In vitro stimulation and inhibition of adrenocorticotropin release by pituitary cells from ovine fetuses and lambs.

Short term cultured pituitary cells from fetal (63-144 days old) and young lambs (30-120 days old) were tested for their in vitro ability to release immunoreactive ACTH and cAMP in response to stimulation by ovine (o) oCRF1-41, arginine vasopressin (AVP), epinephrine, and forskolin, both in the absence and presence of corticosteroids. For each culture, the percentage of corticotrophs was determined by immunocytochemistry using ACTH antibodies, and the net responses (stimulated-baseline) were expressed per 10(5) corticotrophs. During gestation, basal ACTH release did not change significantly except at 125 days where it was 2-fold higher than at other fetal stages. Basal ACTH release was 2-fold higher in lambs than in fetuses. In the presence of oCRF1-41, a significant increase in ACTH secretion over basal value was observed at all stages studied. The maximal response decreased from 7.89 +/- 1.19 ng ACTH 10(5) corticotrophs-1 3 h-1 at 63 days of gestation to 3.49 +/- 0.88 ng at 115 days, then remained fairly constant in prepartum animals and lambs. No significant change in the ED50 was observed. The cAMP output induced by oCRF1-41 decreased progressively between 63 and 133 days of gestation from 10.72 +/- 1.84 to 2.21 +/- 0.62 pmol, then increased in lambs to values similar to that of 63-day-old fetuses. The ACTH response to AVP was higher than that to oCRF1-41 at 115 days, decreased dramatically in late gestation without modification of the ED50, and remained low in lambs. The ACTH response to epinephrine was always very low. Forskolin-induced ACTH release was lower between 115 and 144 days of gestation than at other stages. The synergistic effect of AVP and epinephrine on both cAMP and ACTH productions stimulated by oCRF1-41 decreased at the end of gestation. The ACTH response to each stimulus was inhibited by dexamethasone, cortisol, and corticosterone (10(-9) and 10(-7) M) throughout the period studied. This effect was maximum at 63 days of gestation. cAMP release was not altered by glucocorticoids. These results indicate that 1) maximal ACTH response in vitro of corticotrophs to AVP is achieved during fetal life; 2) the capacity of fetal corticotrophs to release ACTH in response to oCRF1-41 is similar to that of lambs throughout the last month of gestation; and 3) corticotrophs are sensitive to the glucocorticoid-negative feedback at least during the second half of fetal life.

Adrenal adenylate cyclase and steroidogenic activities of 63 day old ovine fetuses: in vitro effects of ACTH1-24 and forskolin.

This study examines the activity of the adenylate cyclase system and that of some enzymes of the steroidogenic pathway of adrenal cells from 62-63 day old ovine fetuses. Synthetic corticotropin (ACTH1-24), cholera toxin and forskolin stimulated both cAMP and corticoid productions by freshly isolated adrenal cells. The cAMP response to ACTH1-24 was lower than that to forskolin. However, forskolin-induced steroidogenesis was significantly lower than the ACTH1-24-induced steroid output. Freshly isolated cells metabolized quickly [14C]-labeled pregnenolone mainly through the 17-deoxy pathway. The amounts of cortisol and of corticosterone formed, in the presence of exogenous pregnenolone, were roughly 15-fold higher than under maximal stimulation by ACTH1-24. When the cells were cultured for 6 days in the absence or presence of ACTH1-24 (10(-8) M) or forskolin (10(-5) M), a small development of the cAMP response to these factors was observed in the course of the experiment. However, the mechanism of this development appeared different, according to the conditions of culture. The amounts of corticosterone secreted on day 6 by ACTH1-24- or forskolin-treated cells were 2- to 4-fold higher than on day 1, whereas cortisol outputs were much lower on day 6 than on day 1. The response to ACTH1-24 of cells maintained in ACTH-free media decreased dramatically during the culture in terms of both cortisol and of corticosterone. On day 6 of the experiment, the metabolism of [14C]pregnenolone was lower than on day 1 under all 3 conditions of culture. Only the 3 beta-hydroxysteroid dehydrogenase/isomerase activity could be maintained by continuous treatment with forskolin. However, both ACTH1-24 and forskolin enhanced the production of pregnenolone from an endogenous substrate. In conclusion, these results present evidence that: 1) the adenylate cyclase system is not a bottleneck in the steroidogenic response to ACTH1-24 of freshly isolated adrenal cells from 62-63 day old ovine fetuses; 2) the main rate-limiting step for steroidogenesis by these cells is the availability of pregnenolone; 3) neither ACTH1-24 nor forskolin is able to maintain the activity of most enzymes involved in the metabolization of pregnenolone by cultured cells while increasing pregnenolone availability; 4) some inhibiting factors are involved in the loss of adrenal cells responsiveness to ACTH between days 50 and 100 of gestation, and they probably act mainly on the adenylate cyclase system.

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.

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.

De novo synthesis of cholesterol by ovine fetal and neonatal adrenocortical cells.

The present study compared the stocks of cholesterol present in adrenal cells from 120-day-old ovine fetuses and from newborn lambs, as well as the capacity of these cells to regulate their stock by de novo synthesis from C2 units. Both free cholesterol and cholesteryl ester concentrations of mitochondrial fractions of adrenal glands from fetuses were lower than those from newborn lambs. In addition, the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) and the cholesterol side-chain cleavage activities were 2- and 5-fold lower, respectively, in fetal than in neonatal adrenals. After 2 days of culture in serum-free media, the cellular contents of cholesterol were similar in control and ACTH1-24-treated cells for both fetuses and newborns. Moreover, ACTH1-24 increased HMG-CoA reductase activity of both fetal and neonatal cells to the same extent. When cells were cultured in the presence of 2% horse serum, the cellular content of cholesterol on day 2 was enhanced only in the case of neonatal cells, but [14C]acetate incorporation in free cholesterol was decreased in both fetal and neonatal cells, while its incorporation in cholesteryl ester was increased. The presence of serum in the medium prevented the enhancing effect of ACTH1-24 on the fetal HMG-CoA reductase activity but had no effect on that of neonatal cells. These data, together with preceding results, suggest that the low stores of cholesterol of ovine fetal adrenal cells result from a limited capacity to synthesize cholesterol and to some alteration in the uptake and/or metabolism of plasma lipoproteins, which in turn might reflect the low steroidogenic capacity of these cells.

Glucocorticoid induction of the maturation of ovine fetal adrenocortical cells.

The aim of the present study was to assess whether glucocorticoids could be directly involved in the maturation of adrenocortical cells from 120-138 days old ovine fetuses. The cAMP response to ACTH1-24 of cells cultured for 24 hours in the presence of ACTH1-24 was 2 fold higher than that of control cells. However, the response of cells cultured in the presence of ACTH1-24 plus metyrapone or aminoglutethimide was lower than that of cells cultured in the presence of ACTH1-24 alone. Cells cultured for 48 hours in the presence of dexamethasone or cortisol released more cAMP than control cells when stimulated by ACTH1-24, but not in response to forskolin. However corticosteroid production stimulated by ACTH1-24, forskolin or dibutyryl cAMP was enhanced by dexamethasone treatment. These results suggest that glucocorticoids can affect the maturation of ovine fetal adrenocortical cells by an auto and/or a paracrine process, and that this effect is exerted, at least, at two different levels in the cell.

In vitro effect of insulin and insulin-like growth factor-I on cell multiplication and adrenocorticotropin responsiveness of fetal adrenal cells.

The present study examined the effects of both insulin and insulin-like growth factor-I (IGF-I) on cell division and specific functions of cultured adrenocortical cells from 100- to 122-day-old ovine fetuses. When culture was performed in a serum-free medium containing transferrin and ascorbic acid, the number of cells increased only slightly (1.2-fold) over a 4-day period. Addition of insulin or IGF-I in the culture medium enhanced the number of cells counted on Day 5. The effect of both peptides was dose-dependent, but 10 ng/ml IGF-I was as potent as 10 micrograms/ml insulin. The acute cyclic adenosine 3',5'-monophosphate (cAMP) and steroidogenic responses to adrenocorticotropin (ACTH1-24) decreased in fetal cells cultured in the absence of insulin or ACTH. Insulin at micromolar concentrations not only prevented this decrease but enhanced the acute ACTH1-24-induced cAMP output on Day 5 over that observed on Day 2. Treatment of fetal cells for 4 days with increasing concentrations of insulin or IGF-I enhanced the acute cAMP and steroidogenic responses (3- to 4-fold) to ACTH1-24 over that of control cells. The ED50 of IGF-I was about 3 ng/ml (congruent to 0.4 nM) whereas that of insulin was about 10 ng/ml (1.7 nM). However, a second plateau was apparent at concentrations of insulin above 1 microgram/ml. The acute cholera toxin stimulation of cAMP production of cells cultured in the absence of insulin or ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of the fetal pituitary-adrenal axis in the sheep.

In the ovine fetus, plasma levels of corticosteroids are very low between 60 and 130 days of gestation, then increase dramatically before birth. ACTH appears to be an important regulating hormone for the fetal adrenal cortex, the sensitivity of which to this hormone increases during late gestation. However, the relationship between immunoreactive ACTH and corticosteroids in the fetus is unclear. We review herein recent work performed in our laboratory on the regulation of ACTH secretion by ovine fetal pituitary cells and on the biochemical modifications responsible for the enhancement of the steroidogenic response to ACTH of fetal adrenal cells. It is suggested that qualitative together with quantitative changes in the pituitary drive to the fetal adrenal has to be taken in account to explain the rise of corticosteroids in prepartum animals. Also extra pituitary hormones may be operating during intrauterine life to regulate fetal adrenal function.

The influence of plasma lipoproteins on steroidogenesis of cultured ovine fetal and neonatal adrenal cells.

The present study examined the effects of serum and lipoproteins on the function of cultured adrenal cells from 115-127-day-old ovine fetuses and from newborn lambs. On day 1 of culture, corticosteroid output was similar in medium containing 2% horse serum or in serum-free medium, both for fetal and neonatal cells. However, on day 5, cells cultured in the absence of serum produced smaller amounts of these steroids than cells maintained in medium containing serum; the difference was more marked under ACTH1-24 stimulation. Conversely, cAMP production was never lower in the absence than in the presence of serum. When stimulated by ACTH1-24 on day 2 of culture, fetal or neonatal adrenal cells incubated in the presence of a saturating concentration of ovine LDL produced more corticosteroids than cells incubated in serum-free medium; HDL also enhanced ACTH1-24-induced steroidogenesis, but to a lesser extent. VLDL was effective only with neonatal cells. In fetal and neonatal cells cultured for 6 days in ACTH-free medium, VLDL and LDL increased ACTH-induced steroidogenesis, but HDL did not. On the other hand, when cells were cultured in the presence of ACTH1-24, LDL and HDL were equipotent in supporting ACTH1-24-induced steroid output. Three major lipoprotein fractions were observed in serum of fetal and newborn lambs. The concentration of cholesterol was very low in the VLDL fraction of fetuses, but it was similar to that of newborns in LDL. Conversely, 4 times more cholesterol was present in HDL of newborns than in HDL of fetuses. These results suggest that: (i) after several days of cell culture, cholesterol availability is an important limiting factor for the steroidogenesis of cells maintained under serum-free conditions; (ii) both an "LDL pathway" and an "HDL pathway" are operating in adrenal cells from fetal as well as newborn sheep; (iii) LDL and HDL are important physiological sources of cholesterol to support steroidogenesis by fetal and neonatal adrenal cells.