............................................................................................................................................................................................................................................. References ..............................................................................................................................................................................................................................................................

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1. Introduction

* Corresponding author. Department of Animal Science, 2471 TAMU, Texas A&M University, College Station, Texas 77843-2471, United States. Tel.: +1 979 862 2659; fax: +1 979 862 3399. E-mail address: [email protected] (F.W. Bazer). http://dx.doi.org/10.1016/j.mce.2014.09.007 0303-7207/© 2014 Elsevier Ireland Ltd. All rights reserved.

Key events affecting the ability of mammals to reproduce begin in utero with differentiation of the gonads and male and female reproductive systems. However, full differentiation of the female reproductive system is a unique postnatal event in mammals that

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is vulnerable to endocrine disrupters such as environmental estrogens and progestins that render the uterus nonfunctional (see Bartol et al., 1988). Endocrine disrupters of uterine adenogenesis have downstream effect to render females subfertile or infertile. This review focuses on endocrine disrupters that prevent development of uterine glands to adversely affect fertility, as well as the importance of the uterus in providing mechanisms for transport of nutrients from the environment into the uterine lumen where they are used via various metabolic pathways to support growth, differentiation and development of the conceptus (embryo/fetus and associated membranes).

2. Endocrine disrupters of uterine adenogenesis and fertility Uterine adenogenesis, the morphological differentiation and development of the uterus, particularly uterine glands, is a unique postnatal event in mammals (see Fig. 1). Adenogenesis is vulnerable to adverse effects of endocrine disrupters in the environment such as progestins and estrogens as reviewed previously (Gray et al., 2001a; Spencer and Bazer, 2004a). Sheep and other ruminant species exposed to forages that contain estrogenic activity experience hyperplasia of endometrial glands, dystocia, prolapse of the uterus, and infertility (Adams, 1995; Lindner, 1976). Experimentally, chronic

Fig. 1. (A) The neonatal ovine uterus on postnatal day (PND) 1 does not have uterine glands, but these develop through buding from the lumenal epithelium and then invasion at glands lined with glandular epithelium that progress through the stroma toward the myometrium while differentiating through branching morphogenesis to fully developed uterine glands by PND56. The glands are shown from Day 10 of the estrous cycle (CD10) and then undergoing hyperplasia between gestational days (GD) 16 and 60 and they hypertrophy as the uterine glands fill with hystotroph between GD 80 and 140 of a 147 day period of gestation. Following parturition, the uterus undergoes rapid involution between post-partum (PP) Days 1–28. (B) The uterus of a normal control adult ewe contains many uterine glands, but a ewe treated with a progestin from PND1-56 fails to develop uterine glands and these ewes do not exhibit normal estrous cycles and they are infertile (see Spencer and Bazer, 2004a).

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opposite the openings of an equal number of uterine glands and transport uterine gland secretions into the fetal–placental circulation (Friess et al., 1981; Knight et al., 1977; Perry and Crombie, 1982). 3. Pregnancy in sheep and pigs

Fig. 2. The uterine microenvironment of histotroph includes various molecules that are secreted or transported into the uterine lumen to stimulate growth and development of the conceptus during the peri-implantation period in some species, up to Day 60 in humans and for the duration of pregnancy in species with epitheliochorial and syndesmochorial placenta such as the pig and sheep, respectively. See Bazer (2013a,b).

exposure of ewe lambs to either a progestin or an estrogen has the epigenetic effect of preventing uterine adenogenesis (Bartol et al., 1988; Carpenter et al., 2003; Hayashi et al., 2004). The result is an adult uterine gland knockout (UGKO) ewe phenotype (see Fig. 1). These ewes are unable to experience normal estrous cycles or support development of the conceptus (embryo and its extra-embryonic membranes) beyond the preimplantation stage of pregnancy (Gray et al., 2001b). The conceptuses in UGKO ewes fail to undergo the transition from spherical to tubular and filamentous forms that must occur between Days 12 and 16 of pregnancy and, therefore, do not secrete sufficient amounts of IFNT for pregnancy recognition signaling (Bazer et al., 2012a). This failure of conceptus development is due to the absence of uterine glandular (GE) epithelial cells that selectively transport or synthesize and secrete substances into the uterine lumen known collectively as histotroph (see Fig. 2). Histotroph contains transport proteins, ions, mitogens, cytokines, lymphokines, enzymes, hormones, growth factors, proteases and protease inhibitors, amino acids, glucose, fructose, vitamins and other substances (Bazer and Johnson, 2014). The morphological development of the uterus and the histoarchitecture of uterine endometria of pigs is also impacted by estrogens (Bartol et al., 1993; Spencer et al., 1993). Female pigs treated with estradiol during the first 60 days of life have uteri with fewer uterine glands and, as adults, they have higher rates of embryonic death losses and produce fewer piglets in their litters (Bartol et al., 2006; Tarleton et al., 2003). Although estrogens serve as the pregnancy recognition signal in pigs (Bazer, 2013b), the timing of endometrial exposure to estrogens is critical since premature administration of estrogen (i.e., before Day 11 of pregnancy) to pregnant pigs alters the pattern of gene expression through the nuclear factor kappa B (NFKB) system (Geisert et al., 2005). The effect of premature exposure of the pig uterus to estrogen is modification of the uterine environment, particularly the window of implantation for the conceptuses, which results in embryonic death later in the peri-implantation period of pregnancy. Humans have invasive implantation; however, histotroph is a primary source of nutrients that support conceptus development during the first trimester of pregnancy (Burton et al., 2002). In livestock species histotroph is essential to support development of the conceptus throughout pregnancy (Gray et al., 2001a). The placentae of ungulates and cetaceans have a true epitheliochorial placenta that includes unique structures referred to as areolae (Steven, 1975). There are several thousand areolae per placenta in pigs, for example, that form

Estimates of embryonic deaths in mammals range from 20% to 40%, with two-thirds of those deaths occurring during the periimplantation period of pregnancy (Nancarrow, 1994). Establishment and maintenance of pregnancy requires appropriate development of the conceptus which must signal pregnancy recognition to ensure maintenance of a functional corpus luteum (CL) on the ovary. The CL secretes progesterone (P4) required for an intrauterine environment that supports implantation, placentation and fetal–placental growth and development (Spencer et al., 2004b). Interactions between the conceptus and cells of the uterus, including uterine luminal (LE), superficial glandular (sGE) and glandular (GE) epithelia and stromal cells, coordinate mechanisms that stimulate: (a) conceptus development, (b) vascular functions and uterine blood flow, (c) water and electrolyte transport, (d) maternal recognition of pregnancy, (e) transport of nutrients such as glucose and amino acids into the uterine lumen, and (f) secretion or selective transport of components of ‘histotroph’ by uterine epithelia into the uterine lumen to meet demands of the conceptus for growth and development (Bazer et al., 2010). Conceptuses may fail to develop appropriately due to their lack of response to components of histotroph or due to deficiencies in components of histotroph required to orchestrate essential developmental events, signaling for pregnancy recognition, implantation and placentation (see Fig. 3). This review focuses on dietary nutrients and endocrine disrupters from the environment. Endocrine disrupters of uterine adenogenesis render the uterus incapable of transporting components of histotroph, such as amino acids and glucose, into the uterine lumen to support conceptus development. Of particular interest are amino acids in histotroph of sheep and pigs, particularly Arg, Leu and Gln, as well as interactions between Arg and secreted phosphoprotein 1 [SPP1, also known as osteopontin (OPN)], that activate MTOR cell signaling that stimulates migration, hypertrophy and hyperplasia of cells of the conceptus (Guertin and Sabatini, 2009; Kim et al., 2010). Arginine (Arg), leucine (Leu) and glutamine (Gln) are abundant in the conceptus (Bazer et al., 2012a; Wu et al., 2013a) and their concentrations, as well as that for SPP1, increase significantly in the uterine lumen during the peri-implantation period of pregnancy (Gao et al., 2009a; Kim et al., 2013). Arg, Leu and Gln are not only substrates for tissue protein synthesis, but they are also regulators of cell signaling pathways required for conceptus survival, growth and development (Wu et al., 2013c, 2013d, 2014). The majority of embryonic mortality in ewes occurs during the peri-implantation period of pregnancy when key physiological events include maternal recognition of pregnancy signaling via interferon tau (IFNT), formation of conceptus mesoderm, elongation of conceptus trophectoderm, formation of trophectoderm binucleate cells, attachment of trophectoderm to uterine LE/sGE, and secretion or selective transport of components of histotroph into the uterine lumen (Bazer et al., 2012a). Elongation of ovine conceptuses is a prerequisite for central implantation and synepitheliochorial placentation which is superficial and noninvasive with increasing apposition and then adhesion between trophectoderm and uterine LE/sGE (Bazer, 2013a). Prenatal fetal death associated with intra-uterine growth restriction (IUGR) and difficulties during lambing are also causes of lamb mortality (Gootwine et al., 2007). Embryonic mortality and the pattern of development and elongation of pig conceptuses are similar to those for sheep conceptuses (Bazer and First, 1983; Bazer and Johnson, 2014; Geisert et al., 1982a, 1982b). Spherical pig blastocysts (0.5–1 mm diameter) shed the zona

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Fig. 3. The peri-implantation of pregnancy in ewes in marked by a rapid transition of hatched blastocysts from spherical to tubular and filamentous forms in response to proteins and select nutrients secreted and/or transported into the uterine lumen. The trophectoderm cells and extra-embryonic endoderm cells undergo proliferation, migration, and cytoskeletal changes to elongate which is critical for secreting interferon tau (IFNT) the signal for pregnancy recognition, as well as implantation. Arginine and secreted phosphoprotein 1 are important for stimulating growth and development of ovine and porcine conceptuses. See Bazer et al. (2012b).

pellucida between Days 6 and 7, expand to 2–6 mm diameter by Day 10, and then elongate rapidly to a filamentous form by Day 16. Pig conceptuses secrete estradiol between Days 11 and 13 to signal pregnancy recognition (Bazer, 2013b). The dramatic changes in morphology of pig and sheep conceptuses precede initial attachment of trophectoderm to uterine LE and initiation of a non-invasive “central-type” implantation (Steven, 1975). It is during this period of morphological and functional transition in livestock species that 30–40% of the conceptuses die, with many failing to elongate and/ or achieve extensive contact of trophectoderm with uterine LE for uptake of components of histotroph from the uterine lumen. Pigs also experience fetal losses during mid-gestation which likely result from inadequate uterine capacity for placentation and/or insufficient elongation of the trophectoderm during the peri-implantation period of pregnancy (Bazer et al., 1969a, 1969b, 2010, 2012b; Fenton et al., 1970; Webel and Dziuk, 1974). Interferon tau is the pregnancy recognition signal in ruminants. However, IFNT also has essential roles in regulating temporal and cell-specific changes in gene expression by uterine LE/sGE that are required for establishment and maintenance of pregnancy. First,

IFNT silences transcription of estrogen receptor alpha (ESR1) to prevent ESR1-dependent expression of the oxytocin receptor (OXTR) gene in uterine LE/sGE which abrogates development of oxytocinmediated pulsatile secretion of luteolytic prostaglandin F2α (PGF) (Bazer et al., 2010). Silencing ESR1 expression by IFNT also prevents estrogens from inducing P4 receptors (PGR) in endometrial epithelia. The absence of PGR in uterine LE/sGE is required for implantation and for expression of genes that are P4-induced or P4induced and further stimulated by IFNT to support of conceptus growth and development (Bazer et al., 2010). Studies of the complex temporal (day of pregnancy) and spatial (cell-specific) regulation of expression of IFNT stimulated genes (ISG) revealed that P4 and IFNT exert effects: (a) on uterine LE/sGE to induce expression of genes [e.g., solute carrier family 7 (cationic amino acid transporter, y + system), member 2 (SLC7A2), cystatin C (CST3), cathepsin L (CTSL), solute carrier family 2 (facilitated glucose transporter), member 1 (SLC2A1), hypoxia-inducible factor 1-alpha (HIF1A), HIF2A, galectin 15 (LGALS15), interferon regulatory factor 2 (IRF2) and gastrin releasing peptide (GRP)] critical to conceptus development and implantation; and (b) on uterine GE and stromal cells to induce

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expression of classical ISGs, [e.g., signal transducer and activator of transcription factor 1 (STAT1), STAT2, interferon regulatory factor 1 (IRF1), IRF9, interferon stimulated gene 15 (ISG15), myxovirus resistance 1, mouse, homolog of (MX1), 2-prime,5-primeoligoadenylate synthetase 1 (OAS1), and radical s-adenosyl methionine domain-containing protein 2 (RSAD2)]. The lack of expression of classical ISGs in uterine LE/sGE is due to the induction of IRF2 in uterine LE/sGE that represses expression of classical ISGs (Choi et al., 2001; Spencer et al., 1998). Therefore, PGR- and STAT1independent cell signaling pathways are responsible for P4induction and IFNT-stimulation of genes by uterine LE/sGE that are in immediate apposition to conceptus trophectoderm. Interferon tau may induce cell signaling in ovine uterine LE/sGE that lack STAT1, STAT2 and IRF9 via alternative cell signaling pathways that include mitogen activated protein kinases (MAPK) and phosphoinositide-3 kinase (PI3K) (Kim et al., 2003; Platanias, 2005). Changes in expression of estrogen receptor alpha (ESR1) and PGR in uterine epithelia and stromal cells of the pig also occur (Bazer, 2013b). ESR1 is expressed by uterine stromal and epithelial cells on Day 1, but only by epithelial cells between Days 5 and 15 postonset of estrus in cyclic pregnant gilts. Epithelial and stromal cells of the pig uterus express PGR between Days 0 and 5 of the estrous cycle and pregnancy, but PGR are expressed primarily by stromal cells between Days 5 and 10, and only by stromal cells between Days 10 and 18 for both cyclic and pregnant pigs. Information on temporal and spatial changes in uterine expression of PGR in the pig uterus beyond Day 18 of gestation is not available. Pig conceptuses secrete estradiol (E2) between Days 10 and 15 for pregnancy recognition, and E2 increases expression of genes in uterine LE which stimulate proliferation, migration, and adhesion of trophectoderm, as well as implantation and conceptus development. Estradiol

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also induces IRF2 in uterine LE/sGE of pigs to restrict expression of classical ISG to uterine GE and stromal cells which results in uterine LE expressing novel genes in response to E2, IFNG and IFND (Johnson et al., 2009). Accordingly, expression of classical ISGs, including Mx, ISG15/17, IRF1, STAT1 and STAT2 is limited to uterine stromal cells in pigs between Days 14 and 18 of pregnancy (Joyce et al., 2007). Estradiol-stimulated genes localized to uterine epithelia in pigs include aldo-keto reductase family 1, member B1(AKR1B1), beta 2 microglobulin (B2M), CD24 antigen (CD24), lysophosphatidic acid receptor (EDG7), fibroblast growth factor 7 (FGF7), IRF2, myxovirus resistance 1, mouse, homolog of (MX1), neuromedin B (NMB), swine leukocyte antigens (SLAs 1, 2,3, 6, 7, 8), solute carrier family 5 (sodium/glucose co-transporter), member 1 (SLC5A1), SPP1, and stanniocalcin (STC1) (Joyce et al., 2007). 3.1. Role of progestamedins In ewes, progesterone is permissive to actions of IFNT. The paradox of mammalian pregnancy is that down-regulation of expression of PGR and ESR1 by uterine epithelia is a prerequisite for uterine receptivity to implantation and expression of genes by uterine LE/sGE and GE that encode for secretory proteins and transporters for molecules critical to conceptus development (Bazer et al., 2012a; see Fig. 4). Endocrine disrupters block or modify uterine adenogenesis; therefore, stromal cell-derived growth factors may exert effects only on uterine LE which is insufficient in its production of components of histotroph required for conceptus development. Down-regulation of PGR correlates with loss of MUC1 on uterine LE that interferes with implantation (Johnson et al., 2001). Further, silencing expression of PGR in ovine uterine epithelia allows P4 to act via PGR-positive uterine stromal cells to induce

Fig. 4. Down-regulation of receptors for progesterone (PGR) is a common feature of pregnancy in mammals. It is required for down-regulation of expression of mucin 1 (Muc-1) which would otherwise block the implantation cascade and down-regulation allows for an increase in expression of genes and proteins by uterine epithelia that are critical to growth and development of the conceptus. With cessation of expression of PGR and estrogen receptor alpha (ESR1) prior to implantation, progesterone then acts via PGR in stromal fibroblasts to stimulate secretion of progestamedins, particularly fibroblast growth factor 10 (FGF10) and hepatocyte growth factor (HGF) in ewes that act via their respective receptors (FGFR2IIIb and MET) to regulate gene expression by uterine epithelia acting in concert with cell signaling pathways stimulated by placental lactogen (CSH1), placental growth hormone (GH) and interferon tau (IFNT). The trophectoderm of the conceptus also expresses receptors for FGF10 and HGF that allow it to respond to these growth factors. For pigs, the uterine luminal epithelium secretes FGF7 that appears to affect gene expression by uterine epithelia and conceptus trophectoderm during the peri-implantation period of pregnancy. The progestamedins (FGF7, FGF10, HGF) allow for cell-specific paracrine signaling for regulation of expression of genes by uterine epithelium and trophectoderm that are critical to growth and development of the conceptus. See Bazer et al. (2010).

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expression of progestamedins, particularly FGF10 that exert paracrine effects on uterine LE/sGE and conceptus trophectoderm that express receptors for FGF10 (FGFR2IIIb) and HGF (MET; protooncogene MET) (Satterfield et al., 2008). In pigs, the established dogma was that FGF7 is a stromal cell derived paracrine mediator of hormone-regulated epithelial growth and differentiation. This dogma was refuted in pigs as FGF7 is expressed by uterine LE, particularly between Days 12 and 15 of the estrous cycle and pregnancy in pigs (Ka et al., 2007). FGF7 transactivates its receptor, FGFR2IIIb, that is expressed by uterine epithelia and conceptus trophectoderm. Estradiol increases FGF7 expression, but only when administered with P4 since P4 must downregulate PGR in uterine LE and GE to allow E2 to increase expression of FGF7 mRNA and protein. FGF7 increases cell proliferation, phosphorylation of FGFR2IIIb, the MAPK cascade and expression of urokinase-type plasminogen activator, a marker for trophectoderm cell differentiation (Ka et al., 2007). Secreted phosphoprotein 1 (SPP1) is expressed initially by uterine LE of pigs in response to estrogen from the conceptuses and it is later also expressed by uterine GE, presumably in response to placental estrogens, as well as progesterone (White et al., 2005). Gilts treated with estradiol on Days 9 and 10 of gestation experienced complete degeneration of their conceptuses by Day 17 of pregnancy. The inappropriate timing of exposure of the uterus to estrogens (i.e., before Day 11 of pregnancy), resulted in down-regulation of 1, 39 and 16 genes on Days 10, 13 and 15, respectively, that included aldose reductase (AKR1B1), SPP1, CD24 antigen (CD24), and neuromedin B (NMB) (Ross et al., 2007). The abberant expression of those genes, particularly SPP1, may account for failure of implantation and conceptus survival beyond Day 17 of pregnancy (Johnson et al., 2009). From about Day 20 of pregnancy, FGF7 expression shifts to uterine GE in pigs in response to P4 and perhaps E2 and it is presumed to affect uterine epithelia and conceptus development throughout pregnancy (Bailey et al., 2010; Ka et al., 2000). The estrogens secreted between Days 15 and 30 of pregnancy in pigs also increase expression of endometrial receptors for prolactin (PRLR) which allow prolactin (PRL) to affect uterine secretory activity (Young et al., 1999).

may also be involved in transport of amino acids (Fredriksson et al., 2008; Hediger et al., 2013). Amino acids are clearly at the forefront of nutrition research to prevent IUGR which has permanent negative impacts on growth and survival of the neonate, as well as development of metabolic diseases and fertility in adulthood (Wu et al., 2006). Amino acids have been classified as nutritionally essential (indispensable) or nonessential (dispensable). Nutritionally essential amino acids (EAA) cannot be synthesized in the body or they are inadequately synthesized de novo by the body to meet metabolic needs and must be provided in the diet to meet requirements (Wu et al., 2013b). Nutritionally nonessential amino acids (NEAA) are synthesized de novo in adequate amounts by the body to meet requirements, but there is increasing evidence that animals (e.g., pigs, sheep, rats, and chickens) and humans cannot synthesize sufficient NEAA for maximum growth or optimal health (Wang et al., 2014; Wu et al., 2014). Conditionally essential amino acids (CEAA) are normally synthesized in adequate amounts by the organism, but must be provided in the diet to meet needs under specific physiological conditions, such as pregnancy and lactation, when rates of utilization are greater than rates of synthesis. Functional amino acids serve as building blocks of proteins and they regulate key metabolic pathways to benefit health, survival, growth, development, lactation, and reproduction in animals and humans (Wu et al., 2010). These unique nutrients include Arg, cysteine (Cys), Gln, Leu, proline (Pro) and tryptophan (Trp). Thus, functional amino acids can be EAA, NEAA or CEAA (Wu et al., 2013b).

4. Nutrients and fertility

5.1. Arginine (see Wu et al., 2009)

Pregnancy, a complex period of human growth, development and imprinting, is influenced significantly by nutrition and metabolism essential for healthy mothers and their offspring during both the neonatal period and into adulthood (Berti et al., 2014). Biological and physiological mechanisms for utilization of nutrients by the mother and for the transfer of nutrients across the placenta and their use by the fetus are now well understood. Berti et al. (2014) concluded that maternal body mass index, gestational weight gain, placental and fetal requirements in relation to adverse pregnancy and long-term outcomes of fetal programming due to nutritional status of the mother and fetus represent an urgent need for research on biochemical mechanisms and pathophysiological events related to maternal–fetal nutrition and offspring health. Nutrients in the environment from foods are critical to the regulation of gene expression and key cellular events required for the establishment and maintenance of pregnancy in mammals. Cells of the body, including those of the uterus and conceptus, sense the availability of nutrients with the mechanistic target of rapamycin (MTOR) pathway being particularly sensitive to amino acid availability for protein synthesis and cellular growth (Kilberg et al., 2005). Among the nutrients in uterine histotroph, amino acids play key roles in stimulating growth and development of the conceptus because they are essential for protein synthesis and activation of cellular functions (Wu et al., 2013a, 2013c). Thus, it is not surprising that of the 395 known members of the solute carrier (SLC) family, 60 transport amino acids and 40 other transporters with unknown substrates

L-Arg is a basic amino acid in physiological fluids, but it is relatively deficient in milk of most mammals (Wu et al., 2014). The intake of Arg in the adult population of the United States of America is 4.4 g/day, with 25%, 20% and 10% of people consuming 7.5 g/day, respectively. Preterm infants who represent 10–12% of newborns exhibit Arg deficiency that results in hyperammonemia and multiorgan dysfunction. Therefore, Arg nutrition is a significant concern in human health, but it is also a growing concern for livestock production because the amount of Arg is inadequate in diets currently designed for maximal growth of milk-fed piglets and maximal reproductive performance of swine (Wu et al., 2010). Arg is considered a NEAA under most conditions; however, under certain conditions such as pregnancy, lactation and growth, Arg must be considered a conditionally EAA since the body cannot synthesize adequate amounts to meet metabolic demands. This is because Arg is the precursor for synthesis of nitric oxide (NO; a key signaling molecule in virtually every cell type) and it regulates vital metabolic pathways beyond protein synthesis (Wu et al., 2014). This review will focus on versatile roles of Arg, as an EAA in growth and development of conceptuses of sheep and pigs (see Fig. 5). The abundance of Arg increases approximately eightfold in the ovine uterine lumen between Days 10 and 15 of pregnancy and is involved in numerous metabolic pathways (Gao et al., 2009a, 2009b, 2009c). This is important since Arg is a precursor for the biosynthesis of NO and polyamines and the dramatic increase in the

5. Amino acids and pregnancy Amino acids may be provided in the diet which is a component of the animals’ environment or synthesized de novo by tissues in an organism. In either case, it is well established that there are key linkages between nutrition and reproductive success in all species. The amino acids may be used in synthesis of proteins and many different molecules in mammals, or they may be metabolized to other molecules required for conceptus development.

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Fig. 5. Arginine can be metabolized to: (1) nitric oxide (NO) by nitric oxide synthase (NOS); (2) polamines (putrescine, spermidine and spermine) via arginase and ornithine decarboxylase (ODC1) and (3) agmatine via arginine decarboxylase (ADC) and agmatine can be metabolized to polyamines by agmatinase. Arginine itself and its metabolic products (NO, polyamines) are known to be essential for embryonic development; however, roles for agmatine have not been defined. See Wang et al. (2014b).

abundance of Arg in the uterine lumen is highly correlated with the morphological transition of conceptuses from the spherical to filamentous forms and the period of signaling for pregnancy recognition by IFNT from conceptus trophectoderm. We also discovered that Arg is converted to agmatine by arginine decarboxylase in the uterus and by conceptus trophectoderm (Wang et al., 2014b). However, the in vivo functions of Arg in conceptuses and uteri of ewes or any mammalian species have not been dissected out from other components in histotroph. Therefore, we conducted studies to gain insight into the functional roles of Arg in early embryonic survival, growth and development. In these studies, we used an in utero morpholino anti-sense oligonucleotide (MAO) loss-of-function approach to knockdown translation of mRNAs for: (1) solute carrier family 7 (cationic amino acid transporter, y + system), member 1 (SLC7A1), the primary transporter for arginine into conceptus trophectoderm; (2) ornithine decarboxylase (ODC1), the rate limiting enzyme in the pathway whereby Arg is converted to polyamines (Arg to ornithine by arginase (ARGI/II) and ornithine to polyamines by ODC1); and (3) nitric oxide synthase 3 (NOS3), the primary NOS isoform in ovine conceptus trophectoderm for production of NO and citrulline. It is practical to use MAO in vivo to target knockdown of mRNA translation specifically in conceptuses because only trophectoderm cells of the conceptuses take up the MAO while uterine epithelial cells do not take up MAO and are, therefore, unaffected with respect to mRNA translation (Wang et al., 2014a, 2014b). 5.2. Solute carrier family 7 (cationic amino acid transporter, Y+ system), member 1 (SLC7A1) Our previous in vivo studies revealed that the major transporter for Arg in ovine conceptus trophectoderm during the periimplantation period of pregnancy is SLC7A1 (Gao et al., 2009b). We conducted an experiment in vivo using MAO-SLC7A1 to knockdown of translation of SLC7A1 mRNA to SLC7A1 protein in sheep conceptus trophectoderm and found no effect on expression of SLC7A1 mRNA or protein in the uterus or the amount of Arg in the uterine lumen. However, in comparison with MAO control conceptuses, the abundance of Arg in MAO-SLC7A1 conceptuses was significantly less which confirmed that we created an Arg-deficient

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ovine conceptus (X. Wang et al., 2014a). Arginine is the common substrate for production of NO via NOS3 (the major isoform in conceptuses), which is essential for angiogenesis and vasodilation, and production of polyamines via arginase and ODC1 for supporting cell proliferation and differentiation. Therefore, we considered downstream pathways of Arg metabolism and found that both ODC1 and NOS3 proteins were down-regulated in MAO-SLC7A1 conceptuses as compared with MAO control conceptuses. These results indicated that pathways for production of NO and polyamines were impaired due to deficiencies in Arg. These results are consistent with additional findings that both citrulline and ornithine decreased significantly in MAO-SLC7A1 conceptuses compared with MAO control conceptuses. Interestingly, the amount of citrulline increased significantly in the uterine lumen of MAO-SLC7A1 ewes even through no differences in arginine and ornithine were detected between MAO-SLC7A1 and MAO control conceptuses. This may be due to the fact that only a small quantity of Arg is metabolized by either NOS3 or the arginase-ODC1 pathway. It is possible that Arg is converted into citrulline in the conceptus or that peptidylarginine deiminase catalyzes the conversion of protein-bound arginine to citrulline. Since amino acids in histotroph are mainly transported from uterine LE and GE into the lumen, and citrulline is generated when Arg is metabolized by NOS3 during NO generation, the increase in citrulline may also reflect a compensatory increase in NO production in uterine epithelia and/or endothelial cells for regulation of angiogenesis and uterine blood flow. However, the abundance of NOS1, NOS2, ADC and AGMAT proteins between MAO-SLC7A1 and MAO control ewes was not different which suggests that these alternative pathways were not activated in response to decreases in Arg in conceptuses deficient in Arg. Nevertheless, there was a significant decrease in agmatine in the uterine lumen and conceptuses indicative of disruption of the alternative pathway for polyamine biosynthesis via ADC and AGMAT and suggesting a critical role for ADC in Arg catabolism and agmatine production in ovine conceptuses. During the peri-implantation period of pregnancy in sheep, requirements for polyamine synthesis switch from uterine LE/GEderived to conceptus-derived polyamines (Wang et al., 2014a). In MAO-SLC7A1 conceptuses, the abundance of polyamines in the uterine lumen was significantly decreased. This result indicates a critical role for SLC7A1 in transport of Arg into conceptus trophectoderm for synthesis of polyamines for proliferation and migration of conceptus trophectoderm for elongation and secretion of IFNT. Glutamine and glutamate are involved in energy metabolism to spare glucose, but they are also precursors for arginine biosynthesis (Self et al., 2004; Wu and Morris, 1998; Wu et al., 1995a, 2011). There was a significant decrease in both glutamine and glutamate in MAO-SLC7A1 conceptuses, but not in uterine flushings between MAO-SLC7A1 and MAO control ewes indicating an irreplaceable role for Arg in conceptus development. This may result from lack of crosstalk between SLC7A1 and transporters of glutamine and glutamate that allows them to provide a compensatory pathway for synthesis of Arg. Glutamine, glutamate and arginine are neutral, acidic, and basic amino acids respectively, and do not share transporters for entry into cells of animals (Wu and Morris, 1998). Novel results from our in vivo study of SLC7A1 protein knockdown were that conceptus development was completely disrupted when the abundance of Arg was significantly decreased in conceptuses. Further, results of this in vivo study revealed an essential role for Arg in survival, growth and development of peri-implantation ovine conceptuses. 5.3. Nitric oxide synthase (NOS3) Studies with mouse embryos in culture revealed that NO regulates early-stage embryonic development with excess NO causing

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apoptosis and a deficiency in NO inhibiting embryonic development (two-cell to blastocyst stage) (Chen et al., 2001; Tranguch et al., 2003). The absence of published evidence for successful creation of an in vivo NO-deficient conceptus model to assess effects of NO insufficiency on conceptus development during the peri-implantation period of pregnancy led us to use MAO to knockdown translation of NOS3 mRNA specifically in ovine conceptus trophectoderm to determine functional roles of NOS3 in NO synthesis from Arg in conceptus development. We found that a deficiency in NO in ovine conceptuses inhibited their development during the periimplantation period of pregnancy. The deficiency of NO in trophectoderm of MAO-NOS3 conceptuses resulted in a morphological delay in development as they were elongated, but smaller, thinner and disorganized compared with MAO-control conceptuses. Both NOS3 mRNA and protein are abundant in trophectoderm and endoderm of peri-implantation ovine conceptuses, whereas NOS1 mRNA and protein are expressed very weakly in conceptuses (Gao et al., 2009c). Clearly, an appropriate supply of NO to the conceptuses is important for ruminants and pigs which have synepitheliochorial and epitheliochorial placentae, respectively, and for conceptuses that undergo rapid elongation during a protracted peri-implantation period. The lack of effect of NOS3 MAO on IFNT production indicated that the developmentally delayed conceptuses are functional with respect to pregnancy recognition signaling. These results support the hypothesis that NO is important for normal growth and development of ovine conceptuses and that the stunted phenotype of MAO-NOS3 treated ovine conceptuses was due to NO insufficiency and downstream adverse effects on such pathways as synthesis of polyamines (Wu et al., 2013c). After confirming MAO delivery and knockdown of NOS3 protein in conceptuses without affecting its expression in the uterus, we studied effects of NOS3 MAO on interactions among SLC7A1, ODC1 and NOS3. Interestingly, SLC7A1 protein was less abundant in MAONOS3 conceptuses, but ODC1 protein was similar for MAO control and MAO-NOS3 conceptuses. These results suggest cross-talk between the Arg transporter SLC7A1 and NOS3 at the protein level with respect to efficiency of transport of Arg into conceptus tissue or the activity of arginase and ODC1 for production of polyamines (Poulin et al., 2012). We next determined actual amounts of amino acids related to NO metabolism in the uterine lumen and their concentration in conceptuses. In comparison with the MAO control, the amounts of Arg, Gln and glutamate (Glu) in the uterine lumen were not affected by knockdown of NOS3 protein in conceptuses. This is because less than 1% of the circulating Arg is used for NO synthesis in mammals in vivo (Wu and Morris, 1998). However, the significant increase in citrulline and significant decrease in ornithine in the uterine lumen of MAO-NOS3 ewes suggest: (1) increased secretion of citrulline into the uterine lumen and/or reduced uptake of citrulline by the conceptus; and (2) decreased secretion of ornithine into the uterine lumen, increased uptake of citrulline by the conceptus and/or increased catabolism of ornithine through the ornithine aminotransferase pathway in the uterine LE/sGE and GE of MAO-NOS3 treated ewes (Wu and Morris, 1998). As citrulline is a co-product of NOS, we determined concentrations of citrulline in conceptuses to gain insight into NO production due to limitations for directly assessing the production of gaseous NO which has a biological half-life of less than 5 s. Our results confirmed that knockdown of NOS3 in conceptuses decreased the abundance of citrulline in conceptuses. Gln, Glu and Arg metabolism is closely linked to Arg homeostasis in mammals. For example, Gln and Glu are involved in energy metabolism to spare glucose and they are precursors for the biosynthesis of Arg in the small intestine (Self et al., 2004; Wu and Morris, 1998; Wu et al., 1995b, 2011). Within the uterine environment, this increases the availability of glucose in the conceptus for metabolism via the pentose-cycle pathway to generate NADPH+,

which is a co-factor for NOS. Arg is the only nitrogenous source for NO production in cells during the peri-implantation period of pregnancy. Accordingly, the concentration of ornithine (produced from Arg hydrolysis by arginase) in MAO-NOS3 conceptuses was significantly less than in MAO-control conceptuses. Decreases in Arg, ornithine, Gln and Glu in MAO-NOS3 conceptuses suggests disruption of pathways for synthesis or transport of those amino acids. NOS3 is localized primarily in membrane-bound caveolae and may affect the transport of basic, neutral, and acidic amino acids by conceptus trophectoderm (Poulin et al., 2012). There were no significant differences in the abundance of NOS1, NOS2, ADC or AGMAT proteins between MAO-NOS3 and MAO control ewes, suggesting that those alternative pathways for production of NO and polyamines were not affected. Moreover, an increase in agmatine in the uterine lumen and conceptuses may account for the smaller and thinner, but elongated MAO-NOS3 conceptus phenotype that produced IFNT. This suggests a role for agmatine as a precursor for synthesis of polyamines required for conceptus elongation and pregnancy recognition signaling during the periimplantation period of pregnancy in sheep. Novel results from the study of MAO-NOS3 conceptuses, a NOdeficient mammalian conceptus model, were that conceptus development was morphologically stunted. This supports results of studies with human embryos indicating a dose-dependent effect of NO on progression of development of embryos to blastocysts (Lipari et al., 2009). This study further dissected out the functional roles of Arg in peri-implantation conceptuses via NO synthesis. Thus, as a functional amino acid that can regulate key metabolic pathways in animals (Wu, 2013) dietary arginine is required by the gestating mother for optimal pregnancy outcomes (Wu et al., 2013d, 2014). Perhaps the most novel finding was the significant decrease in abundances of Arg, ornithine and polyamines in MAO-NOS3 conceptuses and reduced amounts of polyamines in the uterine lumen. Thus, NOS3 may play an important role in regulating transport and metabolism of amino acids in the conceptuses. 5.4. Ornithine decarboxylase (ODC1) Nitric oxide and polyamines (putrescine, spermidine, and spermine) are classical products of Arg catabolism that are critical for placental growth in mammals (Wu et al., 2009); however, we recently discovered that agmatine is also a product of Arg metabolism in uteri of pregnant sheep and agmatine can be converted to polyamines by agmatinase by sheep conceptuses (Wang et al., 2014b). Arg stimulates placental NO production by enhancing expression of GTP cyclohydrolase I (GCH1), the first and rate-controlling enzyme for synthesis of tetrahydrobiopterin (BH4, an essential cofactor for all NOS isoforms). Additionally, glutathione synthesized from glutamate, glycine and cysteine is the major antioxidant in the conceptus (Wu et al., 2009). Transport of amino acids requires multiple specific transporters (Grillo et al., 2008), and IUGR of fetuses is associated with impaired transport of basic, neutral and acidic amino acids by the placenta (Regnault et al., 2002; Wu et al., 2008). Thus, maternal protein nutrition greatly impacts embryonic/fetal survival in pigs (Pond and Wu, 1981; Pond et al., 1969). Along with insulin-like growth factors, vascular endothelial growth factors and other growth factors, NO and polyamines are crucial for angiogenesis, embryogenesis, placental growth, utero-placental blood flows, and transfer of nutrients from mother to fetuses, as well as fetal–placental growth and development (Wu and Meininger, 2009; Wu et al., 2006). In sheep, ornithine decarboxylase (ODC1) is the rate-controlling enzyme for classical de novo biosynthesis of polyamines in mammals; but metabolism of Arg to agmatine via arginine decarboxylase (ADC) and conversion of agmatine to polyamines via agmatinase (AGMAT) is now known to be an alternative pathway for production of polyamines in uteri and conceptuses of sheep and perhaps other

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Fig. 6. In vivo knockdown of ODC1 from Day 8 of pregnancy resulted in developmentally normal [MAO-ODC1(a)] and developmentally retarded [MAO-ODC1(b)] ovine conceptuses on Day 16 of pregnancy. The ODC1 knockdown reduced polyamines, but increased ornithine and citrulline in the uterine lumen and differentially affected de novo synthesis of polyamines in conceptuses via the conventional conversion of ornithine to putrescine by ODC1. (A) Total amounts of polyamines (nmol) in uterine flushes were significantly less in MAO-ODC1(a) and MAO-ODC1(b), as compared with MAO-control conceptuses. (B) The concentrations of polyamines (nmol/g conceptus) in conceptus tissue extracts were not different between MAO control and MAO-ODC1(a) conceptuses, but they were significantly less for MAO-ODC1(b) conceptuses as compared with the other two treatment groups. (C) Total amounts (nmol) of ornithine and citrulline were higher in MAO-ODC1(a) and MAO-ODC1(b) ewes, as compared with MAO control ewes, whereas arginine (nmol) in the uterine lumen was not different among treatment groups. (D) Concentrations of ornithine (nmol/g conceptus) in conceptus tissue extracts were not different among the treatment groups; however, the concentrations of both arginine and citrulline were less for MAO-ODC1(b) conceptuses and concentrations were actually greater for MAO-ODC1(a) than MAO-control conceptuses. Different superscript letters denote significant differences (P < 0.05). Data are presented as means and SEM. (E) Immunohistochemical analyses of NOS3 protein in uteri of ewes from all treatment groups revealed increases in NOS3 protein in uteri from both MAOODC1(a) and MAO-ODC1(b) as compared with MAO control ewes. Sections were not counterstained with hematoxylin and eosin. Mouse IgG (mIgG) served as the negative control. LE, luminal epithelium; GE, glandular epithelium; sGE, superficial glandular epithelium; S, stroma. Width of field, 900 μm. This figure is based on results reported previously (Wang et al., 2014b).

mammals (Wang et al., 2014b). We discovered that a functional ADC/ AGMAT pathway for synthesis of polyamines in mammalian reproductive tissue exists in sheep conceptuses and that this pathway can compensate for loss of ODC1 activity to assure survival and development of some conceptuses. Following in vivo knockdown of translation of mRNA for ODC1 in ovine conceptus trophectoderm using MAO, one-half of the conceptuses were morphologically and functionally normal and the other one-half were abnormal. MAOODC1 knockdown conceptuses that maintained a normal phenotype exhibited an increase in ADC/AGMAT mRNA and protein that compensated for the loss of ODC1 to support polyamine synthesis from Arg. The majority of polyamine synthesis may be via the conventional ODC1-dependent pathway; however, the ADC/AGMATdependent pathway is, at least in sheep, a complimentary pathway

for production of polyamines for supporting survival and development of mammalian conceptuses (see Fig. 6). Agmatine has many functions that may be important for reproduction, but there are no results to define its role in the pregnant uterus other than a precursor for polyamines in the ovine conceptus (Wang et al., 2014b). Agmatine is the decarboxylation product of Arg and an intermediate in polyamine biosynthesis, but it is also a neurotransmitter. In brain, agmatine is synthesized and stored in synaptic vesicles and released by membrane depolarization and inactivated by conversion to putrescine by AGMAT. Agmatine binds α2-adrenergic receptor (ADRA2A) and imidazoline receptor (IRAS) and blocks N-methyl-D-aspartate receptor (NMDR) and other cation ligand-gated channels. Agmatine also inhibits NOS and induces the secretion of some peptide hormones by cells in the hypothalamus

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such as oxytocin and vasopressin (Gorbatyuk et al., 2001). Exogenous agmatine has neuroprotective effects in animal models of ischemia and neurotrauma and it acts via IRAS to stimulate synthesis of eicosanoids and enhance cell migration. Synthesis, metabolism and function of agmatine in animal tissues has been reviewed recently (Molderings and Haenisch, 2012), but little information is available about concentrations of agmatine in animal cells, tissues, or uterine secretions (Dai et al., 2014). Agmatine is formed in mitochondria, and arginase II and mitochondrial NOS (mtNOS) differ from cytosolic forms (Lacza et al., 2003). The NO formed in mitochondria may modulate respiratory rate and ATP synthesis by inhibiting cytochrome c oxidase (Giulivi, 1998) that would otherwise lead to formation of peroxynitrite-induced apoptosis (Ghafourifar et al., 1999). Synthesis of agmatine requires transport of Arg into mitochondria in kidney and brain by an energyindependent mechanism (Dolinska and Albrecht, 1998). We discovered an unusual abundance of Arg in porcine allantoic fluid during early gestation (Wu et al., 1995a, 1995b, 1996). Arginine and ornithine account for 50% and 55% of the total alpha amino-acid nitrogen (the sum of nitrogen in alpha-amino acids) in porcine allantoic fluid on Days 40 and 45 of gestation, respectively. Similarly, members of the Arg family of amino acids are highly abundant in ovine allantoic fluid (e.g., 10 mM citrulline and 25 mM Gln on Day 60 of gestation) (Kwon et al., 2003). The ovine placenta expresses arginase and would rapidly convert arginine to ornithine; therefore, citrulline is highly abundant in allantoic fluid and can be readily converted to arginine for generation of NO and polyamines as required. These observations suggest important biological roles for Arg in growth and development of mammalian conceptuses. Accordingly, rates of NO and polyamine synthesis in both porcine and ovine placentae are highest during early gestation when placental growth is most rapid (Bazer et al., 2012b; Kwon et al., 2004a, 2004b; Wu et al., 2005, 2012). We hypothesize that impaired placental growth (including vascular growth) or function results from reduced placental synthesis of NO and polyamines, thereby contributing to IUGR in both underfed and overfed dams (Wu et al., 2004). Growing evidence from studies with pigs, sheep, and rats support this hypothesis (Wu et al., 2013a, 2013b, 2013c, 2013d). 5.5. Polyamines and trophoblast motility Changes in migration of trophectoderm cells may result from increased expression of ODC1 and polyamine synthesis from Arg, proline and ornithine (Mehrotra and Kitchlu, 1998). Polyamines associate with DNA and nuclear proteins to produce normal chromatin required for gene transcription, proliferation of trophectoderm and formation of multinucleated trophectoderm cells that give rise to giant cells in the placentae of mice (Kwon et al., 2004a). Polyamine cell signaling pathways include tyrosine and mitogen activated protein kinases (MAPK) and proto-oncogenes, c-myc, c-jun, and c-fos (Kwon et al., 2004b). Polyamines also activate MTOR cell signaling to stimulate protein synthesis in porcine trophectoderm cells (Kong et al., 2012). ODC1 is important in the trophectoderm of mouse blastocysts for cell migration, integrin signaling via focal adhesion kinases, cytoskeletal organization, and invasiveness through the uterine luminal epithelium into of the uterine endometrium. Additionally, polyamines stimulate trophectoderm cell migration through modification of beta-catenin phosphorylation and the associated changes in uterine epithelial cells were suggested to be part of a mechanism to allow blastocysts to adhere to uterine LE and undergo implantation in mice (Martin et al., 2003). Synthesis of polyamines is highest in ovine placentomes (uterine caruncle and placental cotyledon) and inter-caruncular endometrium between Days 30 and 60 of gestation when growth and morphological changes in placentomes are most rapid (Bazer et al.,

2012b; Kwon et al., 2004b). High levels of polyamines in ovine placental and endometrial tissues in the second half of pregnancy likely contribute to continued development of the placental vascular bed for increased uterine blood flow to support fetal growth (Kwon et al., 2004b). Similar results have been reported for porcine conceptuses (Wu et al., 2005). Knockout of the Odc1 gene in mice is not lethal until the gastrulation stage of embryogenesis (Pendeville et al., 2001). There is a requirement for polyamines later in embryogenesis as Odc1 null embryos die at the late morula to early blastocyst stages due to apoptotic cell loss in the inner cell mass, but this condition can be rescued by providing putrescine (a precursor of spermidine and spermine) in drinking water of the dam up to the early implantation stage, but not beyond that stage of pregnancy (Pendeville et al., 2001). 5.6. Arginine and steroidogenesis We conducted studies to determine effects of dietary supplementation with Arg on embryonic/fetal survival and growth in gilts (Li et al., 2010, 2014). Gilts were individually supplemented with 0.0%, 0.4%, or 0.8% L-Arg between day of onset of estrus (Days 0) and Day 25 of gestation when they were hysterectomized. Dietary supplementation with 0.4% or 0.8% L-Arg increased concentrations of L-Arg in maternal plasma, as well as vascularity of the chorioallantois. The numbers of corpora lutea (CL) and conceptuses, weights of conceptuses and embryonic mortality did not differ between the 0.4% Arg and control group. However, supplementation with 0.8% L-arginine significantly decreased uterine weight, total number of CL and conceptuses, total fetal weight, total volume of allantoic and amniotic fluids, and concentrations of progesterone in maternal plasma, as well as total amounts of progesterone, estrone and estrone sulfate in allantoic fluid as compared with the control group. Follicular development, ovulation and formation of CL depends on cell signaling via mitogen-activated protein kinases 3 and 1 (also known as extracellular-regulated protein kinases 1 and 2; ERK1/ 2) and liver receptor homolog 1 (Lrh1) (Duggavathi and Murphy, 2009; Duggavathi et al., 2008). Lrh1 is essential for ovulation in mice through a mechanism involving expression of the NOS3 gene; therefore, increased production of NO through Arg supplementation may have inhibited ERK1/2 signaling and Lrh1 function in the porcine ovary to account for the reductions in number of CL and concentrations of progesterone in maternal plasma. A deficiency in Lrh1 may also interfere with progesterone synthesis because of failure of normal expression of the Lrh1 targets including steroidogenic acute regulatory protein and cytochrome P450 side-chain cleavage (Duggavathi and Murphy, 2009; Duggavathi et al., 2008). Thus, strategies for dietary L-Arg supplementation in pigs must consider that ovulation takes place about 44 hours after onset of estrus and that the peri-ovulatory period is vulnerable to adverse effects of NO on follicular maturation and ovulation, as well as steroidogenesis by ovarian follicles, CL and placentae. There are a number of reports indicating that arginine supplementation is effective in enhancing embryonic/fetal survival in pigs under practical production conditions (Wu et al., 2013d). Dietary Arg supplementation beginning as early as Day 14 of pregnancy generally increases placental weight, number of live-born piglets per litter and litter birth weight for liveborn piglets (Li et al., 2014). There are also reports of a reduction in the number of low birth weight piglets which improves preweaning survival and growth of piglets. Similar studies indicate that Arg is truly a functional amino acid for mammalian pregnancy as it has beneficial effects as a dietary supplement to enhance fetal– placental development and reduce IUGR in sheep, rodents and pigs (see Wu et al., 2013d), as well as humans (Shen and Hua, 2011). Arginine stimulates proliferation of ovine trophectoderm cells and the production of IFNT mainly through the polyamine-MTOR rather

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than the NO-MTOR pathway (Wang et al., 2014b). However, research is needed to understand: (1) if effects of Arg are mediated directly or via NO, polyamines, creatine or agmatine; (2) how Arg regulates gene expression; (3) optimum times for Arg supplementation during pregnancy; and (4) effective daily dosages of Arg for each management situation for humans and animals to effectively improve embryonic survival and fetal growth. 6. Glucose and fructose Carbohydrates in the diet, such a glucose, are components of the animals environment whether derived from forages or grains. The ewe, a ruminant that depends primarily on a forage-based diet, is an established animal model for studies of intra-uterine growth restriction during fetal life that predisposes one, through epigenetic effects, to adult onset of metabolic disease (Roberts et al., 2009). In humans, fructose is considered to be an undesirable component of our dietary environment and it is classified by some as an obesogen (Goran et al., 2013). Fructose is among the compounds that disrupt the function and development of adipose tissue and normal metabolism of lipids that increase the risk of obesity and associated diseases. Exposure of the pregnant woman to high fructose intake during critical periods of development of the fetus and neonate may affect lifelong neuroendocrine function, appetite control, feeding behavior, adipogenesis, fat distribution and metabolic systems. These changes ultimately favor the long-term development of obesity and associated metabolic risk. Interestingly, fructose is the major hexose sugar in fetal blood and fetal fluids of ungulate and cetacean species, such as sheep and pigs, but it is largely ignored in research addressing IUGR in sheep as an animal model for IUGR in humans. This appears to be because fructose is not considered to be metabolized via the glycolytic pathway or the Krebs cycle as an energy source. Fructose is the most abundant hexose sugar in fetal fluids of ungulate and cetacean mammals, but is present in very low amounts in fetal fluids of humans, dogs, cats, guinae pigs, rabbits, rats and ferrets (Bacon and Bell, 1946, 1948; Barclay et al., 1949; Goodwin, 1956; Jauniaux et al., 2005; Karvonen, 1949). In general, high levels of fructose are found in fetal blood and fetal fluids of mammals having epitheliochorial and synepitheliochorial placentae that are used extensively as animal models for IUGR (Nathanielsz, 2006; Reynolds et al., 2010; Roberts et al., 2009). The placentae of these mammals contain little or no glycogen, i.e., less than 5% of that in the fetus. In contrast, mammals such as rodents and humans with endotheliochorial and hemochorial placentae (Wooding and Burton, 2008) have fetuses in which glucose is converted to glucose6-phosphate that enters either the pentose phosphate pathway or glycolytic pathway to meet metabolic demands of the rapidly developing conceptus. Studies of pregnant ewes revealed that: (1) glucose not metabolized immediately is rapidly converted to fructose by the placenta; (2) glucose can be transported from fetal to maternal blood, but fructose derived from glucose is not transported from fetal blood into maternal blood; (3) fructose is continuously produced by the placenta independent of glucose concentration in maternal or fetal blood; and (4) the flux of glucose from the maternal to the fetal circulation can be 70 mg/min in hyperglycemic ewes (Alexander et al., 1955a, 1955b; Barclay et al., 1949; Huggett et al., 1951). These results were confirmed in studies using radiolabeled glucose to demonstrate its conversion to radioloabeled fructose by the placenta of pigs (White et al., 1979). The roles of fructose are unclear. Therefore, it has been ignored in studies of metabolic pathways associated with metabolism of hexose sugars since glucose, but not fructose, is metabolized via the glycolytic pathway (Abrams, 1979; Battaglia and Meschia, 1978, 1981; Bell and Ehrhardt, 2002; Hay et al., 1984). Nevertheless, fructose can be utilized for synthesis of ribose sugars

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(precursors for nucleic acids) and generation of reducing equivalents in the form of NADPH+ in the fetal pig (White et al., 1982). Fructose and glucose can be metabolized for synthesis of neutral lipids and phospholipids in heart, liver, kidney, brain and adipose tissue of fetal lambs (Scott et al., 1967). The activities of glucose6-phosphate dehydrogenase, malic enzyme and acetyl-CoA carboxylase in liver are stimulated by glucose in adult rats which increases lipogenesis (Fukuda et al., 1983) and fructose enters adipocytes by both insulin-independent and insulin-insensitive mechanisms (Halperin and Cheema-Dhadli, 1982). Fructose is the primary sugar in blood, allantoic fluid and amniotic fluid of the fetal pig, but it decreases in allantoic fluid as glucose increases between Days 82 and 112 of the 114 day period of gestation (Aherne et al., 1969). The rapid clearance of fructose from blood of piglets by 24 h post-partum indicates that the neonatal piglet is unable to utilize fructose as a potential energy source. In fact, piglets die within 30 h post-partum without a source of glucose for energy and diets containing only fructose as an energy source do not support survival of neonatal piglets (Goodwin, 1957; Steele et al., 1971). This indicates limited catabolism of fructose via hexokinase or fructokinase to produce pyruvate in mammalian fetuses and neonates. We discovered that fructose is actively involved in stimulating cell proliferation and mRNA translation via activation of mechanistic target of rapamycin (MTOR) cell signaling, and synthesis of glycosaminoglycans, specifically hyaluronic acid, via the hexosamine metabolic pathway (Kim et al., 2012). Specifically, fructose stimulates MTOR Complex 1 (MTORC1) that includes the regulatoryassociated protein of mTOR (Raptor), mammalian LST8/G-protein β-subunit like protein (mLST8/GβL) and their AKT1 substrate 1, proline-rich (AKT1S1) and dep domain-containing protein 6 (DEPDC6) involved with nutrient and energy sensing and protein synthesis and responsive to insulin, growth factors, serum, phosphatidic acid, amino acids, and oxidative stress (Hay and Sonenberg, 2004). These findings provided evidence for key roles of fructose in cellular functions and results of detailed analyses of actions of fructose on cell signaling pathways affecting proliferation and mRNA translation in porcine trophectoderm cells. The results support our hypothesis that fructose is metabolized to glucosamine-6-phosphate by GFPT1 of the hexosamine biosynthesis pathway and stimulates the MTOR cell signaling pathway to increase proliferation and mRNA translation of porcine conceptus trophectoderm (pTr) cells (see Fig. 7). Fructose is equivalent to or more stimulatory to pTr cell proliferation than glucose, and fructose induces phosphorylation of RPS6K, RPS6 and EIF4EBP1 in pTr cells. In general, MTOR-RPS6K cell signaling relays signals stimulated by nutrients such as amino acids and mitogens to stimulate cell proliferation, differentiation, and gene expression (Gingras et al., 2004). MTOR also acts in parallel with the PI3K-AKT1 pathway to transduce growth factor signals and regulate common downstream effectors such as RPS6K and EIF4EBP1. Activation of RPS6K, RPS6 and EIF4EBP1 by fructose and glucose is inhibited by LY294002 (inhibits PI3K) and by rapamycin (inhibits MTOR). Thus, fructose likely influences porcine fetal/placental development by activating the MTOR-RPS6K and/or PI3K-AKT pathways during the peri-implantation period of conceptus development. Stimulation of the MTOR signaling pathway by glucose and fructose can also be mediated via the glutamine:fructose-6-phosphate amidotransferase 1 (GFPT1) and the hexosamine pathway (Kim et al., 2012). As a nutrient sensor in the hexosamine biosynthesis pathway and an MTOR upstream effector, fructose-mediated GFPT1 activity is inhibited by azaserine. In a study using pTr cells, azaserine decreased pTr cell proliferation and the abundance of phosphorylated (p) p-RPS6K and p-EIF4EBP1 proteins. In addition, the combined effects of azaserine and LY294002 were more inhibitory to the phosphorylation RPS6K and EIF4EBP1 proteins than the effects of azaserine combined with LY294002 and rapamycin with respect to effects mediated by either glucose or fructose. These results

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Fig. 7. Glucose and fructose can be metabolized by at least three different pathways by porcine trophectoderm cells. Pathway 1 is the hexokinase pathway. Pathway 2 is the fructose kinase pathway. Pathway 3 is the aldolase pathway. The key intermediates are dihydroxyacetone-3-phosphase (DHAP), glyceraldehyde (GAD), glyceraldehyde-3-phosphate (GADP) and the Pentose Cycle via glucose-6-phosphate (Glc6P). Glucose and fructose can be metabolized by glutamine:fructose-6phosphate amidotransferase 1 (GFPT1) to activate the mechanistic target of rapamycin (MTOR) signaling pathway in porcine trophectoderm cells and, in turn, stimulate the MOTR-RPS6K and MTOR-EIF4EBP1 signal transduction cascade to increase proliferation and growth of trophectoderm cells of the conceptus. Legend: Fru, fructose; Glc, glucose; GLUT, glucose/fructose transporter; Glc-6P, glucose-6-phosphate; Fru1P, fructose-6-phosphate; Fructose-6P, GlcN-6P, N-acetylglucosamine-6-phosphate; UDP-GlcNAC, UDP-N-acetylglucosamine; GFPT1, glutamine-fructose-6-phosphate transaminase 1; TSC2, tuberous sclerosis 2; MTOR, mechanistic target of rapamycin; RPS6K, ribosomal protein S6K; RPS6, ribosomal protein S6; EIF4EBP1, eukaryotic translation initiation factor 4E-binding protein 1; AKT1, Proto-oncogenic protein kinase Akt; MAPK/ERK, mitogen-activated protein kinase/extracellular signal-regulated kinase. See Kim et al. (2012).

indicate that fructose stimulates both MTOR-RPS6K and MTOREIF4EBP1 signaling cascades for trophoblast cell growth. The pentose phosphate pathway is responsible for generation of reducing equivalents, in the form of NADPH, used in reductive biosynthesis reactions within cells (e.g. fatty acid synthesis), production of ribose-5phosphate used in the synthesis of nucleotides and nucleic acids, and production of erythrose-4-phosphate (E4P). There is evidence that fructose is utilized for synthesis of nucleic acids and reducing equivalents in the form of NADPH H + in the fetal pig (White et al., 1982) and ovine placenta (Reitzer et al., 1979). Fructose and glucose are equivalent in entering metabolic pathways leading to synthesis of neutral lipids and phospholipids in fetal tissues of domestic animals (Scott et al., 1967). The hepatic activities of glucose-6-phosphate dehydrogenase, malic enzyme and acetyl-CoA carboxylase are stimulated by glucose in adult rats which increases lipogenesis (Fukuda et al., 1983) and fructose enters adipocytes by both insulin-independent and insulin-insensitive mechanisms (Halperin and Cheema-Dhadli, 1982). However, there is considerable evidence that fructose negatively impacts insulin sensitivity in postnatal mammals (Goran et al., 2013). In studies with HeLa cells in medium containing 1 mM glucose or less, 80% is metabolized via glycolysis, but only 4–5% of the glucose enters the

tricarboxylic acid (TCA) cycle. When 2 mM fructose is present in the medium, about 100 times fewer molecules of fructose than glucose were used per mass of cells and glycolytic activity was reduced about 900-fold. Almost all fructose entered the pentose shunt to produce reducing equivalents and nucleic acids necessary for biosynthetic processes. With fructose in the medium, a uterine carcinoma cell line (HeLa cells) used glutamine to provide about 98% of the energy, but fructose had no effect on intracellular levels of ATP or TCA cycle intermediates. At present, the metabolic fate of fructose in the conceptus remains largely unknown. It is possible that the developing conceptuses of ungulates and cetaceans metabolize fructose via the oxidative arm of the pentose shunt to provide substrate for high rates of cell proliferation and associated biosynthetic processes. Energy may be derived from Gln catabolism and it is important to note that Gln is required for conversion of fructose-6-phosphate to glucosamine-6-phosphate (Wu et al., 2011). A study to determine amino acid concentrations in ovine allantoic fluid and amniotic fluid, as well as fetal and maternal plasma in pregnant ewes between Days 30 and 140 of gestation revealed that alanine, Gln, glycine, and serine contributed 50% of total alpha-amino acids in fetal plasma (Kwon et al., 2003). Interestingly, concentrations of alanine, citrulline, and Gln in allantoic fluid increased 20-, 34-, and 18-fold, respectively, between Days 30 and 60 of gestation and were 24.7, 9.7, and 23.5 mM, respectively, during this period of rapid placental growth and development (Bazer et al., 2010). Alanine, citrulline, and Gln accounted for approximately 80% of total alpha-amino acids in allantoic fluid during early gestation in sheep when both concentrations (mg/ ml) and total amounts (mg) of fructose increase rapidly (Kwon et al., 2003). A unique feature of fructose metabolism may be its production by the placenta, along with the utilization of Gln via the hexosamine pathway for synthesis of glycosaminoglycans, e.g., hyaluronic, uridine diphosphate-N-acetyl glucosamine and uridine diphosphateN-acetyl galactosamine that are involved in the synthesis of glycolipids, glycosaminoglycans and proteoglycans critical to cell and tissue functions. Hyaluronic acid and hyaluronidase increase in the uterine lumen of pigs in response to progesterone (Ashworth et al., 1990) which may stimulate angiogenesis (West et al., 1985) and/ or stimulate angiogenesis, morphogenesis and tissue remodeling of the placenta as reported for the human placenta (Ponting and Kumar, 1995). The accumulation of Wharton’s Jelly or hyaluronic acid occurs in the placentae of most mammals and it is localized to the umbilical cord primarily and to placental blood vessels (Mitchell et al., 2003), but it also supports fibroblasts and stem cells within the mesenchymal layer of the placenta (Wang et al., 2004). It is clear that angiogenesis is critical to conceptus development in all species and Kim et al. (2013) reported that fructose is equivalent to glucose for synthesis of glycosaminoglycans such as hyaluronic acid that support angiogenesis, particularly in the placenta of ungulates and cetaceans. Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) is involved in intracellular signaling as a substrate for O-linked N-actetylglucosamine transferases, nuclear pore formation and nuclear signaling and the glucose sensing mechanism, as well as insulin sensitivity of cells (McClain et al., 2002). Uridine diphosphateN-acetylgalactosamine (UDP-GalNAc) is another sugar moiety added to serine or threonine residues that represents the first step in mucin biosynthesis such as O-glycans that impart unique structural features to mucin glycoproteins and membrane receptors, and resistance to thermal changes and proteolysis of some proteins. The O-linked carbohydrate side chains function as ligands for receptors, lymphocyte and leukocyte homing and as signals for protein sorting. Overactivity of the hexosamine pathway results in increased UDPhexosamines which is an important mechanism by which hyperglycemia causes insulin resistance.

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Fructose oxidation to CO2 by placental tissues is about 20% that of glucose, suggesting that the placenta does not use fructose as a primary source of metabolic energy; however, that is based on the assumption that placental cells use aerobic metabolism as their primary means to generate adenosine triphosphate (ATP). High concentrations (2–3 mM; Randall, 1977; Pere, 1995) of lactate are present in plasma of fetal pigs and concentrations of lactate are higher in the umbilical vein than umbilical artery (Pere, 1995) which indicates that the placenta produces lactate, a product of anaerobic glycolysis. Thus, the placenta may use fructose anaerobically to produce lactate instead of CO2. This would be highly inefficient so high concentrations of substrate would be needed which is consistent with the high concentrations of fructose (4–6 mM, Randall, 1977; Pere, 1995) in fetal fluids. The concept of anaerobic metabolism occurring at the same time as limited aerobic metabolism is similar to that proposed by the Warburg hypothesis. The Warburg hypothesis suggests a mechanism whereby early tumor survival results from an increase in anaerobic glycolysis in the anoxic regions of developing tumors. Interestingly, in Warburg’s original investigations, he implicates embryonic development as the source of the idea on the ability of cancer cells to perform anaerobic glycolysis, as indicated in this quote: “On the other hand, we have found that the fermentation of the body cells is greatest in the very earliest stages of embryonal development and that it then decreases gradually in the course of embryonal development” (Warburg, 1956). A recent version of the Warburg hypothesis suggests that the better vascularized regions of tumors metabolize lactate generated anaerobically in oxygen poor regions of the tumor (Draoui and Feron, 2011; Rattigan et al., 2012). According to Leiser and Dantzer (1988), the capillaries of fetal circulation of the pig placenta are concentrated along the epithelial bilayer. For capillaries that do enter the stromal tissue, the blood has been deoxygenated by the fetus, creating an oxygen poor environment within the stroma. After blood passes along the epithelial bilayer it gathers into venules and returns to the fetus. In this description, there is no mechanism for returning oxygenated blood to the stromal capillaries which suggests that the placental stroma is relatively oxygen poor and may use anaerobic glycolysis for generation of metabolic energy. The advantage of this mechanism is that it reduces the requirement of placental tissue for oxygen and spares oxygen for use by the fetus. Also, consistent with the Warburg hypothesis, the epithelial bilayer closer to the source of oxygen may metabolize lactate produced by stromal tissue aerobically. Such lactate shuttling requires monocarboxylic acid (pyruvate) transporters (MCT; Lee et al., 2010), lactate dehydrogenase (Hashimoto et al., 2008), mitochondria and a reliable oxygen source in the receiving cell type. RNA-seq results from trophoblast cells provide evidence for MCT transporters, such as MCT2, LDHA and numerous mitochondrial genes (Vallet et al., 2010). In addition, the proximity of trophoblast epithelial cells to the maternal blood supply provides the most reliable source of oxygen available to the fetal– placental tissues. Thus, stromal cells may rely on anaerobic metabolism and produce lactate, which then diffuses to trophoblast cells or into the fetal blood stream to be further metabolized via aerobic metabolism (see Fig. 8). The generation of lactate by components of the placenta may provide an answer to a previously perplexing question. The two forms of hyaluronidase present in placental tissues of pigs are optimally active at low pH (Vallet et al., 2010) and generation of lactate would reduce the pH to favor high activity of these hyaluronidase isoforms. Activation of hyaluronidase would support changes in placental folding to increase surface area for exchange and production of low molecular weight hyaluronan which is known to be angiogenic (Ashworth et al., 1990; West et al., 1985). Lactate generation by the placenta may also participate in placental development and function by enhancing development of placental folding and an-

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Fig. 8. Nutrients are transported across the uterine luminal epithelium towards the apical surface of the chorionic epithelial cells. The Na+/K+ ATPase pump produces an ion gradient across the chorion to mediate active transport of nutrients into the connective tissue of the chorioallantois. Nutrients then diffuse or are transported into the vasculature within the allantois and then into the general circulation of the conceptus. Nutrients may diffuse to the basal surface of the allantoic epithelium and then be transported by nutrient transporters into the allantoic fluid from which it can be taken up again to enter the circulatory system of the conceptus. Nutrients can be stored in allantoic fluid for transport across the allantoic epithelium into the fetal–placental vasculature by nutrient transporters as there is no evidence for an active Na+/K + ATPase pump on the allantoic membrane at present; however, there may be another mechanism for mediating transport of nutrients into the allantoic vasculature. Glucose and fructose may be converted to carbon dioxide and water by aerobic metabolism, liberating energy in the form of ATP in well vascularized tissues such as the allantois. But, anaerobic glycolysis also generates ATP, and the end result for mammalian cells is typically lactate which may be the case for less well vascularized stromal cells of the pig placenta which is a net producer of lactate and the fetus is a net consumer of lactate. We speculate that tissues, for example stromal cells, in the placenta subjected to low oxygen tension engage in anaerobic metabolism whereas the relatively well oxygenated epithelial cells (chorion and allantois) engage in aerobic metabolism. The synthesis of lactate, a hallmark of anaerobic metabolism, is associated with expression of lactate dehydrogenase A (LDHA) (Draoui and Feron, 2011). Preliminary immunohistochemical analysis indicates that this isoform is localized to placental stromal cells whereas trophectoderm expresses the LDHB form which is associated with cells that convert lactate to pyruvate which then enters aerobic metabolism (J.L. Vallet, 2014). This result supports the hypothesis that a dichotomy exists in pathways for generation of ATP between epithelial and stromal cells in the pig placenta.

giogenesis, enhancing nutrient transport, and playing a role to conserve oxygen for use by the fetus. Glucose induces proliferation of human trophoblast cells through MTOR signaling in a PI3K-independent mechanism that involves activation of MTOR by metabolites of the GFPT1 pathway, particularly UDP-N-acetylglucosamine (Wen et al., 2005). It was proposed that UDP-GlcNAC phosphorylates TSC2, a GTPase activating protein, and p70S6K1, a protein kinase downstream of MTOR, to stimulate trophoblast cell proliferation in response to metabolism of glucose to glucose-6-PO4, fructose-6-PO4 and glucosamine-6-PO4. Fructose can stimulate the MTOR cell signaling pathway to affect cell proliferation, and also be used in the hexosamine pathway for synthesis of hyaluronic acid that can affect angiogenesis and other aspects of fetal–placental development during pregnancy (Kim et al., 2012). 7. Summary This review has focused on uterine adenogenesis in sheep and pigs that is vulnerable to adverse effects of endocrine disrupters in

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the environment such as progestins and estrogens, and dependent on key nutrients in the diet, as well as metabolic pathways during the neonatal period that allows for hormonal regulation of a uterine environment that supports growth and development of the conceptus for a successful outcome of pregnancy. In sheep, progesterone and IFNT regulate expression of genes for transport of nutrients into the uterine lumen as they are required for conceptus development. Arg from the diet or synthesized in the body is transported into the uterine lumen and then into cells of the conceptus by SLC7A1. The Arg is then available for metabolism to NO by NOS3 or to polyamines by arginase and ODC1 or by ADC and AGMAT. Both NO and polyamines are critical for conceptus development and agmatine itself may have direct effects on the conceptus. There was also discussion of carbohydrates in the diets, including fructose which is considered detrimental to health in adults. Nevertheless, fructose can be metabolized via several metabolic pathways to support growth and development of the conceptus. Based on the possible anaerobic and aerobic pathways for utilization of fructose by conceptuses of ungulates and cetaceans, but not adults, the results presented provide some insight into why fructose may increase obesity and insulin insensitivity in adults while enhancing fetal–placental growth. The chronology begins with uterine adenogenesis being adversely affected by endocrine disrupters such as progestins and estrogens during the neonatal period which prevents development of a functional uterus. Due to the failure of uterine adenogenesis, the uterine epithelial cells necessary to secrete and/ or transport nutrients (e.g., Arg, Gln and glucose) into the uterine lumen are not present. Therefore, nutrients are required by the conceptus for metabolism via various metabolic pathways to support normal growth and development beyond the peri-implantation period of pregnancy. Acknowledgements Research in our laboratories was supported by National Research Initiative Competitive Grants from the Animal Reproduction Program (2008-35203-19120, 2009-35206-05211, 2011-6701520067, and 2011-67015-20028) and Animal Growth & Nutrient Utilization Program (2008-35206-18764) of the USDA National Institute of Food and Agriculture, American Heart Association (10GRNT4480020), Texas A&M AgriLife Research (H-8200). The important contributions of our graduate students and colleagues in this research are gratefully acknowledged. References Abrams, R.M., 1979. Energy metabolism. Semin. Perinatol. 3, 109–119. Adams, N.R., 1995. Detection of the effects of phytoestrogens on sheep and cattle. J. Anim. Sci. 73, 1509–1515. Aherne, F., Hays, V.W., Ewan, R.C., Speer, V.C., 1969. Absorption and utilization of sugars by the baby pigs. J. Anim. Sci. 29, 444–450. Alexander, D.P., Andrews, R.D., Huggett, A.S., Nixon, D.A., Widdas, W.F., 1955a. The placental transfer of sugars in the sheep: studies with radioactive sugar. J. Physiol. 129, 352–366. Alexander, D.P., Huggett, A.S., Nixon, D.A., Widdas, W.F., 1955b. The placental transfer of sugars in the sheep: the influence of concentration gradient upon the rates of hexose formation as shown in umbilical perfusion of the placenta. J. Physiol. 129, 367–383. Ashworth, C.J., Fliss, M.F., Bazer, F.W., 1990. Evidence for steroid control of a putative angiogenic factor in the porcine uterus. J. Endocrinol. 125, 15–19. Bacon, J.S., Bell, D.J., 1946. The identification of fructose as a constituent of the foetal blood of the sheep. Biochem. J. 40. Bacon, J.S., Bell, D.J., 1948. Fructose and glucose in the blood of the foetal sheep. Biochem. J. 42, 397–405. Bailey, D.W., Dunlap, K.L., Erikson, D.W., Patel, A., Bazer, F.W., Burghardt, R.C., et al., 2010. Effects of long-term progesterone exposure on porcine uterine gene expression: progesterone alone does not induce secreted phosphoprotein 1 (osteopontin) in glandular epithelium. Reproduction 140, 595–604. Barclay, H., Haas, P., St, G., Huggett, A., King, G., Rowley, D., 1949. The sugar of the foetal blood, the amniotic and allantoic fluids. J. Physiol. 109, 98–102.

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Environmental factors affecting pregnancy: endocrine disrupters, nutrients and metabolic pathways.

Uterine adenogenesis, a unique post-natal event in mammals, is vulnerable to endocrine disruption by estrogens and progestins resulting in infertility...
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