Characterizing the timing of yolk testosterone metabolism and the effects of etiocholanolone on development in avian eggs.

Maternal transfer of steroids to eggs can elicit permanent effects on offspring phenotype. Although testosterone was thought to be a key mediator of maternal effects in birds, we now know that vertebrate embryos actively regulate their exposure to maternal testosterone through steroid metabolism, suggesting testosterone metabolites, not testosterone, may elicit the observed phenotypic effects. To address the role steroid metabolism plays in mediating yolk testosterone effects, we used European starling (Sturnus vulgaris) eggs to characterize the timing of testosterone metabolism and determine whether etiocholanolone, a prominent metabolite of testosterone in avian embryos, is capable of affecting early embryonic development. Tritiated testosterone was injected into freshly laid eggs to characterize steroid movement and metabolism during early development. Varying levels of etiocholanolone were also injected into eggs, with incubation for either 3 or 5 days, to test whether etiocholanolone influences the early growth of embryonic tissues. The conversion of testosterone to etiocholanolone was initiated within 12 h of injection, but the increase in etiocholanolone was transient, indicating that etiocholanolone is also subject to metabolism, and that exposure to maternal etiocholanolone is limited to a short period during early development. Exogenous etiocholanolone manipulation had no significant effect on the growth rate of the embryos or extra-embryonic membranes early in development. Thus, the conversion of testosterone to etiocholanolone may be an inactivation pathway that buffers the embryo from maternal steroids, with any effects of yolk testosterone resulting from testosterone that escapes metabolism; alternatively, etiocholanolone may influence processes other than growth or take additional time to manifest.

Ectoparasites as developmental stressors: Effects on somatic and physiological development.

Developmental stress can alter resource allocation in early life, and in altricial birds with rapid developmental trajectories and high resource demands, nestlings may adjust early resource partitioning to cope with challenging environments. We experimentally manipulated ectoparasite levels in nests and assessed whether ectoparasites affected somatic and physiological development in European starling (Sturnus vulgaris) nestlings. We hypothesized that mites act as developmental stressors in nestlings and predicted that nestlings from infested nests would exhibit either reduced somatic growth, or reduced physiological development, including impaired innate immunity, and would have elevated corticosterone concentrations. We either added ≈200 mites to nests during early incubation, or treated nests with a pesticide, permethrin, to reduce mites and possibly other arthropods. We assessed treatment effects on egg spottiness and mite abundance, and monitored offspring hatching and survival. We also measured somatic growth (mass, tarsus length, and feather growth), hematocrit, immune-related metrics (bacterial killing ability [BKA] and spleen mass), and baseline corticosterone concentrations in response to treatment. Compared with mite treatment, permethrin reduced egg spottiness and mite abundance in nests. Relative to nestlings in mite-reduced nests, nestlings in mite-enhanced nests had lower survival, hematocrit, and corticosterone concentrations. Early in development, nestlings from both treatments exhibited similar rapid somatic growth, yet mite-treated nestlings exhibited lower BKA. Nestlings in both treatments increased BKA across development, despite nestlings in mite-treated nests exhibiting lower mass as nest leaving neared. Overall, we found evidence that mites can act as development stressors, but contrary to our prediction, mites decreased corticosterone concentrations.

Blood-feeding ectoparasites as developmental stressors: Does corticosterone mediate effects of mite infestation on nestling growth, immunity and energy availability?

How resources are distributed to growth and self-maintenance early in life is likely to impact survival and reproduction. Early resource allocation decisions may be particularly critical in altricial birds, as they have rapid developmental trajectories, and may be highly susceptible to environmental factors that can perturb development. The aim of this study was to determine if blood-feeding ectoparasites act as developmental stressors in European starling (Sturnus vulgaris) nestlings, driving a trade-off between growth and immunity. We hypothesized that because ectoparasites compete for resources they would induce growth-immunity trade-offs in parasitized nestlings. We also tested the hypothesis that changes in plasma corticosterone mediate the effects of ectoparasites on growth and immunity. Throughout development we assessed between-nest variation in ectoparasite density, measured growth, and a variety of blood parameters, including plasma corticosterone. We also assessed immune function across development. We found that nestlings from nests with high levels of ectoparasites were smaller, had elevated blood glucose, lower hematocrit levels, and appeared to engage in compensatory growth prior to fledging. They also had elevated innate immune responses early, but reduced responses later relative to nestlings from nests with low levels of ectoparasites. Plasma corticosterone was not affected by ectoparasite load, but did increase with nestling age. Overall, we find evidence that ectoparasites are developmental stressors that affect growth-immunity trade-offs, but their effects do not appear to be mediated by changes in circulating levels of corticosterone.

Real-time monitoring of electrically evoked catecholamine signals in the songbird striatum using in vivo fast-scan cyclic voltammetry.

Fast-scan cyclic voltammetry is a powerful technique for monitoring rapid changes in extracellular neurotransmitter levels in the brain. In vivo fast-scan cyclic voltammetry has been used extensively in mammalian models to characterize dopamine signals in both anesthetized and awake preparations, but has yet to be applied to a non-mammalian vertebrate. The goal of this study was to establish in vivo fast-scan cyclic voltammetry in a songbird, the European starling, to facilitate real-time measurements of extracellular catecholamine levels in the avian striatum. In urethane-anesthetized starlings, changes in catecholamine levels were evoked by electrical stimulation of the ventral tegmental area and measured at carbon-fiber microelectrodes positioned in the medial and lateral striata. Catecholamines were elicited by different stimulations, including trains related to phasic dopamine signaling in the rat, and were analyzed to quantify presynaptic mechanisms governing exocytotic release and neuronal uptake. Evoked extracellular catecholamine dynamics, maximal amplitude of the evoked catecholamine signal, and parameters for catecholamine release and uptake did not differ between striatal regions and were similar to those determined for dopamine in the rat dorsomedial striatum under similar conditions. Chemical identification of measured catecholamine by its voltammogram was consistent with the presence of both dopamine and norepinephrine in striatal tissue content. However, the high ratio of dopamine to norepinephrine in tissue content and the greater sensitivity of the carbon-fiber microelectrode to dopamine compared to norepinephrine favored the measurement of dopamine. Thus, converging evidence suggests that dopamine was the predominate analyte of the electrically evoked catecholamine signal measured in the striatum by fast-scan cyclic voltammetry. Overall, comparisons between the characteristics of these evoked signals suggested a similar presynaptic regulation of dopamine in the starling and rat striatum. Fast-scan cyclic voltammetry thus has the potential to be an invaluable tool for investigating the neural underpinnings of behavior in birds.

The decline in yolk progesterone concentrations during incubation is dependent on embryonic development in the European starling.

Oviparous amniotes, particularly birds, have become model systems in which to study how mothers may utilize steroids to adaptively adjust offspring development. Although there is now ample evidence that maternally derived steroids in the egg at oviposition can influence offspring phenotype, very little is known about how these steroids elicit such effects. Of the major avian steroid hormones found in yolk, progesterone is by far the most abundant at oviposition, but has received little research attention to date. In this study, we examine the metabolism of [(3)H]-progesterone injected into freshly laid European starling eggs throughout the first 5 days of development by characterization of radioactivity within the egg homogenate. We also introduce a technique that utilizes a focal, freeze/thaw cycle to prevent embryonic development and allows us to assess the role of the embryo in metabolizing progesterone during early incubation. Two major findings result. First is that [(3)H]-progesterone is metabolized in eggs possessing a developing embryo, but not in eggs with disrupted embryonic development. Second is that the change in the distribution of radioactivity within eggs possessing an embryo is the result of metabolism of [(3)H]-progesterone to a more polar form that is subsequently conjugated. Together, these data suggest live embryos are necessary for metabolism of progesterone during early incubation, underscoring the potentially important contribution of embryos to functional modulation or mediation of maternal yolk steroid effects.

Embryonic modulation of maternal steroids in European starlings (Sturnus vulgaris).

In birds, maternally derived yolk steroids are a proposed mechanism by which females can adjust individual offspring phenotype to prevailing conditions. However, when interests of mother and offspring differ, parent-offspring conflict will arise and embryonic interests, not those of the mother, should drive offspring response to maternal steroids in eggs. Because of this potential conflict, we investigated the ability of developing bird embryos to process maternally derived yolk steroids. We examined how progesterone, testosterone and oestradiol levels changed in both the yolk/albumen (YA) and the embryo of European starling eggs during the first 10 days of development. Next, we injected tritiated testosterone into eggs at oviposition to characterize potential metabolic pathways during development. Ether extractions separated organic and aqueous metabolites in both the embryo and YA homogenate, after which major steroid metabolites were identified. Results indicate that the concentrations of all three steroids declined during development in the YA homogenate. Exogenous testosterone was primarily metabolized to an aqueous form of etiocholanolone that remained in the YA. These results clearly demonstrate that embryos can modulate their local steroid environment, setting up the potential for parent-offspring conflict. Embryonic regulation must be considered when addressing the evolutionary consequences of maternal steroids in eggs.

Seasonal and individual variation in response to GnRH challenge in male dark-eyed juncos (Junco hyemalis).

Concentrations of gonadal steroids such as testosterone (T) often vary widely in natural populations, but the causes and particularly the consistency of this variation is relatively unexplored. In breeding males of a wild population of the dark-eyed junco (Junco hyemalis), we investigated seasonal and individual variation in circulating T during two breeding seasons by measuring the responsiveness of the HPG axis to a standardized injection of gonadotropin-releasing hormone (GnRH). Individuals were bled prior to and 30min after injection. Pre- and post-challenge levels of T were measured using EIA. Many subjects were sampled repeatedly across multiple breeding stages. Plasma T concentrations nearly doubled in response to GnRH during early spring, but showed significantly smaller increases in later breeding stages. When controlling for seasonal variation in response to challenge, we also found repeatable differences among individuals, indicating individual consistency in the release of T in response to a standardized stimulus. These seasonal and individual differences may arise from comparable variation in responsiveness of the pituitary or a decline in gonadal sensitivity to downstream gonadotropins. In contrast, pre-challenge T showed almost no seasonal changes and did not differ consistently among individuals. To our knowledge, this is the first demonstration of individual repeatability of short-term hormonal changes in a wild population. Such repeatability suggests that hormonal plasticity might evolve in response to changing selection pressures.

Behavioral and physiological responses to experimentally elevated testosterone in female dark-eyed juncos (Junco hyemalis carolinensis).

Testosterone mediates the expression of many fitness-related traits in male vertebrates and is thought to account for numerous sex differences in trait expression. Testosterone is also secreted by females; however, far less is known regarding its effects on female physiology and behavior. Using a bird species in which the effects of testosterone on males are well characterized, the dark-eyed junco (Junco hyemalis), we tested whether an increase in exogenous testosterone in females would alter the phenotypic expression of a suite of behavioral and physiological traits. We found that increased testosterone levels in female dark-eyed juncos led to decreased cell-mediated immune function and increased intrasexual aggression, hypothalamo-pituitary-adrenal (HPA) axis responsiveness, baseline corticosterone and corticosterone-binding globulin (CBG) levels. Furthermore, immunosuppression following testosterone implantation was negatively correlated with total and free testosterone but did not appear to be related to either total or free corticosterone. These results demonstrate that the phenotypic impact of elevated testosterone is not confined to males in dark-eyed juncos, and that the impact in adults can be similar in males and females. We discuss these results in the context of potential endocrine-immune interactions and the evolution of sexual dimorphism.

Percutaneous cement injection into a created cavity for the treatment of vertebral body fracture: preliminary results of a new vertebroplasty technique.

OBJECTIVES: Vertebral body fractures (VBFs) are the most common complication of osteoporosis. Minimally invasive placement of cement to stabilize VBFs results in significant pain reduction and improved performance of daily activities. The authors describe a modified percutaneous vertebroplasty (PV) procedure during which a cavity is created manually in the VBF, allowing the cement to be injected with less resistance. METHODS: Data were gathered from a retrospective chart review from 15 consecutive patients with acute compression VBFs who underwent 33 PV procedures with the Cavity Creation System. Mean follow-up was 30 weeks. Oral opiate intake, quality of life improvement, and visual analog pain scores (VAS) were measured before and 1 month after the procedure. RESULTS: All 15 patients exhibited a reduction in pain VAS (mean reduction 5.9 +/- 2.5). Improvement in quality of life was demonstrated by lower (improved) FACIT scores in the General Activity, Enjoyment of Life, Mood, Normal Work Routine, and Sleep subscales. In addition, opioid use decreased in 10 of the 12 (83%) patients who were taking opioids before surgery. In eight (67%) patients, opioid use decreased by over 50%. Complications included extrusion of cement in two patients (an incidence of 5.7% of the levels operated) and two patients with intraoperative rib fractures. No postoperative neurologic deficits were noted. CONCLUSIONS: The Cavity Creation System is a safe, cost-effective treatment of VBF resulting in good/excellent pain relief and an improved quality of life.

Elevated testosterone reduces choosiness in female dark-eyed juncos (Junco hyemalis): evidence for a hormonal constraint on sexual selection?

Because testosterone (T) often mediates the expression of attractive displays and ornaments, in the absence of constraints sexual selection should lead to an evolutionary increase in male T levels. One candidate constraint would be a genetic correlation between the sexes that leads to a correlated response in females. If increased T in females were to have deleterious effects on mate choice, the effect of sexual selection on male T would be weakened. Using female dark-eyed juncos (Junco hyemalis), we tested whether experimentally enhancing female T would lead to a decrease in discrimination between two classes of males, one treated with T (T-males) and one control (C-males). The two female treatments (T-implanted and C-females) spent equal amounts of time with both classes of males, but T-treated females failed to show a preference for either male treatment, whereas C-females showed a significant preference, albeit in an unexpected direction (for C-males). T-females were less discriminating than C-females, irrespective of the direction of their preference. To our knowledge, this is the first study to show that circulating hormones can alter female choosiness without reducing sexual motivation. Our results suggest that hormonal correlations between the sexes have the potential to constrain sexual selection on males.

Consequences of elevating plasma testosterone in females of a socially monogamous songbird: evidence of constraints on male evolution?

To explore whether selection for testosterone-mediated traits in males might be constrained by costs of higher testosterone to females, we examined the effects of experimental elevation of plasma testosterone on physiological, reproductive, and behavioral parameters in a female songbird, the dark-eyed junco (Junco hyemalis). We used subcutaneous implants to elevate testosterone (T) in captive and free-living female juncos. In captive birds, we measured the effects of high T on body mass, feather molt, and brood patch formation. In the field, we monitored its effects on the timing of egg laying, clutch size, egg size, egg steroid levels, incubation, and nest-defense behavior. Females implanted with testosterone (T-females) had significantly higher circulating levels of testosterone than did control females (C-females). Captive T-females had lower body mass, were less likely to develop brood patches, and delayed feather molt relative to C-females. Among free-living females, the interval between nest completion and appearance of the first egg was longer for T-females than for C-females and egg yolk concentrations of testosterone were higher, but there were no significant differences in estradiol levels, clutch size, or egg size. Incubation and nest defense behavior were also similar between T- and C-females. Our results suggest that selection on males for higher testosterone might initially lead to a correlated response in females producing changes in body mass and feather molt, both of which could be detrimental. Other possible female responses would be delayed onset of reproduction, which might reduce reproductive success, and higher yolk testosterone, which might have either positive or negative effects on offspring development. We found no reason to expect reduced parental behavior by females as a negative fitness consequence of selection for higher testosterone in males.

Play, copulation, anatomy, and testosterone in gonadally intact male rats prenatally exposed to flutamide.

The role of prenatal androgen on the differentiation of sexually dimorphic juvenile play and adult copulatory patterns was evaluated in male offspring of rats injected with 5 mg of the androgen receptor blocker flutamide (4'-nitro-3'-trifluoromethylisobutyranilide) from Days 11-21 of pregnancy. Rough-and-tumble play was incompletely masculinized in flutamide-exposed males at 31 days of age. The copulatory potential tested at 70 days of age was severely attenuated by prenatal flutamide. There was no ejaculatory behavior, low levels of intromissions, and depressed levels of nonintromittive mounting when the animals were tested while gonadally intact. Adult plasma levels of testosterone (T) were not different in flutamide-exposed males and controls, but testicular and epididymal weight, anogenital (AG) distance, and penile length were reduced. While reductions in intromittive mounting and ejaculatory behavior may be due to the abnormalities in the external genitalia, the incomplete masculinization of play and the reduction in nonintromittive mounting probably resulted from effects the androgen antagonist exerted on sexual differentiation of the central nervous system. These data suggest that androgen released prior to birth is needed for the full masculinization of juvenile play behaviors in the rat, just as it is for the adult copulatory pattern.