The fetal adrenals of the rat: correlations between growth, cytology, and hormonal activity, with and without ACTH deficiency.

Growth, ultrastructure, and corticosterone content were studied in the adrenals of rat fetuses in late pregnancy, between Days 19 and 21. In the fetuses of intact mothers, the hypophysial corticostimulating activity decreased between Days 19 and 21. Ablation of the fetal hypothalamus by total removal of the brain, leaving the hypophysis in situ (encephalectomy), induced atrophy of the adrenals, changes in adrenal ultrastructure, and decrease in the adrenal corticosterone content. Such modifications were also observed in response to the ablation of the pituitary gland by decapitation. The hypothalamus was involved in the control of ACTH release in late pregnancy. When mothers were adrenalectomized on Day 14, in the absence of hypothalamic tissue, the pituitary gland of the encephalectomized fetuses showed some corticostimulating activity; indeed, the adrenals of these fetuses were heavier on Day 21 than those of the decapitated fetuses but smaller than those of the littermate controls, and the adrenal corticosterone content was also greater than in the decapitated but less than in the controls. Encephalectomy induced modifications in adrenal cytology less marked than those induced by decapitation. These data suggest that the pituitary gland and the hypothalamo-hypophysial complex are the sites of the corticosteroid-induced inhibition of the corticostimulating activity of the fetal hypophysis.     

CRF activity in fetal rat hypothalamus, in late pregnancy.

The corticosterone content of the adrenal glands was determined in 21-day-old rat fetuses: (1) before and after encephalectomy or decapitation (hypophysectomy); (2) after ACTH treatment of the encephalectomized or decapitated (hypophysectomized) fetuses; and (3) after administration of crude extracts (0.1 N HCl) of hypothalamic or cortical tissue from 20-day-old rat fetuses. A peak in the corticosterone content of the adrenals was observed 10 min after ACTH injection to enceaphlectomized or decapitated fetuses. A rise in corticosterone concentration was noted 5 and 10 min after the encephalectomized fetuses were given an injection of hypothalamic extract. This extract was devoid of appreciable ACTH activity when tested in decapitated fetuses. Cortical extract was inactive in encephalectomized or decapitated fetuses. These data suggest that fetal hypothalamic extract contains some CRF activity and that the fetal pituitary gland is responsive to the CRF.     

Different roles for the pituitary and adrenal cortex in the control of enkephalin peptide localization and cortico-medullary interaction in the sheep adrenal during development.

We have investigated the effect of adrenocorticotrophic hormone (ACTH) replacement after fetal hypophysectomy on the pattern of localization of enkephalin-containing peptides (enkephalins) and phenylethanolamine N-methyltransferase (PNMT) in the fetal sheep adrenal. We have also investigated the relative roles of the fetal pituitary and adrenal cortex in determining the extent of the interdigitation of the peripheral adrenaline (AD)-containing cells of the adrenal medulla with the inner zones of the adrenal cortex in the late gestation fetus. Fetal hypophysectomy (Hx; n = 12) or sham operations (n = 8) were performed at 109-118d. At 138 or 139d, ACTH (1-24) (10.5 micrograms/h) was infused intravenously for 72 h into 4 Hx fetuses (Hx + ACTH group). Saline was infused for 72 h into 4 Hx fetuses (Hx + Sal) and into 4 sham-operated fetal sheep (Intact + Sal). Fetal adrenal glands were collected at autopsy from 141/2d Intact + Sal, Hx + Sal and Hx + ACTH groups, from 4 intact fetal sheep at 145-147d gestation (145/7d Intact group) and 4 Hx fetal sheep at 147-164d gestation (147/64d Hx group). Adrenals were also collected from 4 newborn lambs at 10-12d after birth (10/12d Newborn group). Using the peroxidase-antiperoxidase immunocytochemical staining method, sections of adrenal glands (10-12 microns) from all groups were stained anti-PNMT. Sections of adrenal glands from the 141/2d groups were also stained separately with anti-dopamine-beta-hydroxylase (anti-D beta H) and anti-enkephalin (anti-ENK).(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of adenylate cyclase activity in rat adrenal glands during the perinatal period

Adenylate cyclase activity was studied in crude adrenal membranes from fetal and newborn rats. Basal adenylate cyclase activity was higher in fetal than in newborn rats. ACTH(1-24) (1 mumol/l), guanosine (beta,gamma-imido diphosphate) (Gpp(NH)p) (10 mumols/l) and forskolin (100 mumols/l) stimulated the activity of the enzyme at all stages studied. The sensitivity of the enzyme to ACTH was maximal on days 17 and 19 of gestation. When Gpp(NH)p was added to ACTH(1-24), the response was significantly higher than that induced by Gpp(NH)p alone. Forskolin and Gpp(NH)p alone increased the adenylate cyclase activity and the sensitivity of the enzyme to these compounds was higher in newborn rats than in fetuses. Treatment of 21-day-old rat fetuses with ACTH increased the response of adenylate cyclase to Gpp(NH)p alone or to forskolin whereas treatment with dexamethasone did not modify the response of the enzyme to either Gpp(NH)p alone or forskolin. Our results show that the change in the responsiveness of adenylate cyclase takes place immediately after birth during the first week and ACTH is able to induce a maturation of the fetal adrenal adenylate cyclase system.     

Comparison of the effects of prostaglandin E2, prostacyclin and 1-24 adrenocorticotrophin on plasma cortisol levels of fetal sheep

The changes in plasma cortisol levels in response to intravenous infusions of prostaglandin E2 (PGE2), prostacyclin and 1-24 ACTH have been studied in chronically catheterized fetal sheep during the last third of gestation. All three drugs increased plasma cortisol levels with prostacyclin being sigificantly more potent than either PGE2 or 1-24 ACTH. No interaction between the steroidogenic actions of 1-24 ACTH and either PGE2 or prostacyclin could be demonstrated. The steroidogenic action of PGE2 was not significantly modified by fetal hypophysectomy. It is concluded that neither PGE2 nor prostacyclin is likely to be involved in the enhanced adrenal responsiveness to 1-24 ACTH observed in fetal sheep in the period immediately before birth.     

Responsiveness and maximum secretory capacity of isolated fetal lamb adrenocortical cells throughout the last third of gestation

Although considerable evidence implicates increased fetal adrenal function as a major factor in the initiation of parturition in the sheep, the mechanism responsible for this increased activity has not yet been determined. We have investigated the development of the function of fetal lamb adrenal cortical cells dispersed in vitro. There was no change in the sensitivity of the cells to synthetic ACTH (ACTH1-24), as demonstrated by the concentraton of ACTH1-24 producing a 50% maximum response in corticoid secretion. This finding does not support the suggestion that there is a qualitative change in fetal adrenal receptor function as term approaches. No stimulation of corticoid was observed after the administration of alpha MSH or PRL in vitro at any gestational age or of alpha MSH in vivo in four fetuses at 125--130 days gestation. Both the maximum output and the 50% maximum response in corticoid secretion of adrenal cells from term fetuses were similar to those of adrenal cells from adult ewes. A significant increase in the maximum output of corticoids per cell in response to ACTH1-24 occurs as early as 107 days gestation and continues steadily to term.     

Steroid hydroxylase gene expression in the ovine fetal adrenal gland following ACTH infusion

Between 90 and 120 days of gestation (term = 147 +/- 5), when plasma cortisol concentrations in the fetus are at a minimum, levels of mRNA encoding the steroidogenic enzymes 17 alpha-hydroxylase (P-450(17 alpha] and cholesterol side-chain cleavage (P-450scc) are also very low. Over the following 30 days, P-450(17 alpha) and P-450scc gene expression increases concurrent with increasing fetal cortisol concentration. The hypothesis tested in this study was that cortisol biosynthesis is minimal in the period 90-120 days because of insufficient ACTH. Fetuses were cannulated between 98-102 days of gestation. Following recovery, 7 fetuses received 24-h ACTH infusions (12 micrograms/24 h) and 5 fetuses received 24-h vehicle infusions; 4 ACTH-infused and 4-vehicle-infused fetuses were then sacrificed immediately after cessation of the infusion. The other fetuses were left in utero for 3 days prior to sacrifice. Fetal blood samples were analysed for ACTH and cortisol and the adrenals processed for hybridization histochemistry and Northern blot analysis. ACTH, but not vehicle, induced significant increases in the width of the adrenal cortex and in the levels of P-450(17 alpha) and P-450scc mRNA. Concurrently, fetal plasma ACTH and cortisol concentrations also increased significantly. In adrenals from fetuses left in utero for 3 days after cessation of the ACTH infusion, P-450(17 alpha) and P-450scc mRNA levels returned to control levels. Plasma ACTH and cortisol levels also approximated basal values. P-450c21 mRNA levels did not vary significantly at any time with the treatments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ontogeny of ACTH(1-24) receptors in rat adrenal glands during the perinatal period

Binding of ACTH to receptors was studied on crude adrenal membranes from fetal and newborn rats. 125I-Labelled ACTH(1-24) was used as the radioligand, the steroidogenic potency of which was 100-fold lower than that of unlabelled ACTH(1-24). Binding was specific, rapidly equilibrated and temperature dependent. Scatchard analysis of the binding data revealed a single class of binding sites with a dissociation constant of about 100 nmol/l at all stages of development studied. The concentration of ACTH receptors expressed per mg membrane proteins decreased in fetuses between days 17 and 21 of gestation and remained stable in newborn rats from weeks 1 to 4. The number of ACTH receptors expressed per adrenal increased regularly in fetal and newborn rats. The perinatal evolution of these concentrations of ACTH receptors is related to the increase in the size of the adrenals and the changes in cytoplasmic structures of the adrenocortical cells. When the number of ACTH-binding sites was expressed per microgram DNA, maximum values occurred in fetuses on day 19 of gestation, and minimum values in newborn rats, 1 week after birth. There was an excellent correlation between the plasma levels of immunoreactive ACTH and corticosterone and the number of ACTH receptors per microgram DNA during the perinatal period. Other results suggest that ACTH is able to up-regulate the number of its own receptors.     

Modulation of adrenocorticotropin-stimulated baboon fetal adrenal dehydroepiandrosterone formation in vitro by estrogen at mid- and late gestation

We have reported that ACTH stimulation of dehydroepiandrosterone (DHA) formation by the baboon fetal adrenal at midgestation was suppressed by estrogen. Because fetal adrenal regulation changes with advancing gestation, the action of estrogen on fetal adrenal steroidogenesis may also be dependent on the degree of fetal adrenal maturation. We examined this possibility in the present study by determining the effects of ACTH and estrogen on DHA formation by adrenal cells of fetuses obtained from baboons at mid- and late gestation and from animals administered the antiestrogen MER-25 throughout late gestation. Because low density lipoprotein (LDL) provides substrate for the fetal adrenal, we also determined whether the effect of estrogen was mediated by LDL uptake. Adrenals were removed from baboon fetuses on day 100 (midgestation; n = 7) and day 170 (late gestation; n = 6; term, day 184) of gestation from untreated animals and on day 170 from fetuses whose mothers were treated with MER-25 on days 140-170 (25 mg/kg BW.day; n = 7). Cells were dispersed with 0.2% collagenase and incubated at 37 C for 3 h in 4 ml medium 199 with 10 nM ACTH, 10(-6) M estradiol and/or 500 micrograms LDL. The secretion of DHA into medium was determined by RIA. At midgestation, mean (+/- SE) basal DHA formation (nanograms per 10(5) cells/3 h) was 5.8 +/- 2.1, and DHA was increased (P less than 0.01) by ACTH to 20.0 +/- 5.9. Although estradiol alone had no effect, estradiol prevented the increase in DHA obtained with ACTH. Basal DHA production by adrenals of late gestation (0.7 +/- 0.3 ng/10(5) cells) was lower (P less than 0.01) than at midgestation. ACTH increased (P less than 0.01) DHA in a comparable manner near term in the presence (2.0 +/- 0.4) or absence (1.7 +/- 0.4) of estradiol. Thus, in contrast to day 100, estrogen did not attenuate the action of ACTH on adrenal cells on day 170. In fetal adrenal cells obtained on day 170 from MER-25-treated baboons, DHA formation (1.4 +/- 0.6 ng/10(5) cells) was comparably increased (P less than 0.05) to 2.4 +/- 0.2 and 3.0 +/- 0.5 ng/10(5) cells by ACTH in the absence or presence of estradiol. Thus, ACTH remained effective in enhancing DHA by adrenal cells of fetuses exposed in utero to antiestrogen. DHA formation by adrenals of midgestation was increased (P less than 0.05) to 15.4 +/- 4.8 and 27.4 +/- 7.5 ng/10(5) cells, respectively, by LDL and ACTH plus LDL.(ABSTRACT TRUNCATED AT 400 WORDS)     

alpha-Melanocyte-stimulating hormone and adrenocorticotropin in the regulation of glucocorticoid secretion during the perinatal period in sheep.

To determine the role of other ACTH-like peptides in the regulation of glucocorticoid secretion in fetal sheep, we examined the responses of the adrenal gland of fetal and newborn sheep to comparable single doses of alpha MSH (75 micrograms) or ACTH (50 micrograms) during the last third of gestation and the first month of postnatal life. alpha MSH first increased the plasma glucocorticoid concentration at 121--130 days of gestation [from 16 +/- 1.5 to 36.9 +/- 9 (SE) ng/ml]; the response to alpha MSH persisted on days 131--140 of gestation and during the first month after birth. ACTH first increased the plasma glucocorticoid concentration at 131--140 days of gestation and increased it further in the first month after birth (from 18.9 +/- 3.6 to 97.0 +/- 10 ng/ml). The observations that the adrenal glands of fetuses and newborn lambs responded to alpha MSH at a dose comparable to that of ACTH and that the response to alpha MSH in the fetus preceded the response to ACTH may indicate that adrenal receptors mature during fetal development. These data also suggest that the regulation of the adrenal during fetal life may involve more than one tropic hormone.     

Impact of restriction of placental and fetal growth on expression of 11beta-hydroxysteroid dehydrogenase type 1 and type 2 messenger ribonucleic acid in the liver, kidney, and adrenal of the sheep fetus

We have investigated the effects of fetal growth restriction, induced by restriction of placental growth and function (PR), on 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD-1) and 11betaHSD-2 messenger RNA (mRNA) expression in fetal tissues in the sheep, using Northern blot analysis. Fetal liver, kidney, and adrenals were collected from normally grown fetuses at 90 days (n = 6), 125 days (n = 6), and 141-145 days (n = 7) and from PR fetuses at 141-145 days (n = 6). Expression of 11betaHSD-1 mRNA in the fetal liver increased significantly between 125 days (7.4+/-0.8) and 141-145 days gestation (27+/-5.3). There was also an approximately 2-fold increase in the ratio of 11betaHSD-1 mRNA/18S rRNA expression in the PR group (53.8+/-7.9) compared with that in control animals at 141-145 days gestation. There was a significant decrease in 11betaHSD-2 mRNA in fetal adrenals between 125 days (41.6+/-2.4) and 141-145 days (26.7+/-1.1) gestation, but there was no effect of PR on the expression of adrenal 11betaHSD-2 mRNA. 11betaHSD-2 mRNA expression in the fetal kidney increased between 90 days (16.8+/-1.7) and 141-145 days gestation (31.7+/-4.3), but there was no effect of PR on the levels of 11betaHSD-2 mRNA in the fetal kidney. In summary, 11betaHSD-2 mRNA is differentially regulated in the fetal adrenal and kidney in the sheep fetus during late gestation. There is also a specific increase in the expression of 11betaHSD-1 mRNA in the liver of growth-restricted fetuses in late gestation. This suggests that there is increased hepatic exposure to cortisol in the growth-restricted fetus, which may be important in the reprogramming of hepatic physiology that occurs after growth restriction in utero.     

Plasma profiles of adrenocorticotropic hormone, cortisol, alpha-melanocyte-stimulating hormone, and growth hormone in dogs with pituitary-dependent hyperadrenocorticism before and after hypophysectomy

The 6-h plasma profiles of adrenocorticotropic hormone (ACTH), cortisol, alpha-melanocyte-stimulating hormone (alpha-MSH), and GH were studied in 17 dogs with pituitary-dependent hyperadrenocorticism (PDH) before and after hypophysectomy. The aim of the study was to investigate the relation between the hormone profile characteristics and recurrence of PDH after surgery. The hormones were secreted in a pulsatile fashion. The basal plasma cortisol concentration and area under the curve (AUC) for cortisol were significantly higher in the PDH cases than in eight controls. The characteristics of the plasma profiles of ACTH and alpha-MSH were not significantly different between the PDH cases and the controls. In the PDH cases, less GH was secreted in pulses than in the controls, but the difference was not significant. The basal plasma cortisol concentration, the AUC for ACTH and cortisol, and the pulse frequency of ACTH and cortisol decreased significantly after hypophysectomy for the group of PDH cases. The basal plasma concentrations of ACTH and alpha-MSH, the AUC for alpha-MSH, and the characteristics of the plasma GH profiles of the PDH cases remained unchanged after hypophysectomy. No pulses of alpha-MSH were observed after hypophysectomy. The co-occurrence between the ACTH and cortisol pulses decreased significantly with hypophysectomy. The postoperative pulse frequency of ACTH was the only characteristic with predictive value for the recurrence of PDH after hypophysectomy. The results of this study demonstrate that ACTH, cortisol, alpha-MSH, and GH are secreted in a pulsatile fashion in dogs with PDH. Hypophysectomy effectively reduces the secretion of ACTH and cortisol. The presence of ACTH pulses after hypophysectomy is a risk factor for the recurrence of hyperadrenocorticism.     

Evidence that cortisol inhibits basal adrenocorticotropin secretion in the sheep fetus by 0.70 gestation

To ascertain if reductions in fetal plasma cortisol cause increases in fetal plasma ACTH, we treated pregnant ewes or their fetuses with aminoglutethimide (10 mg/kg BW) and metyrapone (20 mg/kg BW) and measured the hormonal responses with RIAs. When given to fetuses (n = 9) at 0.90 +/- 0.01 gestation (term-145 days), the steroid synthesis inhibitors reduced fetal plasma cortisol from 35.1 +/- 11.9 to 18.5 +/- 6.2 ng/ml (P less than 0.01) and plasma ACTH increased from 37 +/- 7 to 189 +/- 74 pg/ml (P less than 0.02). Thus, late in gestation cortisol from the fetal adrenal suppresses basal fetal ACTH secretion. Blockade of steroid biosynthesis in pregnant ewes carrying intact fetuses at 0.76 +/- 0.02 gestation (n = 11) or adrenalectomized fetuses at 0.81 +/- 0.01 gestation (n = 6) also reduced cortisol and increased ACTH in fetal plasma. In intact fetuses cortisol declined from 9.4 +/- 2.0 to 3.6 +/- 0.9 ng/ml (P less than 0.05), and ACTH increased from 46 +/- 8 to 183 +/- 67 (P less than 0.01); cortisol declined in adrenalectomized fetuses from 2.1 +/- 0.4 to 1.1 +/- 0.3 ng/ml (P less than 0.01), and ACTH increased from 106 +/- 13 to 400 +/- 104 pg/ml (P less than 0.01). Cortisol infusions into intact and adrenalectomized fetuses prevented both the decline in steroid concentration caused by the biosynthesis inhibitors given to the ewe and the increase in fetal plasma ACTH concentration. These data indicate that reductions in plasma cortisol in adrenalectomized fetuses or intact fetuses at a time in development when the fetal adrenal produces little cortisol cause compensatory increases in fetal plasma ACTH concentration. The simplest explanation for these observations is that from approximately 0.70 gestation, basal fetal ACTH secretion is tonically inhibited by cortisol circulating in fetal plasma. This cortisol can originate from sources other than the fetal adrenal.     

The actions of N-terminal fragments of corticotrophin on steroidogenesis in dispersed rat adrenal cells in vitro

The finding that the rat adrenal zona glomerulosa cell shows specific sensitivity to stimulation by alpha-MSH and related peptides has been confirmed both in vivo and in vitro, raising the possibility that alpha-MSH may have a physiological role in the control of glomerulosa function and aldosterone secretion. To define more closely the structural features which confer teh specificity of the glomerulosa response, other ACTH derived peptides have been tested for their specificity of actions on rat adrenal cells in vitro. The peptides tested were ACTH(5-24), ACTH(1-12), ACTH(1-14), ACTH(1-15), ACTH(1-16) and ACTH(1-17). Their actions were compared with those of alpha-MSH and ACTH(1-24). All of the ACTH-derived peptides stimulated glomerulosa corticosterone production with sensitivities similar to that of alpha-MSH; minimum effective concentration was 10 nmol/l. Also, like alpha-MSH, the shorter ACTH peptides stimulated aldosterone production only relatively weakly in these cells from animals on normal sodium intake. Only ACTH(5-24), ACTH(1-16) and ACTH(1-17) stimulated fasciculata/reticularis cells at concentrations up to 1 mumol/1. The actions of all of the shorter peptides were thus unlike those of ACTH(1-24) which stimulates both cell types with approximately equal sensitivity, and which furthermore strongly stimulates aldosterone production. The data suggest that the 18-24 region of the ACTH molecule contains the signal for a fasciculata/reticularis response, and the region 1-13 that for glomerulosa specificity. They confirm the view that, in the rat, alpha-MSH itself may be the specific pituitary glomerulosa-stimulating agent which much experimental work has predicted. They also indicate that synthetic ACTH(1-17) analogues should be used with caution.     

Changes in protein kinase activities in lamb adrenals at late gestation and early postnatal stages

Cytosols prepared from adrenal glands of ovine fetuses (110-144 days of gestation) and of newborn lambs (1-6 days post-partum) were analysed for their protein kinase activities. Two major peaks of casein kinase activities and two major peaks of histone kinase activities were observed in all cytosols of both fetal and neonatal adrenal glands. They were characterized as cAMP-independent casein kinases of type A and type G, and as cAMP-dependent histone kinase isoenzymes of type I and type II. The specific activity of each enzyme increased 2-fold between 118 days of gestation and 6 days post-partum. Casein kinase of the G type was 4-fold higher than casein kinase of the A type; in contrast, the mean ratio of type II to type I histone kinase activity varied between 5- and 12-fold. Infusion of ACTH1-24 (100 micrograms/day) for 5 days to 118- to 128-day-old ovine fetuses in utero increased their plasma corticosteroid levels and the responsiveness of their adrenal cells to stimulation by ACTH1-24 in vitro. In addition, such treatment doubled the specific activity of casein kinases A and G, but had no significant effect on cAMP-dependent histone kinase activities. In relation to current concepts of the role of protein kinases in adult adrenal cells, the present results suggest that casein kinase activities are involved in cell multiplication and differentiation in the fetal adrenal gland. In addition, they show that neither cytosolic histone kinase of type I nor that of type II is likely rate-limiting in the steroidogenic response to ACTH of ovine fetal adrenal cells.     

Effects of dietary sodium restriction on peptide stimulation of aldosterone secretion by the isolated perfused rat adrenal gland in situ: a report of exceptional sensitivity to angiotensin II amide

The effects of prior sodium depletion on the steroidogenic responses of the rat adrenal gland have been investigated using a method of perfusing the isolated adrenal gland of the rat in situ. Secretion rates of aldosterone in response to the known adrenocortical stimulants ACTH, angiotensin II amide and alpha-MSH were measured. In each case, the adrenals from sodium-deplete animals responded to a lower dose of the stimulant than the normal animals. This resulted in a 10-fold increase in sensitivity to ACTH, a 100-fold increase in sensitivity to angiotensin II amide, and a 1000-fold increased sensitivity to alpha-MSH, bringing the threshold concentration required for aldosterone secretion into the physiological range of alpha-MSH concentrations. The perfused adrenal gland is particularly sensitive to angiotensin II amide; a bolus administration of 1 amol gave a significant increase in aldosterone secretion in the sodium-deplete group. These data confirm previous reports of increased adrenal sensitivity to alpha-MSH and angiotensin II in sodium depletion, and also suggest the existence of intraglandular mechanisms for signal amplification which may be involved in mediating the adrenal response to very small concentrations of stimulant.     

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.     

Effects of fetal hypophysectomy and the in vitro treatment by pituitary extracts on the maturation of cultured ovine fetal adrenal cells

In the first series of experiments, the ability of cultured adrenal cells from 113-day-old ovine fetuses to produce both cAMP and corticosteroids in response to ACTH-(1-24) or to fetal (FPE) or newborn acidic pituitary extracts (NPE) was investigated daily. Basal cAMP output did not change during the culture period. When cells from 124-day-old ovine fetuses, or from 5-day-old lambs, were repeatedly stimulated (2 h/day for 4 days) by ACTH-(1-24) or by pituitary extracts, the cAMP responses increased with the same pattern. Outputs on day 4 were 7-fold higher than those on day 1 for ACTH-(1-24)-matured cells or for cells matured by FPE or NPE. The steroid output induced by ACTH-(1-24) or by FPE or NPE developed identically during the experiment to become, on day 4, more than 40-fold higher than on day 1. The response to ACTH-(1-24) on day 5 was also identical both in cAMP and corticosteroids whether the cells had been previously treated with ACTH-(1-24) or with FPE or NPE, In the second set of experiments, adrenal cells from 124-day-old-ovine fetuses either intact of hypophysectomized (Hx) at 118 days of gestation were cultured for 6 days in the absence or presence of ACTH-(1-24). ACTH-(1-24) treatment resulted in a development of cAMP and corticosteroid responses to the hormone which was slower for cells from Hx than cells from control fetuses during the first 3 days of culture. Likewise both cAMP and corticosteroids responses to ACTH-(1-24) of adrenal cells from Hx fetuses cultured for 1 to 3 days in the absence of ACTH were lower than those of cells from control fetuses cultured under the same conditions. These results demonstrate that the pituitary from 124-day-old ovine fetuses contains trophic (and steroidogenic) substances in a sufficient amount to allow in vitro adrenal maturation. Moreover, it appears that high mol wt forms of ACTH, which are most probably extracted by the method we used, did not prevent the in vitro development of the response to ACTH-(1-24). Finally, they show that removal of pituitary hormones in vivo resulted in a decreased potency of fetal adrenal cells to respond in vitro to ACTH-(1-24).