This study was conducted to test the hypothesis that dietary supplementation of arginine, the physiologic precursor of nitric oxide (NO), reduces fat mass in the Zucker diabetic fatty (ZDF) rat, a genetically obese animal model of type-II diabetes mellitus. Male ZDF rats, 9 wk old, were pair-fed Purina 5008 diet and received drinking water containing arginine-HCl (1.51%) or alanine (2.55%, isonitrogenous control) for 10 wk. Serum concentrations of arginine and NO(x) (oxidation products of NO) were 261 and 70% higher, respectively, in arginine-supplemented rats than in control rats. The body weights of arginine-treated rats were 6, 10, and 16% lower at wk 4, 7, and 10 after the treatment initiation, respectively, compared with control rats. Arginine supplementation reduced the weight of abdominal (retroperitoneal) and epididymal adipose tissues (45 and 25%, respectively) as well as serum concentrations of glucose (25%), triglycerides (23%), FFA (27%), homocysteine (26%), dimethylarginines (18-21%), and leptin (32%). The arginine treatment enhanced NO production (71-85%), lipolysis (22-24%), and the oxidation of glucose (34-36%) and octanoate (40-43%) in abdominal and epididymal adipose tissues. Results of the microarray analysis indicated that arginine supplementation increased adipose tissue expression of key genes responsible for fatty acid and glucose oxidation: NO synthase-1 (145%), heme oxygenase-3 (789%), AMP-activated protein kinase (123%), and peroxisome proliferator-activated receptor gamma coactivator-1alpha (500%). The induction of these genes was verified by real-time RT-PCR analysis. In sum, arginine treatment may provide a potentially novel and useful means to enhance NO synthesis and reduce fat mass in obese subjects with type-II diabetes mellitus.

Polyamines (putrescine, spermidine, and spermine) are essential for placental growth and angiogenesis. However, little is known about polyamine synthesis in the porcine placenta during conceptus development. The present study was conducted to test the hypothesis that arginine and proline are the major sources of ornithine for placental polyamine production in pigs. Placentae, amniotic fluid, and allantoic fluid were obtained from gilts on Days 20, 30, 35, 40, 45, 50, 60, 90, and 110 of the 114-day gestation (n = 6 per day). Placentae as well as amniotic and allantoic fluids were analyzed for arginase, proline oxidase, ornithine aminotransferase (OAT), ornithine decarboxylase (ODC), proline transport, concentrations of amino acids and polyamines, and polyamine synthesis using established radiochemical and chromatographic methods. Neither arginase activity nor conversion of arginine into polyamines was detected in the porcine placenta. In contrast, both proline and ornithine were converted into putrescine, spermidine, and spermine in placental tissue throughout pregnancy. The activities of proline oxidase, OAT, and ODC as well as proline transport, polyamine synthesis from proline, and polyamine concentrations increased markedly between Days 20 and 40 of gestation, declined between Days 40 and 90 of gestation, and remained at the reduced level through Day 110 of gestation. Proline oxidase and OAT, but not arginase, were present in allantoic and amniotic fluids for the production of ornithine (the immediate substrate for polyamine synthesis). The activities of these two enzymes as well as the concentrations of ornithine and total polyamines in fetal fluids were highest at Day 40 but lowest at Days 20, 90, and 110 of gestation. These results indicate that proline is the major amino acid for polyamine synthesis in the porcine placenta and that the activity of this synthetic pathway is maximal during early pregnancy, when placental growth is most rapid. Our novel findings provide a new base of information for future studies to define the role of proline in fetoplacental growth and development.

No abstract

Glutamine plays a vital role in fetal carbon and nitrogen metabolism and exhibits the highest fetal:maternal plasma ratio among all amino acids in pigs. Such disparate glutamine levels between mother and fetus suggest that glutamine may be actively synthesized and released into the fetal circulation by the porcine placenta. We hypothesized that branched-chain amino acid (BCAA) metabolism in the placenta plays an important role in placental glutamine synthesis. This hypothesis was tested by studying conceptuses from gilts on Days 20, 30, 35, 40, 45, 50, 60, 90, or 110 of gestation (n = 6 per day). Placental tissue was analyzed for amino acid concentrations, BCAA transport, BCAA degradation, and glutamine synthesis as well as the activities of related enzymes (including BCAA transaminase, branched-chain alpha-ketoacid dehydrogenase, glutamine synthetase, glutamate-pyruvate transaminase, and glutaminase). On all days of gestation, rates of BCAA transamination were much greater than rates of branched-chain alpha-ketoacid decarboxylation. The glutamate generated from BCAA transamination was primarily directed to glutamine synthesis and, to a much lesser extent, alanine production. Placental BCAA transport, BCAA transamination, glutamine synthesis, and activities of related enzymes increased markedly between Days 20 and 40 of gestation, as did glutamine in fetal allantoic fluid. Accordingly, placental BCAA levels decreased after Day 20 of gestation in association with a marked increase in BCAA catabolism and concentrations of glutamine. There was no detectable catabolism of glutamine in pig placenta throughout pregnancy, which would ensure maximum output of glutamine by this tissue. These novel results demonstrate glutamine synthesis from BCAAs in pig placentae, aid in explaining the abundance of glutamine in the fetus, and provide valuable insight into the dynamic role of the placenta in fetal metabolism and nutrition.

Postnatal uterine development involves differentiation and development of the endometrial glandular epithelium from the luminal epithelium as well as development of the mesenchyme into the endometrial stroma and myometrium. This period of development is critical because exposure of neonates to endocrine disruptors compromises reproductive cycles and pregnancy in the adult. However, the hormonal, cellular, and molecular mechanisms regulating postnatal uterine development remain largely unknown. In order to identify candidate genes and gene networks that regulate postnatal uterine development, uteri were collected from CD-1 outbred mice on postnatal days (PND) 3, 6, 9, 12, and 15, and gene expression profiling was conducted using Affymetrix mouse genome U74Av2 GeneChips in study 1. Of the approximately 12,000 genes analyzed, 9002 genes were expressed in the uterus and expression of 3012 genes increased or decreased 2-fold during uterine development. In study 2, the uterine epithelium was enzymatically separated from the stroma/myometrium on PNDs 3, 6, and 9, and gene expression profiling was conducted using CodeLink UniSet Mouse I Expression Bioarrays. Results from these two studies support the hypothesis that postnatal uterine development is a complex process involving overlapping positive and negative changes in uterine epithelial and stromal/myometrial gene expression. Candidate genes regulating uterine development encode secreted factors (Wnt5a, Wnt7a), transcription factors (Hoxa10, Hoxa11, Hoxd10, MSX-1), enzymes (matrix metalloproteinases, cathepsin, carbonic anhydrase), growth factors (IGF-II, IGF binding proteins), and components of the extracellular matrix (osteopontin) to name a few. The candidate genes and gene networks identified by transcriptional profiling provide an important foundation to discern and understand mechanisms regulating postnatal uterine morphogenesis.

Porcine trophoblast attachment to the uterine surface is associated with increased conceptus and endometrial production of prostaglandins. Conceptus secretion of estrogen on Day 12 of gestation is important for establishment of pregnancy; however, early (Days 9 and 10) exposure to exogenous estrogens results in embryonic mortality. Present studies established the temporal and spatial pattern of endometrial PTGS1 (prostaglandin-endoperoxide synthase 1) and PTGS2 expression during the estrous cycle and early pregnancy and determined the effect of early estrogen treatment on endometrial PTGS expression in pregnant gilts. Endometrial PTGS1 mRNA expression increased 2- to 3-fold after Day 10 of the estrous cycle and pregnancy, whereas PTGS2 mRNA expression increased 76-fold between Days 5 and 15 of the estrous cycle and pregnancy. Increased expression of the PTGS2 transcript was detected in the lumenal epithelium after Day 10 in both cyclic and pregnant gilts. There was a 10- and 20-fold increase in endometrial PTGS2 protein expression between Days 5 and 18 of the estrous cycle and pregnancy respectively. Administration of estrogen on Days 9 and 10 of gestation increased endometrial PTGS2 mRNA and protein on Day 10, but decreased PTGS2 mRNA and protein in lumenal epithelium (LE) on Day 12 of gestation compared to vehicle-treated gilts. The present study demonstrates that an increase in uterine epithelial PTGS2 expression occurs after Day 10 of the estrous cycle and early pregnancy in the pig. The conceptus-independent increase in the uterine LE indicates that a novel pathway exists for endometrial induction PTGS2 expression before conceptus elongation and attachment to the uterine surface. Epithelial expression of PTGS2 may serve as one of the signals for placental attachment and embryo survival in the pig. Early administration of estrogen on Days 9 and 10 of pregnancy alters endometrial PTGS2 mRNA and protein expression, which may, at least in part, represent a mechanism by which endocrine disruption of pregnancy causes total embryonic loss during implantation in the pig.

No abstract

Secreted phosphoprotein 1 (SPP1, osteopontin) is the most highly upregulated extracellular matrix/adhesion molecule/cytokine in the receptive phase human uterus, and Spp1 null mice manifest decreased pregnancy rates during mid-gestation as compared with wild-type counterparts. We hypothesize that Spp1 is required for proliferation, migration, survival, adhesion, and remodeling of cells at the conceptus-maternal interface. Our objective was to define the temporal/spatial distribution and steroid regulation of Spp1 in mouse uterus during estrous cycle and early gestation. In situ hybridization localized Spp1 to luminal epithelium (LE) and immune cells. LE expression was prominent at proestrus, decreased by estrus, and was nearly undetectable at diestrus. During pregnancy, Spp1 mRNA was not detected in LE until day 4.5 (day 1Zvaginal plug). Spp1-expressing immune cells were scattered within the endometrial stroma throughout the estrous cycle and early pregnancy. Immunoreactive Spp1 was prominent at the apical LE surface by day 4.5 of pregnancy and Spp1 protein was also co-localized with subsets of CD45-positive (leukocytes) and F4/80-positive (macrophages) cells. In ovariectomized mice, estrogen, but not progesterone, induced Spp1 mRNA, whereas estrogen plus progesterone did not induce Spp1 in LE. These results establish that estrogen regulates Spp1 in mouse LE and are the first to identify macrophages that produce Spp1 within the peri-implantation endometrium of any species. We suggest that Spp1 at the apical surface of LE provides a mechanism to bridge conceptus to LE during implantation, and that Spp1-positive macrophages within the stroma may be involved in uterine remodeling for conceptus invasion.