Immunolocalisation of P450(C17) in the fetal sheep adrenal gland during gestation and in response to ACTH and glucocorticoid administration.

Abstract

In sheep, increased output of cortisol from the fetal adrenal gland is critical to organ maturation and parturition. Cortisol synthesis is determined in part by the activity of P450(C17) enzyme. We have used immunohistochemistry and Western immunoblotting to examine the distribution of P450(C17) in the ovine fetal adrenal during gestation, and after ACTH or dexamethasone administration to fetuses between Days 125 and 130. The patterns were compared with changes in 3beta-hydroxysteroid dehydrogenase (3beta-HSD) localisation and levels. Adrenal tissue was obtained from four fetuses at each of Days 63-65, 100, 125-130 and term (>140 days). Further animals were chronically catheterised and infused with ACTH, dexamethasone or saline for 96 h beginning on Day 125. Immunohistochemistry for P450(C17), 3beta-HSD, and phenylethanolamine-N-methyl transferase (PNMT) was conducted using standard techniques. At Day 63-65 of pregnancy immunoreactive (ir-)P450(C17) was present in cords of cells throughout the adrenal gland. Ir-P450(C17) was reduced or was undetectable at Day 100, but had increased by Day 125-130, and was present throughout the zona fasciculata of the adrenal cortex of term animals. An increase in P450(C17) protein was also seen between Day 100 and 125 by Western blotting, and after ACTH treatment. Dexamethasone administration led to a marked reduction in ir-P450(C17) levels. In contrast, ir-3beta-HSD was present in the fetal adrenal cortex between Day 100 and term, and was less affected by ACTH or dexamethasone treatment. We conclude that P450(C17) in the fetal sheep adrenal is responsive to regulation by ACTH, and that changes in its levels correlate with previously reported alterations in patterns of cortisol output by the fetal adrenal gland.