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-mu-g/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-mu-m) 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). Morphometric analyses were carried out on mid-glandular sections stained with anti-PNMT and an index of the interdigitation between the adrenal cortex and adrenal cortex and adrenal medulla was calculated (i.e. interdigitation index = the ratio of the perimeter of the adrenal medulla to the perimeter of a circle of the same area as the adrenal medulla). In the 141/2d Hx + Sal and the 141/2d Hx + ACTH groups, more central adrenomedullary cells were stained with anti-PNMT than in the intact group. In the 141/2d Intact + Sal group, staining with anti-ENK was observed in the peripheral rim of adrenal medulla cells which were adjacent to and interdigitated with the cells of the adrenal cortex. In the intact and Intact + SAL group, the intensity of staining with anti-ENK in the central noradrenaline (NA)-containing region was variable with many unstained cells present. In the 141/2d Hx + Sal and Hx + ACTH groups, however, staining for enkephalins was present throughout the entire adrenal medulla. The interdigitation index of the adrenal medulla and the adrenal cortex was significantly increased in the 10/12d Newborn (3.00 +/- 0.07), 145/7d Intact (2.68 +/- 0.07), and the 141/2d Hx + ACTH (2.62 +/- 0.06) when compared to the 147/64d Hx (1.42 +/- 0.03), 141/2d Intact + Sal (1.29 +/- 0.02) and 141/2d Hx + Sal (1.15 +/- 0.01) groups (p < 0.05). When the data were combined for all fetal and newborn lamb adrenals there was a positive correlation between the area of the adrenal cortex and the interdigitation index (r = 0.802; P < 0.001). In summary, we found that the pattern of localization of the enkephalins and PNMT in the fetal adrenal was similar in the 141/2d Hx + Sal and Hx + ACTH groups, therefore it would appear that the pituitary does not act via the adrenal cortex to suppress proENK A and PNMT gene expression in the centre of the adrenal medulla in the late gestation sheep fetus. The adrenal cortex, however, influences the extent of interdigitation between the peripheral rim of AD containing cells and the inner zone of the adrenal cortex, in the sheep adrenal gland during development.
