In VitroMaturation of Steroidogenic Capacity of Ovine Fetal and Neonatal Adrenal Cells*

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Abstract
The in vitro production of total corticosteroids, corticosterone (B), and cortisol (F) by cultured adrenocortical cells from fetuses (115-120 days old) and newborn lambs exposed to ACTH-(1-24) was investigated in relation to the time of culture. Corticosteroids were measured by using an antibody against F, whereas B and F separately were each assayed by specific antibodies after their separation by TLC. Basal corticosteroid production by fetal cells increased .apprx. 5-fold between days 1 and 6 of culture, whereas the production of B and F increased from 3 .+-. (SE) 0.3 to 194 .+-. 25 and 3.9 .+-. 0.3 to 17.4 .+-. 1.9 [ng (8 .times. 105 cells)-1 2 h-1], respectively. ACTH-(1-24) stimulation on day 6 further increased the production of corticosteroid (3.5-fold) and B (2-fold), but had no effect on that of F. When cells were exposed to ACTH-(1-24) from the 1st day of culture on, there was a dramatic enhancement of F production which became, by day 6, 100 times higher (1746 .+-. 149) than in the case of cells maintained in ACTH-free medium. Basal corticosteroid production by nontreated neonatal adrenal cells increased between days 1 and 6; this was mainly due to B, the production of which increased from 10.1 .+-. 0.2 to 447 .+-. 35. ACTH-(1-24) stimulation of these cells on day 6 increased 2-fold the secretion of F and 1.5-fold that of B. ACTH-(1-24) treatment (2 h/day for 5 days) of neonatal cells increased 11-fold their production of F as compared with cells cultured in ACTH-free medium, and to a lesser extent that of B. In the 2nd set of experiments, pregnenolone (8 .times. 10-5 M) was added to the medium and its conversion to B and F was measured. On day zero the production of B and F by untreated fetal cells was 31 and 16 times higher than that of cells incubated without pregnenolone but stimulated by ACTH-(1-24); however, on day 6 the increase was only 2 and 5 times higher, respectively. On day 6 production of B and F by ACTH-treated cells was modestly increased by the addition of pregnenolone. The capacity of freshly isolated neonatal adrenal cells (day zero) to convert pregnenolone to B and F was 10- and 150-fold higher, respectively, than that of cells from fetuses. ACTH-(1-24) treatment of neonatal cells maintained their capacity to produce F and increased 3-fold the production of B. These results show a spontaneous development of some enzymes involved in steroidogenesis of fetal adrenal cells cultured in ACTH-free medium. During fetal life some factor inhibits the development of these enzymes. In vitro ACTH-(1-24) treatment accelerates the spontaneous maturation of cultured adrenocortical cells and specifically increases their 17.alpha.-hydroxylase activity.