Elevated plasma corticosterone decreases yolk testosterone and progesterone in chickens: linking maternal stress and hormone-mediated maternal effects

Despite considerable research on hormone-mediated maternal effects in birds, the underlying physiology remains poorly understood. This study investigated a potential regulation mechanism for differential accumulation of gonadal hormones in bird eggs. Across vertebrates, glucocorticoids can suppress reproduction by downregulating gonadal hormones. Using the chicken as a model species, we therefore tested whether elevated levels of plasma corticosterone in female birds influence the production of gonadal steroids by the ovarian follicles and thus the amount of reproductive hormones in the egg yolk. Adult laying hens of two different strains (ISA brown and white Leghorn) were implanted subcutaneously with corticosterone pellets that elevated plasma corticosterone concentrations over a period of nine days. Steroid hormones were subsequently quantified in plasma and yolk. Corticosterone-implanted hens of both strains had lower plasma progesterone and testosterone levels and their yolks contained less progesterone and testosterone. The treatment also reduced egg and yolk mass. Plasma estrogen concentrations decreased in white Leghorns only whereas in both strains yolk estrogens were unaffected. Our results demonstrate for the first time that maternal plasma corticosterone levels influence reproductive hormone concentrations in the yolk. Maternal corticosterone could therefore mediate environmentally induced changes in yolk gonadal hormone concentrations. In addition, stressful situations experienced by the bird mother might affect the offspring via reduced amounts of reproductive hormones present in the egg as well as available nutrients for the embryo.     

Hormone-mediated maternal effects in birds: mechanisms matter but what do we know of them?

Over the past decade, birds have proven to be excellent models to study hormone-mediated maternal effects in an evolutionary framework. Almost all these studies focus on the function of maternal steroid hormones for offspring development, but lack of knowledge about the underlying mechanisms hampers further progress. We discuss several hypotheses concerning these mechanisms, point out their relevance for ecological and evolutionary interpretations, and review the relevant data. We first examine whether maternal hormones can accumulate in the egg independently of changes in hormone concentrations in the maternal circulation. This is important for Darwinian selection and female physiological trade-offs, and possible mechanisms for hormone accumulation in the egg, which may differ among hormones, are reviewed. Although independent regulation of plasma and yolk concentrations of hormones is conceivable, the data are as yet inconclusive for ovarian hormones. Next, we discuss embryonic utilization of maternal steroids, since enzyme and receptor systems in the embryo may have coevolved with maternal effect mechanisms in the mother. We consider dose-response relationships and action pathways of androgens and argue that these considerations may help to explain the apparent lack of interference of maternal steroids with sexual differentiation. Finally, we discuss mechanisms underlying the pleiotropic actions of maternal steroids, since linked effects may influence the coevolution of parent and offspring traits, owing to their role in the mediation of physiological trade-offs. Possible mechanisms here are interactions with other hormonal systems in the embryo. We urge endocrinologists to embark on suggested mechanistic studies and behavioural ecologists to adjust their interpretations to accommodate the current knowledge of mechanisms.     

Organizational effects of maternal testosterone on reproductive behavior of adult house sparrows

Despite the well-known, long-term, organizational actions of sex steroids on phenotypic differences between the sexes, studies of maternal steroids in the vertebrate egg have mainly focused on effects seen in early life. Long-term organizational effects of yolk hormones on adult behavior and the underlying mechanisms that generate them have been largely ignored. Using an experiment in which hand-reared house sparrows (Passer domesticus) from testosterone- or control-treated eggs were kept under identical conditions, we show that testosterone treatment in the egg increased the frequency of aggressive, dominance, and sexual behavior of 1-year-old, reproductively competent house sparrows. We also show that circulating plasma levels of progesterone, testosterone, 5alpha-dihydrotestosterone, and 17beta-estradiol did not differ between treatment groups. Thus, a simple change in adult gonadal hormone secretion is not the primary physiological cause of long-term effects of maternal steroids on adult behavior. Rather, differences in adult behavior caused by exposure to yolk testosterone during embryonic development are likely generated by organizational modifications of brain function. Furthermore, our data provide evidence that hormone-mediated maternal effects are an epigenetic mechanism causing intra-sexual variation in adult behavioral phenotype.     

Sex differences in yolk hormones depend on maternal social status in Leghorn chickens (Gallus gallus domesticus)

Maternal hormones are known to be present in avian eggs and can have beneficial effects on chick development. Recently, differences in avian yolk steroid concentrations between the sexes have been demonstrated, and in this context steroids have been proposed to be part of the avian sex-determining mechanism. In our study, we show that it is very unlikely that androgen concentrations alone are the decisive part of the sex-determining mechanism. We found that sex-specific differences in the yolk hormones strongly depend on the social rank of the mother. First, dominant females, but not subdominant females, allocated significantly more testosterone to male eggs than to female eggs. Second, subordinate females increased the testosterone concentrations of female eggs. This pattern of yolk hormone deposition can be functionally explained. In polygynous species such as the chicken, reproductive success is more variable in males than in females. Parental investment in sons or daughters is therefore expected to occur in direct relation to parental rearing capacities. We found that the social status of a hen was indeed negatively correlated with her maternal capacities (for example, body mass, egg mass). Differential androgen deposition might thus provide a mechanism for adaptive maternal investment depending on both the sex of the egg and the social status of the mother.     

Experimental evidence for genetic heritability of maternal hormone transfer to offspring

In many animal species, embryos are exposed to maternal hormones that affect their development. Maternal hormone transfer varies with environmental conditions of the mother and is often interpreted as being shaped by natural selection to adjust the offspring to prevailing environmental conditions. Such hormone transfer requires genetic variability, which has not yet been experimentally demonstrated. Our study reports direct evidence for additive genetic variance of maternal androgens through a bidirectional selection on yolk testosterone (T) levels in Japanese quail. Lines selected for high egg T (HET) and low egg T (LET) concentration differed in yolk levels of this androgen, resulting in high realized heritability (h2 = 0.42)Correlated responses to selection on other gonadal hormones indicated that selection specifically targeted biologically active androgens. Eggs of HET quail contained higher androstenedione and lower estradiol concentrations than did those of LET quail, with no line differences in yolk progesterone concentration. Plasma T concentrations in adult females were not affected by selection, seriously challenging the hypothesis that transfer of maternal hormones to offspring is constrained by hormone levels in a mother's circulation. Our results suggest that transfer of maternal T represents an indirect genetic effect that has important consequences for the evolution of traits in offspring.     

Maternal dietary carotenoids mitigate detrimental effects of maternal GnRH on offspring immune function in Japanese quail Coturnix japonica

Maternal resources deposited in eggs can affect the development of several offspring phenotypic traits and result in trade‐offs among them. For example, maternal androgens in eggs may be beneficial to offspring growth and competitive ability, but detrimental to immunocompetence and oxidative stress. In contrast, maternal antioxidants in eggs may be beneficial if they mitigate oxidative stress and immunosuppressive effects of androgens. We investigated possible interactive effects of maternal steroids and carotenoids on aspects of offspring physiology and phenotype, by simultaneously manipulating levels of androgens (via gonadotropin‐releasing hormone, GnRH‐challenges) and carotenoids (via diet supplementation) in captive female Japanese quail Coturnix japonica during egg laying. Carotenoid supplementation of hens, which elevates yolk concentrations of carotenoid and vitamins A and E, enhanced egg hatching success, offspring survival to age 15 d, and size of the bursa of Fabricius in offspring. In contrast, repeated maternal GnRH challenges, which elevated yolk testosterone concentrations, enhanced offspring neonatal size, but negatively affected bursa size. However, interaction among the treatments suggests that the positive effect of maternal carotenoid supplementation on plasma bactericidal capacity was mediated by maternal GnRH challenges. Chicks originating from carotenoid‐supplemented hens were less immunosuppressed than those originating from carotenoid‐supplemented + GnRH‐challenged hens, which were less immunosuppressed than chicks from GnRH‐challenged females not supplemented with carotenoids. Females availability of carotenoid enriched diets allows them to enhance the development of offspring immune system via carotenoids and vitamins deposited in egg yolks and offset detrimental effects of androgens deposited by GnRH‐challenged females.     

Carotenoid supplementation and GnRH challenges influence female endocrine physiology, immune function, and egg-yolk characteristics in Japanese quail (Coturnix japonica)

Androgens and carotenoids circulating in plasma affect the physiology and behavior of vertebrates. Much is known about control mechanisms and functions of each of these substances, yet their interactive effects are not well understood. Here we examine possible additive, multiplicative, and interactive effects of testosterone and carotenoids on female endocrine physiology, immunocompetence, and investment in eggs by simultaneously manipulating levels of testosterone [via gonadotropin releasing hormone (GnRH) challenges] and carotenoids (via diet supplementation) in captive female Japanese quail (Coturnix japonica). Females were randomly assigned to one of four treatments: carotenoid supplementation, GnRH challenge, GnRH challenge?+?carotenoid supplementation, or control. Carotenoid supplementation significantly increased circulating plasma carotenoid levels and acquired immune system performance, but not innate immunity. GnRH challenges elevated circulating testosterone and carotenoid levels, and induced immunosuppression in females. However, females in the GnRH challenge?+?carotenoid supplementation treatment had higher cell-mediated immune responses than control females and similar responses to those of carotenoid-supplemented females. Hence, availability of carotenoids in female quail seemed to counteract immunosuppressive effects of GnRH challenges. Our results provide further evidence for synergistic effects of carotenoids and testosterone on endocrine physiology and immune function in female birds. Elevated plasma testosterone or carotenoids levels resulted in increased deposition of those compounds to eggs, respectively. Furthermore, because we found that concentrations of testosterone and carotenoids in yolks were correlated within each treatment group, differential deposition of hormones and carotenoids in eggs may not only respond to surrounding social and environmental conditions, but also to other components of the egg.     

Do group size and laying order influence maternal deposition of testosterone in smooth-billed ani eggs?

The avian egg contains maternal hormones that affect behavior, growth, morphology, and offspring survival. Evidence to date suggests that patterns of yolk androgen deposition could provide females with a means to manipulate sibling competition and, thereby, increase their fitness. We examined yolk testosterone (T) concentrations in eggs of the smooth-billed ani (Crotophaga ani) to understand patterns of androgen deposition in eggs of this plural-breeding joint-nesting cooperatively breeding species. We tested the hatching asynchrony adjustment hypothesis, which states that increases in yolk androgen levels over the laying sequence function to mitigate the disadvantage of being a later-hatched chick in species without adaptive brood reduction. We also investigated the effect of group size on yolk T deposition to test the hypothesis that females in multi-female groups could give a competitive edge to their own chicks by depositing higher T levels in their eggs. Predictions of the hatching asynchrony adjustment hypothesis were supported in both single- and multi-female groups as yolk testosterone levels increased from early- to late-laid eggs. This suggests that ani females can influence nestling competition and chick survival by within-clutch differential T allocation. Unexpectedly, we did not observe an effect of group size on yolk T deposition. Yolk testosterone concentrations may not be a mere reflection of a female's hormonal status as female plasma circulating levels of T did not vary in the same direction as yolk T levels. Results of this study therefore support the idea that females may adaptively manipulate chick behavior through hormonal deposition in eggs.     

Multiple pathways of maternal effects in black-headed gull eggs: constraint and adaptive compensatory adjustment

We investigated in the black-headed gull whether female deposition of antioxidants and immunoglobulins (enhancing early immune function), and testosterone (suppressing immune function and increasing early competitive skills) correlate suggesting that evolution has favoured the mutual adjustment of different pathways for maternal effects. We also took egg mass, the position of the egg in the laying sequence and offspring sex into account, as these affect offspring survival. Yolk antioxidant and immunoglobulin concentrations decreased across the laying order, while yolk testosterone concentrations increased. This may substantially handicap the immune defence of last-hatched chicks. The decrease in antioxidant levels was greater when mothers had a low body mass and when the increase in testosterone concentrations was relatively large. This suggests that female black-headed gulls are constrained in the deposition of antioxidants in last-laid eggs and compensate for this by enhanced testosterone deposition. The latter may be adaptive since it re-allocates the chick's investment from costly immune function to growth and competitive skills, necessary to overcome the consequences of hatching late from an egg of reduced quality.     

Negative effects of yolk testosterone and ticks on growth in canaries

Maternal yolk hormones in bird eggs are thought to adjust the offspring to the post-hatching environment. This implies that the effects of maternal yolk hormones should vary with the post-hatching environment, but to date such context-dependency has largely been ignored. We experimentally increased yolk testosterone concentrations in canary eggs and simultaneously manipulated the post-hatching context via an experimental tick-infestation of the chicks. This allows us to evaluate the context-dependency of hormone-mediated maternal effects, as it has previously been shown that ectoparasites alter the maternal yolk androgen deposition. The experimental tick infestation reduced growth in chicks from sham-treated eggs, indicating harmful effects of this ectoparasite in canaries. Chicks from testosterone-treated eggs were not affected in their development by ticks, suggesting lower ectoparasite vulnerability. But this may also be due to the fact that experimentally elevated yolk testosterone levels impaired growth even under parasite-free conditions. This contrasts previous studies, but these studies often manipulated first laid eggs, while we used eggs of subsequent laying positions. Later laid eggs are presumably of lower quality and contain higher yolk testosterone concentrations. Thus, the effects of elevated yolk testosterone on growth may be dose-dependent or vary with the egg quality, suggesting prenatal context-dependency.     

Maternal adjustment or constraint: differential effects of food availability on maternal deposition of macro‐nutrients,steroids and thyroid hormones in rock pigeon eggs

In oviparous species like birds, eggs provide the direct environment in which embryos are developing. Mothers may adjust different egg components in different ways in reaction to environmental cues either to adjust offspring development or because of constraints. In this study, we investigated the effects of food quality and quantity before and during egg laying on three different aspects of egg quality: macro‐nutrients (egg and yolk mass), androgens (testosterone and androstenedione), and thyroid hormones (3,5,3′‐triiodothyronine, T3 and l ‐thyroxine, T4), using the rock pigeon (Columba livia). As expected, egg and yolk mass were significantly reduced for the eggs laid under the poor‐food condition, indicating a maternal trade‐off between offspring and self in allocating important resources. We did not find any significant change in yolk testosterone or their within‐clutch pattern over the laying sequence. This is consistent with the fact that, in contrast with nutrients, these hormones are not costly to produce, but does not support the hypothesis that they play a role in adjusting brood size to food conditions. In contrast, we found that T3 levels were higher in the egg yolks under the poor‐food condition whereas the total T4 content was lower. This change could be related to the fact that iodine, the critical constituent of thyroid hormones, might be a limiting factor in the production of this hormone. Given the knowledge that food restriction usually lead to reduction of circulating T3 levels, our results suggested that avian mothers can independently regulate its concentrations in their eggs from their own circulation. The study demonstrates that environmentally induced maternal effects via the egg can be a result of a combination of constrained resources and unconstrained signals and that thyroid hormones might be an interesting case of both. Therefore, this hormone and the interplay of different maternal effects on the offspring phenotype deserve much more attention.     

Dietary daidzein influences laying performance of ducks (Anas platyrhynchos) and early post-hatch growth of their hatchlings by modulating gene expression

Our previous studies demonstrated that dietary supplementation of daidzein improves egg production in duck breeders during late periods of the laying cycle. The present study was aimed to clarify whether the growth of ducklings hatched from eggs laid by daidzein-treated hens would be affected, and to elucidate the mechanisms underlying potential trans-generational effects, by determining changes of hormone levels and mRNA expression of relevant genes. Daidzein was added to the basal diet of 415-day-old duck breeders at the level of 5 mg/kg. During 9 weeks of daidzein treatment, laying rate increased by 7.70%, average egg mass tended to increase, whereas yolk/albumen ratio decreased significantly. These changes were accompanied by significantly elevated plasma T4 and E2 levels, enhanced gonadotropin releasing hormone (GnRH) mRNA, but diminished estrogen receptor (ER)-beta mRNA expression in hypothalamus of daidzein-treated hens. Ducklings hatched from daidzein-treated eggs were significantly smaller at hatching, but they caught up with their control counterparts by 4 weeks of age. Serum levels of T4, pituitary GH, hepatic GH receptor (GHR) and type-1 IGF receptor (IGF-1R) mRNA expression were all suppressed markedly in the daidzein-treated group at hatching, but this suppression proved to be temporary, as at 4 weeks of age, expression levels of all investigated genes were restored. However, it is noteworthy that at 4 weeks of age an obvious down-regulation of hypothalamic GnRH mRNA expression was detected in ducklings maternally exposed to daidzein. Our results provide evidence that maternal exposure to daidzein affects post-hatch growth in the duck with accompanying changes in the secretion of metabolic hormones and expression of growth-related genes. Although the negative effect of maternal daidzein on embryonic growth could be eliminated 4 weeks after hatching, the long-term effect of maternal daidzein on reproductive function is not to be ignored and awaits further investigation.     

The effect of maternal state on the steroid and macronutrient content of lesser black-backed gull eggs

It has been proposed that female birds can influence the phenotype of their offspring by provisioning eggs with variable amounts of nutrients and maternal hormones. Egg quality is strongly influenced by maternal body reserves and the amount of food available at the time of egg formation. This study investigated the effects of maternal state and food availability on the capacity of female lesser black-backed gulls Larus fuscus to provision their eggs with macronutrients and steroid hormones. Maternal state was reduced by increasing egg-production effort, whereas extra food was provided to reverse this effect. Compared with eggs of first clutches, eggs of experimentally induced replacement clutches exhibited a lower yolk/albumen ratio and contained more yolk testosterone. During one of the three years in which the study was performed, replacement eggs also contained more 17β-estradiol. Food provisioning during the relaying interval did not affect changes in yolk/albumen ratio or steroid concentrations, but fed females produced bigger eggs in their replacement clutch. This study demonstrates significant within-female consistency in egg size, macronutrient content, and yolk steroid concentration, and it shows that these egg characteristics are influenced by maternal state, food availability, and the timing of breeding.     

Consequences of prenatal androgen exposure for the reproductive performance of female pheasants (Phasianus colchicus)

Maternal hormones in vertebrate eggs can mediate important forms of maternal effects. However, the function of hormone transfer to the eggs is still debated, especially because long-term fitness consequences have been little studied. We investigated the effect of prenatal exposure to physiologically elevated yolk testosterone (T) levels on reproduction of female pheasants (Phasianus colchicus) in captivity. We found that females hatching from T-injected eggs (T-females) had a lower egg-laying rate than controls, and their eggs were more frequently infertile than those laid by control females. There were no effects of prenatal maternal treatment on egg size and yolk T concentration, but eggs carrying a female embryo laid by T-females had smaller yolks than eggs with a male embryo, while there was no sex difference in yolk size among the eggs laid by control females. Progeny sex ratio was unaffected by maternal treatment. These findings suggest that the transfer of high androgen levels to the eggs by the mother is constrained by complex trade-offs between direct effects on her daughters' reproduction and by trans-generational differential consequences on male and female descendants.     

Manipulative signals in family conflict? On the function of maternal yolk hormones in birds

Maternal hormones in the yolk of birds' eggs have been a focus of attention in behavioral and evolutionary ecology stimulated by the pioneering work of Hubert Schwabl. Since then, knowledge of both the factors that influence maternal deposition patterns and their consequences for offspring development has accumulated rapidly. To date, the field has been dominated by the idea that mothers use yolk hormones to adaptively adjust offspring development, a view that assigns control over hormone deposition and its effects on the offspring to the mother. This neglects the possibility that the evolutionary interests of the mother and offspring differ. When there is such parent-offspring conflict, the offspring are selected to respond to the hormones in a way that is adaptive for themselves rather than for the mother. Moreover, sexual conflict between the parents over parental investment may shape the evolution of yolk hormone deposition: females may manipulate the male's contribution to parental care through the effect of yolk hormones on offspring begging, competitiveness, and developmental rate. We therefore suggest that for a full understanding of the evolution of hormone-mediated maternal effects, it is essential to study both fitness consequences and physiological mechanisms and constraints from the perspective of all family members.     

Correlated evolution of maternally derived yolk testosterone and early developmental traits in passerine birds

Recent studies on hormone-mediated maternal effects in birds have highlighted the influence of variable maternal yolk androgen concentration on offspring phenotype, particularly in terms of early development. If genetic differences between laying females regulate variation in yolk hormone concentration, then this physiological maternal effect is an indirect genetic effect which can provide a basis for the co-evolution of maternal and offspring phenotypes. Thus, we investigated the evolutionary associations between maternally derived yolk testosterone (T) and early developmental traits in passerine birds via a comparative, phylogenetic analysis. Our results from species-correlation and independent contrasts analyses provide convergent evidence for the correlated evolution of maternal yolk T concentration and length of the prenatal developmental period in passerines. Here, we show these traits are significantly negatively associated (species-correlation: p<0.001, r2=0.85; independent contrasts: p=0.005). Our results highlight the need for more studies investigating the role of yolk hormones in evolutionary processes concerning maternal effects.     

Maternally derived testosterone and 17beta-estradiol in the eggs of Arctic-breeding glaucous gulls in relation to persistent organic pollutants

It is largely unknown if and how persistent organic pollutants (POPs) affect the transfer of maternal hormones to eggs. This occurs despite an increasing number of studies relating environmental conditions experienced by female birds at the time of egg formation to maternal hormonal effects. Here we report the concentrations of maternal testosterone, 17beta-estradiol and major classes of POPs (organochlorines, brominated flame retardants and metabolically-derived products) in the yolk of unincubated, third-laid eggs of the glaucous gull (Larus hyperboreus), a top-predator in the Arctic marine environment. Controlled for seasonal and local variation, positive correlations were found between the concentrations of certain POPs and testosterone. Contaminant-related changes in the relative concentrations of testosterone and 17beta-estradiol were also observed. In addition, yolk steroid concentrations were associated with contaminant profiles describing the proportions of different POPs present in the yolk. Eggs from nests in which two sibling eggs hatched or failed to hatch differed in POP profiles and in the relative concentrations of testosterone and 17beta-estradiol. Although the results of this correlative study need to be interpreted with caution, they suggest that contaminant-related changes in yolk steroids may occur, possibly affecting offspring performance over and above toxic effects brought about by POPs in eggs.     

Stress hormones: a link between maternal condition and sex-biased reproductive investment

In species where offspring fitness is sex-specifically influenced by maternal reproductive condition, sex allocation theory predicts that poor-quality mothers should invest in the evolutionarily less expensive sex. Despite an accumulation of evidence that mothers can sex-specifically modulate investment in offspring in relation to maternal quality, few mechanisms have been proposed as to how this is achieved. We explored a hormonal mechanism for sex-biased maternal investment by measuring and experimentally manipulating baseline levels of the stress hormone corticosterone in laying wild female European starlings (Sturnus vulgaris) and examining effects on sex ratio and sex-specific offspring phenotype adjustment. Here we show that baseline plasma corticosterone is negatively correlated with energetic body condition in laying starlings, and subsequent experimental elevation of maternal baseline plasma corticosterone increased yolk corticosterone without altering maternal condition or egg quality per se. Hormonal elevation resulted in the following: female-biased hatching sex ratios (caused by elevated male embryonic mortality), lighter male offspring at hatching (which subsequently grew more slowly during postnatal development), and lower cell-mediated immune (phytohemagglutinin) responses in males compared with control-born males; female offspring were unaffected by the manipulation in both years of the study. Elevated maternal corticosterone therefore resulted in a sex-biased adjustment of offspring quality favorable to female offspring via both a sex ratio bias and a modulation of male phenotype at hatching. In birds, deposition of yolk corticosterone may benefit mothers by acting as a bet-hedging strategy in stochastic environments where the correlation between environmental cues at laying (and therefore potentially maternal condition) and conditions during chick-rearing might be low and unpredictable. Together with recent studies in other vertebrate taxa, these results suggest that maternal stress hormones provide a mechanistic link between maternal quality and sex-biased maternal investment in offspring.     

Individual variation in testosterone and parental care in a female songbird; The dark-eyed junco (Junco hyemalis)

When competition for sex-specific resources overlaps in time with offspring production and care, trade-offs can occur. Steroid hormones, particularly testosterone (T), play a crucial role in mediating such trade-offs in males, often increasing competitive behaviors while decreasing paternal behavior. Recent research has shown that females also face such trade-offs; however, we know little about the role of T in mediating female phenotypes in general, and the role of T in mediating trade-offs in females in particular. Here we examine the relationship between individual variation in maternal effort and endogenous T in the dark-eyed junco, a common songbird. Specifically, we measure circulating T before and after a physiological challenge (injection of gonadotropin releasing hormone, GnRH), and determine whether either measure is related to provisioning, brooding, or the amount of T sequestered in egg yolk. We found that females producing more T in response to a challenge spent less time brooding nestlings, but provisioned nestlings more frequently, and deposited more T in their eggs. These findings suggest that, while T is likely important in mediating maternal phenotypes and female life history tradeoffs, the direction of the relationships between T and phenotype may differ from what is generally observed in males, and that high levels of endogenous T are not necessarily as costly as previous work might suggest.