Fig 1: Steroidogenic enzyme localization and expression in the fetal adrenal. a CYP11A1 (brown) was localized primarily in the inner fetal zone (yellow arrow) with some cells in the definitive zone (black arrow) exhibiting weak immunostaining. b CYP17A1 (brown) was expressed throughout the adrenal, while (c) CYP21A2 (brown) was localized primarily in the fetal zone (yellow arrow), although protein expression was also seen in the definitive zone (black arrows, inset). d CYP11A1 (cyan) and CYP21A2 (magenta) co-localize in some cells of the fetal zone (yellow arrow), but most cells only express one of these enzymes with CYP21A2 the predominant enzyme. e HSD3B was predominantly localized to the outer definitive zone (black arrow), although it was also present in some cells within the fetal zone of adrenals at 12 weeks of gestation (f; yellow arrow). These HSD3B-positive cells in the fetal zone were not observed after 13 weeks of gestation. g Representative Western blot showing CYP11A1, CYP17A1, CYP21A2 and the loading control ß-actin. Molecular weight markers are labelled on the left of the image. h–j Expression levels of CYP11A1, CYP17A1 and CYP21A2 protein in the fetal adrenal during the second trimester. Data points from individual fetuses are shown. Black lines show the generalized linear regression, and grey fill denotes the confidence interval (0.95) around the regression. Expression of CYP11A1 was unaffected by gestational age, while both CYP17A1 (P = 0.004) and CYP21A2 (P = 0.04) showed increased expression between 12 and 19 weeks