Chorionic somatomammotropin RNA interference alters fetal liver glucose utilization

Chorionic somatomammotropin (CSH) is a placenta-specific hormone associated with fetal growth, and fetal and maternal metabolism in both humans and sheep. We hypothesized that CSH deficiency could impact sheep fetal liver glucose utilization. To generate CSH-deficient pregnancies, day 9 hatched blastocysts were infected with lentiviral particles expressing CSH-specific shRNA (RNAi) or scramble control shRNA (SC) and transferred to synchronized recipients. CSH RNAi generated two distinct phenotypes at 135 days of gestational age (dGA); pregnancies with IUGR (RNAi-IUGR) or with normal fetal weight (RNAi-NW). Fetal body, fetal liver and placental weights were reduced (P < 0.05) only in RNAi-IUGR pregnancies compared to SC. Umbilical artery plasma insulin and insulin-like growth factor 1 (IGF1) concentrations were decreased, whereas insulin receptor beta (INSR) concentration in fetal liver was increased (P < 0.05) in both RNAi phenotypes. The mRNA concentrations of IGF1, IGF2, IGF binding protein 2 (IGFBP2) and IGFBP3 were decreased (P < 0.05) in fetal livers from both RNAi phenotypes. Fetal liver glycogen concentration and glycogen synthase 1 (GYS1) concentration were increased (P < 0.05), whereas fetal liver phosphorylated-GYS (inactive GYS) concentration was reduced (P < 0.05) in both RNAi phenotypes. Lactate dehydrogenase B (LDHB) concentration was increased (P < 0.05) and IGF2 concentration was decreased (P < 0.05) in RNAi-IUGR fetal livers only. Our findings suggest that fetal liver glucose utilization is impacted by CSH RNAi, independent of IUGR, and is likely tied to enhanced fetal liver insulin sensitivity in both RNAi phenotypes. Determining the physiological ramifications of both phenotypes, may help to differentiate direct effect of CSH deficiency or its indirect effect through IUGR.

IMPACT OF CHORIONIC SOMATOMAMMOTROPIN RNA INTERFERENCE ON UTERINE BLOOD FLOW AND PLACENTAL GLUCOSE UPTAKE IN THE ABSENCE OF INTRAUTERINE GROWTH RESTRICTION

Chorionic Somatomammotropin (CSH) is one of the most abundantly produced placental hormones, yet its exact function remains elusive. Near-term (135 dGA), CSH RNA interference (RNAi) results in two distinct phenotypes: 1) pregnancies with intrauterine growth restriction (IUGR), and 2) pregnancies with normal fetal and placental weights. Here we report the physiological changes in CSH RNAi pregnancies without IUGR. The trophectoderm of hatched blastocysts (9 dGA) were infected with lentiviral-constructs expressing either a scrambled control (Control RNAi) or CSH-specific shRNA (CSH RNAi), prior to transfer into synchronized recipient ewes. At 126 dGA, Control RNAi (n = 6) and CSH RNAi (n = 6) pregnancies were fitted with maternal and fetal catheters. Uterine and umbilical blood flows were measured at 132 dGA and nutrient uptakes were calculated by the Fick principle. Control RNAi and CSH RNAi pregnancies were compared by analysis of variance, and significance was set at P ≤ 0.05. Absolute (ml/min) and relative (ml/min/kg fetus) uterine blood flows were reduced (P ≤ 0.05) in CSH RNAi pregnancies, but umbilical flows were not impacted. The uterine artery-to-vein glucose gradient (mmol/l) was significantly (P ≤ 0.05) increased. The uteroplacental glucose uptake (μmol/min/kg placenta) was increased (P ≤ 0.05), whereas umbilical glucose uptake (μmol/min/kg fetus) was reduced. Our results demonstrate that CSH RNAi has significant physiological ramifications, even in the absence of IUGR, and comparing CSH RNAi pregnancies exhibiting both IUGR and non-IUGR phenotypes may help determine the direct effects of CSH and its potential impact on fetal development.

152 Impact of chorionic somatomammotropin RNA interference on uterine blood flow and placental glucose uptake in the absence of intrauterine growth restriction

Chorionic somatomammotropin (CSH) is one of the most abundant hormones produced by the sheep placenta, yet the exact function of CSH has been elusive. Previously we reported the use of in vivo RNA interference (RNAi) to assess the impact of CSH deficiency on placental and fetal growth in sheep. Near-term (135 dGA), there are two distinct CSH RNAi phenotypes: 1) pregnancies with intrauterine growth restriction (IUGR), and 2) pregnancies with normal fetal and placental weights. This study describes physiological changes in the latter phenotype. To generate the CSH RNAi pregnancies, the trophectoderm of hatched blastocysts (9 dGA) were infected with lentiviral-constructs expressing either a scrambled control (NTS) or CSH-specific shRNA (CSH RNAi), prior to transfer into synchronized recipient ewes. At 120 dGA, 6 NTS and 6 CSH RNAi pregnancies were fitted with maternal and fetal catheters. Uterine and umbilical blood flows were measured utilizing the 3H2O transplacental diffusion technique at 132 dGA, and nutrient uptakes were calculated by the Fick principle. Resulting data were analyzed by Student’s t-test and significance was set at P ≤ 0.05. CSH RNAi tended (P ≤ 0.10) to reduce placentome weight with no effect on fetal weight. Absolute (ml/min) and relative (ml/min/kg fetus) uterine blood flows were reduced (P ≤ 0.05) in CSH RNAi pregnancies, but umbilical flows were not impacted. The uterine artery-to-vein glucose gradient (mmol/l) was significantly (P ≤ 0.05) increased, whereas the gradients for taurine and glycine were reduced (P ≤ 0.05). Uteroplacental glucose uptake (mmol/min/kg placenta) was increased 27% (P ≤ 0.05), whereas umbilical glucose uptake (mmol/min/kg fetus) was reduced 13%. This cohort demonstrates that even in the absence of IUGR, CSH deficiency has significant physiological ramifications, and the investigation of CSH RNAi pregnancies exhibiting both IUGR and non-IUGR phenotypes may help determine the direct effects of CSH and its potential impact on fetal programming. Supported by NIH R01 HD093701.

PSII-39 Trophectoderm-specific RNA interference of chorionic somatomammotropin alters glucose metabolism in sheep fetal liver

Chorionic somatomammotropin (CSH) is a placenta-specific hormone and secreted into both fetal and maternal circulation. Reduced maternal CSH is observed with intrauterine growth restriction (IUGR) in both humans and sheep, and it has long been held that CSH modulates maternal and fetal metabolism. We hypothesized that CSH deficiency, created by RNA interference (RNAi), could impact fetal liver glucose metabolism. To generate CSH-deficient pregnancies, day 9 hatched blastocysts were infected with lentiviral particles expressing CSH-specific shRNA (RNAi) or scramble control shRNA (SC) and transferred to synchronized recipients. CSH RNAi generated two distinct phenotypes at 135 dGA; CSH RNAi pregnancies with IUGR (RNAi-IUGR; n = 8) or without IUGR (RNAi; n = 8). Data from both RNAi phenotypes were compared separately with SC using Welch’s t-test. Liver and placental weights were reduced (P &lt; 0.05) in RNAi-IUGR pregnancies, but not in RNAi pregnancies, as compared to SC (n = 8). Umbilical artery plasma insulin and insulin-like growth factor 1 (IGF1) concentrations were decreased (P &lt; 0.05), whereas insulin receptor beta (IRβ) concentration, as determined by Western immunoblot analysis, in fetal liver was increased (P &lt; 0.05) in both RNAi phenotypes. Fetal liver glycogen quantity was also increased (P &lt; 0.05) in both RNAi phenotypes. Glycogen synthase-1 (GYS-1) concentration in fetal liver was increased (P &lt; 0.05) in both RNAi phenotypes, whereas there was no change in GYS-2 concentration. Phosphorylated-GYS (inactive GYS) was reduced (P &lt; 0.05) in fetal livers for both RNAi phenotypes. Lactate dehydrogenase beta (LDHβ) concentration was increased (P &lt; 0.05) and IGF2 concentration was decreased (P &lt; 0.05) in RNAi-IUGR fetal livers only. From these results we conclude that fetal liver glucose metabolism is impacted by CSH RNAi, independent of IUGR, and is likely tied to enhanced insulin sensitivity in both CSH RNAi phenotypes. Differences between the two phenotypes may help differentiate direct and indirect effects of CSH. Supported by NIH R01 HD093701.

PSII-35 Melatonin supplementation and restricted nutrition do not affect chorionic somatomammotropin (CSH) concentration in ovine placenta from mid- to late- gestation

Melatonin plays a role as a vasodilator. Vasoactive and angiogenic factors are expressed by placental binucleate cells (BNC) and produce chorionic somatomammotropin (CSH), known to impact fetal and placental growth. We hypothesized that melatonin supplementation and restricted nutrition from mid- to late-gestation would alter CSH concentration and some characteristics of BNC in placenta. At day 50 of gestation, ewes carrying singletons were randomly assigned to a 2 × 2 factorial design and were fed either an adequate (ADQ; 100% NRC; n = 15) or restricted (RES; 60% NRC; n = 15) diet supplemented with 0 (CON, n = 14) or 5 mg of melatonin (MEL; n = 16). Placentomes were collected on day 130 of gestation and preserved in formalin for histological analysis. Cotyledon (COT) were snap frozen for western immunoblotting analyses. Tissue sections were stained using biotinylated Dolichos Biflurus (DBA; a marker of fetal membrane) lectin and fluorescein labeled Texas red-avidin and fluorescein labeled Griffonia Simplifolica (BS) lectin (a marker of BNC). The number, area, and diameter of BNC in COT were determined by image analysis. For immunoblotting, protein was extracted from COT in SDS phosphate buffer, loaded equally, and separated on 12.5% polyacrylamide gels. Protein was transferred to PVDF membranes and incubated with rabbit anti-CSH. Bands were visualized and imaged. Data were analyzed using Proc Mixed procedure of SAS. Melatonin supplementation and restricted nutrition did not affect BNC number, area, or diameter, or CSH protein expression. While we reject our hypothesis that melatonin supplementation and nutrient restriction would alter the CSH concentration and BNC characteristics in COT, we continue to evaluate if the BNC produce angiogenic or vasoactive factors that may influence placental and mammary gland functions in sheep.

Chorionic somatomammotropin impacts early fetal growth and placental gene expression

Several developmental windows, including placentation, must be negotiated to establish and maintain pregnancy. Impaired placental function can lead to preeclampsia and/or intrauterine growth restriction (IUGR), resulting in increased infant mortality and morbidity. It has been hypothesized that chorionic somatomammotropin (CSH) plays a significant role in fetal development, potentially by modifying maternal and fetal metabolism. Recently, using lentiviral-mediated in vivo RNA interference in sheep, we demonstrated significant reductions in near-term (135 days of gestation; dGA) fetal and placental size, and altered fetal liver gene expression, resulting from CSH deficiency. We sought to examine the impact of CSH deficiency on fetal and placental size earlier in gestation (50 dGA), and to examine placental gene expression at 50 and 135 dGA. At 50 dGA, CSH-deficient pregnancies exhibited a 41% reduction (P ≤ 0.05) in uterine vein concentrations of CSH, and significant (P ≤ 0.05) reductions (≈21%) in both fetal body and liver weights. Placentae harvested at 50 and 135 dGA exhibited reductions in IGF1 and IGF2 mRNA concentrations, along with reductions in SLC2A1 and SLC2A3 mRNA. By contrast, mRNA concentrations for various members of the System A, System L and System y+ amino acid transporter families were not significantly impacted. The IUGR observed at the end of the first-third of gestation indicates that the near-term IUGR reported previously, began early in gestation, and may have in part resulted from deficits in the paracrine action of CSH within the placenta. These results provide further compelling evidence for the importance of CSH in the progression and outcome of pregnancy.

Development of ovine chorionic somatomammotropin hormone-deficient pregnancies

Intrauterine growth restriction (IUGR) is a leading cause of neonatal mortality and morbidity. Chorionic somatomammotropin hormone (CSH), a placenta-specific secretory product found at high concentrations in maternal and fetal circulation throughout gestation, is significantly reduced in human and sheep IUGR pregnancies. The objective of this study was to knock down ovine CSH ( oCSH) expression in vivo using lentiviral-mediated short-hairpin RNA to test the hypothesis that oCSH deficiency would result in IUGR of near-term fetal lambs. Three different lentiviral oCSH-targeting constructs were used and compared with pregnancies ( n = 8) generated with a scrambled control (SC) lentiviral construct. Pregnancies were harvested at 135 days of gestation. The most effective targeting sequence, “target 6” (tg6; n = 8), yielded pregnancies with significant reductions ( P ≤ 0.05) in oCSH mRNA (50%) and protein (38%) concentrations, as well as significant reductions ( P ≤ 0.05) in placental (52%) and fetal (32%) weights compared with the SC pregnancies. Fetal liver weights were reduced 41% ( P ≤ 0.05), yet fetal liver insulin-like growth factor-I ( oIGF1) and -II mRNA concentrations were reduced ( P ≤ 0.05) 82 and 71%, respectively, and umbilical artery oIGF1 concentrations were reduced 62% ( P ≤ 0.05) in tg6 pregnancies. Additionally, fetal liver oIGF-binding protein ( oIGFBP) 2 and oIGFBP3 mRNA concentrations were reduced ( P ≤ 0.05), whereas fetal liver oIGFBP1 mRNA concentration was not impacted nor was maternal liver o IGF and o IGFBP mRNA concentrations or uterine artery oIGF1 concentrations ( P ≥ 0.10). Based on our results, it appears that oCSH deficiency does result in IUGR, by impacting placental development as well as fetal liver development and function.

Identification of functional CCAAT/enhancer-binding protein and Ets protein binding sites in the human chorionic somatomammotropin enhancer sequences

The human chorionic somatomammotropin (CS) A and B genes (listed asCSH1andCSH2in the HUGO database) are highly expressed in placenta. A 241 bp potent enhancer, nucleotides (nts) 1–241, located at the 3′ end of theCS-Bgene (CS-Benh) stimulates promoter activity specifically in placental trophoblast cellsin vitro. Strong activity is exerted by a 23 bp element within the CS-Benh (nts 117–139), shown to interact with transcription enhancer factor (TEF) members of the transcription enhancer activator (TEA) DNA-binding domain-containing family. An identical TEF element is present in the homologous (97.5%) CS-Aenh; however, a few nucleotide differences suppress its activity. Previously, we identified regulatory sequences distinct from the TEF element within an 80 bp modulatory domain (nts 1–80) in the CS-Benh. Using structural and functional assays we now show that CCAAT/enhancer-binding protein (C/EBP) binding sites exist in the 80 bp modulatory domains of both enhancers, and an Elk-1 binding site exists in the modulatory domain of the CS-Aenh. C/EBPα or C/EBPβ strongly repressedCSp.CATactivity but stimulatedCSp.CAT.CS-Benhactivity. In contrast, the equivalentCS-Aenhancer sequences were unable to relieve promoter repression. Elk-1 overexpression also resulted in differential effects on the CS-Aenh versus CS-Benh. Finally, we provide evidence for the association of C/EBPβ with theCS-AandCS-Bgenes in human placental chromatin, including differential involvement of C/EBPβ with the CS-Aenh versus the CS-Benh, and therefore consistent with the notion that these are regions of regulatory significancein vivo. We conclude that members of the C/EBP and Ets families can differentially modulate CS-Benh and CS-Aenh activity.