Invited review: Adrenocortical function in avian and non-avian reptiles: Insights from dispersed adrenocortical cells.

Herein we review our work involving dispersed adrenocortical cells from several lizard species: the Eastern Fence Lizard (Sceloporus undulatus), Yarrow's Spiny Lizard (Sceloporus jarrovii), Striped Plateau Lizard (Sceloporus virgatus) and the Yucatán Banded Gecko (Coleonyx elegans). Early work demonstrated changes in steroidogenic function of adrenocortical cells derived from adult S. undulatus associated with seasonal interactions with sex. However, new information suggests that both sexes operate within the same steroidogenic budget over season. The observed sex effect was further explored in orchiectomized and ovariectomized lizards, some supported with exogenous testosterone. Overall, a suppressive effect of testosterone was evident, especially in cells from C. elegans. Life stage added to this complex picture of adrenal steroidogenic function. This was evident when sexually mature and immature Sceloporus lizards were subjected to a nutritional stressor, cricket restriction/deprivation. There were divergent patterns of corticosterone, aldosterone, and progesterone responses and associated sensitivities of each to corticotropin (ACTH). Finally, we provide strong evidence that there are multiple, labile subpopulations of adrenocortical cells. We conclude that the rapid (days) remodeling of adrenocortical steroidogenic function through fluctuating cell subpopulations drives the circulating corticosteroid profile of Sceloporus lizard species. Interestingly, progesterone and aldosterone may be more important with corticosterone serving as essential supportive background. In the wild, the flux in adrenocortical cell subpopulations may be adversely susceptible to climate-change related disruptions in food sources and to xenobiotic/endocrine-disrupting chemicals. We urge further studies using native lizard species as bioindicators of local pollutants and as models to examine the broader eco-exposome.

Functional remodeling of adrenal steroidogenic tissue by food deprivation in the lizard, Sceloporus undulatus.

The present study examined how food availability interacts with age to modulate lizard adrenal steroidogenic function at the cellular level. Adult male and juvenile male and female Eastern Fence Lizards (Sceloporus undulatus) underwent a period of food deprivation with or without a shorter re-feeding period. Lizards maintained on a full feeding regimen served as the controls. Across the feeding regimens, plasma corticosterone of adult lizards was unchanged whereas that of food-deprived juvenile lizards was increased nearly 7 times and this increase was normalized by a short re-feeding period. Freshly dispersed adrenocortical cells derived from these lizards were incubated with ACTH and the production of selected steroids was measured by highly specific radioimmunoassay. Net maximal steroid rates of juvenile cells were 161% greater than those of adult cells. Adult and juvenile progesterone rates were consistently suppressed by food deprivation (by nearly 48%) and were normalized by a re-feeding period, whereas divergent effects were seen with corticosterone and aldosterone rates. Food deprivation suppressed corticosterone rates of adult cells by 43% but not those of juvenile cells. In a reciprocal manner, food deprivation had no significant effect on aldosterone rates of adult cells, but it suppressed those of juvenile cells by 52%. A short re-feeding period normalized most rates in both adult and juvenile cells and further augmented the adult aldosterone rate by 54%. The effect of the feeding regimens on ACTH sensitivity varied with life stage and with steroid. The overall sensitivity of adult cells to ACTH was nearly three times that of juvenile cells. Collectively, the data presented here and data from previous work indicate that food restriction/deprivation in Sceloporus lizard species causes a functional remodeling of the adrenocortical tissue. Furthermore, life stage adds more complexity to this remodeling.

Modulation of adrenal steroidogenesis by testosterone in the lizard, Coleonyx elegans.

Our previous work with adrenocortical cells from several Sceloporus lizard species suggests that gonadal hormones influence the steroidogenic capacity and the sensitivity to ACTH. However, there are discrepancies in these cellular response parameters suggesting that the effects of gonadal hormones on adrenocortical function vary with species, sex, age, season, and environmental/experimental conditions. To gain further insight into these complex interactions, here we report studies on Coleonyx elegans, Eublepharidae (Yucatán Banded Gecko), which is only distantly related to Sceloporus lizards via a basal common ancestor and in captivity, reproduces throughout the year. We hypothesized that a more constant reproductive pattern would result in less variable effects of gonadal hormones on adrenocortical function. Reproductively mature male geckos were orchiectomized with and without replacement of testosterone (300 µg) via an implanted Silastic® tube. Reproductively mature intact female geckos received implants with and without testosterone. After 11 weeks, adrenocortical cells were isolated from these lizards and incubated with corticotropin (ACTH) for 3 h at 28 °C. Three adrenocortical steroids, progesterone, corticosterone and aldosterone, were measured by highly specific radioimmunoassays. The production rate of each steroid was statistically analyzed using established software and net maximal rate (by subtracting the basal rate) in response to ACTH was determined. In general, corticosterone predominated and comprised ∼83% of the total net maximal rate, followed by progesterone (∼14%) and aldosterone (∼3%). Compared to the functional responses of adrenocortical cells derived from other lizards thus far, adrenocortical cells from C. elegans exhibited a depressed steroid response to ACTH and this depressed response was more pronounced in male cells. In addition, other sex differences in cellular response were apparent. In female cells, the net maximal rates of progesterone, corticosterone and aldosterone were, respectively, 161, 122 and 900% greater than those in intact-male cells. In contrast, cellular sensitivity to ACTH, as determined by the half-maximally effective steroidogenic concentration (EC50) of ACTH, did not differ between intact-male and intact-female adrenocortical cells. Treatment effects were most striking for corticosterone, the putative, major glucocorticoid in lizards. Orchiectomy caused an increase in the net maximal corticosterone rate equivalent to that of intact-female cells. Testosterone maintenance in orchiectomized lizards completely suppressed the stimulatory effect of orchiectomy. However, orchiectomy with or without testosterone maintenance did not alter cellular sensitivity to ACTH. The effect of testosterone supplementation in intact females, although suppressive, was notably different from its effect in orchiectomized males. Its effect on the net maximal corticosterone rate was relatively modest and did not completely "masculinize" the greater rate seen in intact-female cells. However, testosterone supplementation dramatically suppressed the basal corticosterone rate (by 82%) and enhanced the overall cellular sensitivity to ACTH by 150%, two effects not seen in cells derived from testosterone-treated orchiectomized lizards. Collectively, these findings clearly indicating that the gonad directly or indirectly regulates lizard adrenocortical cell function. Whereas other gonadal or extra-gonadal factors may play a role, testosterone appears to be an essential determinant of the observed sex differences in adrenocortical function.

Effects of food restriction on steroidogenesis in dispersed adrenocortical cells from Yarrow's Spiny Lizard (Sceloporus jarrovii).

Changes in energy balance can lead to functional alterations at all levels of the hypothalamic-pituitary-adrenal (HPA) axis. However, relatively little is known about how energy balance affects functional properties of adrenocortical cells themselves. We investigated effects of restricted food intake on sensitivity to ACTH and rates of steroidogenesis in adrenocortical cells isolated from growing female and male Yarrow's Spiny Lizards (Sceloporus jarrovii). At the end of the feeding regimen, we assayed acute (3h) progesterone (P(4)), corticosterone (B), and aldosterone (ALDO) production in response to ACTH in dispersed adrenocortical cells. Food restriction depressed growth rate by about 50% in both males and females but did not alter baseline plasma B measured at 10 weeks in either sex. At the cellular level, food restriction had the following effects: (1) increased basal B production in both sexes and basal ALDO production in males, (2) increased net maximal rates of production of P(4), B, and ALDO in response to ACTH, and (3) no overall effect on adrenocortical cellular sensitivity to ACTH. There were modest sex differences: overall rates of P(4) production were 46% greater in cells from females than from males, and in response to food restriction, the net maximal rate of ALDO production was 50% greater in cells from males than from females. Our results demonstrate that food restriction in S. jarrovii increases adrenocortical cellular rates of steroid production without affecting overall cellular sensitivity to ACTH.

Gonadal modulation of in vitro steroidogenic properties of dispersed adrenocortical cells from Sceloporus lizards.

Effects of adrenal corticosteroids on reproductive and endocrine functions of the gonads are well known, but reciprocal effects of gonadal hormones on the hypothalamo-pituitary-adrenal (HPA) axis and on adrenocortical steroidogenesis in particular have received much less attention. We investigated effects of gonadectomy and testosterone (T) replacement on adrenocortical cell function in a year-long field study of male Sceloporus undulatus (Eastern Fence Lizard) and in a shorter term laboratory study with male Sceloporus jarrovii (Yarrow's Spiny Lizard). We also compared females to males in Sceloporus virgatus (Striped Plateau Lizard) and investigated effects of gonadectomy in short-term laboratory experiment on females of this species. As measured by in vitro production of progesterone (P(4)), corticosterone (B), and aldosterone (ALDO), sensitivity of adrenocortical cells to corticotrophin (ACTH) was lower in control males than females of S. virgatus. In S. jarrovii males, cellular sensitivity to ACTH was reduced by orchiectomy but was not restored to levels of intact controls by T replacement. By contrast, in S. undulatus, cellular sensitivity to ACTH was not affected by orchiectomy alone but was reduced by T replacement in orchiectomized males. Maximal rates of steroid production were less consistently affected by experimental treatments, but were lower in males than in females of S. virgatus and were dramatically reduced by T replacement in orchiectomized S. undulatus males. Overall, our experiments clearly demonstrate two distinct sources of variation in functional capacities of dispersed adrenocortical cells isolated from Sceloporus lizards: (1) naturally occurring differences between males and females (Carsia and John-Alder, 2003), and (2) species-dependent changes in response to surgical gonadectomy with or without exogenous testosterone. Sex differences and functional lability in adrenocortical cells are probably widespread among vertebrates and may be an important component of variation in output of the HPA.

Natriuretic peptides are negative modulators of adrenocortical cell function of the eastern fence lizard (Sceloporus undulatus).

Elucidation of the role of natriuretic peptides (NPs) in vertebrate adrenal steroidogenesis has been facilitated by the use of freshly dispersed adrenocortical cells. Our recent characterization of lizard adrenocortical cells [Carsia, R.V., John-Alder, H.B., 2003. Seasonal alterations in adrenocortical cell function associated with stress-responsiveness and sex in the Eastern Fence Lizard (Sceloporus undulatus). Horm. Behav. 43, 408-420] provided the opportunity to examine the influence of atrial natriuretic peptides (ANPs) and related NPs on reptilian adrenal steroidogenesis at the cellular level. In the present report, the action of NPs on lizard adrenal steroidogenesis was investigated using freshly dispersed adrenocortical cells derived from the Eastern Fence Lizard (Sceloporus undulatus). Basal production rates of aldosterone and corticosterone and maximal angiotensin II (ANG II)-induced production rates of these corticosteroids were inhibited with high efficacy (75-90%) by rat ANP at potencies of 0.4-0.7 nM. By contrast, rat ANP had no effect on maximal production rates of these corticosteroids in response to a maximal steroidogenic concentration of adrenocorticotropin (ACTH; 1 nM). However, rat ANP inhibited aldosterone and corticosterone production rates in response to a half-maximal steroidogenic concentration of ACTH (10 pM; approximately 50 pg/ml), albeit with less efficacy ( approximately 50%) and potency (approximately 6 nM) than for ANG II. Rat and eel ANP and rat and chicken brain natriuretic peptide (BNP) were equally efficacious at inhibiting maximal ANG II-induced aldosterone and corticosterone production but with different potencies. The order of inhibitory potency was rat ANP = chicken BNP > eel ANP > rat BNP. However, a specific peptide ligand for the NP clearance receptor was without effect. This study indicates that ANP and related NPs are efficacious inhibitors of lizard adrenal steroidogenesis by acting directly at the level of the adrenocortical cell.

Seasonal alterations in adrenocortical cell function associated with stress-responsiveness and sex in the eastern fence lizard (Sceloporus undulatus).

We characterized steroidogenic properties of dispersed adrenocortical cells from field-active male and female eastern fence lizards (Sceloporus undulatus) to investigate whether alterations in cell function could, in part, explain seasonal variation in baseline and stress-induced plasma corticosterone (B). Lizards were collected during the breeding and postbreeding seasons and shortly prior to hibernation. Dispersed cells in vitro produced B, aldosterone (ALDO), and progesterone in response to 8-Br-cAMP, 25-(OH)cholesterol, adrenocorticotropin (ACTH; as little as 100 fM), and angiotensin II. Maximal progesterone, B, and ALDO responses to ACTH were roughly 1000%, 500%, and 100% greater than corresponding basal values. Angiotensin II was an effective steroidogenic stimulant but much less so than ACTH. Corticosteroid production exhibited considerable steroid-specific variation among seasons. Maximal ACTH-induced B production was lower in the postbreeding season than at either of the other two measurement points, essentially opposite to the pattern for ALDO. Males and females generally produced B at similar rates, but ALDO and progesterone showed numerous sex differences that usually covaried between the two steroids. Cellular sensitivity to 25-(OH)cholesterol and angiotensin II showed few sex differences or seasonal changes. In contrast, sensitivity to ACTH decreased markedly from the breeding to the postbreeding season in males, corresponding to the decrease in stress-responsiveness, and in both sexes was considerably lower prior to hibernation than during the breeding season. Under some conditions, plasma B may be limited by the production capacity of adrenocortical cells. In summary, seasonal variations in body condition, reproductive activity, and baseline and stress-induced plasma B may be attributed at least in part to alterations in adrenocortical cell steroidogenic function.