HMGA2 is regulated by LIN28 and BRCA1 in human placental cells.

The chromatin associated transcription factor HMGA2 is a downstream target of let-7 miRNAs and binds to chromatin to regulate gene expression. Inhibition of let-7 miRNAs by RNA-binding proteins LIN28A and LIN28B is necessary during early embryogenesis to ensure stable expression of HMGA2. In addition to LIN28, HMGA2 is regulated by a BRCA1/ZNF350/CtIP repressor complex. In normal tissues, the BRCA1/ZNF350/CtIP complex binds to the HMGA2 promoter to prevent transcription. However, in many cancers the oncomiR miR-182 targets BRCA1, preventing BRCA1 translation and allowing for increased HMGA2. Little is known about the regulation of HMGA2 during early placental development; therefore, we hypothesized that both LIN28 and BRCA1 can regulate HMGA2 in placental cells. Using siRNA and CRISPR gene editing techniques, we found that knockdowns of both LIN28A and LIN28B increase HMGA2 levels in ACH-3P cells. These cells also demonstrated deficiencies in cell differentiation, seemingly differentiating solely towards the syncytiotrophoblast sublineage, secreting higher amounts of hCG, and displaying upregulated ERVW-1. Additionally, we found that a knockout of both LIN28A and LIN28B caused a significant increase of miR-182 and a decrease in BRCA1 allowing HMGA2 mRNA levels to increase and protein levels to remain the same. Using chromatin immunoprecipitation, we saw binding of the BRCA1 repressor complex to HMGA2. We also saw a decrease in binding to HMGA2's promoter in the LIN28A/B knockout cells. These findings suggest a novel role for BRCA1 during early human placental development.

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.

Duration of maternal undernutrition differentially alters fetal growth and hormone concentrations.

To investigate the impact of duration of maternal undernutrition in twin sheep pregnancies, ewes were either fed 100% (C) or 50% of their nutrient requirements from 28 to 78 d gestational age (dGA) and readjusted to 100% beginning at 79 dGA (LC) or continuously restricted from 28 to 135 dGA (LL). Weights of the fetus, empty carcass, brain, and liver were greater in the LC than LL fetuses at 135 dGA (P ≤ 0.05). Although umbilical vein (UmV) glucose concentrations did not differ, the UmV:umbilical artery (UmA) glucose gradient was smaller (0.26 ± 0.03 vs 0.38 ± 0.03 and 0.39 ± 0.04 mmol L(-1); P ≤ 0.05) in LL than C and LC fetuses, respectively. Umbilical vein concentrations of IGF-1 were less (46.7 ± 5.62 vs 74.3 ± 6.71 ng/mL; P ≤ 0.05) in LL than LC fetuses. Additionally, LL fetuses tended (P ≤ 0.10) to have lower UmA concentrations of insulin (0.24 ± 0.13 vs 0.70 ± 0.15 ng/mL) and IGF-1 (66.6 ± 7.51 vs 91.4 ± 8.97 ng/mL) than LC fetuses. Although most of the observed differences occurred between LC and LL pregnancies, LC fetuses tended (P ≤ 0.10) to have greater UmV and UmA pCO2 than C fetuses. Furthermore, the UmV:UmA O2 content gradient tended to be greater (5.02 ± 0.43 vs 3.41 ± 0.47; P ≤ 0.10) in C than LL fetuses. UmA placental lactogen also tended to be greater (46.6 ± 4.40 vs 31.1 ± 4.69 ng/mL; P ≤ 0.10) in LL than C fetuses. These data suggest that in twin pregnancies, maternal undernutrition followed by realimentation induces a different fetal outcome compared with continuous nutrient restriction, and both may differ physiologically from control fed pregnancies.

Effects of overfeeding naturally-mated adolescent ewes on maternal, fetal, and postnatal lamb growth.

The objective of this study was to evaluate the effects of overfeeding naturally-mated adolescent ewes (Ovis aries) on maternal, fetal, and postnatal lamb growth, hormone concentrations, and lamb carcass characteristics. Two experiments were conducted in which singleton-bearing adolescent ewes were fed a diet containing 2.72 Mcal/kg ME at a rate which met NRC gestational age requirements (MN; n = 10 in Exp. 1, n = 7 in Exp. 2) or were fed the same diet ad libitum (15% refusal rate) throughout gestation (HN; n = 7 in Exp. 1, n = 6 in Exp. 2). Ewe BW was greater (P < 0.05) for HN than MN ewes beginning on 75 d and 52 d of gestation for Exp. 1 and 2, respectively. Final BCS was greater (P ≤ 0.05) for HN than MN ewes in both experiments; 3.5 vs. 3.0, respectively, for Exp. 1, and 4.8 vs. 2.9, respectively, for Exp. 2. Fasting maternal blood insulin concentrations were greater (P ≤ 0.05) in HN ewes near term (135 d of gestation), whereas fasting maternal glucose concentrations were greater (P ≤ 0.05) during most of the second half of gestation in HN ewes, for both experiments. Gestation length did not differ (P = 0.69) between treatments in Exp. 1, but in Exp. 2, HN ewes had shorter (P = 0.01) gestation lengths (144 vs. 149 d) and had increased (P = 0.002) dystocia scores. Fetal abdominal circumference was greater (P < 0.05) in lambs from MN than HN ewes at 97 d of gestation in Exp. 1 (20.8 vs. 17.4 cm) but did not differ (P = 0.94) between treatments at 95 d of gestation in Exp. 2 (averaging 20.5 cm). There were no differences (P ≥ 0.15) in lamb BW, abdominal circumference, crown-rump length, and biparietal distance at birth; or in postnatal BW and plasma concentrations of glucose, insulin, and lactate in either experiment. There were no differences (P ≥ 0.18) in HCW, dressing percentage, LM area, fat thickness, or KPH between treatments in Exp. 2. Although there was no difference (P ≥ 0.31) between treatments in concentrations of IGF1 or IGF2 mRNA in liver samples collected at harvest, lambs from MN ewes had greater (P ≤ 0.05) concentrations of IGF1R and INSR mRNA, suggesting long-term effects of maternal diet on postnatal hepatic function. In conclusion, excess nutrition during gestation in naturally-mated adolescent ewes did not affect birth weight or postnatal performance of offspring.

Effects of ractopamine and trenbolone acetate implants with or without estradiol on growth performance, carcass characteristics, adipogenic enzyme activity, and blood metabolites in feedlot steers and heifers.

Two experiments were conducted to evaluate effects of ractopamine (RAC) and steroidal implant treatments on performance, carcass traits, blood metabolites, and lipogenic enzyme activity in feedlot cattle. In Exp. 1, yearling steers (n = 486; initial BW = 305 kg) were used in a 3 × 3 factorial arrangement of RAC doses of 0 (R0), 100 (R100), or 200 (R200) mg·steer(-1)·d(-1) fed for 28 d and implant regimens (implant-reimplant) of no implant-no reimplant (NI-NI), 120 mg of trenbolone acetate (TBA) and 24 mg of estradiol-17ß (E17B)-no implant (RS-NI), or 80 mg of TBA and 16 mg of E17B followed by 120 mg of TBA and 24 mg of E17B (RI-RS). Except for KPH and skeletal maturity score, no RAC × implant interactions were noted (P > 0.10). Carcasses from R200 were 6.3 kg (P = 0.042) heavier than those from R0. Marbling, calculated empty body fat (EBF), and USDA quality grade did not differ (P > 0.10) among RAC treatments. The RI-RS steers had 12.6 kg (P = 0.001) and 41.1 kg (P < 0.001) greater HCW than RS-NI and NI-NI, respectively. Despite no difference (P > 0.10) in EBF, marbling score was decreased for RI-RS (P < 0.001) and RS-NI (P = 0.001) relative to NI-NI, resulting in 14.6 and 11.4 percentage unit fewer USDA Prime and Choice carcasses with RI-RS (P = 0.008) and RS-NI (P = 0.039) than with NI-NI. In Exp. 2, heifers (n = 48; initial BW = 347 kg) were used in a 3 × 2 factorial arrangement of RAC doses of 0 (R0) or 250 (R250) mg·heifer(-1)·d(-1) and implant regimens of none (NI), 200 mg of TBA (TO), or 200 mg of TBA and 20 mg of E17B (TE). Blood samples were collected at various times during the feeding period, and subcutaneous adipose samples were collected on d 119. For growth and carcass measurements, no RAC × implant interactions (P > 0.10) were detected. The RAC-supplemented heifers had greater HCW (P < 0.10) with no difference in marbling score. For implant regimens, TE heifers had greater HCW than the NI (P = 0.001) and TO (P = 0.037) heifers. Although EBF did not differ among implant treatments (P > 0.10), TE (P = 0.021) and TO (P = 0.039) had fewer Choice carcasses than NI. Heifers with implants had decreased cortisol and increased IGF-1 and NEFA (P < 0.10) compared with NI heifers. An implant × RAC interaction was detected (P = 0.001) for serum urea nitrogen (SUN), with TE and RAC-supplemented heifers having decreased SUN. These data suggest that the effects of implant and RAC on growth and carcass traits are independent and that USDA quality grade and marbling score can differ significantly among carcasses with similar calculated EBF values.

Assessing gene function in the ruminant placenta.

The placenta provides the means for nutrient transfer from the mother to the fetus, waste transfer from the fetus to the mother, protection of the fetus from the maternal immune system, and is an active endocrine organ. While many placental functions have been defined and investigated, assessing the function of specific genes expressed by the placenta has been problematic, since classical ablation-replacement methods are not feasible with the placenta. The pregnant sheep has been a long-standing animal model for assessing in vivo physiology during pregnancy, since surgical placement of indwelling catheters into both maternal and fetal vasculature has allowed the assessment of placental nutrient transfer and utilization, as well as placental hormone secretion, under unanesthetized-unstressed steady state sampling conditions. However, in ruminants the lack of well-characterized trophoblast cell lines and the inefficiency of creating transgenic pregnancies in ruminants have inhibited our ability to assess specific gene function. Recently, sheep and cattle primary trophoblast cell lines have been reported, and may further our ability to investigate trophoblast function and transcriptional regulation of genes expressed by the placenta. Furthermore, viral infection of the trophoectoderm layer of hatched blastocysts, as a means for placenta-specific transgenesis, holds considerable potential to assess gene function in the ruminant placenta. This approach has been used successfully to "knockdown" gene expression in the developing sheep conceptus, and has the potential for gain-of-function experiments as well. While this technology is still being developed, it may provide an efficient approach to assess specific gene function in the ruminant placenta.

Effect of dietary supplemental vitamin A concentration on performance, carcass merit, serum metabolites, and lipogenic enzyme activity in yearling beef steers.

A randomized complete block design experiment with 360 single-source black yearling steers (average BW = 316.1 +/- 9.1 kg) fed a 91% concentrate (steam-flaked corn base) diet was conducted to evaluate the effects of supplemental vitamin A (0, 1,103, 2,205, 4,410, or 8,820 IU/kg of dietary DM) on plasma and liver vitamin A and E concentrations, lipogenic enzyme activity, marbling score, and performance of yearling steers. Final BW (586, 580, 590, 585, and 584 kg for 0, 1,103, 2,205, 4,410, and 8,820 IU of supplemental vitamin A/kg of DM, respectively) did not differ (P = 0.39) among treatments. Feed efficiency, ADG, and daily DMI did not differ (P > 0.10) among treatments within each 28-d period or for the overall experiment. From d 57 to slaughter, average DMI (10.33, 10.28, 10.57, 9.75, and 10.22 kg/steer daily for 0, 1,103, 2,205, 4,410, and 8,820 IU of vitamin A/kg of DM, respectively) was less (P < 0.02) by steers receiving 4,410 IU of supplemental vitamin A/kg of dietary DM than for steers in the other treatments. Furthermore, DMI was greater (P = 0.06) for steers receiving 2,205 IU of supplemental vitamin A/kg of dietary DM than for steers receiving 8,820 IU of supplemental vitamin A/kg of DM. Marbling score, HCW, LM area, and 12th-rib fat thickness did not differ (P > 0.10) among treatments. Similarly, the percentage of carcasses grading >or=USDA Choice (62.6, 52.8, 64.0, 58.4, and 58.4% for 0, 1,103, 2,205, 4,410, and 8,820 IU of vitamin A/kg of DM, respectively), Select, or 0.10) among treatments. Except for d 56 (P = 0.050; r = 0.18 for liver retinol), no correlations (P > 0.10) between marbling score and any plasma or liver tissue retinol or alpha-tocopherol concentrations or vitamin A intake were found, and no differences (P > 0.10) in lipogenic enzyme activity were detected among treatments. Taken together with previous and concurrent research, results of this experiment suggest that vitamin A supplementation at a concentration up to twice the NRC recommendation has little effect on performance, marbling, or lipogenic enzyme activity in adipose tissue samples in yearling feedlot steers, and that 2,205 IU of supplemental vitamin A/kg of DM (20,000 IU/steer daily) or less is adequate to meet the vitamin A requirements of finishing beef cattle.

Expression of enzymes regulating placental ammonia homeostasis in human fetal growth restricted pregnancies.

Functional placental insufficiency results in impaired feto-placental exchange, and subsequently in fetal growth restriction (FGR). We hypothesized that reductions in placental amino acid transporter activities in FGR pregnancies may be accompanied by abnormal expression of placental ammonia-handling enzymes. Term placentas were obtained from growth restricted (N=11) and normal (N=17) human pregnancies, and examined for glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutaminase (GA) mRNA and protein expression. Northern and Western blots were normalized on human actin mRNA and protein expression. For GA, the presence of mRNA coding the kidney isoform, and the absence of mRNA coding the liver isoform of the enzyme were demonstrated in the human placenta. In FGR pregnancies, placental expression of GDH mRNA was reduced (P<0.05) compared to normal pregnancies (1.576+/-0.144 vs. 2.092+/-0.177, respectively; mean+/-SE), whereas GS and GA mRNA expression was not different between the two types of pregnancy. GDH protein expression were also reduced (P<0.05) in FGR placentas compared to normal placentas (1.055+/-0.079 vs. 1.322+/-0.053, respectively; mean+/-SE). The GS and GA protein expression was not different in FGR pregnancies. Our data indicate that in cases of FGR, glutamate-to-oxoglutarate transformation in the placenta is limited, yet glutamine synthesis from and decomposition to glutamate seems to be preserved. This may reflect down-regulation of GDH in response to decreased fetal liver output and reduced umbilical artery glutamate concentrations in human FGR pregnancies.

Specificity protein-1 and -3 trans-activate the ovine placental lactogen gene promoter.

The proximal promoter (-383/+16) of the ovine placental lactogen (oPL) gene provides trophoblast-specific expression in vitro. Footprint 6 (FP6; -319/-349) lies within this region, and transfection of two-base pair mutations across FP6 into BeWo cells identified potential binding sites for CCAAT-enhancer binding protein (CEBP) and specificity proteins (Sp). Transfection of CEBP dominant negative or over-expression constructs did not impact transactivation of the proximal promoter. However, Sp1 and Sp3 over-expression constructs increased (p

The pregnant sheep as a model for human pregnancy.

Successful outcome of human pregnancy not only impacts the quality of infant life and well-being, but considerable evidence now suggests that what happens during fetal development may well impact health and well-being into adulthood. Consequently, a thorough understanding of the developmental events that occur between conception and delivery is needed. For obvious ethical reasons, many of the questions remaining about the progression of human pregnancy cannot be answered directly, necessitating the use of appropriate animal models. A variety of animal models exist for the study of both normal and compromised pregnancies, including laboratory rodents, non-human primates and domestic ruminants. While all of these animal models have merit, most suffer from the inability to repetitively sample from both the maternal and fetal side of the placenta, limiting their usefulness in the study of placental or fetal physiology under non-stressed in vivo conditions. No animal model truly recapitulates human pregnancy, yet the pregnant sheep has been used extensively to investigate maternal-fetal interactions. This is due in part to the ability to surgically place and maintain catheters in both the maternal and fetal vasculature, allowing repeated sampling from non-anesthetized pregnancies. Considerable insight has been gained on placental oxygen and nutrient transfer and utilization from use of pregnant sheep. These findings were often confirmed in human pregnancies once appropriate technologies became available. The purpose of this review is to provide an overview of human and sheep pregnancy, with emphasis placed on placental development and function as an organ of nutrient transfer.

Ribonucleic acid interference: a new approach to the in vivo study of gene function.

The definition of hormone function was classically accomplished by ablation-replacement studies. However, as our knowledge of the complexity of hormones and growth factors has grown, it has become increasingly difficult to clearly define the necessity and function of many of the hormones, growth factors, and regulatory proteins under investigation. The use of homologous recombination within mouse embryonic stem cell lines allows functional gene ablation and has been used extensively during the past 15 yr to define specific gene function. The use of similar methodologies in livestock species has yet to yield an efficient approach. In contrast, the parallel development of our understanding of naturally occurring RNA interference, along with the development of efficient virus-based vectors for gene transfer, holds great potential for effectively "knocking down" specific gene function. Short-hairpin (sh) RNA-encoding cassettes, typically consisting of inverted repeats separated by a loop sequence and followed by a short poly(T) string to terminate transcription, are inserted downstream of an RNA polymerase III promoter within the viral vector of choice. Several viral vectors are useful for delivery of shRNA expression cassettes, each with particular attributes. Adenovirus- and lentivirus-derived vectors provide a high rate of infectivity in most mammalian cell types, with lentiviral vectors allowing stable integration into the host genome if the study of long-term effects is needed. Upon transcription, a shRNA is generated, and the loop is recognized by the processing enzyme Dicer, generating guide sequences. Guide sequences are incorporated into the RNA-induced silencing complex, which targets mRNA for degradation if recognized by the guide sequence. For each mRNA of interest, design and testing of a number of shRNA, along with adequate controls, are required to identify the most efficient construct before proceeding to in vivo use. This technology may become the method of choice for defining gene function in livestock.

Fetoplacental transport and utilization of amino acids in IUGR--a review.

Amino acids have multiple functions in fetoplacental development. The supply of amino acids to the fetus involves active transport across and metabolism within the trophoblast. Transport occurs through various amino acid transport systems located on both the maternal and fetal facing membranes, many of which have now been documented to be present in rat, sheep and human placentas. The capacity of the placenta to supply amino acids to the fetus develops during pregnancy through alterations in such factors as surface area and specific time-dependent transport system expression. In intrauterine growth restriction (IUGR), placental surface area and amino acid uptakes are decreased in human and experimental animal models. In an ovine model of IUGR, produced by hyperthermia-induced placental insufficiency (PI-IUGR), umbilical oxygen and essential amino acid uptake rates are significantly reduced in the most severe cases in concert with decreased fetal growth. These changes indicate that severe IUGR is likely associated with a shift in amino acid transport capacity and metabolic pathways within the fetoplacental unit. After transport across the trophoblast in normal conditions, amino acids are actively incorporated into tissue proteins or oxidized. In the sheep IUGR fetus, however, which is hypoxic, hypoglycemic and hypoinsulinemic, there appear to be net effluxes of amino acids from the liver and skeletal muscle, suggesting changes in amino acid metabolism. Potential changes may be occurring in the insulin/IGF-I signaling pathway that includes decreased production and/or activation of specific signaling proteins leading to a reduced protein synthesis in fetal tissues. Such observations in the placental insufficiency model of IUGR indicate that the combination of decreased fetoplacental amino acid uptake and disrupted insulin/IGF signaling in liver and muscle account for decreased fetal growth in IUGR.

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 morbidity 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.

Ruminant models of prenatal growth restriction.

Intrauterine growth restriction (IUGR) is a significant health issue that not only affects infant mortality and morbidity, but may also predispose individuals to coronary heart disease, diabetes, hypertension and stroke as adults. The majority of IUGR pregnancies in humans are characterized by asymmetric fetal growth, resulting from inadequate nutrient transfer to the fetus. Furthermore, most of these pregnancies involve functional placental insufficiency, and may also show altered umbilical velocimetry. As the severity of IUGR increases, the fetus becomes increasingly hypoxic, hypoglycaemic and acidotic. In addition, placental transfer or utilization of some amino acids is known to be altered in IUGR pregnancies. Although a great deal has been learned from clinical studies of human IUGR, appropriate animal models are required to define completely the mechanisms involved in the development of IUGR. The pregnant sheep is a long-standing model for placental-fetal interactions, and fetal growth restriction can be induced in pregnant sheep by maternal nutrient restriction, maternal nutrient excess, administration of glucocorticoid, utero-placental embolization, carunclectomy and maternal hyperthermia. Although all of these sheep models are capable of inducing fetal growth restriction, the degree of restriction is variable. This review compares these sheep models of IUGR with the characteristics of human IUGR.

Placental expression of VEGF, PlGF and their receptors in a model of placental insufficiency-intrauterine growth restriction (PI-IUGR).

Placental development requires adequate and organized interaction of vascular growth factors and their receptors, including vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). Both VEGF and PlGF, acting through the tyrosine kinase receptors VEGFR-1 and VEGFR-2, have been implicated in playing a role in ovine placental vascular development. The present studies describe the placental expression of components of the VEGF family at two maturational time points (55 and 90 days post coitus, dpc) in a hyperthermic-induced ovine model of placental insufficiency-intrauterine growth restriction (PI-IUGR). Both caruncular and cotyledonary VEGF and PlGF mRNA concentration increased with gestational age (P< 0.05), whereas only cotyledonary VEGF and PlGF protein concentration increased over gestation (P< 0.002). At 55 dpc, VEGF mRNA concentration was elevated in hyperthermic (HT) ewes, compared to control thermoneutral (TN) animals (TN; 0.52+/-0.08 vs HT; 1.27+/-0.17 VEGF/GAPDH, P< 0.001). At 90 dpc, expression of PlGF and VEGF mRNA was not altered by the HT treatment. Both TN cotyledonary VEGFR-1 and VEGFR-2 mRNA expression levels rose significantly over the period studied (P< 0.05 and P< 0.01 respectively). Receptor mRNA concentration in HT cotyledonary tissue was significantly reduced at 90 dpc (VEGFR-1; TN 0.21+/-0.02 vs HT 0.11+/-0.01 VEGFR-1/actin, P< 0.05, VEGFR-2; TN 0.18+/-0.05 vs HT 0.07+/-0.01 VEGFR-2/actin, P< 0.01). Soluble VEGFR-1 (sVEGFR-1) mRNA was not detected in these tissues. These alterations in growth factor and growth factor receptor mRNA expression, as a result of environmental heat stress early in placental development, could impair normal placental vascular development. Furthermore, alterations in VEGF, VEGFR-1 and VEGFR-2 mRNA expression, during the period of maximal placental growth, may contribute to the development of placental insufficiency, and ultimately intrauterine growth restriction.

Placental development in normal and compromised pregnancies-- a review.

Intrauterine growth restriction (IUGR) is a significant cause of infant mortality and morbidity. It is now clear that IUGR infants exhibit higher rates of coronary heart disease, type 2-diabetes, hypertension and stroke as adults. Therefore, fetal growth not only impacts the outcome of the perinatal period, but also impacts adult well-being. The etiologies of IUGR are numerous, but are often associated with abnormalities in placental structure and function. The process of implantation and placentation requires the production of a plethora of growth factors, cell-adhesion molecules, extracellular matrix proteins, hormones and transcription factors. Many of these exhibit altered expression within the placenta of IUGR pregnancies. However, it has been difficult to fully assess their role during the development of placental insufficiency (PI) in the human, underscoring the need for animal models. Using an ovine model of PI-IUGR we have observed changes in the expression of vascular endothelial growth factor, placental growth factor, their common receptors, as well as angiopoietin 2 and its receptor, Tie 2. We found that changes in these growth factors can be associated with both acute and chronic changes in placental vascular structure and function. These studies and others are providing needed insight into the developmental chronology of placental insufficiency.

Transcriptional regulation in the placenta during normal and compromised fetal growth.

The placenta synthesizes a number of cytokines and growth factors that are involved in the establishment, maintenance or regulation of pregnancy. Included are interferons, placental lactogens, other members of the growth hormone/prolactin gene family, leptin, and an array of angiogenic growth factors. While their roles in pregnancy differ, in their absence pregnancy is either lost or compromised. Therefore an understanding of the cell-specific transcriptional regulation of these genes is imperative if we are ever to alter their expression to benefit pregnancy progression. Our understanding of transcriptional regulation in the placenta is still in its infancy, and there appears to be considerable divergence in the transcriptional regulation of these genes between species, as well as between the various cytokine genes being examined. For example, while there are some commonalities in the regulation of human, rodent and ruminant placental lactogens, there are differences that require the study of placental lactogen gene regulation across species. However, one common theme that is emerging with the angiogenic growth factors, such as vascular endothelial growth factor and the angiopoietins, is the transcriptional control of these genes by oxygen tension within the placenta. Examination of transcriptional regulation in normal and compromised pregnancies will provide additional insight in this area.

Cotyledon and binucleate cell nitric oxide synthase expression in an ovine model of fetal growth restriction.

Heat exposure early in ovine pregnancy results in placental insufficiency and intrauterine growth restriction (PI-IUGR). We hypothesized that heat exposure in this model disrupts placental structure and reduces placental endothelial nitric oxide synthase (eNOS) protein expression. We measured eNOS protein content and performed immunohistochemistry for eNOS in placentas from thermoneutral (TN) and hyperthermic (HT) animals killed at midgestation (90 days). Placental histomorphometry was compared between groups. Compared with the TN controls, the HT group showed reduced delivery weights (457 +/- 49 vs. 631 +/- 21 g; P < 0.05) and a trend for reduced placentome weights (288 +/- 61 vs. 554 +/- 122 g; P = 0.09). Cotyledon eNOS protein content was reduced by 50% in the HT group (P < 0.03). eNOS localized similarly to the vascular endothelium and binucleated cells (BNCs) within the trophoblast of both experimental groups. HT cotyledons showed a reduction in the ratio of fetal to maternal stromal tissue (1.36 +/- 0.36 vs. 3.59 +/- 1.2; P< or = 0.03). We conclude that eNOS protein expression is reduced in this model of PI-IUGR and that eNOS localizes to both vascular endothelium and the BNC. We speculate that disruption of normal vascular development and BNC eNOS production and function leads to abnormal placental vascular tone and blood flow in this model of PI-IUGR.

Novel activator protein-2alpha splice-variants function as transactivators of the ovine placental lactogen gene.

Activator protein-2 (AP-2) has been implicated as a transactivator of the human and ovine placental lactogen (oPL) genes. Transcriptional enhancement through an AP-2 cis-acting element has been described for other genes expressed in the placenta, but the AP-2 isoform enhancing expression is species dependent. Transactivation of the oPL minimal promoter (-124 bp to +16 bp) by AP-2 was confirmed by mutational analysis in transiently transfected human choriocarcinoma cells (BeWo). AP-2alpha was localized in ovine chorionic epithelial cells by immunohistochemistry and a 3-kb transcript was identified by Northern hybridization. Four nearly full-length AP-2 cDNAs were isolated from an ovine placenta cDNA library. Nucleotide sequencing these cDNAs revealed that the AP-2 mRNA expressed in the ovine placenta shares identity with human AP-2alpha, but variations in the predicted N-terminus were observed, and three unique AP-2alpha splice-variants were identified. Expression of AP-2alpha variants in HepG2 cells, devoid of endogenous AP-2, indicates that enhancement through the AP-2 element in the oPL gene minimal promoter was variant dependent. RNA transcripts for all of the ovine AP-2alpha splice-variants were confirmed in ovine placenta by RT-PCR, and homologs for two variants were found in human placenta. However, only one AP-2alpha transcript, which shares identity to Xenopus AP-2alpha, was expressed in BeWo cells. Immunoblot analysis confirmed AP-2alpha variants in ovine chorionic binucleate cell nuclear extracts, one of which migrates similar to the AP-2alpha variant identified in BeWo cell nuclear extracts. These data indicate the presence of new mammalian AP-2alpha splice-variants that augment transactivation of the oPL gene in ovine chorionic binucleate cells.

Restriction of fetal growth has a differential impact on fetal prolactin and prolactin receptor mRNA expression.

Prolactin is present in the fetal circulation and prolactin receptors are expressed in a wide range of fetal tissues. The factors which regulate the synthesis and secretion of prolactin, and the expression of its receptors before birth, are poorly understood. We have investigated whether experimental restriction of placental growth in the sheep has an impact on the prolactin axis in the growth restricted fetus. The majority of uterine endometrial caruncles were removed before pregnancy in 10 ewes (placental restriction; PR group). Placental, fetal liver and kidney weights were reduced in the PR compared to the control group (n = 10). The ratio of fetal prolactin mRNA : 18S rRNA was significantly lower (P < 0.01) in the PR group (1.83 +/- 0.45, n = 6) than in the control group (4.11 +/- 0.54, n = 6). The ratio of prolactin mRNA : 18S rRNA in the fetal pituitary was positively correlated with fetal and with placental weight. Using stepwise linear regression, it was determined that the level of fetal prolactin mRNA : 18S rRNA expression was best described (as judged by the maximum adjusted R2) by prolactin mRNA: 18 S rRNA = - 3.0378 + 0.17 PO2 + 2.772 glucose (adjusted R2 = 0.765, F = 17.53, P < 0.001). Fetal plasma prolactin concentrations were significantly reduced (P < 0.05) in the PR group compared to control animals between 109 and 141 days gestation. Fetal prolactin receptor (PRLR) mRNA transcripts encoding long (PRLR1) and short forms (PRLR2) of PRLR were present in the liver and kidney of animals in the PR and control groups at 140-141 days gestation. PR did not alter the levels of PRLR1 or PRLR2 mRNA in the fetal liver or kidney. The suppression of the synthesis and secretion of prolactin in the growth restricted fetus may limit the action of prolactin on the growth and metabolism of key fetal organs during suboptimal intrauterine conditions