Expression of II β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) in the human fetal adrenal

OBJECTIVE: To understand better the steroidogenic capacity of the human fetal adrenal (HFA), we evaluated the expression of 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) in the fetal zone and neocortex of the HFA using a specific RNase protection assay. METHODS: Adrenal glands were obtained at the time of elective termination of pregnancy. Whole adrenals (n = 7) were frozen in liquid nitrogen, and subsequently total RNA extraction was performed by tissue homogenization followed by guanidinium/chloroform purification. In addition, RNA was obtained from separated fetal zone (n = 4) and neocortex (n = 4) tissues obtained by dissection. RNase protection assays were then performed using radiolabeled complementary RNA probes generated by T7 RNA polymerase directed against transcripts for CYP11B1, CYP11B2, and actin, the latter of which was used as a control for RNA integrity. Transcripts also were examined using a reverse transcription polymerase chain reaction (RT-PCR) protocol specific for CYP11B1 or CYP11B2. RESULTS: The RNase protection assay was designed to distinguish specific bands that corresponded to CYPI1B1 (232 bp), CYP11B2 (262 bp), and actin (221 bp). RNA isolated from whole HFA was observed to have high levels of CYP11B1 transcript, whereas CYP11B2 was not detected. Dissected neocortex and fetal zones were found to contain transcript for CYP11B1 using both the RNase protection assay and RT-PCR analysis. In contrast, using the RNase protection assay, CYP11B2 mRNA was not observed in the RNA from the fetal zone, but after prolonged exposure there was a band corresponding in size to CYP11B2 observed in RNA from the neocortex. Using the more sensitive RT-PCR method, transcript for CYP11B2 was found in both neocortex and fetal zone. CONCLUSION: The HFA expresses low levels of CYP11B2 in accordance with its low production of mineralocorticoid. The expression of CYP11B1 in the fetal zone is intriguing because this enzyme is not necessary for the production of C₁₉ steroids. Definition of the molecular mechanisms controlling expression of the CYP11B genes will be necessary to determine why the HFA differentially expresses these isoenzymes.