Fig 1.
Comparison of histological and functional ZG.
H&E staining and CYP11B2 immunolabelling (brown) of consecutive adrenal sections reveal a weak degree of superimposition of histological and functional ZG. indicates CYP11B2 cell clusters migrating into the zona fasciculata. - - - delineates the histological boundary between zona glomerulosa (above) and zona fasciculata (below). (A-D) 22-year-old man. (E-H) 66-year-old man. The sections are matched in consecutive pairs, such as A-B and C-D for the 22-year-old and, E-F and G-H for the 66-year-old adrenal. * identifies common structures on consecutive sections.
Fig 2.
Co-immunofluorescence of CYP11B1 and CYP11B2 demonstrates adrenal remodelling.
Illustrative merge photographs of CYP11B2 (red) and CYP11B1 (green) combined with nuclear staining with DAPI (blue) showing that the pattern of expression of the steroidogenic enzymes does not support the zonation defined by histological criteria in the adrenal cortex of a 68-year-old woman (A-D).
Fig 3.
CYP11B1 and CYP11B2 positive cells are interwoven throughout the adrenal cortex.
(A-F) Double immunofluorescence staining reveals the occurrence of CYP11B2 positive cells (red) in the ZF (CYP11B1 positive; green) in the adrenal cortex of a 49-year-old man. (A-B) In the inner cortex, clusters of CYP11B2 positive cells are intermingled with CYP11B1 expressing cells. (C-F) Both CYP11B1 and CYP11B2 positive cells participate in the formation of baskets found in the histological ZG.
Fig 4.
Phenotype stability of ectopically localized CYP11B2-positive cells.
Expression of 3β-hydroxysteroid dehydrogenase type II (HSD3β2), CYP11B2, and 17α-hydroxylase (17CYPA1) in the adrenal gland from a 45-year-old woman (A) and a 66-year-old man (B).
Fig 5.
Ectopic localization of steroidogenic cells in the adrenal gland.
(A) CYP11B2 immunostaining reveals that organized islets or isolated functional ZG cells can be identified in the vicinity of the central vasculature in the adrenal cortices of a 49-year-old man (a) and a 52-year-old woman (b). The left composite microphotographs show immunoreactivity for CYP11B2 throughout the cortex. (B) Immunostaining for SF1 highlights the occurrence of steroidogenic cells in the medulla (M). The adrenal medulla appears to be invaded by single or clusters of SF1-expressing cells. (a) 30-year-old man. (b) 72-year-old woman. (C) Double immunofluorescence for Tyrosine hydroxylase (TH) and either CYP11B2 or CYP11B1. The CYP11B2 (a) and CYP11B1 (b) positive cells are found within the adrenal medulla in close contact with the chromaffin cells. (a) 45-year-old man. (b) 64-year-old woman.
Fig 6.
Dab2 expression is not restricted to the functional ZG.
Triple immunofluorescence for CYP11B1, CYP11B2 and Dab2. Dab2 expression is observed throughout the adrenal cortex independently of the donor age and steroidogenic cell type. (A) 42-year-old man, Cap: Capsule. (B) 81-year-old man.
Fig 7.
KCNJ5 expression in the normal human adrenal cortex.
Double immunofluorescence of KCNJ5 and CYP11B2 reveals that KCNJ5 is highly expressed in aldosterone producing cells (A, B, C). Intense KCNJ5 immunolabelling is observed in CYP11B2 cells arranged in large (A) or small (B) APCC or isolated in the deep cortex (C). Cell nuclei were visualised with DAPI. (A) 42-year-old man. (B) 45-year-old man. (C) 52-year-old woman.
Fig 8.
Proliferation occurs throughout all zones of the cortex irrespective of the functional status of the steroidogenic cells.
(A, B) illustrative microphotographs showing the distributions of CYP11B2 and Ki-67 immunostainings in consecutives sections of two human adrenal cortices ((A) 68-year-old man. (B) 70-year-old woman). * denotes common structures on consecutive sections. (C) Double immunofluorescence for CYP11B1 (red) and Ki-67 (green) in the adrenal cortex of a 34-year-old man. Cell nuclei were visualised with DAPI (blue). (D) Box and whisker plot of Ki-67 percentage immunopositivity showing higher value associated with CYP11B1 expressing cells (**** P<0.0001).
Fig 9.
Wnt/β-catenin pathway is activated in normal human adrenal gland.
Immunostaining for β-catenin (A and B) allows detection of the protein throughout the cortex at the membrane and cytoplasmic/nucleus levels while β-catenin expression in chromaffin cells of the medulla (M) is restricted to the membrane (A, lower microphotographs). Lef-1 expression is detected in the nucleus of cortical cells (C and D) and not in chromaffin cells (C, lower microphotographs). indicates cluster of cortical cells expressing β-catenin (A) or Lef-1 (C) in the medulla and in the vicinity of the central vasculature of the adrenal gland. (A and C) 30-year-old man. (B and D) 70-year-old man.