Effects of testosterone on urogenital tract morphology and androgen receptor expression in immature Eastern Fence lizards (Sceloporus undulatus).

In non-avian reptiles, the onset of sexual dimorphism of the major structures of the urogenital tract varies temporally relative to gonadal differentiation, more so than in other amniote lineages. In the current study, we used tonic-release implants to investigate the effects of exogenous testosterone (T) on postnatal development of the urogenital tract in juvenile Eastern Fence Lizards (Sceloporus undulatus) to better understand the mechanisms underlying the ontogeny of sexual differentiation in reptiles. We examined gonads, mesonephric kidneys and ducts (male reproductive tract primordia), paramesonephric ducts (oviduct primordia), sexual segments of the kidneys (SSKs), and hemiphalluses to determine which structures were sexually dimorphic independent of T treatment and which structures exhibited sexually dimorphic responses to T. To better understand tissue-level responsiveness to T treatment, we also characterized androgen receptor (AR) expression by immunohistochemistry. At approximately 4 months after hatching in control animals, gonads were well differentiated but quiescent; paramesonephric ducts had fully degenerated in males; mesonephric kidneys, mesonephric ducts, and SSKs remained sexually undifferentiated; and hemiphalluses could not be everted in either sex. Exogenous T caused enlargement, regionalization, and secretory activity of the mesonephric ducts and SSKs in both sexes; enlargement and regionalization of the oviducts in females; and enlargement of male hemipenes. The most responsive tissues exhibited moderate but diffuse staining for AR in control lizards and intense nuclear staining in T-treated lizards, suggestive of autoregulation of AR. The similarity between sexes in the responsiveness of the mesonephric ducts and SSK to T indicates an absence of sexually dimorphic organizational effects in these structures prior to treatment, which was initiated approximately 2 months after hatching. In contrast, the sex-specific responses in oviducts and hemipenes indicate that significant organization and/or differentiation had taken place prior to treatment.

Species differences in hormonally mediated gene expression underlie the evolutionary loss of sexually dimorphic coloration in Sceloporus lizards.

Phenotypic sexual dimorphism often involves the hormonal regulation of sex-biased expression for underlying genes. However, it is generally unknown whether the evolution of hormonally mediated sexual dimorphism occurs through upstream changes in tissue sensitivity to hormone signals, downstream changes in responsiveness of target genes, or both. Here, we use comparative transcriptomics to explore these possibilities in 2 species of Sceloporus lizards exhibiting different patterns of sexual dichromatism. Sexually dimorphic S. undulatus develops blue and black ventral coloration in response to testosterone, while sexually monomorphic S. virgatus does not, despite exhibiting similar sex differences in circulating testosterone levels. We administered testosterone implants to juveniles of each species and used RNAseq to quantify gene expression in ventral skin. Transcriptome-wide responses to testosterone were stronger in S. undulatus than in S. virgatus, suggesting species differences in tissue sensitivity to this hormone signal. Species differences in the expression of genes for androgen metabolism and sex hormone-binding globulin were consistent with this idea, but expression of the androgen receptor gene was higher in S. virgatus, complicating this interpretation. Downstream of androgen signaling, we found clear species differences in hormonal responsiveness of genes related to melanin synthesis, which were upregulated by testosterone in S. undulatus, but not in S. virgatus. Collectively, our results indicate that hormonal regulation of melanin synthesis pathways contributes to the development of sexual dimorphism in S. undulatus, and that changes in the hormonal responsiveness of these genes in S. virgatus contribute to the evolutionary loss of ventral coloration.

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.

Living on the edge: Glucocorticoid physiology in desert iguanas (Dipsosaurus dorsalis) is predicted by distance from an anthropogenic disturbance, body condition, and population density.

Ecological factors, such as habitat quality, influence the survival and reproductive success of free-living organisms. Urbanization, including roads, alters native habitat and likely influences physiology, behavior, and ultimately Darwinian fitness. Some effects of roads are clearly negative, such as increased habitat fragmentation and mortality from vehicle collision. However, roads can also have positive effects, such as decreasing predator density and increased vegetation cover, particularly in xeric habitats due to increased water run-off. Glucocorticoids are metabolic hormones that reflect baseline metabolic needs, increase in response to acute challenges, and may mediate endogenous resource trade-offs between survival and reproduction. Here we examined circulating concentrations of corticosterone (baseline and stress-induced) in desert iguanas (Dipsosaurus dorsalis) in relation to the distance from a major anthropogenic disturbance, a high-traffic road in Palm Springs, CA. Additionally, we analyzed body condition and population density as additional predictors of glucocorticoid physiology. Surprisingly, we found lower baseline CORT levels closer to the road, but no effect of distance from road on stress-induced CORT or stress responsiveness (difference between baseline and stress-induced concentrations). Both population density and body condition were negative predictors of baseline CORT, stress-induced CORT, and stress responsiveness. Given the known effect of roads to increase run-off and vegetation density, increased water availability may improve available forage and shade, which may then increase the carrying capacity of the habitat and minimize metabolic challenges for this herbivorous lizard. However, it is important to recognize that surfaces covered by asphalt are not usable habitat for iguanas, likely resulting in a net habitat loss.

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.

Sex hormones and the development of sexual size dimorphism: 5α-dihydrotestosterone inhibits growth in a female-larger lizard (Sceloporus undulatus).

Sexual differences in adult body size [sexual size dimorphism (SSD)] and color (sexual dichromatism) are widespread, and both male- and female-biased dimorphisms are observed even among closely related species. A growing body of evidence indicates testosterone can regulate growth, thus the development of SSD, and sexual dichromatism. However, the mechanism(s) underlying these effects are conjectural, including possible conversions of testosterone to estradiol (E2) or 5α-dihydrotestosterone (DHT). In the present study, we hypothesized that the effects of testosterone are physiological responses mediated by androgen receptors, and we tested two specific predictions: (1) that DHT would mimic the effects of testosterone by inhibiting growth and enhancing coloration, and (2) that removal of endogenous testosterone via surgical castration would stimulate growth. We also hypothesized that females share downstream regulatory networks with males and predicted that females and males would respond similarly to DHT. We conducted experiments on eastern fence lizards (Sceloporus undulatus), a female-larger species with striking sexual dichromatism. We implanted Silastic® tubules containing 150 µg DHT into intact females and intact and castrated males. We measured linear growth rates and quantified color for ventral and dorsal surfaces. We found that DHT decreased growth rate and enhanced male-typical coloration in both males and females. We also found that, given adequate time, castration alone is sufficient to stimulate growth rate in males. The results presented here suggest that: (1) the effects of testosterone on growth and coloration are mediated by androgen receptors without requiring aromatization of testosterone into E2, and (2) females possess the androgen-receptor-mediated regulatory networks required for initiating male-typical inhibition of growth and enhanced coloration in response to androgens.

Nutritional modulation of IGF-1 in relation to growth and body condition in Sceloporus lizards.

Nutrition and energy balance are important regulators of growth and the growth hormone/insulin-like growth factor (GH/IGF) axis. However, our understanding of these functions does not extend uniformly to all classes of vertebrates and is mainly limited to controlled laboratory conditions. Lizards can be useful models to improve our understanding of the nutritional regulation of the GH/IGF-1 axis because many species are relatively easy to observe and manipulate both in the laboratory and in the field. In the present study, the effects of variation in food intake on growth, body condition, and hepatic IGF-1 mRNA levels were measured in (1) juveniles of Sceloporus jarrovii maintained on a full or 1/3 ration and (2) hatchlings of Sceloporus undulatus subjected to full or zero ration with or without re-feeding. These parameters plus plasma IGF-1 were measured in a third experiment using adults of S. undulatus subjected to full or zero ration with or without re-feeding. In all experiments, plasma corticosterone was measured as an anticipated indicator of nutritional stress. In S. jarrovii, growth and body condition were reduced but lizards remained in positive energy balance on 1/3 ration, and hepatic IGF-1 mRNA and plasma corticosterone were not affected in comparison to full ration. In S. undulatus, growth, body condition, hepatic IGF-1 mRNA, and plasma IGF-1 were all reduced by zero ration and restored by refeeding. Plasma corticosterone was increased in response to zero ration and restored by full ration in hatchlings but not adults of S. undulatus. These data indicate that lizards conform to the broader vertebrate model in which severe food deprivation and negative energy balance is required to attenuate systemic IGF-1 expression. However, when animals remain in positive energy balance, reduced food intake does not appear to affect systemic IGF-1. Consistent with other studies on lizards, the corticosterone response to reduced food intake is an unreliable indicator of nutritional stress. Further studies on ecologically relevant variation in food intake are required to establish the importance of nutrition as an environmental regulator of the GH/IGF axis. Within the range of positive energy balance, the potential involvement of molecular signals in growth regulation requires further investigation.

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.

A chromosome-level genome assembly for the eastern fence lizard (Sceloporus undulatus), a reptile model for physiological and evolutionary ecology.

BACKGROUND: High-quality genomic resources facilitate investigations into behavioral ecology, morphological and physiological adaptations, and the evolution of genomic architecture. Lizards in the genus Sceloporus have a long history as important ecological, evolutionary, and physiological models, making them a valuable target for the development of genomic resources. FINDINGS: We present a high-quality chromosome-level reference genome assembly, SceUnd1.0 (using 10X Genomics Chromium, HiC, and Pacific Biosciences data), and tissue/developmental stage transcriptomes for the eastern fence lizard, Sceloporus undulatus. We performed synteny analysis with other snake and lizard assemblies to identify broad patterns of chromosome evolution including the fusion of micro- and macrochromosomes. We also used this new assembly to provide improved reference-based genome assemblies for 34 additional Sceloporus species. Finally, we used RNAseq and whole-genome resequencing data to compare 3 assemblies, each representing an increased level of cost and effort: Supernova Assembly with data from 10X Genomics Chromium, HiRise Assembly that added data from HiC, and PBJelly Assembly that added data from Pacific Biosciences sequencing. We found that the Supernova Assembly contained the full genome and was a suitable reference for RNAseq and single-nucleotide polymorphism calling, but the chromosome-level scaffolds provided by the addition of HiC data allowed synteny and whole-genome association mapping analyses. The subsequent addition of PacBio data doubled the contig N50 but provided negligible gains in scaffold length. CONCLUSIONS: These new genomic resources provide valuable tools for advanced molecular analysis of an organism that has become a model in physiology and evolutionary ecology.

Testosterone Reduces Growth and Hepatic IGF-1 mRNA in a Female-Larger Lizard, Sceloporus undulatus: Evidence of an Evolutionary Reversal in Growth Regulation.

Previous research has demonstrated that testosterone (T) can inhibit growth in female-larger species and stimulate growth in male-larger species, but the underlying mechanisms of this regulatory bipotentiality have not been investigated. In this study, we investigated the effects of T on the expression of hepatic insulin-like growth factor-1 (IGF-1) mRNA and circulating IGF-1 hormone in Sceloporus undulatus, a species of lizard in which females grow faster to become larger than males and in which T inhibits growth. Experiments were performed in captivity on mature female and male adults in the asymptotic phase of their growth curve and on actively growing, pre-reproductive juveniles. In adult males, the expression of hepatic IGF-1 mRNA increased following surgical castration and returned to control levels with T replacement; in intact adult females, exogenous T had no effect on IGF-1 mRNA expression. In juveniles, T significantly reduced both growth and the expression of hepatic IGF-1 mRNA to similar extents in intact females and in castrated males. The relative inhibitory effects of T on mRNA expression were greater in juveniles than in adults. Plasma IGF-1 hormone was about four times higher in juveniles than in adults, but T had no significant effect on IGF-1 hormone in either sex or in either age group. Our finding of inhibition of the expression of hepatic IGF-1 mRNA stands in contrast to the stimulatory effects of T in the published body of literature. We attribute our novel finding to our use of a species in which T inhibits rather than stimulates growth. Our findings begin to explain how T has the regulatory bipotentiality to be stimulatory in some species and inhibitory in others, requiring only an evolutionary reversal in the molecular regulation of growth-regulatory genes including IGF-1. Further comparative transcriptomic studies will be required to fully resolve the molecular mechanism of growth inhibition.

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.

Testosterone inhibits growth in juvenile male eastern fence lizards (Sceloporus undulatus): implications for energy allocation and sexual size dimorphism.

In the eastern fence lizard, Sceloporus undulatus, female-larger sexual size dimorphism develops because yearling females grow faster than males before first reproduction. This sexual growth divergence coincides with maturational increases in male aggression, movement, and ventral coloration, all of which are influenced by the sex steroid testosterone (T). These observations suggest that male growth may be constrained by energetic costs of activity and implicate T as a physiological regulator of this potential trade-off. To test this hypothesis, we used surgical castration and subsequent administration of exogenous T to alter the physiological and behavioral phenotypes of field-active males during the period of sexual growth divergence. As predicted, T inhibited male growth, while castration promoted long-term growth. Males treated with T also exhibited increased daily activity period, movement, and home range area. Food consumption did not differ among male treatments or sexes, suggesting that the inhibitory effects of T on growth are mediated by patterns of energy allocation rather than acquisition. On the basis of estimates derived from published data, we conclude that the energetic cost of increased daily activity period following T manipulation is sufficient to explain most (79%) of the associated reduction in growth. Further, growth may have been constrained by additional energetic costs of increased ectoparasite load following T manipulation. Similar studies of the proximate behavioral, ecological, and physiological mechanisms involved in growth regulation should greatly improve our understanding of sexual size dimorphism.

A comparative test of adaptive hypotheses for sexual size dimorphism in lizards.

It is commonly argued that sexual size dimorphism (SSD) in lizards has evolved in response to two primary, nonexclusive processes: (1) sexual selection for large male size, which confers an advantage in intrasexual mate competition (intrasexual selection hypothesis), and (2) natural selection for large female size, which confers a fecundity advantage (fecundity advantage hypothesis). However, outside of several well-studied lizard genera, the empirical support for these hypotheses has not been examined with appropriate phylogenetic control. We conducted a comparative phylogenetic analysis to test these hypotheses using literature data from 497 lizard populations representing 302 species and 18 families. As predicted by the intrasexual selection hypothesis, male aggression and territoriality are correlated with SSD, but evolutionary shifts in these categorical variables each explain less than 2% of the inferred evolutionary change in SSD. We found stronger correlations between SSD and continuous estimates of intrasexual selection such as male to female home range ratio and female home range size. These results are consistent with the criticism that categorical variables may obscure much of the actual variation in intrasexual selection intensity needed to explain patterns in SSD. In accordance with the fecundity advantage hypothesis, SSD is correlated with clutch size, reproductive frequency, and reproductive mode (but not fecundity slope, reduced major axis estimator of fecundity slope, length of reproductive season, or latitude). However, evolutionary shifts in clutch size explain less than 8% of the associated change in SSD, which also varies significantly in the absence of evolutionary shifts in reproductive frequency and mode. A multiple regression model retained territoriality and clutch size as significant predictors of SSD, but only 16% of the variation in SSD is explained using these variables. Intrasexual selection for large male size and fecundity selection for large female size have undoubtedly helped to shape patterns of SSD across lizards, but the comparative data at present provide only weak support for these hypotheses as general explanations for SSD in this group. Future work would benefit from the consideration of alternatives to these traditional evolutionary hypotheses, and the elucidation of proximate mechanisms influencing growth and SSD within populations.

Hormones, performance and fitness: Natural history and endocrine experiments on a lizard (Sceloporus undulatus).

We used the "morphology-performance-fitness" paradigm (Arnold, 1983) as our framework to investigate endocrine control of performance and fitness in Sceloporus undulatus (Eastern Fence Lizard). Focusing on males, we used the "natural experiments" of seasonal, sexual, and developmental variation in growth and in exercise endurance to identify testosterone and corticosterone as potential modulators of performance and related traits of interest. We followed with experimental manipulations of testosterone to investigate functional relationships, both in the laboratory and in the field. Further, we used focal observations and demographic studies, coupled with genetic determination of paternity, to test associations between performance and fitness, measured as reproductive success. We found that in males, endurance and plasma concentrations of testosterone and corticosterone are at their peaks in the spring breeding season, when lizards are most actively engaged in patrolling home ranges and in reproductive behavior. At that time, plasma concentrations of testosterone are correlated with body size; plasma concentrations of corticosterone and parameters of home range, including area and the number of overlapped females, are correlated with home-range overlap between males and females. During prereproductive development, males (but not females) experience a maturational increase in plasma testosterone. At about the same time, they become more active, expand their home ranges, and grow less quickly than do females, suggesting a trade-off in the allocation of energy, mediated by testosterone. Experimentally, testosterone has positive effects on fitness by stimulating endurance and reproductive activity and increasing home-range area, but it exacts costs in fitness by increasing ectoparasitism, decreasing growth, and decreasing survivorship. We found evidence of selection on body size, endurance, and home-range size (and thus access to potential mates). Despite having positive effects on performance traits, plasma concentrations of testosterone were not correlated with number of offspring sired by males. However, we found a strong correlation between the level of plasma corticosterone and the number of offspring sired. We also found evidence of size-assortative mating, indicating that for males, both the number and the size (and thus, fecundity) of their mates increase with body size. Our studies exemplify the power of natural history combined with experimental endocrine manipulations to identify hormonal regulators of performance and linkages to fitness. Furthermore, our results illustrate ecological and evolutionary significance of individual variation in endocrine traits.

Testosterone stimulates the expression of a social color signal in Yarrow's Spiny Lizard, Sceloporus jarrovii.

The sex steroid testosterone has been shown to regulate the development of male-specific coloration in many organisms that exhibit sexual dichromatism, but the role of testosterone is less certain for species in which both sexes express bright coloration. Lizards in the genus Sceloporus possess bright blue patches on their throats and abdomens. These patches, which are used in social signaling, are often regulated by testosterone and are consequently expressed only in males of most species. However, Yarrow's Spiny Lizard (Sceloporus jarrovii, Cope 1875) exhibits a derived condition in which both sexes express bright blue ventral patches despite dramatic sexual differences in circulating testosterone levels throughout postnatal ontogeny. In this study, we used surgical castration and hormone replacement in juvenile males to test the hypothesis that testosterone stimulates the expression of blue ventral coloration in S. jarrovii. In two separate experiments conducted in captivity and the natural field environment, we found that surgical castration decreased the hue and saturation while increasing the brightness of blue throat and abdominal patches. Castration also decreased the amount of black pigment bordering the blue throat patch. Treatment of castrated males with exogenous testosterone restored all aspects of ventral coloration to values similar to those of intact control males. Early organizational effects of testosterone during prenatal development may lead to the expression of blue coloration in both sexes, but the results of our present experiments indicate that subsequent effects of testosterone during sexual maturation further enhance the coloration of males.

Proximate developmental mediators of sexual dimorphism in size: case studies from squamate reptiles.

Sexual dimorphism in size (sexual size dimorphism; SSD) is nearly ubiquitous, but the relative importance of genetic versus environmental control of SSD is not known for most species. We investigated proximate determinants of SSD in several species of squamate reptiles, including three species of Sceloporus lizards and the diamond-backed rattlesnake (Crotalus atrox). In natural populations of these species, SSD is caused by sexual differences in age-specific growth. Males and females, however, may often share similar potentials for growth: growth is strongly responsive to the availability of food, and sexual differences in growth can be greatly suppressed or completely absent under common environmental conditions in the laboratory. Sexually divergent growth is expressed in natural environments because of inherent ecological differences between males and females and because of potential epigenetic effects of sex-specific growth regulators. In field-active Sceloporus, sexual differences in growth rate are associated with sexual divergence in plasma testosterone. Experiments confirm that testosterone inhibits growth in species in which females are larger (for example, S. undulatus and S. virgatus) and stimulates growth in those in which males are larger (for example, S. jarrovii). Interestingly, however, sexual divergence in plasma testosterone is not accompanied by divergence in growth in S. jarrovii or in male-larger C. atrox in the laboratory. Furthermore, experimental effects of castration and testosterone replacement on growth are not evident in captive S. jarrovii, possibly because growth effects of testosterone are superseded by an abundant, high-quality diet. In female-larger S. undulatus, growth may be traded-off against testosterone-induced reproductive costs of activity. In male-larger species, costs of reproduction in terms of growth are suggested by supplemental feeding of reproductive female C. atrox in their natural environment and by experimental manipulation of reproductive cost in female S. jarrovii. Growth costs of reproduction, however, do not contribute substantially to the development of SSD in male-larger S. jarrovii. We conclude that the energetic costs of testosterone-induced, male reproductive behavior may contribute substantially to the development of SSD in some female-larger species. However, despite strong evidence that reproductive investment exacts a substantial cost in growth, we do not support the reproductive cost hypothesis as a general explanation of SSD in male-larger species.

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.

Testosterone has opposite effects on male growth in lizards (Sceloporus spp.) with opposite patterns of sexual size dimorphism.

Sexual size dimorphism (SSD) has received considerable attention from evolutionary biologists, but relatively little is known about the physiological mechanisms underlying sex differences in growth that lead to SSD. Testosterone (T) stimulates growth in many male-larger vertebrates, but inhibits growth in the female-larger lizard Sceloporus undulatus. Thus, opposite patterns of SSD may develop in part because of underlying differences in the hormonal regulation of male growth. In the present study, we examined the effects of T on male growth in two sympatric congeners with opposite patterns of SSD (S. virgatus: female-larger; S. jarrovii: male-larger). During the mating season, yearling males of both species have higher plasma T levels than females, but whereas yearling males of S. virgatus grow only half as fast as females, yearling males of S. jarrovii grow more quickly than females. Thus, we hypothesized that T inhibits growth in yearling S. virgatus males, but promotes growth in yearling S. jarrovii males. In support of this hypothesis, we found that castrated (CAST) males of S. virgatus grew faster than castrated males given T implants (TEST). In contrast, TEST males of S. jarrovii grew faster than CAST males. Our results provide the first direct evidence for opposite effects of T on male growth in closely related species with opposite patterns of SSD. We speculate that growth inhibition by T reflects an energetic trade-off between growth and reproductive investment, and propose that such ;costs' of male reproduction may help explain the evolution of female-larger SSD in Sceloporus.