Russell V. Anthony

The relationship between transplacental O2 diffusion and placental expression of PlGF, VEGF and their receptors in a placental insufficiency model of fetal growth restriction

Placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) are involved in placental angiogenesis through interactions with the VEGFR‐1 and VEGFR‐2 receptors. The placenta of pregnancies whose outcome is fetal growth restriction (FGR) are characterized by abnormal angiogenic development, classically associated with hypoxia. The present study evaluated the near‐term expression of this growth factor family in an ovine model of placental insufficiency–FGR, in relationship to uteroplacental oxygenation. Compared to controls, FGR pregnancies demonstrated a 37 % increase in uterine blood flow (FGR vs. control, 610.86 ± 48.48 vs. 443.17 ± 37.39 ml min−1 (kg fetus)−1; P < 0.04), which was associated with an increased maternal uterine venous PO2 (58.13 ± 1.00 vs. 52.89 ± 1.26 mmHg; P < 0.02), increased umbilical artery systolic/diastolic ratio (3.90 ± 0.33 vs. 2.12 ± 0.26, P < 0.05), and fetal hypoxia (arterial PO2; 12.79 ± 0.97 vs. 18.65 ± 1.6 mmHg, P < 0.005). Maternal caruncle PlGF mRNA was increased in FGR (P < 0.02), while fetal cotyledon VEGF mRNA was reduced (P < 0.02). VEGFR‐1 mRNA was also reduced in FGR fetal cotyledon (P < 0.001) but was not altered in caruncle tissue. Immunoblot analysis of PlGF and VEGF demonstrated single bands at 19 000 and 18 600 Mr, respectively. Caruncle PlGF concentration was increased (P < 0.04), while cotyledon VEGF was decreased (P < 0.05) in FGR placentae. The data establish that uterine blood flow is not reduced in relationship to metabolic demands in this FGR model and that the transplacental PO2 gradient is increased, maintaining umbilical oxygen uptake per unit of tissue. Furthermore, these data suggest that an increased transplacental gradient of oxygen generates changes in angiogenic growth factors, which may underline the pathophysiology of the post‐placental hypoxic FGR.

Investigating the causes of low birth weight in contrasting ovine paradigms

Intrauterine growth restriction (IUGR) still accounts for a large incidence of infant mortality and morbity worldwide. Many of the circulatory and transport properties of the sheep placenta are similar to those of the human placenta and as such, the pregnant sheep offers an excellent model in which to study the development of IUGR. Two natural models of ovine IUGR are those of hyperthermic exposure during pregnancy, and adolescent overfeeding, also during pregnancy. Both models yield significantly reduced placental weights and an asymmetrically growth‐restricted fetus, and display altered maternal hormone concentrations, indicative of an impaired trophoblast capacity. Additionally, impaired placental angiogenesis and uteroplacental blood flow appears to be an early defect in both the hyperthermic and adolescent paradigms. The effects of these alterations in placental functional development appear to be irreversible. IUGR fetuses are both hypoxic and hypoglycaemic, and have reduced insulin and insulin‐like growth factor‐1 (IGF‐1), and elevated concentrations of lactate. However, fetal utilization of oxygen and glucose, on a weight basis, remain constant compared with control pregnancies. Maintained utilization of these substrates, in a substrate‐deficient environment, suggests increased sensitivities to metabolic signals, which may play a role in the development of metabolic diseases in later adult life.

Uterine Vein Infusion of Interferon Tau (IFNT) Extends Luteal Life Span in Ewes

Interferon tau (IFNT) from the ovine conceptus has paracrine actions on the endometrium that alter release of prostaglandin F2alpha (PGF) and protect the corpus luteum (CL). Antiviral activity in uterine vein blood and expression of interferon-stimulated genes (ISGs) in CL is greater in pregnant than in nonpregnant ewes. We hypothesized that IFNT contributes to antiviral activity in uterine vein blood and has endocrine actions on the CL. Preadsorption of IFNT with antiserum against recombinant ovine (ro) IFNT revealed that antiviral activity in uterine vein blood from pregnant ewes was mediated by IFNT. Endocrine actions of IFNT were examined after infusing either roIFNT or bovine serum albumin (BSA; 200 μg/24 h; mini-osmotic pump) into the uterine vein of nonpregnant ewes from Day 10 to Day 11 postestrus. The abundance of ISG15 mRNA and protein was greater in CL (P < 0.05) from ewes receiving 24-h roIFNT infusion compared to that from ewes receiving 24-h BSA infusion. Injection of PGF at 12 h following insertion of mini-osmotic pumps resulted in a decline in serum progesterone concentrations 6 through 12 h later in BSA-infused ewes; however, in roIFNT-infused ewes, a similar decline in progesterone concentrations at 6 h was followed by recovery to control values at 12 h. Ewes then received infusions (200 μg/day) of either roIFNT or BSA for 7 days beginning on Day 10 of the estrous cycle. All BSA-infused ewes returned to estrus by Day 19, whereas 80% of roIFNT-infused ewes maintained luteal-phase concentrations of progesterone through Day 32. In conclusion, IFNT is released from the uterus into the uterine vein and acts through an endocrine mechanism to induce ISGs in the CL and delay luteolysis.

Prolactin Receptor Gene Expression and Foetal Adipose Tissue

We have investigated the effects of increasing gestational age, maternal undernutrition or restricted placental growth on prolactin receptor (PRLR) gene expression in perirenal adipose tissue collected from foetal sheep during late gestation (term=147 d±3 d of gestation). Foetal nutrient supply was reduced by either restriction of placental growth following removal of endometrial caruncles before mating or by reducing maternal feed intake by 50% from 115 d of gestation. Total RNA was extracted from adipose tissue taken from foetal sheep between 90 and 145 d of gestation, and only at 141–145 d in placentally restricted, nutrient restricted and control foetuses. Messenger RNAs encoding the long (PRLR1) and short (PRLR2) forms of the PRLR and glyceraldehyde‐phosphate‐dehydrogenase (GAPDH) were detected and quantified in a ribonuclease protection assay using an antisense RNA probe complementary to ovine PRLR2 and GAPDH. There was a 7.5‐fold increase in the amount of perirenal adipose tissue between 90 and 125 d of gestation, compared with a 1.3‐fold increase between 125 and 145 d of gestation. The abundance of mRNA encoding PRLR1 and PRLR2 in perirenal adipose tissue increased 10‐ and sixfold, respectively, between 90 and 125 d of gestation, and then declined by 145 d of gestation. Both placental restriction and maternal undernutrition significantly reduced foetal adipose tissue deposition. The abundance of PRLR1 but not PRLR2 mRNA was reduced in adipose tissue from the placentally restricted group, where as GAPDH mRNA was three times higher than in controls. In contrast, maternal undernutrition from 115 d of gestation did not affect PRLR1, PRLR2 or GAPDH mRNA expression in foetal adipose tissue. It is concluded that during the period of rapid deposition of perirenal adipose tissue, there is a concomitant increase in PRLR gene expression. This indicates that prolactin may play an important role in the growth and maturation of foetal adipose tissue which occurs before birth.

Expression of miRNAs in ovine fetal gonads: potential role in gonadal differentiation.

BackgroundGonadal differentiation in the mammalian fetus involves a complex dose-dependent genetic network. Initiation and progression of fetal ovarian and testicular pathways are accompanied by dynamic expression patterns of thousands of genes. We postulate these expression patterns are regulated by small non-coding RNAs called microRNAs (miRNAs). The aim of this study was to identify the expression of miRNAs in mammalian fetal gonads using sheep as a model.MethodsWe determined the expression of 128 miRNAs by real time PCR in early-gestational (gestational day (GD) 42) and mid-gestational (GD75) sheep ovaries and testes. Expression data were further examined and validated by bioinformatic analysis.ResultsExpression analysis revealed significant differences between ovaries and testes among 24 miRNAs at GD42, and 43 miRNAs at GD75. Bioinformatic analysis revealed that a number of differentially expressed miRNAs are predicted to target genes known to be important in mammalian gonadal development, including ESR1, CYP19A1, and SOX9. In situ hybridization revealed miR-22 localization within fetal testicular cords. As estrogen signaling is important in human and sheep ovarian development, these data indicate that miR-22 is involved in repressing estrogen signaling within fetal testes.ConclusionsBased on our results we postulate that gene expression networks underlying fetal gonadal development are regulated by miRNAs.

An animal model of placental insufficiency-induced intrauterine growth restriction.

Intrauterine growth restriction (IUGR), often associated with functional placental insufficiency, results in increased perinatal mortality and morbidity. For obvious reasons, many questions regarding the progression of IUGR pregnancies cannot be addressed experimentally in humans, predicating the use of animal models. Although no animal model fully recapitulates human pregnancy, the pregnant sheep has been used extensively to investigate maternal-fetal interactions. In sheep, surgical placement of catheters in both the maternal and fetal vasculature allows repeated sampling from nonanesthetized pregnancies. Considerable insight has been gained on placental oxygen and nutrient transfer and utilization from use of pregnant sheep, often confirmed in the human once appropriate technologies became available. This review will focus on one sheep model, used to examine the impact of placental insufficiency-induced IUGR on oxygen and nutrient transport and utilization.

Altered placental and fetal expression of IGFS and IGF-binding proteins associated with intrauterine growth restriction in fetal sheep during early and mid-pregnancy

The insulin-like growth factors (IGFs) are postulated to be altered in association with the development of intrauterine growth restriction (IUGR). The present studies examined placental and fetal hepatic mRNA concentration of components of the IGF system at two time points (55 and 90 d gestational age, dGA; Term 147 dGA) in a hyperthermia (HT)-induced sheep model of placental insufficiency-IUGR. Maternal plasma insulin and IGF-I were constant at 55 and 90 dGA and were unaffected by treatment. Umbilical vein insulin concentrations tended to be reduced at 90 dGA following HT exposure. Caruncle IGF-I mRNA was increased at 90 dGA in HT placentae (p < 0.05), while cotyledon concentrations were constant over gestation and unaltered by treatment. In control cotyledons, IGF-II mRNA concentration increased (p < 0.01) and IGFBP-3 decreased between 55 and 90 dGA (p < 0.01). Cotyledon IGF-II and caruncle IGFBP-4 mRNA were elevated at 55 dGA in HT placentae compared with control (p < 0.01 and p < 0.05 respectively). Fetal hepatic IGF-I, IGFBP-2, -3 and -4 concentrations rose over gestation (p < 0.05), but there were no treatment effects. These data suggest that changes in placental IGF expression in early and mid gestation may predispose the pregnancy to placental insufficiency, resulting in inadequate substrate supply to the developing fetus later in gestation.

Placental expression of angiopoietin-1, angiopoietin-2 and tie-2 during placental development in an ovine model of placental insufficiency-fetal growth restriction.

Fetal growth restriction (FGR) is associated with increased perinatal morbidity and mortality, and often results from functional placental insufficiency. Placentation requires extensive vasculogenesis and subsequent angiogenesis, in both maternal and fetal tissues. Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2) are angiogenic growth factors expressed in the placenta, and compete for binding to a common receptor, Tunica interna endothelial cell kinase-2 (Tie-2). Our objective was to examine Ang-1, Ang-2 and Tie-2 expression in ovine placental tissue obtained from normal and FGR pregnancies throughout gestation. Fetal cotyledon and maternal caruncle tissue concentrations of Ang-1, Ang-2 and Tie-2 mRNA were assessed by real-time reverse transcriptase-polymerase chain reaction and protein concentrations were assessed by Western immunoblot analysis, at 55, 90 and 135 d gestational age (dGA). Concentrations of Ang-1, Ang-2 and Tie-2 mRNA in FGR fetal cotyledons were increased at 55 dGA, and Tie-2 mRNA concentrations were decreased in FGR fetal cotyledons and maternal caruncles at 135 dGA. Immunoblot analysis demonstrated increased concentrations of Ang-2 in the fetal cotyledon at 55 dGA, and lower concentrations at 135 dGA. In contrast, concentrations of Tie-2 were increased at 90 dGA, but tended to decrease at 135 dGA in FGR maternal caruncles. The changes observed during early- to mid-gestation may result in increased branching angiogenesis, but may also set the stage for increased nonbranching angiogenesis during late gestation, altered placental architecture and placental insufficiency that result in FGR.

The expression of ovine placental lactogen, StAR and progesterone-associated steroidogenic enzymes in placentae of overnourished growing adolescent ewes

Overnourishing pregnant adolescent sheep promotes maternal growth but reduces placental mass, lamb birth weight and circulating progesterone. This study aimed to determine whether altered progesterone reflected transcript abundance for StAR (cholesterol transporter) and the steroidogenic enzymes (Cyp11A1, Hsd3b and Cyp17). Circulating and placental expression of ovine placental lactogen (oPL) was also investigated. Adolescent ewes with singleton pregnancies were fed high (H) or moderate (M) nutrient intake diets to restrict or support placental growth. Experiment 1: peripheral progesterone and oPL concentrations were measured in H (n=7) and M (n=6) animals across gestation (days 7-140). Experiment 2: progesterone was measured to mid- (day 81; M: n=11, H: n=13) or late gestation (day 130; M: n=21, H: n=22), placental oPL, StAR and steroidogenic enzymes were measured by qPCR and oPL protein by immunohistochemistry. Experiment 1: in H vs M animals, term placental (P<0.05), total cotyledon (P<0.01) and foetal size (P<0.05) were reduced. Circulating oPL and progesterone were reduced at mid- (P<0.001, P<0.01) and late gestation (P<0.01, P<0.05) and oPL detection was delayed (P<0.01). Experiment 2: placental oPL was not altered by nutrition. In day 81 H animals, progesterone levels were reduced (P<0.001) but not related to placental or foetal size. Moreover, placental steroidogenic enzymes were unaffected. Day 130 progesterone (P<0.001) and Cyp11A1 (P<0.05) were reduced in H animals with intrauterine growth restriction (H+IUGR). Reduced mid-gestation peripheral oPL and progesterone may reflect altered placental differentiation and/or increased hepatic clearance respectively. Restricted placental growth and reduced biosynthesis may account for reduced progesterone in day 130 H+IUGR ewes.

Temporal Release, Paracrine and Endocrine Actions of Ovine Conceptus-Derived Interferon-Tau During Early Pregnancy

ABSTRACT The antiviral activity of interferon (IFN) increases in uterine vein serum (UVS) during early pregnancy in sheep. This antiviral activity in UVS collected on Day 15 of pregnancy is blocked by anti-IFN-tau (anti-IFNT) antibodies. Conceptus-derived IFNT was hypothesized to induce IFN-stimulated gene (ISG) expression in endometrium and extrauterine tissues during pregnancy. To test this hypothesis, blood was collected from ewes on Days 12–16 of the estrous cycle or pregnancy. Serum progesterone was >1.7 ng/ml in pregnant (P) and nonpregnant (NP) ewes until Day 13, then declined to <0.6 ng/ml by Day 15 in NP ewes. A validated IFNT radioimmunoassay detected IFNT in uterine flushings (UFs) on Days 13–16 and in UVS on Days 15–16 of pregnancy. IFNT detection in UF correlated with paracrine induction of ISGs in the endometrium and occurred prior to the inhibition of estrogen receptor 1 and oxytocin receptor expression in uterine epithelia on Day 14 of pregnancy. Induction of ISG mRNAs in corpus luteum (CL) and liver tissue occurred by Day 14 and in peripheral blood mononuclear cells by Day 15 in P ewes. Expression of mRNAs for IFN signal transducers and ISGs were greater in the CL of P than that of NP ewes on Day 14. It is concluded that: 1) paracrine actions of IFNT coincide with detection of IFNT in UF; 2) endocrine action of IFNT ensues through induction of ISGs in peripheral tissues; and 3) IFNT can be detected in UVS, but not until Days 15–16 of pregnancy, which may be limited by the sensitivity of the IFNT radioimmunoassay.