Diurnal rhythms in plasma melatonin concentrations in the fetal sheep and pregnant ewe during late gestation

We have measured plasma melatonin (MT) concentrations in the pregnant ewe and fetal sheep during 24-h periods between 114 and 142 days gestation. There was a clear diurnal rhythm in the plasma MT concentrations in both the ewe and fetus from 114 days gestation. Blood samples were also collected from the pregnant ewe and fetus during the day every 2-3 days from 112 days gestation to term. There was no gestational age trend in maternal or fetal day time plasma MT concentrations during late pregnancy. To establish whether there was transplacental transfer of MT, pregnant ewes were injected with [3H]MT, and total radioactivity (disintegrations per min) was measured in maternal and fetal arterial plasma and in amniotic fluid collected before and for 1 h after the [3H]MT injection. Two minutes after [3H]MT injection, radioactivity was detected in both maternal and fetal sheep plasma. Extraction of fetal plasma with chloroform indicated that [3H]MT accounted for 48.0 +/- 7.2 (SE) % of total radioactivity at 2 min after the injection. In one pregnant ewe infused with unlabeled MT (0.3 microgram/ml saline.min for 20 min) maternal and fetal plasma MT concentrations increased within 6 min after the start of the MT infusion. We conclude that there is a diurnal rhythm in the plasma concentrations of MT in the fetal lamb and pregnant ewe between 114 and 142 days gestation, and that MT crosses the ovine placenta from the maternal to the fetal circulation. Therefore, the MT present in the fetal sheep circulation may be solely of maternal origin or it may be derived from both fetal and maternal sources.     

Diurnal variations in plasma concentrations of cortisol, prolactin, growth hormone and glucose in the fetal sheep and pregnant ewe during late gestation

We have measured fetal and maternal plasma concentrations of cortisol, prolactin, GH and glucose in samples collected during a 24-h period in 14 animals between 127 and 142 days of gestation. There was a significant increase in both the mean daily plasma cortisol concentration and mean daily coefficient of variation (C.V.) of plasma cortisol concentrations after 135 days of gestation. There was also a significant variation in the fetal plasma cortisol concentrations with a peak occurring at 19.00 h. There was a significant sinusoidal diurnal rhythm in the plasma prolactin concentrations in both the fetal sheep and pregnant ewe and the maximal prolactin concentrations occurred between 19.00 and 23.00 h (fetal) and 21.00 and 01.00 h (maternal). Although no significant diurnal variation was detected in fetal plasma GH concentrations, there was a significant sinusoidal diurnal rhythm in the plasma GH concentrations of the pregnant ewe and the maximal maternal GH concentrations occurred between 21.00 and 01.00 h. Both the fetal and maternal plasma glucose concentrations showed a significant sinusoidal diurnal rhythm. The maximal maternal and fetal glucose concentrations were measured between 21.00 and 01.00 h and between 23.00 and 03.00 h respectively. We have therefore established that diurnal variations in plasma cortisol and prolactin concentrations exist prenatally. Whether the presence of such hormonal rhythms reflects the activity of an endogenous fetal circadian pacemaker remains to be established.     

Maternal pinealectomy abolishes the diurnal rhythm in plasma melatonin concentrations in the fetal sheep and pregnant ewe during late gestation

We have investigated the effect of pinealectomy of ewes in pregnancy on the presence of the diurnal rhythm in fetal and maternal plasma concentrations of melatonin. Six ewes were pinealectomized between 104 and 118 days of gestation. Fetal and maternal blood samples were collected during 24-h periods between 125 and 140 days of gestation in the pinealectomized ewes and in an intact control (n = 4). There was a significant diurnal rhythm in both fetal and maternal plasma concentrations of melatonin in the control group. In this group, the fetal and maternal plasma melatonin concentrations were significantly higher in the dark (128.4 +/- 6.2 and 192.2 +/- 10.7 pmol/l respectively) than in the light (46.2 +/- 4.2 and 25.8 +/- 2.1 pmol/l respectively). However there was no diurnal rhythm in either the fetal or maternal plasma melatonin concentrations in the pinealectomized group between 125 and 140 days of gestation. In contrast to the control animals, there was also no light-dark difference in the fetal or maternal plasma melatonin concentrations in four pinealectomized animals sampled frequently in the 3-7 days preceding delivery (mean length of gestation 146.5 days). However, in the pinealectomized sheep there was a gradual increase in the combined light-dark fetal plasma melatonin concentrations during late gestation from 27.9 +/- 2.8 pmol/l (at 15-20 days before delivery) to 95.2 +/- 14.1 pmol/l on the day of delivery. We have therefore demonstrated that the maternal pineal is the major source of the diurnal rhythm in maternal and fetal plasma melatonin concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of adrenocorticotrophin secretion by catecholamines in the pregnant and foetal sheep.

The effect of adrenaline on the maternal and foetal plasma ACTH concentration of twelve pregnant sheep with chronically implanted vascular catheters has been studied. Adrenaline infused into the jugular vein of the ewe or foetus produced carotid arterial adrenaline concentrations of 1-9 ng/ml. The foetal plasma ACTH was 253 +/- 73 pg/ml and it showed a fivefold increase during adrenaline infusion; the ACTH concentration achieved was proportional to the plasma adrenaline. In the ewe plasma ACTH was 99 +/- 23 pg/ml. During adrenaline infusion to the ewe this rose by an amount dependent on the adrenaline concentration achieved and there was also a rise in foetal plasma ACTH but no consistent change in foetal plasma adrenaline. There was no reproducible change in plasma corticosteroid concentration during adrenaline infusion into the foetus but a rise in maternal plasma corticosteroid concentration during infusion into the ewes. Because the adrenaline concentrations achieved during the infusions were within the physiological range, the results indicate that circulating catecholamines may directly or indirectly influence the concentration of ACTH in the circulation. Also, physiological rises in plasma catecholamines in pregnant animals may stimulate the release of ACTH from the foetal pituitary.     

Effect of morphine and naloxone on plasma prolactin concentrations in the fetal sheep and pregnant ewe during late gestation

This study investigated the effects of intrafetal morphine or naloxone administration on fetal and maternal plasma prolactin concentrations in the sheep during late pregnancy (117-143 days gestation). After intravenous morphine (3 mg/kg) there was a significant increase (p less than 0.05) in fetal plasma prolactin concentrations which was sustained for 180 min post-injection. There was no significant effect of either gestational age (125-133 days compared to 134-143 days gestation) or repeated administration (up to 3 treatments) of morphine on the fetal prolactin response to morphine. Intrafetal administration of naloxone (3.8 mg/kg bolus + 9.9 mg/kg/60 min), blocked the fetal prolactin response to morphine. Maternal plasma concentrations of prolactin were significantly increased (p less than 0.05) at 180 min after the intrafetal morphine bolus. When naloxone alone was infused, there was no change in fetal plasma prolactin concentrations, but there was a significant fall (p less than 0.05) in maternal plasma prolactin from 25 min after the start of the naloxone infusion. Thus, acute administration of morphine is associated with fetal and maternal hyperprolactinaemia. Although the endogenous opioids do not appear to mediate basal prolactin secretion in the fetus, they may have a role in the control of prolactin release in the pregnant ewe during late gestation.     

Prostaglandin production at the onset of ovine parturition is regulated by both estrogen-independent and estrogen-dependent pathways

A current hypothesis of ovine parturition proposes that fetal adrenal cortisol induces placental E2 production, which, in turn, triggers intrauterine PG production. However, recent evidence suggests that cortisol may directly increase PG production in trophoblast-derived tissues. To separate cortisol-dependent and estrogen-dependent PG production in sheep intrauterine tissues, we infused singleton, chronically catheterized fetuses beginning on day 125 of gestation (term, 147-150 days) with 1) cortisol (1.35 mg/h; n = 5); 2) cortisol and 4-hydroxyandrostendione, a P450aromatase inhibitor (4-OHA: 1.44 mg/h; n = 5); 3) saline (n = 5); or 4) saline and 4-OHA (n = 5). Fetal and maternal arterial blood samples were collected at 12-h intervals starting 24 h before infusion and continuing during treatment for 80 h or until active labor. Uterine contractility was measured by electromyogram recording of myometrial activity. Plasma E2, progesterone (P4), PGE2, and 13,14-dihydro- 15-keto-PGF2alpha were quantified by RIA. PGHS-II messenger RNA (mRNA) and protein expression were determined by in situ hybridization and Western blot analysis, respectively. Data were analyzed by ANOVA (P < or = 0.05). Labor-type uterine contractions were present after 68 h of cortisol infusion and had increased significantly by 80 h. Labor-type uterine contractions were induced after 68 h of cortisol plus 4-OHA infusion, but the contraction frequency remained less than that in the cortisol-treated animals. Fetal cortisol infusion increased fetal and maternal plasma E2 concentrations and decreased the maternal plasma P4 concentration significantly; concurrent 4-OHA infusion attenuated the increase in fetal and maternal plasma E2, but not the decrease in maternal plasma P4. The fetal plasma PGE2 concentration increased after both cortisol and cortisol plus 4-OHA infusion. The maternal plasma 13,14-dihydro-15-keto-PGF2alpha concentration rose after fetal cortisol infusion, but not after cortisol plus 4-OHA infusion. Placental trophoblast PGHS-II mRNA and protein expression were increased significantly after both cortisol and cortisol plus 4-OHA infusion. Endometrial PGHS-II mRNA and protein expression increased after cortisol infusion, but not after cortisol plus 4-OHA infusion. Plasma steroid and PG concentrations, uterine activity pattern, and intrauterine PGHS-II expression were not altered in either control group. We conclude that these data suggest distinct pathways of intrauterine PG synthesis: a cortisol-dependent/E2-independent mechanism within trophoblast tissue leading to elevations in fetal plasma PGE2, and an E2-dependent mechanism within maternal endometrium that leads to increased maternal plasma PGF2alpha and appears necessary for uterine activity and parturition.     

Maintaining fetal normoglycemia prevents the increase in myometrial activity and uterine 13,14-dihydro-15-keto-prostaglandin F2 alpha production during food withdrawal in late pregnancy in the ewe

Food deprivation during pregnancy leads to an increase in maternal and fetal prostaglandin (PG) production and increased uterine contractility. We investigated the effect of maintaining fetal normoglycemia during food withdrawal-induced maternal hypoglycemia on uterine 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) production and myometrial activity in late pregnant sheep. Pregnant sheep were surgically instrumented with fetal and maternal catheters and electromyogram leads under halothane anesthesia. Maternal and fetal blood plasma samples were obtained once a day at 0900 h, 24 h before (baseline sample) and after 48 h of food withdrawal. Food, but not water, was withdrawn from ewes in group I (n = 5). During food withdrawal in group II (n = 5), glucose was infused into a fetal vein to maintain fetal normoglycemia. All data were normalized to the concentration in the baseline sample in each animal as 100%. After 48 h of food withdrawal, maternal whole blood glucose fell by 42.2 +/- 4.4% (mean +/- SEM: group I) and 31.4 +/- 6.2% (group II). These values were not significantly different. Fetal blood glucose fell by 40.4 +/- 5.7% (group I). In group II, fetal blood glucose was maintained in the normal range (99.6 +/- 1.6% of baseline). Maternal uterine electromyogram activity, uterine venous estrone sulfate, and uterine veno-arterial difference in PGFM rose significantly during food withdrawal in group I ewes, but not in group II ewes. Maternal and fetal arterial plasma ACTH and cortisol did not change in group II animals. We conclude that maintenance of fetal normoglycemia during 48 h of food withdrawal in sheep prevents the increase in myometrial activity, maternal plasma estrogens, and uterine PGFM production during food withdrawal in late pregnancy.     

Androstenedione treatment of pregnant baboons at 0.7-0.8 of gestation promotes a premature forward shift in the nocturnal maternal plasma estradiol surge relative to progesterone and increases myometrial contraction activity

Androstenedione treatment of pregnant monkeys at 0.8 of gestation reproduces endocrine, biophysical, and biochemical changes similar to those measured during spontaneous, term labor in the pregnant monkey. In the pregnant baboon, the spontaneous onset of labor at term has been attributed to a forward shift in the nocturnal estradiol surge relative to that of progesterone in maternal plasma. This study investigated whether androstenedione treatment of the pregnant baboon at 0.7-0.8 of gestation promotes a premature forward shift in the nocturnal surge of maternal plasma estradiol relative to progesterone and whether this shift is associated with premature increases in nocturnal myometrial activity. Eight pregnant baboons were prepared surgically under general anesthesia with vascular catheters and myometrial electromyogram electrodes between 121 and 139 days of gestation (term is ca. 185 days). Catheters were maintained patent by continuous infusion of heparinized saline from the time of surgery until one of two treatments began following at least 9 days of postoperative recovery. In four baboons (Group I), the saline administration was replaced by a continuous infusion of 10% intralipid vehicle during Day 1 of the experimental protocol. During Day 2 and Day 3, the intralipid infusion was switched for a continuous infusion of androstenedione dissolved in intralipid set at a low (0.8 mg x kg(-1) x h(-1)) and at a high (1.6 mg x kg(-1) x h(-1)) dose, each delivered for 24 h. The other four pregnant baboons (Group II) received 10% intralipid vehicle for Days 1, 2, and 3 of the experimental protocol. One baboon from Group I received an additional dose of 0.4 mg x kg(-1) x h(-1) for 24 h before the low and the high dose of androstenedione. In each baboon, during each experimental day, maternal arterial blood samples (1 ml) were taken at 1 h intervals for 12 h, starting 3 h before the onset of darkness in the animal's environment, for measurement of maternal plasma estradiol and progesterone concentrations via RIA. Myometrial contractions were counted during each night-time period of the experimental protocol. All pregnant baboons demonstrated increases in maternal plasma estradiol and progesterone concentrations at night-time. Androstenedione had a dose-dependent effect in elevating day-time maternal plasma estradiol concentrations and in promoting a forward shift in the nocturnal surge of maternal plasma estradiol without affecting the nocturnal progesterone profile in maternal plasma. Maternal treatment with androstenedione also led to an increase in nocturnal myometrial contraction activity. We conclude that androstenedione treatment of the pregnant baboon at 0.7-0.8 of gestation promotes a premature forward shift in the nocturnal estradiol surge relative to that of progesterone in maternal plasma and that this shift is associated with an increase in nocturnal myometrial contraction activity, in a similar way to that measured during spontaneous onset of labor at term in this species.     

Nutritional regulation of insulin-like growth factor-binding protein gene expression in the ovine fetus and pregnant ewe.

The factors controlling the synthesis and degradation of the insulin-like growth factor-binding proteins (IGFBPs) during pregnancy are poorly understood. To clarify the roles of nutritional factors in the regulation of fetal and maternal IGFBP production, we examined the effects of fasting, refeeding, and glucose administration on plasma IGFBP concentrations and hepatic IGFBP mRNA levels in fetal lambs and pregnant ewes (n = 24). Maternal fasting for 3 days in late gestation stimulated a 50-100% increase in maternal plasma BP-1 concentrations (P < 0.05) and a 2- to 3-fold increase in fetal plasma BP-1 (P < 0.05), as determined by densitometric analysis of Western ligand blots. Fasting also stimulated a 40-70% increase in maternal plasma BP-2 concentrations (P < 0.05), but had no significant effect on fetal plasma BP-2 levels. Levels of hepatic BP-1 mRNA in the fetus and pregnant ewe during fasting paralleled plasma BP-1 levels, suggesting that fasting modulates fetal and maternal plasma BP concentrations at least in part through effects on hepatic gene expression. The effects of fasting on both mRNA and plasma levels of BP-1 and BP-2 were reversed by 3 days of refeeding and were prevented by glucose infusion during fasting. When ewes were made hyperglycemic by the infusion of hypertonic glucose, plasma BP-1 and BP-2 concentrations varied inversely with blood glucose concentrations. In addition, hyperglycemia reduced maternal liver BP-1 and BP-2 mRNA levels and fetal BP-1 mRNA levels by 50-65%. Direct administration of hypertonic glucose to the fetus decreased fetal plasma BP-1 levels acutely and reduced fetal BP-1 mRNA levels by 57%, but had no effect on fetal plasma BP-2 or fetal hepatic BP-2 mRNA levels. These findings indicate that glucose and other nutritional factors regulate gene expression and plasma levels of BP-1 and BP-2 in the pregnant ewe and BP-1 in the fetal lamb. The changes in expression of these IGFBPs during fasting and hyperglycemia may play roles in adaptation of the pregnant mother and fetus to metabolic stress.     

Melatonin increases activities of glutathione peroxidase and superoxide dismutase in fetal rat brain

Melatonin is a powerful scavenger of oxygen free radicals. In humans, melatonin is rapidly transferred from the maternal to the fetal circulation. To investigate whether or not maternal melatonin administration can protect the fetal rat brain from radical-induced damage by increasing the activities of antioxidant enzymes, we administered melatonin to pregnant rats on day 20 of gestation. Melatonin (10 mg/kg) was injected intraperitoneally at daytime (14:00 hr) and, to remove the fetuses, a laparotomy was performed at 1, 2, or 3 hr after its administration. We measured the melatonin concentration in the maternal serum and in fetal brain homogenates and determined the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in fetal brain homogenates. Melatonin administration markedly increased melatonin concentrations in the maternal serum and fetal brain homogenates, with peak levels achieved 1 hr after melatonin administration (serum: 538.2+/-160.7 pM/mL; brain homogenates: 13.8+/-2.8 pM/mg protein). Between 1 and 3 hr after melatonin administration, GSH-Px activity in fetal brain homogenates increased significantly (P<0.01). Similarly, SOD activity increased significantly between 1 and 2 hr after melatonin administration (P<0.01). These results indicate that melatonin administration to the mother increases antioxidant enzyme activities in the fetal brain and may thereby provide indirect protection against free radical injury. Thus, melatonin may potentially be useful in the treatment of neurodegenerative conditions that may involve excessive free radical production, such as fetal hypoxia and preeclampsia.     

Can maternal stress alter fetal adrenocorticotropin secretion?

The purpose of this study was to assess the role of increases in maternal plasma cortisol, within the range observed after stress, in the control of fetal ACTH responses to subsequent fetal stress. We infused cortisol or vehicle into the inferior venae cavae of pregnant ewes which, with their fetuses (120-130 days gestation), had been chronically catheterized. Five-hour maternal cortisol infusions produced increases in maternal plasma cortisol smaller in magnitude than those induced by hypoxia stress and resulted in physiological increases in fetal plasma cortisol. One hour after the end of the maternal cortisol infusion, fetal ACTH secretion was stimulated by infusion of sodium nitroprusside into the fetal inferior vena cava. After maternal cortisol infusion, the fetal ACTH and cortisol responses to fetal hypotension were inhibited. We conclude that increases in maternal cortisol alters the fetal ACTH response to stress.     

Fetal plasma insulin and thyroid hormone levels during acute in utero ethanol exposure in a maternal-fetal sheep model

The effects of acute in utero ethanol (ETOH) treatment on basal and stimulated thyroid and insulin levels in fetal plasma were studied in chronically cannulated fetal sheep. In test situations, pregnant ewes (0.78-0.88 gestation) which were chronically cannulated received 2 g/kg ETOH [25% (vol/vol) in isotonic saline] for 2 h; this was followed by a maintenance iv infusion of 0.13 g/kg ETOH. Control animals received isovolemic infusions of isotonic saline. Fetal arterial plasma samples were obtained after the 2-h infusion, and basal levels of T3, T4, glucose, and insulin were measured. The 2-h ETOH infusion did not influence fetal basal plasma T3, T4, insulin, or glucose. Fetal thyroid responses to an intraarterial injection of 0.01, 0.10, 1.00, or 10.00 micrograms/kg TRH or of 5 mU/kg TSH through the fetal catheters were studied in the presence or absence of high plasma ETOH concentrations. Fetal T4 or T3 levels during the 4 h following any of these stimuli were not significantly different in ethanol-treated and control animals. The effects of acute ETOH exposure on insulin responses to a glucose challenge were studied in six chronically cannulated ewes and their fetuses using a cross-over experimental design. After the 2-h ETOH infusion, ewes received a bolus injection of 600 mg/kg 50% glucose, followed by a 1-h infusion of 624 mg/kg 50% glucose and 0.13 g/kg ETOH. In control situations, ewes received saline plus glucose. Acute ETOH treatment did not influence maternal or fetal plasma glucose levels at any time, but enhanced both maternal and fetal insulin responses to glucose. Total insulin release, as measured by the area under the insulin response curve, was greater during ETOH exposure in both mother (ETOH, 4740 +/- 1475 microU/ml X min; control, 2807 +/- 766 microU/ml X min; P = 0.05) and fetus (ETOH, 562 +/- 94 microU/ml X min; control, 363 +/- 46 microU/ml X min; P less than 0.05). Thus acute in utero ETOH exposure does not diminish plasma levels of either thyroid hormones or insulin, two important hormones for fetal growth and development. However, ethanol exposure enhances the insulin response to increases in blood glucose in both mother and fetus.     

Prostaglandin mediates premature delivery in pregnant sheep induced by estradiol at 121 days of gestational age

The experiments reported here were designed for both in vivo and in vitro approaches in the same animals to obtain a better picture of the role of estrogen in the control of parturition. Chronically catheterized pregnant ewes were treated with vehicle (n = 5) or estradiol (n = 6), 5 mg twice a day, im for 2 d starting at d 119 of gestation. Maternal and fetal plasma estradiol, progesterone, and cortisol were measured by RIA and maternal plasma prostaglandin (PG) F2alpha was measured by enzyme immunoassay. Intrauterine PG H synthase 2 mRNA and protein and placental P450(c17)alpha hydroxylase mRNA were determined by Northern, in situ hybridization, Western blot analysis, and immunocytochemistry. Data were analyzed by ANOVA.Five of six estradiol-treated ewes delivered their fetuses within 48 h; however, the placenta was still retained 5-6 h after fetal delivery. Both maternal plasma estradiol and PGF2 alpha increased significantly in the estradiol-treated group. Maternal and fetal plasma progesterone and cortisol were not altered in either group. There were significant increases of PGH synthase 2 mRNA and protein in myometrium, endometrium, and maternal placenta but not in fetal placenta in estradiol-treated ewes. Placental P450(c17)alpha hydroxylase mRNA was not detectable in vehicle or estradiol-treated groups.Estradiol can, in the absence of increase in plasma cortisol, stimulate uterine PG production and induce labor, resulting in fetal delivery in the sheep. Failure of placental delivery after estradiol treatment suggests that estradiol alone is insufficient to stimulate some of the key changes required to complete delivery at the stage of gestation studied.     

High-density lipoproteins (HDL) stimulate placental lactogen secretion in pregnant ewes: further evidence for a role of HDL in placental lactogen secretion during pregnancy

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 less than 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 less than 0.001). In contrast, the maternal infusion of lipoprotein-free plasma proteins or saline had no effect on maternal plasma PL concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Melatonin modulates the fetal cardiovascular defense response to acute hypoxia

Experimental studies in animal models supporting protective effects on the fetus of melatonin in adverse pregnancy have prompted clinical trials in human pregnancy complicated by fetal growth restriction. However, the effects of melatonin on the fetal defense to acute hypoxia, such as that which may occur during labor, remain unknown. This translational study tested the hypothesis, in vivo, that melatonin modulates the fetal cardiometabolic defense responses to acute hypoxia in chronically instrumented late gestation fetal sheep via alterations in fetal nitric oxide (NO) bioavailability. Under anesthesia, 6 fetal sheep at 0.85 gestation were instrumented with vascular catheters and a Transonic flow probe around a femoral artery. Five days later, fetuses were exposed to acute hypoxia with or without melatonin treatment. Fetal blood was taken to determine blood gas and metabolic status and plasma catecholamine concentrations. Hypoxia during melatonin treatment was repeated during in vivo NO blockade with the NO clamp. This technique permits blockade of de novo synthesis of NO while compensating for the tonic production of the gas, thereby maintaining basal cardiovascular function. Melatonin suppressed the redistribution of blood flow away from peripheral circulations and the glycemic and plasma catecholamine responses to acute hypoxia. These are important components of the fetal brain sparing response to acute hypoxia. The effects of melatonin involved NO‐dependent mechanisms as the responses were reverted by fetal treatment with the NO clamp. Melatonin modulates the in vivo fetal cardiometabolic responses to acute hypoxia by increasing NO bioavailability.     

The Endocrinology of equine parturition

Delivery of viable young requires co-ordination of fetal maturation with the onset of labour at term. In turn, this depends on a cascade of fetal and maternal endocrine events. The sequence of these events is broadly similar in most mammals but there are differences in placental hormone synthesis and in the timing and magnitude of key prepartum endocrine changes between species. In most farm animals, maternal progesterone (P4) levels decline and oestrogen levels increase in the last 5 - 10 days before delivery in response to activation of the fetal hypothalamicpituitary-adrenal (HPA) axis and increased fetal cortisol concentrations. This cortisol surge is also responsible for fetal maturation and increasing uteroplacental prostaglandin (PG) synthesis. In the mare, there is little, if any, P4 in the maternal plasma during late gestation and both progestagens and oestrogens are produced by a feto-placental unit which uses precursors supplied by the fetus to synthesise a range of C21 and C18 steroids, many of which are unique to the horse. Regulation of uterine quiescence and activation is, therefore, complex in the mare near term. Indeed, total progestagen concentrations rise and total oestrogen levels fall in the mare during the last 20 - 30 days of gestation and only show the changes typical of impending parturition in other species in the last 24 - 48 h before delivery. Fetal cortisol concentrations also rise late in gestation in the horse compared to other species. In common with other species, the prepartum endocrine cascade appears to begin in the fetal horse with activation of the fetal HPA axis but, initially, the primary product of the fetal equine adrenal appears to be pregnenolone (P5) and not cortisol. This leads to increased progestagen production by the uteroplacental tissues, which maintains uterine quiescence in the face of increasing uterine stretch caused by the rapidly growing fetus. Very close to term in association with increasing fetal ACTH levels, the fetal equine adrenals appear to switch to producing cortisol. This late cortisol surge induces a period of rapid fetal maturation and may also contribute to increased uteroplacental oestradiol-17 beta and PG production. The fall in P5 availability may reduce uteroplacental progestagen production and lift the block on myometrial contractility. Finally, increased PG secretion activates myometrial contractions, which stimulate oxytocin release via a neuroendocrine reflex. The endocrine regulation of equine parturition, therefore, involves progestagens, oestrogens, PGs and oxytocin as in other species. However, further studies are required to establish the causes and consequences of the rise and fall in maternal progestagens and the extent to which initiation of equine labour depends on the fetal HPA axis.     

Neuropeptide Y in the sheep fetus: effects of acute hypoxemia and dexamethasone during late gestation

Plasma concentrations of neuropeptide Y (NPY) were measured in pregnant ewes and their fetuses under basal conditions and in response to acute hypoxemia during late gestation. The effects of fetal treatment with dexamethasone on these NPY responses were also examined. Under general anesthesia, 10 Welsh Mountain ewes and their fetuses were chronically instrumented between 117-120 days gestation (dGA; term is approximately 145 dGA) with vascular and amniotic catheters, and an ultrasonic probe around a femoral artery of each fetus. At 124 dGA, five fetuses were continuously infused i.v. with dexamethasone for 48 h at a rate of 1.73 +/- 0.16 microg x kg(-1) x h(-1) while the remaining five fetuses received vehicle at the same rate. At 126 dGA, 45 h from the onset of either infusion, 1 h of materno-fetal hypoxemia was induced by reducing maternal FiO2. During normoxia, maternal plasma NPY concentrations were three times those measured in fetal plasma in both groups. During hypoxemia, PaO2 fell to similar levels in the control and dexamethasone-treated groups in both mothers and fetuses. In control animals, there was a significant increase in the NPY concentration in fetal, but not maternal, plasma during hypoxemia. Fetal treatment with dexamethasone significantly enhanced the fetal NPY response to acute hypoxemia but had no effects on basal NPY levels in the fetal or maternal plasma or on the maternal response to acute hypoxemia. These data show: 1) differences between the maternal and fetal plasma NPY response to maternal inhalation hypoxia; 2) that NPY may play a role in mediating fetal defense responses to acute hypoxemia; and 3) that fetal exposure to glucocorticoids modifies the fetal plasma NPY response to acute hypoxemia.     

Physiological and regulatory roles of activin A in late pregnancy

Unexplained fetal death in utero in late pregnancy represents an increasing proportion of perinatal deaths. It has been assumed that critical hypoxia is the likely mechanism underlying these losses, but the lack of a physiological marker has hampered both confirmation and prediction which could lead to timely intervention. In this paper, we report studies on hypoxia that we have performed in chronically cannulated late pregnant sheep, complemented by parallel investigations undertaken in human pregnancies. Our initial studies were directed towards determining activin secretion in the fetus and mother during late gestation, and immediately after fetal surgery using a sheep model. This led us to propose that there may be a relationship between hypoxia and activin A, follistatin and prostaglandin (PG) release from the feto-placental unit. Subsequent studies have been directed towards examining this potential relationship in sheep and in humans with compromised pregnancies. As a result of these studies, we have identified a potential mechanism by which activin A may be involved in regulating the response of the fetus to hypoxic insult. Activin A and follistatin concentrations increased in late gestation in ovine maternal plasma and in fetal fluids. Feto-placental hypoxemia or maternal isocapnic hypoxemia, leading to fetal hypoxia, were specific triggers for an acute increase in fetal activin A and follistatin concentrations during late gestation. The source and secretion of activin A, follistatin, and the associated release of PGE_2, from within the feto-placental unit varied according to the site of the insult. The concomitant secretion of activin A and PGE₂ into the fetal circulation and amniotic fluid during reduced uterine blood flow provides an insight into the physiological regulatory mechanisms that might be involved. Changes observed in maternal activin A concentrations in mid and late gestation in the human may also be associated with fetal compromise. In human pregnancies, elevated activin A concentrations were observed in maternal plasma in mid and late gestation, in association with severe pre-eclampsia and with severe fetal growth restriction, compared to those observed in pregnancies with constitutionally small, healthy fetuses. Activin A was also elevated in maternal and arterial cord plasma in women at term during labour and immediately prior to undergoing emergency Caesarean section for failure to progress. These findings offer exciting new possibilities to gain insights into the mechanisms that underlie the maintenance of fetal wellbeing and provide a rationale for the potential that activin A may prove to be a useful clinical marker of fetal distress.     

Suppression subtractive hybridization identified a marked increase in thrombospondin-1 associated with parturition in pregnant sheep myometrium

Changes in several extracellular matrix proteins, such as fibronectin in fetal membrane and cervical collagens, occur at term and preterm delivery. However, no studies have evaluated changes in extracellular matrix proteins, in relation to myometrial activation recorded using invasive techniques during parturition in any species. We used suppression subtractive hybridization (SSH), to demonstrate the dramatic increases in one extracellular matrix protein, thrombospondin-1 (TSP1), in the pregnant ovine myometrium associated with parturition. Myometrial poly-A- RNA, extracted from term control ewes not in labor at 143-147 days of gestational age (dGA, n = 4), and from ewes in spontaneous term labor (STL) at 145-147 dGA (n 4), was subjected to SSH to construct a subtracted myometrial complementary DNA library. A complementary DNA clone from myometrial subtracted library, representing differentially expressed gene in the pregnant sheep myometrium during STL, was identified as TSP1 by sequence analysis and Blastn search. This cloned TSP1 was used to perform Northern blot analysis on total RNA isolated from five early controls, not in labor, at 130 dGA, five pregnant ewes in betamethasone-induced premature labor (BPL) at 130 dGA (betamethasone administered i.v. to the fetus at 0.48 mg over 48 h), six term-control-not-in-labor ewes at 143-147 dGA, and six pregnant ewes in STL. Northern blot analysis demonstrated that TSP1 increased significantly, associated with both BPL and STL. TSP1 protein level paralleled the increase of TSP1 messenger RNA during BPL and STL. To determine whether increase in this gene paralleled the increased strength of myometrial contractility, six ewes were treated with nimesulide, a selective PG synthase 2 inhibitor, at 147-148 dGA. Nimesulide infusion to the ewe i.v. to inhibit myometrial contraction (30 mg bolus, followed by 5 h infusion, 30 mg/h) commenced 9 h after onset of labor at 147-148 dGA. TSP1 in the myometrium decreased when myometrial contraction was inhibited by nimesulide. Both in situ hybridization and immunocytochemistry demonstrated that fibroblasts and the smooth muscle cells contained TSP1 messenger RNA and protein. TSP1 was also localized in the extracellular matrix. Our conclusions are: 1) our data provide the first evidence that changes in TSP1 are associated with myometrial activation in pregnant sheep during term and preterm labor; 2) myometrial fibroblasts and the smooth muscle cells are responsible for producing TSP1; and 3) SSH is a powerful technique that enables us to study differentially regulated genes during labor.     

Responses of the fetal pituitary-adrenal axis to acute and chronic hypoglycemia during late gestation in the sheep

We investigated the response of the fetal pituitary-adrenal axis to acute and chronic hypoglycemia before and after the normal prepartum activation of this axis at around 135 days gestation (term = 147 +/- 3 days). Pregnant ewes were either well nourished (control group; n = 22) or undernourished (UN; 50% reduction in maternal nutrient intake; n = 23) during the last 30 days of pregnancy. Acute hypoglycemia was induced by intrafetal administration of insulin between 125 and 130 days gestation (control, n = 7; UN, n = 12) and between 138 and 141 days gestation (control, n = 6; UN = 9). Fetal plasma glucose concentrations were significantly lower (P < 0.005) in the UN compared with the control group throughout the insulin infusion period at both gestational age ranges. In the control group, there was no fetal ACTH response to insulin infusion before 135 days gestation, but there was a significant (P < 0.001) response after 136 days gestation. In the UN group, there was a significant ACTH response to insulin infusion both before and after 135 days gestation, and there was no difference in the fetal ACTH response between the two gestational age ranges. The plasma cortisol responses to insulin were greater (P < 0.001) after 136 days compared with before 135 days gestation in both the UN and control groups. In the control group there was no significant relationship between basal fetal plasma ACTH and glucose concentrations between 115-135 days gestation or between 136-145 days gestation. In the UN group, fetal glucose ranged from 0.5-2.0 mM, and plasma ACTH and glucose concentrations were inversely related at 115-135 days gestation [log ACTH = -0.31 (glucose) + 2.21; r = -0.37; P < 0.001] and at 136-145 days gestation [log ACTH = -0.40 (glucose) + 2.50; r = -0.54; P < 0.001]. When the UN and control groups were combined, fetal plasma ACTH concentrations were significantly greater (F = 13.5; P < 0.05) when plasma glucose concentrations were less than 1.0 mM at either 115-135 days or 136-147 days gestation. Similarly, fetal plasma cortisol concentrations were also significantly greater (F = 18.7; P < 0.05) when plasma glucose concentrations were less than 1.0 mM at each gestational age range. Therefore, there is an increased sensitivity of the fetal hypothalamo-pituitary axis to acute falls in glucose concentrations below 1.2 mM after 135 days compared with earlier in gestation. The fetal hypothalamo-pituitary axis can respond, however, when plasma glucose concentrations fall below 1.0 mM, before and after 135 days gestation, independently of whether the low glucose concentrations are a consequence of insulin-induced hypoglycemia or maternal nutrient restriction.