Gender and gonadal status differences in zona reticularis expression in marmoset monkey adrenals: Cytochrome b5 localization with respect to cytochrome P450 17,20-lyase activity

Abstract

Neonatal marmosets express an adrenal fetal zone comparable to humans. While adult males fail to express a functional ZR, with barely detectable blood DHEA levels, females produce higher levels of DHEA than males in adulthood. We investigated the presence of a putative functional ZR in adult female marmosets. In contrast to males, immunohistochemical analysis showed the ZR marker cytochrome b5 was elevated in the innermost zone in cycling females (compared to testis-intact males), further elevated in the adrenals from anovulatory females, and substantially elevated and continuous in ovariectomized females. As a functional test in vivo, following overnight dexamethasone treatment, cycling and anovulatory females showed higher levels of DHEA relative to males, but DHEA failed to increase in response to ACTH. In direct contrast, while ovariectomized females exhibited lower initial DHEA levels, clear increases were detectable after ACTH administration (p<0.05), suggesting an adrenal origin. The apparent differences in cytochrome b5 expression between groups were also further verified by Western blotting of adrenal microsomes, and compared to 17,20-lyase activity; the two parameters were positively correlated (p<0.01) across multiple treatment groups. We conclude that the cycling female marmoset expresses a rudimentary ZR with at least a capacity for DHEA production that becomes significantly ACTH-responsive after anovulation. Expression of cytochrome b5 in this region may be directly or indirectly controlled by gonadal function, and is, at least in part, a critical determinant in the development of an adrenal ZR that is more defined and significantly ACTH-responsive.

Figure 1. The adrenocortical steroid biosynthesis pathway in human and nonhuman primates

Cortisol is predominantly synthesized through the Δ-5 pathway [pregnenolone (P5) converted to 17alpha-hydroxypregnenolone (17OHP5) converted to 17alpha-hydroxyprogesterone (17OHP4) converted to 11-deoxycortisol converted to cortisol]. High dehydroepiandrosterone (DHEA) production requires metabolism of P5 through the Δ-5 pathway, in the face of low 3beta-hydroxysteroid dehydrogenase (3β-HSD) expression and high cytochrome b5 (cyt_b5) and cytochrome P450 oxido-reductase (POR) expression. 17alpha-hydroxylase/17,20-lyase cytochrome P450 (CYP17) and 3β-HSD are critically positioned for the biosynthesis of 17OHP5, DHEA, 17OHP4, and androstenedione (A4). These same enzymes are also critically positioned for the biosynthesis of cortisol. Sulfotransferase (SULT2A1).

Figure 2. Immunohistochemical analysis of marmoset adrenal sections

Primary antibody specificities are as labeled in each panel. Immunodetectable protein is indicated by brown stain while counterstain color is blue. (A) Dominant female (n=6; aged 24–48 months), (B) Subordinate female (n=3; aged 30–60 months), (C) Ovariectomized female (n=6; aged 48–84 months). Cap (capsule), med (medulla). 10X Magnification.

Figure 3. Marmoset adrenal microsomal protein expression as detected by Western blot

(A) Stripblots for CYP17 and cyt_b5 are shown, with molecular weights (right). Abbreviations are: adult testis-intact male (M), dominant female (DF), and ovariectomoized female (OF). (B) Quantified protein expression in adrenal microsomes from adult testis-intact males (n=6, aged 60–132 months), ovary-intact, dominant females (DomF; n=6, aged 24–132 months), and ovariectomized females (OvxF; n=6, aged 72–132 months). * significant difference from male (p<0.01), + significant difference from dominant female (p<0.01).

Figure 4. Regression of marmoset adrenal microsomal cyt_b5 expression vs. 17,20-lyase activity

Cytochrome b5 expression (from Fig. 4) vs. 17,20-lyase activity (values in text) data were compared for microsomal preparations from the following groups: Open circles: testis-intact males, black squares: ovary-intact, dominant females, black triangles: ovariectomized females. Regression analysis revealed a correlation of r²=0.355, (p<0.01).

Figure 5. Inefficiency of the adrenal zona fasciculata in marmosets

Corticosterone production, as a fraction of cortisol production, was calculated as percentages from the data obtained in Table 1. Abbreviations are: dominant female (DF), ovariectomized female (OF), subordinate female (SF), testis-intact male (IM). * significant difference from dominant (p<0.05).