Steroidogenic activity of hACTH and related peptides on the human neocortex and fetal adrenal cortex in organ culture.

Explants prepared from the neocortex and the fetal zone of the human fetal adrenal (gestational age 13 to 18weeks) were maintained under conditions of organ culture for 7 to 9 days during which time they were exposed to hACTH and various related peptides. Corticotrophic activity was monitored by the daily release of dehydroepiandrosterone sulfate (3beta-hydroxy-5-androsten-17-one, 3-sulfate; DHA-S) and cortisol as quantified by radioimmunoassay, hACTH (2.2 x 10(-9) - 2.2 x 10(-8)M) was the most active in sustaining steroidogenesis by both neocortical and fetocortical cells. alpha-MSH possessed similar properties but not at concentrations lower than 10(-6)M, whereas CLIP (4.4 x 10(-9) - 1.1 x 10(-7)M), the 18-39 C-terminal moiety of ACTH, was devoid of activity. Corticotrophic activity with respect to fetocortical explants appeared to be that of maintenance of function best illustrated by dehydroepiandrosterone sulfate biosynthesis, while enhancement of steroidogenesis was observed in the neocortex as manifested by cortisol release. Although not eliminating the possible existence of a specific fetal corticotrophin related to ACTH1-39, the data indicate that hACTH is capable of regulating steroidogenesis in the fetal zone which is primarily geared to the formation of dehydroepiandrosterone sulfate.

Short term culture of human midterm and term placenta: parameters of hormonogenesis.

Monolayer cultures of human midterm and term placentae have been established following trypsin dispersion of placental minces. Maintenance of endocrine function was monitored by the concentrations of specific hormones in the culture media. At either gestational age the cultures 1) secret estradiol-17beta(1) and estrone (in a ratio of about 1:20) and aromatize 3H- or 14C-dehydroepiandrosterone sulfate and 14C-androstenedione, estrogen production being markedly enhanced by addition of dehydroepiandrosterone (10(-6)7) to the culture medium; 2) metabolize 3H-pregnenolone to progesterone and 14C-cortisol to cortisone; and 3) produce increasing amounts of chorionic gonadotropin and decreasing amounts of placental lactogen during the first week in culture. It is proposed that the model is highly suited to the study of factors affecting hormonogenesis by the human placenta whether they be of maternal or of fetal origin.

Exploration of the human fetal pituitary adrenal axis: stimulation of cortisol and dehydroepiandrosterone sulfate biosynthesis by homologous pituitary in organ culture.

Adrenals obtained from human abortices at midpregnancy were kept under conditions of tissue culture and the production of cortisol and dehydroepiandrosterone sulfate (3beta-hydroxy-5-androsten-17-one, 3-sulfate; DHA-S) monitored by radioimmunoassays for up to 2 weeks. Basal production of dehydroepiandrosterone sulfate was considerably higher than that of cortisol. alpha1-24corticotrophin, alpha1-39corticotrophin, (a long acting porcine corticotrophin) at the concentration of 1 mU/ml of culture medium in both instances, and dibutyryl cyclic AMP (1mM) enhanced the production of both steroids some 3 to 30 times above control values. Medium harvested from homologous pituitary cultures had comparable corticotrophic activity. It is concluded that at midpregnancy the regulation of corticoidogenesis implies the existence of a corticotrophic factor either identical or closely related to ACTH.

Preliminary observations of bovine adrenal fasciculata-reticularis cells in monolayer culture: steroidogenesis, effect of ACTH and cyclic AMP.

Bovine adrenocortical cells dispersed by trypsin digestion of fasciculata-reticularis minces were maintained in monolayer culture for up to 6 weeks. During the first week cells grown in medium containing ACTH (1 mU/ml) secreted steroids at a rate 10 to 20-fold greater than control cultures, cortisol accounting for 80-90% of the corticotrophic response. Using tracer amounts of [3H] progesterone and [3H] pregneolone, the major products were cortisol, corticosterone, 11-deoxycortisol and 11-deoxycorticosterone in decreasing order of magnitude. After 10 to 15 days in culture steroidogenesis was no longer enhanced by ACTH. This was concomitant with an apparent loss of 11 beta-hydroxylase activity which was mainly manifested by a sharp increase in the formation of 44-deoxycortisol. Short-term incubations of these cells during the first week in culture provided evidence that steroidogenesis was related to ACTH concentrations (from 0.1 to 100 muU/ml) and stimulated by dibutyryl cyclic AMP, the corticotrophic responses being further enhanced by theophylline (0.5to 50 mumoles/5 ml). Exposure of the cells to ACTH (50 muU/ml) resulted in a rapid increase in intracellular cyclic AMP contractions concomitant with a progressive increase in the corticosteroids released into the medium.

Biogenesis of corticosteroids in monolayer cultures of human foetal adrenal cells.

Human foetal adrenal cells were grown in monolayer culture and their steroidogenic capacity observed for up to a month. The cells produced a complex array of steroids and some of their ester sulphates from endogenous as well as from [14C] and [3H] precursors. ACTH stimulated corticoidogenesis, particularly cortisol secretion, and markedly enhanced the incorporation of progesterone and pregnenolone into cortisol. Following incubation with the same precursors, large amounts of radioactivity remained water soluble. From the butanol extractable material of this fraction, dehydroepiandrosterone sulphate was characterized as the main metabolite of pregnenolone and corticosterone and 11-deoxycorticosterone sulphates as the main metabolites of progesterone. With time in culture there was a decrease in steroidogenesis as well as a steady decline in responsiveness to ACTH, mainly manifested by cortisol secretion. The medium from homologous foetal pituitary cultures stimulated cortisol production by the human adrenal cell monolayer.

Insulin antagonism of cortisol action on lechithin synthesis by cultured fetal lung cells.

In monolayer cel cultures from late gestation (28-day) rabbit fetal lungs, cortisol (5.5 X 10(-6)M) enhances lecithin synthesis and reduced cellular growth. The addition of insulin (25-100 muU/ml) abolishes the stimulatory effect of cortisol on lecithin synthesis but does not affect its growth-inhibiting activity.

Effects of cortisol on serially propogated fibroblast cell cultures derived from the rabbit retal lung and skin.

Cortisol affects the growth of serially propogated, fibroblast cell cultures derived from the rabbit fetal lung in a manner which is dependent upon the gestational age of the material used: early in gestation (20 days), the hormone (10(-7)-10(-5) M) stimulates [6-3H]thymidine incorporation into DNA, while in late gestation (28 days), cortisol (10(-7) and 10(-6) M) inhibits this process. Cultures derived from the rabbit fetal skin are inhibited by cortisol (10(-5) M) at both gestational ages. Fibroblasts derived from lung, but not from skin, efficiently convert cortisone to cortisol and this activity increases with advancing gestation. Cortisol does not affect the incorporation of [3H]choline into lecithin by confluent cultures of any of the fibroblast types studied.

The rabbit fetal lung as a glucocorticoid target tissue.

Fetal lung cells from 28 day gestation rabbits cultured in the presence of cortisol (5.5 times 10 - minus 6M) or dexamethasone (5.5 times 10- minus 8M) incorporated [3-H] choline into lecithin to a significantly greater extent than did control cultures. 11-Deoxycortisol, 21-deoxycortisol and 11beta-hydroxyprogesterone, at a concentration of 5.5 times 10- minus 5 M, had no effect on lecithin synthesis. However, when lung cells were simultaneously exposed to these steroids and to cortisol at the concentrations quoted, [3-H] choline incorporation was reduced to control values. Cortisone (5.5 times 10- minus M) also enhanced lecithin synthesis, the activity of the steroid likely being related to the capacity of the lung cells to convert cortisone to cortisol. This hypothesis was supported by the observations that 11-ketoprogesterone (1.3 times 10- minus 5M), which totally inhibited the conversion of cortisone to cortisol and which had no effect of its own on [3-H] choline incorporation, inhibited the effect of cortisone on lecithin synthesis but not that of cortisol. These data support the view that glucocorticoids affect lung cell maturation in a manner comparable to the interaction of other steroid hormones with their target tissues. The capacity of the fetal lung to convert cortisone to cortisol may be physiologically significant in light of the high concentration of 11-oxo-steroids in the fetal circulation throughout pregnancy.

Evidence for different gestation-dependent effects of cortisol on cultured fetal lung cells.

The effect of cortisol (5.5 muM) on primary monolayer cultures of trypsin-dispersed lung cells from rabbit fetuses of 20-28 days gestation was monitored with respect to (a) cellular growth as determined by DNA content after 72 h, at which time all cultures were in the exponential phase of growth, and (b) cellular maturation as reflected by the incorporation of [(14)C]-palmitate into saturated lecithin and its release into the culture medium. Cortisol significantly increased growth in cultures prepared from 20 day (control: 59.8+/-8.9 nmol DNA/flask; cortisol: 118.7+/-15.7, P < 0.001) and 22 day (control: 69.2+/-17.2; cortisol: 106.7+/-13.3, P < 0.001) fetuses but had no effect on the growth of cells from 24 or 26 day fetuses. At 28 days, the effect was reversed, cortisol reducing growth by a factor of two (control: 42.0+/-8.5; cortisol: 19.3+/-4.0, P < 0.001). Incorporation of palmitate into lecithin was expressed as picomoles incorporated per micromole DNA per flask, thus correcting for differences in the number of cells. Cortisol had no effect on palmitate incorporation until day 26, at which time it caused a slight increase (control: 51.2+/-5.5: cortisol: 72.8+/-16.2, P < 0.01) which became very striking by day 28 (control: 19.7+/-3.1; cortisol; 286.8+/-47.0, P < 0.001). The proportion of recovered radiolabeled lecithin that was disaturated rose with gestational age from 72% at 20 days to 98% at 28 days. Saturated lecithin made up over 90% at the two gestational ages (26 and 28 days) where cortisol increased palmitate incorporation. In contrast, cortisol had no effect on the incorporation of palmitate into sphingomyelin at any of the gestational ages studied.The results suggest that cortisol may increase fetal pulmonary cellular growth in early gestation while enhancing maturation and slowing growth as term approaches.