Plasticity of the zona reticularis in the adult marmoset adrenal cortex: voyages of discovery in the New World.

Adrenarche in humans occurs at the age of 5-7 years, yet the process by which dehydroepiandrosterone (DHEA) biosynthesis in the adrenal zona reticularis (ZR) increases so dramatically remains as a matter of debate. One suggestion is that increased DHEA production by P450c17 (CYP17A1 as listed in HUGO Database) in the ZR results from a coincident fall in the expression of HSD3B, which would otherwise compete for pregnenolone substrate. Nonetheless, studies of human and rhesus adrenal show that cytochrome b5 (CYTB5) expression increases in the ZR with DHEA biosynthesis, and cloned human and rhesus P450c17 show selective increases in 17,20-lyase activity in the presence of CYTB5. The marmoset, a New World primate, expresses a fetal zone during development which regresses after birth. Adult males, however, do not develop an obvious functional ZR, while females develop a ZR in a manner that depends on their social/gonadal status. In all social and physiologic states, changes in marmoset ZR function relate directly to changes in the expression of CYTB5. Recent cloning and expression of marmoset P450c17 also show that while amino acid sequence homology is in the order of approximately 85% of that found in human and rhesus sequences, and basal lyase activity is low compared with rhesus, all previously described amino acids critical to human 17,20-lyase activity are completely conserved. Furthermore, the 17,20-lyase activity of the marmoset P450c17 clone is dramatically increased by addition of CYTB5. We propose that these combined data from the marmoset model provide further compelling evidence that the control of ZR CYTB5 expression is a key determinant of ZR function.

The developmental increase in adrenocortical 17,20-lyase activity (biochemical adrenarche) is driven primarily by increasing cytochrome b5 in neonatal rhesus macaques.

Adrenarche is thought to be experienced only by humans and some Old World primates despite observed regression of an adrenal fetal zone and establishment of a functional zona reticularis (ZR) in other species like rhesus macaques. Adrenal differentiation remains poorly defined biochemically in nonhuman primates. The present studies defined ZR development in the neonatal rhesus by examining androgen synthetic capacity and factors affecting it in rhesus and marmoset adrenals. Western immunoblots examined expression of 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17), cytochrome b5 (b5), and 3beta-hydroxysteroid dehydrogenase (3betaHSD), among other key enzymes. 17,20-lyase activity was quantified in adrenal microsomes, as was the contribution of b5 to 17,20-lyase activity in microsomes and cell transfection experiments with rhesus and marmoset P450c17. Expression of b5 increased from birth to 3 months, and was positively correlated with age and 17,20-lyase activity in the rhesus. Recombinant b5 addition stimulated 17,20-lyase activity to an extent inversely proportional to endogenous levels in adrenal microsomes. Although 3betaHSD expression also increased with age, P450c17, 21-hydroxylase cytochrome P450, and the redox partner, reduced nicotinamide adenine dinucleotide phosphate-cytochrome P450 oxidoreductase, did not; nor did recombinant cytochrome P450 oxidoreductase augment 17,20-lyase activity. Cotransfection with b5 induced a dose-dependent increase in dehydroepiandrosterone synthesis by both nonhuman primate P450c17 enzymes. We conclude that the increase in 17,20-lyase activity characteristic of an adrenarche in rhesus macaques is driven primarily by increased b5 expression, without the need for a decrease in 3betaHSD, as suggested from human studies. The rhesus macaque is a relevant and accessible model for human ZR development and adrenal function.

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.

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.

Male marmoset monkeys express an adrenal fetal zone at birth, but not a zona reticularis in adulthood.

Neonatal human males produce high levels of dehydroepiandrosterone (DHEA) and its sulfo-conjugated form (DS) that decline within a few months of birth, due to regression of the adrenal fetal zone (FZ). Adult male humans and rhesus monkeys produce C19 steroids in abundance from the adrenal zona reticularis (ZR). Male marmoset monkeys produce DS at birth, but unlike humans and rhesus monkeys, do not produce comparable amounts of DHEA and DS in adulthood. To determine whether male marmosets express a functional ZR in adulthood, we examined adult and neonatal male marmosets for the presence of a ZR and FZ, respectively. Exogenous ACTH failed to stimulate DHEA or DS in adults, and dexamethasone treatment failed to suppress DHEA and DS, although cortisol levels changed as expected. In steroidogenic tissues, the key proteins necessary to synthesize C19 steroids from pregnenolone are P450c17, 3beta-hydroxysteroid dehydrogenase (3beta-HSD), nicotinamide adenine dinucleotide phosphate (reduced) oxido-reductase cytochrome P450 (reductase), and cytochromeb5 (cytb5). Adult adrenal cross sections showed P450c17 and reductase protein expression throughout the cortex but showed no expected decrease in 3beta-HSD and increase in cytb5 in the innermost region. Western analysis confirmed these data, demonstrating comparable P450c17 expression to rhesus monkeys, but not cytb5. HPLC analysis revealed similar 17alpha-hydroxylase action on pregnenolone for adult marmoset and rhesus adrenal microsomes but greatly diminished 17,20-lyase activity in marmosets. Neonatal marmoset adrenals exhibited staining indicative of a putative FZ (with P450c17, reduced 3beta-HSD and increased cytb5). We conclude that neonatal marmosets exhibit a C19 steroid-secreting FZ similar to humans, but adult males fail to acquire a functional ZR.

Isolation of marmoset P450c17 cDNA and gene regulation in vitro.

Marmosets express a fetal zone during adrenal development but fail to produce significant amounts of C19 steroids in adulthood. It is not clear however if P450c17 regulation is different from that in humans/rhesus or the primary sequence is altered. To this end we isolated marmoset and rhesus adrenocortical cells, and treated the cells with known regulators of P450c17 expression for 48 h. P450c17 protein increased with Forskolin (F) treatment, but was marginally inhibited by AII (A) and TPA (T) alone. Combined A + F and T + F dramatically ablated the F response. Cortisol levels (EIA) increased upon F treatment and were inhibited by A and T. Combination of treatments partially inhibited the F-induced response. The protein-coding region of marmoset and rhesus P450c17 cDNAs were then isolated from adrenals using RT-PCR/TA cloning. Marmoset P450c17 shows one amino acid deletion but otherwise shares 90.6% and 91.4% homologies with the human and rhesus cDNA sequences, and 82.4% and 85% homologies with the human and rhesus predicted AA sequences, respectively. Since marmoset adrenocortical cells exhibit similar endocrine function to rhesus, impaired 17,20-lyase activity in the adult marmoset adrenal may in part be due to differences in the primary sequence.

Expression of AT1-R in marmoset whole adrenal glands and adrenocortical cells in culture.

The marmoset adrenal is interesting as it is developmentally similar to humans and other primates, but in adulthood 17,20-lyase activity is very low. One possible explanation is an altered regulation of P450c17 expression by AT1-R. We investigated the expression and zonal distribution of the AT1-R in marmoset adrenal glands and adrenocortical cells in culture since it is known that AT1-R is a regulator of P450c17 expression and activity. AT1-R was expressed strongly in the ZG and to diminishing degrees through the remainder of the cortex. There was negative expression in a putative ZI. Dispersed adrenocortical cells retained AT1-R protein as detected by ICC. Cells cultured for several days and treated in serum-free media for 48 h maintained AT1-R expression, as measured by western. However, at that 48 h time point, treatment with Forsk, AII, TPA, or the combination of A+F or T+F did not appear to effect AT1-R expression. We conclude that marmoset adrenals express AT1-R similarly to humans and other higher mammals, adrenocortical cells retain AT1-R expression in culture, and consistent with other adrenal cell culture models, AT1-R expression is not lost in response to agonists long term.

Variations in adrenal androgen production among (nonhuman) primates.

The synthesis and secretion of the adrenal androgens dehydroepiandrosterone (DHEA) and its sulfate (DS) is a phenomenon apparently unique to humans and nonhuman primates. It occurs at three life stages: in utero from the fetal zone (FZ) cells of the developing adrenal cortex, during adolescence with the onset of adrenarche and the development of the zona reticularis (ZR), and in ever decreasing amounts from the ZR with aging (adrenal senescence). Insufficient data exist to know if any single nonhuman primate exactly mirrors human adrenal androgen secretion through all three life stages, and detailed morphological, biochemical, and endocrinologic studies are required to do so. Androgen synthesis requires that cells express three key enzymes, 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17), nicotinamide-adenine dinucleotide phosphate (NADPH)-cytochrome P450 oxidoreductase (CPR), and cytochrome b5, and that they do not express 3beta-hydroxysteroid dehydrogenase (3beta-HSD). Cytochrome b5 has emerged as a particularly useful marker of androgen synthetic potential. Although a reliable index of the rate of adrenal androgen secretion, DS concentrations may not accurately reflect total adrenal androgen output because rates and routes of androgen metabolism may vary greatly among species. Based on the very limited available data, the most promising nonhuman primate models are marmosets for the human FZ, chimpanzees for human adrenarche, and macaques and baboons for mature ZR function that declines with senescence.