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The intravenous infusion of ornithine (0·5–0·9 g/kg) into five fetal sheep of 116–141 days gestation caused no significant change in concentrations of fetal plasma placental lactogen (PL) or GH as determined by specific homologous radioimmunoassays. In contrast, the intravenous infusion of ornithine (0·3–0·5 g/kg) into three of the ewes caused a 144·5 ± 74·7 (s.e.m.)% increase in maternal plasma PL concentrations and a 255·2 ± 55·0% increase in maternal GH concentrations. Fetal PL concentrations remained unchanged despite the large increase in maternal PL concentrations. This study, which indicates a differential effect of ornithine on PL and GH secretion in the mother and fetus, suggests that the factors regulating PL and GH secretion in the mother and fetus are distinct.
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Experiments utilizing RNA interference technology were performed to determine whether the forkhead transcription factor FOXO1A, a member of the FOXO family of proteins, plays a critical role in the induction of human uterine decidualization. Human decidual fibroblast cells were decidualized in vitro for 6 days with medroxyprogester-one, estradiol, and dibutyryl cAMP in the presence or absence of a highly specific FOXO1A small interfering RNA (siRNA) that inhibits FOXO1A mRNA and protein expression by more than 80%. RNA and proteins were extracted from the cells at 0, 2, 4, and 6 days. FOXO1A and IGFBP-1 proteins were determined by immunoblotting; and intracellular mRNA levels for several decidualization marker genes were determined by real-time PCR. Exposure of the cells to FOXO1A siRNA in five separate experiments resulted in a 40–75% inhibition of prolactin, IGFBP-1, tissue inhibitor of metalloproteinase 3 (TIMP3), somatostatin and endometrial bleeding-associated factor (EBAF) mRNAs, all of which are markedly induced during the decidualization process. In contrast, actin and GAPDH mRNA levels did not change during decidualization. The inhibition of mRNA levels was first noted at day 2 and persisted for the remainder of each experiment. Western blot analysis indicated that the FOXO1A siRNA inhibited IGFBP-1 protein expression by 60–80%. Decidual fibroblast cells exposed in an identical manner to a control RNA that had no effect on FOXO1A expression caused only a 0–15% inhibition of the marker genes and IGFBP-1 protein. Taken together, these findings strongly suggest a critical role for FOXO1A in the induction of human decidualization.
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ABSTRACT
Previous studies from our laboratory showed that high-density lipoproteins (HDL) stimulate the release of human placental lactogen (PL) from cultured trophoblast cells from normal pregnant women. To determine whether HDL stimulates PL secretion in vivo, ovine HDL was infused over 2–5 min into 11 pregnant ewes (22 separate experiments) at 86–130 days of gestation via an indwelling catheter into the maternal jugular vein. The HDL, freshly prepared from the plasma of pregnant ewes by differential flotation ultracentrifugation, was greater than 99% purified as judged by SDS-PAGE. Plasma samples were obtained from the ewes before and at 0·5-h intervals for 6 h following the infusions and were assayed for PL by a specific homologous radioimmunoassay. The maternal infusion of HDL at doses of 302–784 mg (5·3–13·8 mg/kg body weight) stimulated significant increases in maternal plasma PL concentrations in six out of eight experiments (six ewes), and the infusion of 108–264 mg (1·9–4·6 mg/kg) stimulated plasma PL concentrations in two out of six experiments. In contrast, HDL at doses < 100 mg were without effect in eight experiments. The response to the HDL infusions was characterized by a sustained increase in plasma PL concentrations beginning 1·5–2·5 h after the infusions, reaching a maximum 274·2 ± 21·9% of the baseline value (P<0·001). In contrast, the maternal infusion of lipoprotein-free plasma proteins or saline had no effect on maternal plasma PL concentrations. Although the infusion of HDL into pregnant ewes stimulated an increase in maternal plasma PL concentrations, the infusion of HDL (0·8–22·0 mg/kg) into three fetuses in seven separate experiments had no effect on fetal plasma PL concentrations. The demonstration that HDL stimulates an increase in plasma PL concentrations in pregnant ewes strongly supports a novel physiological role for HDL in the regulation of PL secretion.
Journal of Endocrinology (1989) 120, 423–427
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Arginine is a potent stimulus to the secretion of placental lactogen (PL) as well as GH and prolactin in sheep. To determine whether other amino acids of the urea cycle also affect PL secretion, ornithine (25 g) and citrulline (20 g) were infused intravenously into four pregnant ewes over a period of 0·5 h. In eight experiments, ornithine stimulated PL secretion by 124 ± 25 (s.e.m.) % with the initial increase occurring by 1 h after the start of each infusion. Plasma GH concentrations increased from 3·4 ± 1·1 to 24·5 ± 5·9 ng/ml and plasma prolactin concentrations increased from 58·8 ± 12·8 to 310·7 ± 88·4 ng/ml. Citrulline, on the other hand, had no consistent effect on PL secretion. Plasma GH concentrations following the infusion of citrulline, however, increased by 15–20 ng/ml in two of four experiments and plasma prolactin concentrations decreased by 73·2 ± 3·2% in all four experiments.The results indicate that ornithine is also a potent stimulus to the secretion of PL, GH and prolactin and suggest that arginine-induced PL secretion may result from the conversion of arginine to ornithine.
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ABSTRACT
The relationships between plasma concentrations of energy substrates and placental lactogen (PL) were investigated in pregnant ewes. In successive hourly samples of plasma PL, concentrations varied by ± 30% but were not related to general activity or feeding of the ewes or the time of day. Fasting ewes for 72 h did not alter the pattern or the mean PL titres. Insulin-induced acute hypoglycaemia, hyperglycaemia and decreases or increases in free fatty acids (FFA) all failed to alter PL levels significantly during 5-h post-treatment periods. These experiments demonstrate that PL secretion in the ewe fluctuates markedly and is unaffected by changes in plasma glucose or FFA concentrations.
J. Endocr. (1987) 114, 391–397
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Abstract
During human placental differentiation, mononuclear cytotrophoblast cells fuse and differentiate into syncytiotrophoblast cells. Although syncytiotrophoblast cells have been shown to express interleukin-1α (IL-1α), IL-1β and IL-6, the pattern of expression of these cytokines during placental differentiation is unknown. We have examined the expression of IL-1α, IL-1β and IL-6 mRNA during differentiation of cytotrophoblast cells in culture. IL-1α, IL-1β and IL-6 mRNA levels were determined by semiquantitative reverse transcription-PCR analysis using glyceraldehyde phosphate dehydrogenase as an internal control. All three cytokine mRNA levels decreased markedly during trophoblast differentiation. After 6 days in culture, when almost all the cytotrophoblast cells had fused and differentiated into syncytiotrophoblast cells, the amounts of IL-1α, IL-1β and IL-6 mRNA were decreased by 87·1, 72·1 and 60·9% respectively. Exogenous IL-6 had differential effects on cytokine mRNA expression. When added to placental cultures during the first 6 days of culture, IL-6 markedly inhibited IL-6, IL-1α and IL-1β mRNA expression. However, when added to the cells during days 6–9 of culture, when most of the cells were syncytiotrophoblast cells, IL-6 stimulated IL-lα and IL-1β mRNA expression. The results of these studies indicate that IL-1α, IL-1β and IL-6 mRNA expression decreases markedly during cytotrophoblast differentiation in vitro and that the regulation of trophoblast cytokine mRNA levels changes during differentiation.
Journal of Endocrinology (1995) 147, 487–496
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Serum somatomedin C concentrations in fetal sheep and pregnant ewes from days 51 to 149 of gestation were determined by specific radioimmunoassay. In the fetus (n = 61 samples) serum somatomedin C concentrations, fitted to a regression curve, increased significantly with advancing gestation from 0·44 units/ml at 51 days to 3·99 units/ml at 149 days, an increase of 806% (P < 0·001). In the pregnant ewes (n = 14 samples), serum somatomedin C did not change during gestation. The temporal relationship between the marked increase in fetal somatomedin C concentrations and the acceleration of fetal growth is consistent with the hypothesis that somatomedin is important in the stimulation of fetal growth.
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ABSTRACT
To determine whether arachidonic acid stimulates the secretion of ovine placental lactogen (oPL), arachidonic acid was infused as an intravenous bolus into pregnant ewes and fetuses. Plasma oPL concentrations were determined in mothers and fetuses before and for 5 h after infusion. The administration of 12·5 mg arachidonic acid (0·15−0·2 mg/kg, n = 11 experiments) to the pregnant ewes caused an increase in maternal plasma oPL concentrations of 73·9±15·6% (s.e.m.) and 60·8±18·1% above the pretreatment concentrations at 4 and 5 h respectively P<0·01 in each instance). The infusion of 25 mg arachidonic acid (n = 8) caused increases of 96·0±19·1% and 100·3±26·4% (P<0·005), and the stimulation was not inhibited by the cyclo-oxygenase inhibitors indomethacin and ibuprofen. In contrast to arachidonic acid, vehicle alone or palmitic acid had no effects on plasma oPL concentrations. Despite the increase in maternal plasma oPL concentrations, plasma oPL concentrations in the fetus remained unchanged after the maternal infusions. The infusion of arachidonic acid (0·5–1·5 mg/kg) directly into six fetuses had no effects on either fetal or maternal oPL concentrations. These studies indicate that (1) arachidonic acid stimulates maternal plasma oPL concentrations but has no effect on fetal oPL concentrations and (2) the stimulation of oPL secretion is not due to the conversion of arachidonic acid to prostaglandins or other cyclo-oxygenase products.
J. Endocr. (1985) 106, 43–47
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SUMMARY
The concentrations of ovine placental lactogen (oPL) have been determined in maternal plasma, umbilical cord plasma, and allantoic fluid by an homologous radioimmunoassay for oPL which is sensitive to 0·1 ng hormone. Ovine placental lactogen was first detected in maternal plasma at 41–50 days of gestation and reached a peak concentration of 2547 ± 226 (s.e.m.) ng/ml at 121–130 days in ewes with singleton gestations. The oPL concentration in cord plasma was 336·4 ± 60·3 ng/ml and in allantoic fluid was 29·6 ± 6·4 ng/ml. After surgical removal of the placenta, oPL disappeared from maternal plasma with a half-life of 29·1 ± 1·3 min.
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A heterologous radioimmunoassay for the measurement of somatomedin C in sheep serum has been developed using purified125I-labelled human somatomedin C and an antiserum to human somatomedin C. It was observed that the GH dependence of somatomedin C could not be verified when unprocessed sheep sera were assayed, because of interference by somatomedin binding proteins. After incubation of samples at pH 3·8, however, concentrations of somatomedin C mirrored GH status. Assay results from sera after prolonged exposure to acid agreed with results from samples which had undergone acidification and gel chromatography. Using the methods developed in this study, 12 sera from normal sheep had a mean concentration of somatomedin C of 2·35 ± 0·27 (s.e.m.) units/ml while nine sera from hypophysectomized sheep contained 0·45± 0·04 units/ml (P < 0·001), and one serum from a ewe with a prolonged GH excess associated with a pituitary tumour had a value of 6·9 units/ml. The radioimmunoassay resulting from these studies should provide a tool for investigation of the physiological control of somatomedin C in sheep.
