Effect of fetal adrenalectomy on messenger ribonucleic acid for proopiomelanocortin in the anterior pituitary and for corticotropin-releasing hormone in the paraventricular nucleus of the ovine fetus

In the ovine fetus, adrenalectomy at 90-120 days gestational age (dGA) results in a gradual increase in basal concentrations of fetal plasma ACTH beginning at approximately 122 dGA. Bilateral adrenalectomy at 116-119 dGA also results in an increase in POMC mRNA in the fetal pituitary. It is not known whether both the paraventricular nuclei (PVN) of the hypothalamus and the anterior pituitary of the ovine fetus are responsive in late gestation to the removal of cortisol negative feedback. The purpose of this study was to determine the subsequent effect of fetal adrenalectomy at 118-121 dGA on the CRH mRNA content in fetal PVN and on POMC mRNA in the fetal anterior pituitary at 134 dGA. Mature Rambouellet-Columbia cross-bred ewes (n = 10), bred on a single occasion only and carrying fetuses of known gestational ages, were used. Both fetal adrenal glands were exposed via a retroperitoneal approach and removed [adrenalectomized (ADX); n = 5]. In control fetuses (CONT; n = 5) adrenal glands were exposed and isolated, but not removed. At 134 dGA, fetal plasma cortisol concentrations were significantly greater in CONT fetuses (7.2 +/- 2.5 ng/ml) than in ADX fetuses (mean +/- SD, 1.97 +/- 0.9 ng/ml; P less than 0.025). At 134 dGA the fetal PVN was removed by micropunching, and the anterior pituitary was separated from neurointermediate and posterior lobes after necropsy. Total RNA was prepared by the guanidium isothiocyanate-cesium chloride method and subjected to Northern analysis using specific cDNA probes to CRH and POMC. After autoradiography, quantification of mRNA was performed by scanning densitometry. Quantities of specific hybridization signal for POMC and CRH were normalized to the content of actin mRNA in each individual sample. RNA prepared from PVN exhibited a single specifically hybridizing band for CRH of approximately 1300 nucleotides. RNA prepared from anterior pituitary exhibited a single specifically hybridizing band for POMC at approximately 1300 nucleotides. Anterior pituitary POMC mRNA was significantly increased (P less than 0.025) in ADX fetuses (236 +/- 32% of CONT). CRH mRNA in PVN was greater in ADX fetuses than in CONT fetuses (P less than 0.05; mean +/- SEM, 179 +/- 21% of CONT). Adrenalectomy in fetal sheep significantly increased expression of CRH and POMC. We conclude that the increased levels of mRNA for CRH and POMC indicate that both the fetal PVN (CRH) and the anterior pituitary (POMC) are responsive to removal of the primary source of circulating glucocorticoid at this gestational age.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hypothalamic paraventricular nuclear lesions delay corticotroph maturation in the fetal sheep anterior pituitary.

The purpose of this study was to determine whether normal morphological development occurs in pituitary corticotrophs deprived of products of the hypothalamic paraventricular nucleus (PVN), e.g. corticotropin releasing hormone and arginine vasopressin (AVP), after PVN lesions. In addition, we have attempted to ascertain if the neurophysin/AVP-positive fibers innervating the fetal sheep anterior pituitary are affected by PVN lesions. The experimental groups consisted of fetal sheep in which 1) hypothalamic PVN lesions were placed at 118-122 days gestation (dGA) and the fetuses subsequently harvested while still in utero at 157 dGA or more (PVNX; n = 5); 2) sham PVN lesions were placed at 118-122 dGA and subsequently harvested as newborn lambs immediately after birth at 146.5 +/- 0.9 (mean +/- SEM) dGA combined with two uninstrumented fetuses harvested at 144 dGA or more but not in labor (perinatal; n = 6); and 3) no instrumentation was placed, and the fetuses were harvested at 120 dGA (control; n = 4). Two ACTH-immunoreactive cell types were seen in the anterior pituitary: 1) fetal cells: large and variably stained, often columnar, occurring in clusters and arranged in palisades; and 2) adult cells: smaller, darkly staining, and angular, occurring singly or in small groups. Quantification of the distribution of the two ACTH cell types was performed by scanning sections from a one in six series from each pituitary and estimating the percent area of each section in the well that showed adult type staining only. The observer was blind to the treatment group assignment of the sections. The estimated percentages of the portion of the pituitaries of each group that contained adult-type cells only were as follows: PVNX, 42.8 +/- 10.0%; perinatal, 90.9 +/- 2.1%; and control, 3.7 +/- 1.1% (mean +/- SEM; P less than 0.05 for all comparisons). There were no qualitative differences between all groups in the appearance of neurophysin-positive fibers innervating the anterior pituitary. AVP staining was strong in the internal zone of the median eminence in all groups, but was absent in the external zone of PVNX fetuses only. The intermediate pituitary lobes stained darkly in all groups. We conclude that lesions of the PVN at 120 dGA delay development of fetal pituitary corticotrophs, but have no effect on the presence of neurophysin-positive nerve fibers in the anterior pituitary.     

Effect of implantation of dexamethasone adjacent to the paraventricular nucleus on messenger ribonucleic acid for corticotropin-releasing hormone and proopiomelanocortin during late gestation in fetal sheep.

Glucocorticoids act upon the hypothalamic paraventricular nucleus (PVN) and anterior pituitary in a classic negative feedback loop to regulate ACTH biosynthesis and secretion. Evidence exists to indicate that glucocorticoid feedback may be attenuated during late gestation in the sheep fetus to allow the preterm rise in fetal plasma cortisol necessary for parturition in this species. The present studies were undertaken to determine the effect of glucocorticoids placed adjacent to the fetal PVN on messenger RNA (mRNA) for CRH in the PVN and mRNA for POMC in the anterior pituitary during late gestation. We performed our studies at two critical stages during late gestation to determine if gestational age related changes occur in the efficacy of negative feedback regulation of expression of CRH and subsequently POMC. Dexamethasone (DEX) implants were placed bilaterally 2 mm lateral to the fetal PVN at 105 to 107 days gestational age (dGA; group I, n = 4) and 121-123 dGA (group II; n = 4). Gestational-age matched, sham implanted fetuses were used as controls (CONT) for both groups (n = 4 per group). Fetuses were recovered at 126-128 (group I) and 136 dGA (group II). Fetal PVN were isolated by micropunching, and the anterior pituitary was separated from neurointermediate and posterior lobes after necropsy. Total RNA was subjected to Northern analysis using specific complementary DNA probes to CRH and POMC, and specific message was normalized to actin mRNA content in each individual sample. Anterior pituitary POMC mRNA was not different in DEX fetuses compared to CONT for either group I (78 +/- 26% of CONT; mean +/- SEM) or group II (84 +/- 17% of CONT). PVN CRH mRNA content was lower in DEX fetuses in group I (28 +/- 14% of CONT; P less than or equal to 0.01) and group II (65 +/- 12% of CONT; P less than or equal to 0.01). The degree to which DEX suppressed mRNA for CRH was greater in group I compared to group II (P less than or equal to 0.05). We conclude that 1) CRH expression in the PVN of fetal sheep is suppressible by glucocorticoids; 2) suppression can occur directly at the level of the PVN and 3) that the efficacy of negative feedback decreases with increasing gestational age. Furthermore, the lack of effect of hypothalamic administration of DEX on anterior pituitary POMC mRNA indicates that basal expression of POMC in fetal sheep may be independent from support from the PVN at this stage of gestation.     

Regulation of proopiomelanocortin messenger ribonucleic acid levels in the ovine fetal anterior pituitary in vitro

Parturition in sheep is dependent upon maturation of the fetal hypothalamo-pituitary-adrenocortical (HPA) axis. Anterior pituitary expression of the ACTH precursor, proopiomelanocortin (POMC), increases during the final days of gestation in spite of exponentially increasing fetal plasma cortisol levels. Lesion of the hypothalamic paraventricular nucleus prevents the late gestation increase in POMC mRNA. The purpose of this study was to examine glucocorticoid, corticotropin releasing factor (CRF) and arginine vasopressin (AVP) regulation of POMC mRNA levels in fetal anterior pituitary corticotropes in vitro and to address potential interactions between glucocorticoids and neuropeptides in regulating POMC. Anterior pituitaries from fetal sheep at two gestational ages (dGA; 118–125 dGA, n=9; 140–144 dGA, n=7) were enzymatically dispersed. POMC mRNA levels were determined at 24, 48 and 72 h post-dispersion. CRF, AVP and dexamethasone (DEX) regulation of POMC mRNA were determined at 24 and 72 h post-dispersion. The capacity of CRF and AVP to modulate DEX suppression of POMC mRNA levels was also examined. POMC mRNA was elevated at 24 h (P<0.01) and 48 h (P<0.05) post-dispersion compared to 0 h (immediately post-dispersion) in 140–144 dGA but not 118–125 dGA corticotropes. DEX suppressed POMC mRNA in a dose-dependent manner (when administered at 24 h post-dispersion) in the 140–144 dGA anterior pituitary cells but not 118–125 dGA anterior pituitary cells. Administration of DEX (10 nM) at 0 h prevented the increase in POMC mRNA levels observed at 24 h post dispersion in the 140–144 dGA group. Neither CRF nor AVP (administered at either 24 or 72 h post-dispersion) altered POMC mRNA levels in either 118–125 or 140–144 dGA anterior pituitary cells. Continuous exposure of anterior pituitary cells with either CRF or AVP (50 pM) through 96 h increased (P<0.05) POMC mRNA. No synergistic or additive effects were observed with CRF and AVP. Four hour pretreatment with CRF but not AVP (100 nM at 24 h post-dispersion) attenuated (P<0.05) DEX suppression of POMC mRNA levels in 140–144 dGA corticotropes. In conclusion, our results indicate that direct glucocorticoid suppression of POMC expression in fetal sheep initiates between 120 and 140 dGA, coincident with the period of gestation when fetal plasma cortisol is exponentially rising. Further, while short duration exposure of fetal corticotropes to either CRF or AVP had no effect on POMC mRNA, CRF appears capable of interfering with glucocorticoid suppression of POMC mRNA. The latter observation provides a potential mechanism via which the fetal PVN may counter rising fetal plasma cortisol concentrations resulting in the previously observed late gestation increase in anterior pituitary POMC mRNA.     

Proopiomelanocortin processing in the anterior pituitary of the ovine fetus after lesion of the hypothalamic paraventricular nucleus

The hypothalamic-pituitary-adrenocortical axis plays an essential role in the maturation of fetal organs and, in sheep, birth. Lesioning the paraventricular nucleus (PVN) in fetal sheep prevents adrenocortical maturation and parturition without altering plasma immunoreactive ACTH concentrations. The purpose of this study was to determine the effect of PVN lesion on anterior pituitary processing of proopiomelanocortin (POMC) to ACTH, plasma concentrations of ACTH and ACTH precursors (POMC; 22-kDa proACTH), and expression of subtilisin-like prohormone convertase 3 (SPC3) in corticotropes in fetal sheep. PVN lesion did not affect anterior pituitary POMC and 22-kDa proACTH levels, whereas ACTH was significantly affected. The ACTH precursor (POMC plus 22-kDa proACTH) to ACTH ratio in the anterior pituitary was significantly increased after PVN lesion. Post-PVN lesion, fetal plasma ACTH(1-39), was below the limit of detection, whereas ACTH precursors (POMC plus 22-kDa proACTH) were not affected. In the inferior region of the anterior pituitary, 40-50% of corticotropes had detectable SPC3 hybridization signal, and PVN lesion did not change the extent of colocalization of POMC and SPC3, or SPC3 mRNA levels within corticotropes. Neither the percent of corticotropes in the superior region containing SPC3 hybridization (7-12%) or hybridization signal strength was altered in response to PVN lesion. In conclusion, the fetal PVN is necessary for sustaining adequate anterior pituitary processing of POMC to ACTH and ACTH release needed for maturing the adrenal cortex in the sheep fetus.     

Effect of bilateral lesions of the ovine fetal hypothalamic paraventricular nuclei at 118-122 days of gestation on subsequent adrenocortical steroidogenic enzyme gene expression.

Fetal adrenal steroid hydroxylase activity and messenger RNA (mRNA) expression increases concurrent with the preterm rise in fetal plasma cortisol during late gestation in sheep. By placing bilateral lesions of the fetal paraventricular nuclei (PVN) we have previously demonstrated that the fetal PVN is necessary for the initiation of parturition, the late gestation preparturient increase in fetal plasma cortisol and ACTH, and ACTH secretion in response to fetal hypoxemia and hypotension. The purpose of this study was to determine the role of the fetal PVN in the late gestation increase in expression of mRNA for 17 alpha-hydroxylase (P-450(17)alpha), side-chain cleavage (P-450SCC), 11 beta-hydroxylase (P-450(11)beta), 21 hydroxylase (P-450C21), and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in the fetal adrenal. Ovine fetuses were subjected to bilateral lesions of the PVN (Lx; n = 4) or sham lesions (Sh; n = 4) at 118-122 days gestational age (dGA). Lx fetuses were recovered by cesarean section at greater than or equal to 157 dGA; Sh fetuses were recovered immediately postbirth at normal term (146.5 +/- 0.9 dGA). In addition, uninstrumented fetuses were obtained at 145-147 dGA by cesarean section (n = 3). RNA obtained from individual fetal adrenals was subjected to Northern analysis. Lx of the fetal PVN decreased (P less than or equal to 0.05) mRNA for P-450(17)alpha and P-450SCC but did not affect adrenocortical mRNA for P-450C21, P-450(11)beta, or 3 beta-HSD compared to Sh. To determine if the differences observed between Lx and Sh for P-450(17)alpha and P-450SCC mRNA were due to the process of labor, we compared uninstrumented 145-147 dGA to Sh. No differences in adrenal mRNA content were observed for P-450(17)alpha or P-450SCC between these groups. We conclude that in late gestation fetal sheep an intact fetal PVN is necessary for normal gene expression of adrenocortical P-450(17)alpha and P-450SCC while P-450(11)beta, P-450C21, and 3 beta-HSD may be primarily regulated by factors not dependent upon a functional PVN.     

Effect of maternal nutrient restriction in early gestation on development of the hypothalamic-pituitary-adrenal axis in fetal sheep at 0.8-0.9 of gestation

The fetal hypothalamic-pituitary-adrenal (HPA) axis has numerous key roles in development. Epidemiological data have linked adverse prenatal nutrition with altered organ development and increased incidence of disease in adult life. We studied HPA axis development in resting and stimulated states in late gestation fetal sheep, following 15% reduction in maternal nutritional intake over the first 70 days of gestation (dGA). Fetuses from control (C) and nutrient-restricted (R) ewes were chronically catheterised and response profiles for ACTH and cortisol were determined at 113-116 and 125-127 dGA after administration of corticotrophin releasing hormone (CRH) and arginine vasopressin (AVP). At 126-128 dGA cortisol profiles were also determined following ACTH administration. Basal ACTH and cortisol concentrations were not different between C and R fetuses. In R fetuses, ACTH response to CRH+AVP was significantly smaller at 113-116 dGA (P<0.01), and cortisol responses were smaller at both 113-116 dGA (P<0.01) and 125-127 dGA (P<0.0001). Cortisol response to ACTH was also smaller in R fetuses (P<0.001). We conclude that, in late gestation fetal sheep, pituitary and adrenal responsiveness is reduced following modest maternal nutrient restriction in early gestation.     

Characterization of high affinity receptors for insulin-like growth factors I and II on rat anterior pituitary cells

Primary cultures of rat anterior pituitary cells were assessed for the presence of specific receptors for insulin and for the somatomedin peptides, insulin-like growth factors I and II (IGF-I and IGF-II). Specific binding per 100,000 pituitary cells averaged 9.45 +/- 1.69% (mean +/- SD) for [125I]IGF-II, 0.83 +/- 0.06% for [125I]IGF-I, and only 0.11% for [125I]insulin, IGF-II was twice as potent as IGF-I in displacing [125I]IGF-II, while insulin was totally nonreactive, IGF-I was 5-fold more potent than IGF-II at displacing [125I]IGF-I and 1000-fold more potent than insulin. Scatchard analysis of [125I]IGF-II binding revealed a curvilinear plot, which could be resolved into a high affinity receptor with a Ka of 7.0 X 10(8) M-1 and 120,000 receptor sites/cell, and a low affinity receptor with a Ka of 1.1 X 10(8) M-1 and 720,000 receptor sites/cell. The existence of abundant high affinity somatomedin receptors (especially for IGF-II) on rat anterior pituitary cells is consistent with a potential role for these peptides in the regulation of GH secretion.     

Opioid receptors are present in the hypothalamus but not detectable in the anterior pituitary of the developing ovine fetus.

Exogenous opioids stimulate adrenocorticotropic hormone (ACTH) release in fetal sheep after day 125 of pregnancy (term 145 days), but not at day 110. To determine if the different response is due to an alteration in opioid-binding sites and to examine sites of opioid action on ACTH release, we established an in vitro binding assay to study changes and characteristics of opioid receptor binding sites in ovine fetal hypothalamus and pituitary during development. [3H]-naloxone was used as the radiolabelled ligand. The binding of [3H]-naloxone to hypothalamic membrane preparations was specific, saturable with respect to [3H]-naloxone concentration, and linear with the hypothalamic membrane protein content. Binding assays were conducted on tissues collected from fetuses at three gestational ages: days 110-115 and 125-130 and term. In all cases, Scatchard analysis revealed a single class of binding sites with high binding affinity (0.9-1.2 nM) which did not differ significantly with gestational ages. The binding capacity increased significantly from 45.4 +/- 2.5 fmol/mg protein at days 110-115 to 76.8 +/- 2.3 fmol/mg at days 125-130, but did not change further in term fetuses. When anterior pituitaries from the three groups of fetuses were processed and analyzed in a similar manner, no detectable binding was found. These results indicate (1) that endogenous opioid peptides may act at the hypothalamus rather than pituitary to regulate hypothalamic-pituitary-adrenal activity in the ovine fetus, and (2) the developmental changes in the binding capacity may contribute, in part, to the altered ACTH response reported in previous in vivo studies.     

Involvement of vasopressin in the down-regulation of pituitary corticotropin-releasing factor receptors after adrenalectomy

The role of vasopressin (VP) in the regulation of pituitary corticotropin-releasing factor (CRF) receptors was studied by examining the effects of adrenalectomy and VP infusion on pituitary CRF receptors in genetically VP-deficient rats (di/di) and Long-Evans control rats. Binding studies with [125I]Tyr-ovine CRF in 30,000 X g anterior pituitary membrane-rich fractions revealed similar characteristics for the CRF receptors in Long-Evans and di/di rats, with Kd values of 2.4 +/- 0.6 and 1.9 +/- 0.2 nM, respectively, and receptor concentrations of 278 +/- 31 and 286 +/- 43 fmol/mg, respectively. Two days after adrenalectomy, the pituitary CRF receptor concentration decreased by 72 +/- 4.2% in Long-Evans rats, but by only 20.3 +/- 5.6% in di/di rats. CRF receptor affinity was unchanged after adrenalectomy (Kd = 1.7 +/- 0.5 nM; n = 8). To determine whether VP deficiency is responsible for the smaller decrease in CRF receptor in di/di rats, the effect of exogenous VP infusion (100 ng/min) by sc osmotic minipumps was studied in adrenalectomized di/di rats. Two days after adrenalectomy, pituitary CRF receptors were reduced by 21 +/- 8% in control di/di rats, whereas a 77.7 +/- 1.8% decrease was observed in VP-infused di/di rats, comparable to the effect of adrenalectomy in Long-Evans rats. VP infusion also caused a significant 35 +/- 2% decrease in CRF receptors in the pituitaries of sham-operated di/di rats, with no change in CRF receptor affinity. In Sprague-Dawley rats, VP or CRF infusion (100 ng/min) decreased pituitary CRF receptors by 14 +/- 1.9% and 46 +/- 3%, respectively. However, the combined infusion of both peptides caused a 65% +/- 4.2 decrease, similar to that observed after adrenalectomy. In vitro incubation of quartered pituitaries with VP or CRF for 4 h reduced CRF receptors by 23.1 +/- 8.2% and 38.2 +/- 3.8%, respectively, while simultaneous preincubation with both peptides was followed by a decrease of 55.3 +/- 5.3%. These findings indicate that increased hypothalamic release of VP contributes to the down-regulation of pituitary CRF receptors after adrenalectomy.     

Pulmonary epithelial liquid absorption,expressed in relation to alveolar surface area,is reduced in fetal lambs following in utero tracheal occlusion

We examined the effect of accelerated lung growth, induced by in utero tracheal occlusion (TO), on lung liquid uptake in near-term fetal sheep. In utero TO was performed in five fetal sheep at 110 days of gestation (term, approximately 145 days); six SHAM operated fetuses served as controls. The rate of liquid movement across the pulmonary epithelium was measured, using a previously established technique, in anesthetized fetal sheep between 133-137 days of gestation during a 2-hr adrenaline infusion (0.50 microg/min/kg, I.V.) and while lung luminal pressure was maintained at 5 mmHg.The rate of fetal lung liquid uptake was linear in all fetuses (mean r(2) < 0.97, n = 11). Mean values of lung liquid uptake expressed in relation to dry lung weight and luminal surface area of the right lung were significantly lower in TO fetuses (1.8 +/- 0.3 mL/hr/g and 1.0 +/- 0.2 mL/hr/m(2)) than in SHAM fetuses (2.6 +/- 0.2 mL/hr/g and 1.8 +/- 0.1 mL/hr/m(2)); surface area of the right lung was 140% greater in TO fetuses than in SHAM fetuses. There was a linear relationship between lung liquid uptake and pulmonary epithelial surface area in SHAM animals, but not in TO fetuses.We hypothesize that loss of alveolar epithelial type-II cells induced by increased levels of fetal lung expansion may impair alveolar liquid clearance in the perinatal period.     

The ontogeny of somatotropic binding sites in ovine hepatic membranes

The binding of human [125I]GH, ovine [125I]GH, bovine [125I]GH, and ovine [125I]PRL, to hepatic microsomal membranes (100,000 g) prepared from fetal, neonatal, and infant lambs and adult ewes has been examined. The specific binding of hGH increases (P less than 0.01) from 3.4 +/- 0.8% in the fetus (n = 7) to 20.0 +/- 2.1% (n = 6) in lambs at least 6 days postpartum. The binding of oGH is low in the fetus (0.7 +/- 0.2%; n = 13) and neonatal lamb (1.3 +/- 0.5%; n = 5) and increases (P less than 0.01) in older lambs (14.6 +/- 4.7%; n = 6) and adult sheep (14.9 +/- 5.3%; n = 4). Similarly, the binding of bGH is less (P less than 0.05) to fetal tissues. In contrast, the binding of oPRL is similar in fetal and postnatal preparations. Cross-reaction studies suggest that the binding of GHs is to a site with lactogenic characteristics in the fetus. In contrast, in lambs at least 4 days postpartum and in adult sheep, binding is to a site with somatogenic characteristics. The inability to detect somatogenic sites in the fetal liver is not due to saturation by endogenous GH or chorionic somatomammotropin, as the binding characteristics do not change after MgCl2 pretreatment of the membrane fractions. No change in binding is observed 25 days after fetal decapitation at 69 days (n = 3), suggesting that circulating GH does not down-regulate the fetal GH receptor. These observations suggest an immaturity of the somatogenic receptor in the ovine fetal liver and its appearance in the perinatal period. Immaturity of this receptor is likely to be the basis for the lack of a major effect of fetal GH on fetal somatic growth.     

Effect of bilateral splanchnic nerve section on adrenal function in the ovine fetus

The effect of bilateral splanchnic nerve (SPLX) section in fetal sheep [116-121 days gestational age (dGA)] on subsequent adrenocortical function was investigated. Fetal cortisol release was stimulated by 1) ACTH-(1-24) administration (100 ng/min for 15 min) at 126-129 and at 132-135 dGA, and 2) nitroprusside-induced hypotension (50% reduction in fetal arterial blood pressure for 10 min) at 129-132 and 136-139 dGA. No differences were observed between SPLX and control fetuses (CONT) in basal arterial plasma concentrations of cortisol from 126-141 dGA. A significant effect of fetal age on basal cortisol was observed in both SPLX and CONT fetuses from 126-141 dGA. A significant (P less than or equal to 0.05) increase in fetal arterial concentrations of cortisol was achieved by ACTH-(1-24) infusion in SPLX and CONT and did not differ between groups at either 126-129 or 132-135 dGA. Hypotension induced a significant increase in fetal plasma cortisol concentrations in SPLX and CONT fetuses (P less than or equal to 0.05). SPLX fetuses secreted significantly less cortisol in response to hypotension than CONT fetuses at both 129-132 and 136-139 dGA (P less than 0.05). Fetal arterial plasma concentrations of immunoreactive ACTH in response to hypotension were not different between CONT and SPLX fetuses. We estimated the half-life of endogenous fetal plasma cortisol after both hypotension and infusion of ACTH-(1-24). There were no differences between either method of inducing cortisol release on the endogenous cortisol half-life, nor were any differences in cortisol half-life observed between SPLX and CONT. At 136-139 dGA the adrenomedullary response to hypotension was abolished in SPLX, confirming completeness of denervation. In conclusion, the splanchnic nerves do not appear to be involved in the normal increase in basal fetal plasma cortisol observed in late gestation in fetal sheep. However, splanchnic nerve modulation of secretion of cortisol in response to stress may be involved in the increased fetal adrenal sensitivity to stress observed late in gestation.     

The contribution of local tissue thyroxine monodeiodination to the nuclear 3,5,3'-triiodothyronine in pituitary, liver, and kidney of euthyroid rats.

The contributions of local T4 monodeiodination and plasma T3 to the nuclear T3 of anterior pituitary, liver, and kidney were measured in euthyroid rats. After injection of [125I]T4, there was a gradual increase in the quantity of plasma [125I]T3 in excess of injected contaminant, which peaked at approximately 12 h after injection and remained a constant fraction of plasma [125I]T4 (2.8 X 10(-3) after that time. In the nuclei of anterior pituitary tissue, there was also a slow increase in locally produced [125I]T3 (in excess of that which could be accounted for by plasma [125I]T3) which appeared to peak at about 16 h after [125I]T4 administration. The ratio of nuclear [125I]T3 generated intracellularly to plasma [125I]T4 was constant at 18 and 24 h after T4 injection and was 13 +/- 2 X 10(-3) in nuclei of pituitary, 2.0 +/- 0.4 x 10(-3) in liver, and 0.47 +/- 0.1 x 10(-3) in kidney (all values are mean +/- SD). This locally generated T3 resulted in a markedly higher nuclear to plasma (N:P) ratio for [125I]T3 than for injected [131I]T3 in the same animals. The N:P ratio for [125I]T3 at equilibrium after injected T4 was 2.4 +/- 0.6, 0.47 +/- 0.09, and 0.10 +/- 0.03 (nanograms of T3 (mg DNA)-1/ng T3 ml-1) in pituitary, liver, and kidney. Comparable values for [131I]T3 N:P ratios were 0.47 +/- 0.14 (pituitary), 0.18 +/- 0.01 (liver), and 0.036 +/- 0.008 (kidney). Using RIA values for plasma T4 and T3 concentrations in these rats and maximal nuclear T3-binding capacities estimated in parallel experiments, the gravimetric quantities of nuclear T3 derived from plasma T3 and from local T4 to T3 monodeiodination were estimated and expressed as the percentage of saturation of T3 receptors. Seventy-eight percent of nuclear T3 receptor sites in anterior pituitary were occupied with one-half of the nuclear T3 derived directly from plasma T3 and the other half from intrapituitary T4 monodeiodination. Local T4 monodeiodination provided only 28% and 14%, respectively, of the nuclear T3 in liver and kidney, and the nuclear receptors of these tissues were about 50% saturated. Since our previous studies have shown that the occupancy of the pituitary nuclear T3 receptors may regulate TSH release, these data provide a mechanism by which TSH secretion could be altered by changes in either plasma T3 or T4, whereas nuclear T3 in liver and kidney is predominantly a function of the plasma T3 concentration.     

Arginine vasopressin and corticotropin releasing factor: binding to ovine anterior pituitary membranes

In the sheep, in contrast to the rat, arginine vasopressin (AVP) is a more potent stimulus to ACTH secretion from the anterior pituitary (AP) than CRF. To further explore this difference, we have compared [3H]AVP and [125I]-[Nle21 Tyr32]ovine CRF binding in membranes prepared from rat and sheep AP. Between species, no difference in affinity of binding was found for either ligand. In contrast, the concentration of AVP receptors in sheep AP was twice that in rat, whereas that of CRF receptors was only one tenth. AVP receptor concentration in sheep AP was not altered by chronic (10 day) dexamethasone administration, but fell to 60% of control after chronic (60 day) hypothalamo-pituitary-disconnection. The increased level of AVP receptors and the much lower level of CRF receptors in sheep compared with rat may thus provide an explanation for our previous findings of increased sensitivity to AVP and a very poor response to CRF in stimulating ACTH release from the sheep AP. In addition the finding that AVP receptor numbers are reduced in the hypothalamo-pituitary-disconnected sheep suggests that hypothalamic factors may play a role in regulating AVP receptor concentration in the ovine AP gland.     

Synergism between systemic corticotropin-releasing factor and arginine vasopressin on adrenocorticotropin release in vivo varies as a function of gestational age in the ovine fetus

In sheep, parturition is associated with maturation of fetal pituitary-adrenal function, and with rises in the concentrations of ACTH and cortisol (F) in fetal plasma. We examined the hypothesis that pituitary ACTH output in response to arginine vasopressin (AVP) and CRF separately and together might change during late pregnancy as a function of fetal age. Fetal sheep were chronically catheterized, and bolus iv injections of equimolar AVP, CRF, AVP plus CRF, or saline (controls) were given on days 110-115, 125-130, and 135-140 of gestation. AVP evoked significant rises in plasma ACTH on days 110-115 and 125-130, but not on days 135-140. After AVP, the peak plasma concentrations of ACTH were attained at 5-10 min, and basal (preinjection) values were reestablished by 30-60 min. After CRF treatment, plasma ACTH rose progressively throughout the 240 min of the study. Evidence was obtained in support of an increase in pituitary responsiveness to CRF between days 110-115 and 125-130 and a decrease in response on days 135-140, when basal F concentrations were higher. The ACTH response to AVP, relative to that to CRF, was greatest in the youngest fetuses. On days 110-115 only, CRF and AVP showed a synergistic response in ACTH output, especially during the first 30 min after agonist injection. Plasma F rose in response to the changes in endogenously released ACTH in a manner consistent with progressive fetal adrenal maturation between days 110-140 of pregnancy. We conclude that in vivo the ovine fetal pituitary responds separately and synergistically to AVP and CRF on days 110-115 of gestation, but the relative role of AVP in stimulating ACTH release decreases with progressive gestational age.     

Effects of synthetic corticotropin-releasing factor and dopamine on the release of immunoreactive beta-endorphin/beta-lipotropin and alpha-melanocyte-stimulating hormone from human fetal pituitaries in vitro

The effects of synthetic corticotropin-releasing factor (CRF) and dopamine on immunoreactive beta-endorphin/beta-lipotropin (i beta-END/LPH) and alpha MSH release were studied in superfused human fetal pituitary glands. CRF (20 ng) stimulated the release of i beta-END/LPH in four anterior hemipituitaries from fetuses older than 20 weeks in gestation. There was no effect on three anterior hemipituitaries from fetuses of 19-20 weeks gestation. CRF had no effect on i beta-END/LPH or alpha MSH secretion from neurointermediate lobes regardless of fetal age. Dopamine (10(-6) M) had no effect on i beta-END/LPH or alpha MSH secretion from either anterior or neurointermediate lobes. The data suggest that anterior pituitary responsiveness to CRF develops at about 20 weeks gestation and that fetal neurointermediate lobe secretion of peptides is not regulated by CRF.     

Characteristics and developmental changes of corticotrophin-releasing hormone-binding sites in the fetal sheep anterior pituitary gland.

The responses of the fetal sheep pituitary to corticotrophin-releasing hormone (CRH) change during gestation with maximum output of ACTH around days 120-130, and decreased ACTH output near term. However, there is no information available concerning the extent to which these responses may be modulated by alterations in the number of CRH receptors. Therefore we measured specific CRH-binding sites, and changes in binding characteristics in membrane preparations from fetal sheep anterior pituitaries collected at days 65-70, 85-88, 100-110, 125-130 and at term (approximately 145 days). Binding assays were carried out using 125I-labelled Tyr-ovine CRH (125I-Tyr-oCRH), incubated with crude membrane fractions for 90 min at 22 degrees C. Binding was time- and temperature-dependent, linear with protein concentration, saturable and specific for oCRH. Scatchard analysis of binding data for individual tissues revealed a single class of CRH-binding sites with high affinity (Kd congruent to 1 nmol/l) that did not change significantly with gestational age. However, the number of CRH-binding sites increased progressively from days 65-70 to a maximum at days 125-130, then decreased at term. These results demonstrate the presence of specific CRH-binding sites in the fetal sheep anterior pituitary. Furthermore, the change in CRH receptor number with advancing pregnancy follows a similar time-course to the changes reported previously in responsiveness of the fetal sheep anterior pituitary to exogenous CRH stimulation in vivo. These results suggest that alterations in CRH receptor number may contribute to changes in responsiveness of the fetal sheep anterior pituitary to CRH during gestation.