Effects of early gestation GH administration on placental and fetal development in sheep.

Ovine GH (oGH) is synthesized in placental tissue during maximal placental growth and development. Our objectives were to localize oGH mRNA in the placenta, and study the impact of exogenous GH on twin pregnancies during the normal window (35-55 days of gestational age; dGA) of placental expression. In situ hybridization localized oGH mRNA in uterine luminal epithelium but not in tissues of fetal origin. While maternal GH and IGF-I concentrations were increased (P<0.001) approximately tenfold, uterine, uterine fluid, placental, and fetal weights were unaffected by treatment at either 55 or 135 dGA. Fetal length, liver weight, and liver weight per kg of body weight were unaffected by maternal GH treatment. However, in the cotyledon, IGF-binding protein (BP)-1 and IGFBP-4 mRNA concentrations were increased (P<0.05), while IGFBP-2 mRNA was decreased (P<0.05). The concentration of mRNA for IGFBP-3 was unaffected by treatment. Within the caruncle, IGFBP-1 mRNA was decreased (P<0.05), while IGFBP-3 and IGFBP-4 mRNA were increased (P<0.05), and IGFBP-2 mRNA was unchanged due to GH treatment. While our data indicate that elevated maternal GH and IGF-I concentrations during early and mid-gestation do not enhance placental and fetal growth in twin pregnancies, localization of GH mRNA in uterine luminal epithelium could explain GHs transitory expression from 35 to 55 dGA, since by the end of this period the majority of the uterine luminal epithelium has fused with chorionic binucleate cells forming the placental syncytium.

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.

Enhanced expression of uridine diphosphate-N-acetylglucosaminyl transferase (OGT) in a stable, tetracycline-inducible HeLa cell line using histone deacetylase inhibitors: kinetics of cytosolic OGT accumulation and nuclear translocation.

We have created a stable, tetracycline-inducible HeLa cell line that overexpresses murine uridine diphosphate-N-acetylglucosaminyl transferase (OGT). Tetracycline increased cytosolic OGT activity about 4-fold in a dose-dependent manner (ED(50)=0.03 microg/ml) with enhanced activity observable at 8h and maximal activity observable by 40h. Enhanced OGT activity was due to overexpression of OGT protein as determined by Western analysis. Trichostatin A (TSA), a potent and specific histone deacetylase inhibitor (HDI), markedly enhanced tetracycline-induced OGT gene expression, resulting in a >10-fold increase in OGT activity (>50-fold compared to that of uninduced cells). Other HDIs such as butyrate (ED(50)=1.6mM) and propionate (ED(50)=8mM) were similarly effective, but less potent than TSA (ED(50)=120 nM). We next examined the appearance of recombinant OGT in cytosol and nucleosol at various times (10 min to 6h) after inducing OGT gene. Within 2h, recombinant OGT was detected by Western analysis in both cytosol and nucleosol. This indicates rapid biosynthesis and accumulation of recombinant OGT in the cytosol and subsequent nuclear translocation. Entry of OGT into the nucleus was closely correlated with enhanced O-linked glycosylation of nuclear proteins, indicating that recombinant OGT was enzymatically active. The ability to rapidly induce OGT expression in a stable cell line provides an excellent model system to study the mechanism(s) underlying OGT nuclear translocation and a useful system to elucidate the cascade of signaling events related to O-linked glycosylation.

Measurement of UDP-N-acetylglucosaminyl transferase (OGT) in brain cytosol and characterization of anti-OGT antibodies.

UDP-N-acetylglucosaminyl transferase (OGT) catalyzes O-linked glycosylation of cytosolic and nuclear proteins, but enzyme studies have been hampered by the lack of a rapid, sensitive, and economical OGT assay. Employed assay methods typically involved the use of HPLC, formic acid, and large amounts of expensive radiolabeled [3H]UDP-N-acetylglucosaminyl ([3H]UDP-GlcNAc). In the current study, we have developed an OGT assay that circumvents many of these problems through four critical assay improvements: (1) identification of an abundant and enriched source of OGT enzyme (rat brain tissue), (2) utilization of a rapid method for efficiently removing salts and sugar nucleotides from cytosol (polyethylene glycol precipitation of active enzyme), (3) expression of a recombinant p62 acceptor substrate designed to facilitate purification (polyhistidine metal-chelation site), and (4) development of two alternative methods to rapidly separate free [3H]UDP-GlcNAc from 3H-p62ST acceptor peptide (trichloroacetic acid precipitation and metal-chelation affinity purification). To study the enzymology of OGT, independent of potential regulatory proteins within cytosol, we also developed and characterized an alternate OGT assay that uses antibody-purified OGT as the enzyme source. The major advantage of this assay lies in the ability to measure OGT in the absence of other cytosolic proteins.