Epidermal growth factor acts as a corticotropin-releasing factor in chronically catheterized fetal lambs.

Epidermal growth factor (EGF) has been reported to stimulate adrenocorticotropin hormone (ACTH), growth hormone and prolactin secretion from pituitary tissue in vitro, and in large doses evokes ACTH secretion in adult sheep in vivo. In order to assess a possible role for EGF in the pituitary hyperfunction characteristic of the in utero fetus, we measured changes in plasma immunoreactive ACTH concentrations after acute administration of saline, purified mouse EGF or synthetic ovine corticotropin releasing factor (CRF) to chronically catheterized fetal sheep. Both CRF and EGF were associated with increases in plasma immunoreactive ACTH concentrations. Peak values 60 min after 10-micrograms injections of either EGF or CRF increased from baseline ACTH values of 61 +/- 11 pg/ml to 191 +/- 37 and 178 +/- 25 pg/ml, respectively. Dose-response studies indicate that at low doses (less than 20 micrograms) EGF is as potent a stimulus for ACTH release as CRF. EGF infusion was not associated with detectable changes in circulating CRF, catecholamines, arginine vasopressin levels, or plasma growth hormone concentrations. We speculate that EGF may be important in the regulation of pituitary function in the developing mammalian fetus.

Distribution of thyrotropin-releasing hormone (TRH) and precursor peptide (TRH-Gly) in adult rat tissues.

TRH (pGlu-His-Pro-NH2) arises from the post-translational processing of a larger precursor peptide containing multiple copies of the TRH progenitor sequence, Gln-His-Pro-Gly. Concentrations of TRH and its precursor peptide (TRH-Gly) were determined in serum and a variety of tissues of the rat using specific RIA systems. TRH and TRH-Gly immunoreactivities were detectable in almost all tissues studied. TRH was distributed mainly in neural tissues, with the highest mean concentration (126 pg/mg tissue) in hypothalamus. In extra-neural tissues, mean TRH levels ranged from 0.6-4.8 pg/mg tissue; the mean serum concentration was 12.4 pg/ml. In contrast to the distribution of TRH, relatively higher mean TRH-Gly concentrations were observed in serum (76.5 pg/ml) and in extraneural tissues, including prostate (83.3 pg/mg tissue), spleen (19.0 pg/mg), adrenal (16.2 pg/mg), kidney (13.3 pg/mg), and gastrointestinal tract (6.3-19.8 pg/mg). Among brain tissues, the TRH-Gly concentration was highest in pituitary gland (13.1 pg/mg). The mean ratio of TRH-Gly/TRH concentrations was less than 1 in neural tissues and pancreas. The lowest ratio (0.04) was observed in hypothalamus, and the highest ratio (66) in prostate gland. Assuming that tissue TRH-Gly levels reflect TRH synthesis, these results suggest that 1) the processing of TRH-Gly to TRH varies among tissues, 2) TRH-Gly to TRH conversion occurs most efficiently in neural tissues, and 3) TRH-Gly to TRH conversion may be a rate-limiting step in TRH biosynthesis.

Biochemical and ontogenic characterization of thyroxine 5'-monodeiodinase in brown adipose tissue from fetal and newborn lambs.

We characterized T4 5'-monodeiodinase (5'-MDI) and its ontogenic changes in brown adipose tissue (BAT) of fetal lambs from a gestational age of 99 days to the newborn period. BAT 5'-MDI activity in fetal or newborn lamb was markedly enhanced by dithiothreitol (DTT); a near-maximum stimulation was reached at a DTT concentration of 1 mM. The enzyme is heat-labile (inactivated when heated to 56 C for 5 min) and has a pH optimum at 6.5. In the presence of 1 mM DTT, BAT 5'-MDI was extremely sensitive to propylthiouracil (PTU): 0.04 mM PTU resulted in 94% inhibition of the T4 to T3-converting activity. An increase of DTT concentration to 20 mM in the incubation media reduced the inhibition by 0.04 mM PTU to 59%. Kinetic analysis, in the presence of 4 mM DTT, revealed a Michaelis-Menten constant (Km) of 1.43 microM for T4 and an inhibitory constant (Ki) of 22 microM for PTU. A marked and progressive rise of BAT 5'-MDI was noted from the 99-day fetus to the newborn period [6.0 vs. 94 +/- (SE) 12 pmol T3/mg protein X h]. The total sulfhydryl content in BAT varied from 3.8 to 5.4 mmol/kg wet weight equivalent, whereas nonprotein sulfhydryl content varied from 0.5 to 0.9 mmol/kg in groups of lambs between 99 days and the newborn period, without a significant change with age. These results indicate that 5'-MDI in fetal lamb BAT increases progressively between 99 days gestation and the neonatal period. Ovine fetal BAT 5'-MDI is highly sensitive to the inhibitory effect of PTU at low DTT concentration (1 mM) and is relatively insensitive to PTU only at high DTT concentration (20 mM). Finally, 5'-MDI in BAT from the fetal lamb reaches maximal activity near term and seems similar in its biochemical characteristics to the 5'-MDI of liver, kidney, or thyroid.

Two pathways for thyroxine 5'-monodeiodination in brown adipose tissue in fetal sheep: ontogenesis and divergent responses to hypothyroidism and 3,5,3'-triiodothyronine replacement.

Thermogenesis in rat brown adipose tissue (BAT) is thyroid hormone responsive. Rat BAT expresses a type II 5'-iodothyronine monodeiodinase (5'MDI) which mediates local T3 production from T4. Earlier studies show that BAT from fetal and newborn sheep contains a high Km type I, instead of type II, 5'MDI. To better characterize the 5'MDI of ovine fetal BAT, we studied the in vitro monodeiodination of [125I]T4 at a low substrate concentration (2 nM) and in the presence of 1 mM propylthiouracil in BAT homogenates of control and thyroidectomized fetuses at different gestational ages as well as in newborn lambs. Thyroidectomies were performed at three gestational ages: 99-107 days (group 1), 129-132 days (group 2), and 115-117 days (group 3A). Animals were studied 8-13 days after surgery. A significant increase in the activity of a low Km T4 5'MDI was noted in BAT from hypothyroid fetuses at all three gestational ages. This low Km activity was similar to the type II enzyme in rat BAT and brain in that the activity was also T3 resistant. A gradual rise in BAT type II 5'MDI activity was measured between 99 days gestation and term (150 days). These results indicate that ovine BAT contains two distinct iodothyronine 5'-monodeiodinating activities, one with a high Km and another with a low Km. The latter, resembling the type II 5'MDI in rat brain and BAT, is increased in ovine hypothyroid BAT. The former predominates in euthyroid tissue and is similar to the type I 5'MDI characterized in rat liver, kidney, and thyroid. We speculate that BAT type II 5'MDI may be important for neonatal BAT thermogenesis, while the type I enzyme may play a significant role in the increase in serum T3 concentration that occurs at birth.

A 3,3'-diiodothyronine sulfate cross-reactive compound in serum from pregnant women.

Recently, we found high serum/urine concentrations of 3,3'-diiodothyronine sulfate (T2S) in both fetal and maternal sheep. In the present study, a RIA was employed to detect and measure serum T2S in women of different gestational ages and after delivery. Results were compared with values in nonpregnant women. In maternal serum, we identified a material that cross-reacts with T2S antibody, but is not T2S. Its concentration increased with the progression of pregnancy. The exact chemical structure of the T2S-like material (which we designated compound W) is unclear. It is immunologically (or chemically) similar to T2S, but does not cochromatograph with synthetic T2S in high pressure liquid chromatography. The serum concentrations of compound W were expressed as T2S equivalents (nanomoles per L +/- SE). Serum compound W concentrations were slightly elevated in women during the first trimester compared to those in nonpregnant women (0.73 +/- 0.04 vs. 0.17 +/- 0.02 nmol/L; P < 0.01). There was a moderate and progressive rise in the compound W concentration between 14-35 weeks gestation. The maternal serum compound W concentration then rapidly peaked before parturition (36-40 weeks gestation, 3.49 +/- 0.49 nmol/L; 27-35 weeks, 1.67 +/- 0.11 nmol/L; P < 0.01). After parturition, maternal serum levels of compound W decreased from 2.61 +/- 0.18 nmol/L (n = 25) to 1.47 +/- 0.12 nmol/L (n = 18) at 1 day, 0.89 +/- 0.07 nmol/L (n = 15) at 3 days, and 0.33 +/- 0.03 nmol/L (n = 8) at 7 days. hCG increased serum concentrations of T2S-cross-reactive material 6.2-fold (P < 0.01) in nonpregnant women. In summary, whereas hCG stimulation may account for some increase in maternal serum concentrations of this T2S-like material in the first trimester, the more rapid increase in maternal serum compound W concentrations during the late third trimester are probably related to changes that occur in fetal thyroid hormone economy. It is speculated that placental transfer and transformation of fetal T3 may be related to the rise in the level of T2S-like compound W in the serum of pregnant women.

Differential response of ovine placental lactogen levels in maternal and fetal circulations following single umbilical artery ligation in fetal sheep.

We investigated circulating maternal and fetal serum concentrations of ovine placental lactogen (oPL) following single umbilical artery ligation (SUAL) at 108 to 114 days' gestation. Ovine placental lactogen was isolated and purified from placental cotyledons, and a radioimmunoassay developed using previously described methods. Intrauterine growth retardation (IUGR) was manifest as increasing fetal brain-to-liver weight ratio with increasing duration of survival following SUAL. During the first five to seven days following SUAL, circulating oPL levels in ewes with SUAL fetuses were significantly reduced when compared with levels in ewes with control fetuses. In contrast, oPL levels in SUAL fetuses were significantly increased above levels in control fetuses for the first five to seven days following surgery. Fetal ovine growth hormone levels were elevated in SUAL fetuses, while ovine prolactin levels were similar in the two groups. IUGR was associated with mild fetal acidosis and fetal plasma CAT levels which were similar in SUAL and control fetuses. No correlation was found between fetal pH or CAT and fetal oPL levels. These findings are consistent with the view that circulating levels of oPL in the mother are related to the mass of functioning trophoblast. Elevated fetal oPL levels following SUAL may result from acute placental ischaemia with alterations in placental lactogen secretion at the maternofetal interface.

Postnatal lung responses and surfactant function after fetal or maternal corticosteroid treatment.

To evaluate the effect of dose and route of administration of betamethasone on subsequent postnatal lung function, pregnant ewes were randomized at 127 days gestation to receive maternal or fetal intramuscular doses of 0.2 or 0.5 mg/kg body wt betamethasone or saline. At delivery 24 h later, preterm lambs were treated with surfactant and ventilated for 4 h. The lambs exposed to 0.5 mg/kg betamethasone by either the maternal or fetal route had higher Po2 values, lung volumes, dynamic compliances, and ventilatory efficiency indexes, as well as lower ventilatory pressure requirements, than did control animals (P < 0.05). There were no consistent improvements in postnatal lung function for the 0.2 mg/kg dose given to the fetus or ewe. However, measurements of radiolabeled protein in the total lung were decreased in all treatment groups (P < 0.01). Surfactant that was recovered from all groups of lambs and fractionated to isolate the large-aggregate fraction improved lung volumes in preterm rabbits to a greater degree than the surfactant used to treat the lambs (P < 0.05). Surfactant recovered from both groups treated with 0.5 mg/kg betamethasone was less sensitive to inactivation by plasma than was surfactant from the 0.2 mg/kg groups or the controls (P < 0.01). Fetal or maternal treatment with 0.5 mg/kg betamethasone improved postnatal lung function and increased the resistance of surfactant to inactivation.

Postnatal lung function and protein permeability after fetal or maternal corticosteroids in preterm lambs.

We evaluated postnatal lung function and intravascular albumin loss to tissues of 123-days-gestation preterm surfactant-treated and ventilated lambs 15 h after direct fetal (n = 8) or maternal (n = 9) betamethasone treatment or saline placebo (n = 9). The betamethasone-treated groups had similar increases in dynamic compliances, ventilatory efficiency indexes, and lung volumes relative to controls (P < 0.05). The losses of 125I-labeled albumin from blood, a marker of intravascular integrity, and the recoveries of 125I-albumin in muscle and brain were similar for control and betamethasone-exposed lambs. Betamethasone-treated lambs had lower recoveries of 125I-albumin in lung tissues and in alveolar washes than did controls (P < 0.01). Although blood pressures were higher for the treated groups (P < 0.05), all groups had similar blood volumes, cardiac outputs, and organ blood flows. Maternal or fetal treatment with betamethasone 15 h before preterm delivery equivalently improved postnatal lung function, reduced albumin recoveries in lungs, and increased blood pressures. However, prenatal betamethasone had no effects on the systemic intravascular losses of albumin or did not change blood volumes.

Effect of interval from fetal corticosteriod treatment to delivery on postnatal lung function of preterm lambs.

The effect of altering the interval from treatment to delivery on postnatal lung function of the preterm lamb is unknown. We treated groups of 8-10 singleton fetal sheep with 0.5 mg/kg betamethasone by fetal injection and evaluated postnatal lung function 40 min after preterm delivery at 123 days gestation 2 days after treatment or at 128 days gestation 2, 4, and 7 days after treatment relative to groups of 4-8 saline-injected control animals. At 123 days, betamethasone significantly improved arterial PCO2, dynamic thoracic compliance, and ventilatory efficiency index and doubled lung gas volume relative to a control group. Fetal treatment with betamethasone 2, 4, or 7 days before delivery at 128 days also improved these same indicators of lung function relative to controls, and the magnitude of the improvements was the same for all indicators and independent of treatment-to-delivery interval. Betamethasone suppressed the normal postnatal increase in plasma cortisol after 2 and 4 days of exposure but not after 7 days of exposure. Betamethasone also increased fetal and postnatal triiodothyronine concentrations after 2 days of exposure but not at 4 or 7 days of exposure. Although the hormone effects were transient, postnatal lung functional responses to betamethasone persisted over the 2- to 7-day interval from treatment to delivery.

Exogenous surfactant function in very preterm lambs with and without fetal corticosteroid treatment.

We have asked whether the function of a bovine source surfactant frequently used clinically (Survanta) could be enhanced after exposure to the very preterm lung when the surfactant was subsequently tested in vivo in preterm surfactant-deficient rabbits. We also evaluated whether there would be effects resulting from fetal treatment with 0.5 mg/kg betamethasone given 48 h before delivery of lambs at 121 days gestational age. The fetal corticosteroid treatment significantly improved gas exchange, increased compliance, increased functional residual capacity, decreased vascular-to-alveolar protein leak, and increased static lung volumes. However, surfactant from both groups of lambs, recovered by alveolar wash and subsequently fractionated to recover the large-aggregate functional surfactant, was equivalent in function to the Survants given to the lambs when tested in preterm surfactant-deficient rabbits. Addition of plasma to Survanta resulted in high minimum surface tensions in vitro, and this inhibition could be prevented by supplementation of the Survanta with 5 or 10% sheep surfactant. No activation occurred with treatment of the very preterm lung, a result consistent with the lung being too immature to contribute components to the surfactant used for treatment. Fetal corticosteroid treatment had no effect on surfactant function at this gestational age.

Thyroid hormone effects on neonatal thermogenesis.

It is clear that thyroid hormones modulate non-shivering heat production of the newborn mammal. While some obvious thyroid hormone effects on thermogenesis can be demonstrated in adult, cold-acclimated or hibernating animals, these findings cannot be directly applied to the newborn, in which changes in amount and composition of brown adipose tissue, as well as sympathetic and thyroid system maturational events, are occurring. Thyroid hormones do influence the prenatal development and subsequent responses of brown adipose tissue in neonates through primary actions on brown adipose tissue iodothyronine 5'monodeiodinase and mitochondrial uncoupling protein. Secondary effects involving brown adipocyte growth, lipid composition, oxidation proteins, and sympathoadrenal activity are of lesser importance in thyroid hormone modulation of newborn thermogenesis. The acute surge in thyroid hormones that occurs at birth seems of limited significance with regard to neonatal thermogenesis. The stimulation of sympathoadrenal activity at the time of birth, as well as continued in situ conversion of T4 to T3 in the brown adipocyte, are of critical importance in stimulating and modulating heat production in the newborn.

Preterm betamethasone treatment of fetal sheep: outcome after term delivery.

OBJECTIVES: Although antenatal corticosteroids improve outcomes for preterm newborns, negative effects could result if preterm delivery does not occur. We investigated whether betamethasone treatment of preterm fetal sheep would alter cardiovascular, renal, and lung function after delivery at term. METHODS: Preterm fetal lambs were randomized at 126-128 days' gestation to receive single doses of saline (n = 6) or 0.5 mg/kg betamethasone (n = 7) by ultrasound-guided fetal intramuscular injection. The lambs were delivered by cesarean at term, 20 days after fetal treatment, then ventilated for 4 hours to evaluate lung, cardiovascular, renal, and endocrine newborn adaptive responses, as well as responses to mild hypoxia. RESULTS: Body and organ weights (brain, lung, heart, kidney, adrenal) were similar in the two groups. Values for blood gases and pH, mean arterial pressures, heart rates, glomerular filtration rates, renal osmolar clearance, and plasma cortisol, angiotensin II, epinephrine, and norepinephrine levels were similar between groups for 3 hours after delivery and before hypoxia. A 20-minute period of mild hypoxia resulted in increases in catecholamines, arginine vasopressin, and atrial natruretic factor in both betamethasone-treated and control lambs. However, hypoxia did not alter cardiovascular or lung function in either group. After reversal of hypoxia, measured physiologic parameters did not differ between groups. Kidney Na, K-adenosine triphosphatase activity was significantly higher for the betamethasone-treated lambs. CONCLUSION: Preterm fetal betamethasone administration does not alter neonatal pulmonary, cardiovascular, renal, or endocrine physiology after term delivery or in response to mild hypoxia.

Do breast milk derived hormones play a role in neonatal development?

Many hormones and several growth factors are present in significant concentrations in breast milk. That these substances are of physiologic significance has been suggested by many investigators. This review summarizes the biological roles of thyroid hormones and epidermal growth factor (EGF) derived from breast milk in a variety of developing mammals.

Diagnosis and management of altered fetal thyroid status.

Recent advances in the understanding of the normal process of maturation of fetal thyroid function now make possible the in utero diagnosis of fetal system abnormalities. The approach to the fetus at risk for altered thyroid function is discussed in the context of normal thyroid development and emphasis is given to the fetus presenting with goiter.

Development of the thyroid.

The fetal hypothalamic-pituitary-thyroid axis develops autonomously of maternal influence. System ontogenesis begins with the appearance and histological development of the thyroid and pituitary glands followed by development of the hypothalamus and the pituitary portal vascular system. Hypothalamic-pituitary control of thyroid function matures during the last half of human fetal development. Thyroid hormones undergo several types of biochemical transformations in tissues, including deiodination, side-chain metabolism, and conjugation with sulphate or glucuronide. Enzyme-mediated monodeiodination is the most important pathway. The first step in T4 metabolism is either outer-ring monodeiodination to active T3 or innerring monodeiodination to inactive rT3. Most T4 is metabolized to rT3 in fetal tissues and/or placenta and rT3 is the major circulating T4 metabolite in the fetus. Selective tissues, such as brain, can monodeiodinate T4 to T3, and this T3 is available for local action. Nuclear thyroid hormone receptors mature at different times in different tissues. Receptors appear earlier in brain than in liver and local T3 production and action may be important in fetal brain development. Most thyroid hormone actions, however, appear in the perinatal period, and infants with thyroid agenesis appear normal at birth and develop normally with prompt neonatal diagnosis and treatment. Premature infants, particularly those less than 30-32 weeks' gestational age, have an immature thyroid system and manifest a state of transient hypothalamic-pituitary TSH deficiency. This does not require treatment. Infants with primary hypothyroidism, either due to thyroid dysgenesis or to thyroid dyshormonogenesis, by contrast, require prompt diagnosis and treatment. Rarely an infant is born with permanent TSH deficiency with or without other pituitary hormone deficiencies. These infants also require prompt treatment. Mothers with thyroid disease or a history of thyroid disease and with IgG autoantibodies to thyroid gland TSH receptors may deliver infants with hypothyroidism or hyperthyroidism due to transplacental passage of the receptor-blocking or receptor-stimulating autoantibodies. These infants also require careful evaluation and management.

Serum thyroid hormones and tissue 5'-monodeiodinase activity in acutely thyroidectomized newborn lambs.

After either total thyroidectomy or sham operation in full-term fetal sheep, fetuses were delivered and serial blood samples were obtained for measurements of thyroxine (T4), triiodothyronine (T3), and catecholamines. Despite comparable serum T4 values (T4 means +/- SE, sham 7.1 +/- 0.6 micrograms/dl, thyroidectomized 6.8 +/- 0.7 micrograms/dl at 60 min after birth), serum T3 values were lower in the thyroidectomized animals (T3 means +/- SE, thyroidectomized 39 +/- 4.8 ng/dl, sham 153 +/- 20.1 ng/dl at 60 min after birth). Four hours after birth, the animals were killed with an intravenous overdose of barbiturate. Brain, thyroid, liver, kidney, and brown adipose tissues were dissected and analyzed for thyroxine 5'-monodeiodinase (5'-MDI) activity in vitro. 5'-MDI activity was comparable in all tissues from sham-operated and thyroidectomized lambs. Plasma epinephrine and norepinephrine concentrations, mean arterial pressure, mean pulse, rectal temperature, and arterial blood gas values were similar in the two groups of animals. These data support the hypothesis that the thyroid gland is the major source of T3 for the T3 surge in the immediate newborn period. They also indicate that the neonatal T3 surge has limited immediate metabolic significance in euthyroid newborns.

Ontogeny of thyrotropin releasing hormone and precursor peptide in the rat.

Thyrotropin releasing hormone (TRH) and its precursor peptide pGlu-His-Pro-Gly (TRH-Gly) were measured in serum and in a variety of tissues of developing rats using specific RIA. TRH and TRH-Gly immunoreactivities were detected in most tissues. TRH concentrations were highest in pancreas, in which mean (+/- SEM) TRH concentrations were 138 +/- 20 pmol/g wet tissue 2 d before birth and 644 +/- 80 and 586 +/- 86 pmol/g, respectively, 2 and 5 d after birth. Hypothalamic TRH levels gradually increased from 4 d before birth (12 +/- 2.5 pmol/g) to 77 d of postnatal age (348 +/- 33 pmol/g). Hypothalamic concentrations were lower than levels in pancreas until 13 d of age. The mean serum TRH level at 2 d was 80 +/- 20 pmol/L and fell to the adult range by 21 d. TRH-Gly concentrations were highest in small gut (371 +/- 64 pmol/g) during the neonatal period, falling gradually to adult levels (33 +/- 4.8 pmol/g) by 35 d. Mean hypothalamic TRH-Gly concentrations increased to a peak of 62 +/- 4.5 pmol/g at 13 d, falling thereafter. High TRH-Gly concentrations (greater than 100 pmol/g) also were observed in pancreas (at d 2), kidney, and pituitary gland (at d 21). Serum TRH-Gly concentrations were highest (mean 417 +/- 26 pmol/L) on the 2nd postnatal day and gradually decreased to the adult level by 35 d. Changes in the TRH-Gly/TRH ratio were inversely correlated with tissue TRH concentrations in hypothalamus, pancreas, and liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Ontogeny of thyroid hormone effect on tissue 5'-monodeiodinase activity in fetal sheep.

Most of the thyroxine (T4) in fetal mammals is deiodinated to the inactive metabolite, reverse triiodothyronine (rT3), via an iodothyronine 5-monodeiodinase in fetal tissues. Maturation of the tissue 5'-monodeiodinase (MDI) enzymes required for conversion of T4 to active triiodothyronine (T3) in the rat, an altricial species, occurs in the postnatal period. To characterize fetal maturation of the enzymes for active T3 production in a precocial species, 5'-MDI activities were measured in liver, kidney, and brain tissue homogenates of ovine fetuses 13 days after total thyroidectomy (Tx) conducted at gestational ages of 99-107 or 129-132 days. Sham-operated twin fetuses served as controls. Hepatic type I 5'-MDI activity was not significantly lowered by Tx in group I but was significantly lower after Tx in group II fetuses. Renal type I 5'-MDI was not affected by Tx in either group. Type II 5'-MDI activity in cerebral cortex was significantly elevated after Tx in both groups I and II fetuses. Tissue sulfhydryl contents were similar in liver, kidney, and cerebral cortex from control and Tx fetuses in group I. These data indicate that hypothyroidism induced early in the third trimester is associated with increased brain type II 5'-MDI activity without significant change in liver or kidney type I 5'-MDI. Late third trimester hypothyroidism is associated with decreased type I 5'-MDI activity in liver homogenates as well as increased type II 5'-MDI activity in brain tissue.(ABSTRACT TRUNCATED AT 250 WORDS)