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The effect of transdermal corticosterone application on plasma corticosterone levels in pregnant Lacerta vivipara
Comparative Biochemistry and Physiology Part A 134 (2003) 497–503
The effect of transdermal corticosterone application on plasma corticosterone levels in pregnant Lacerta vivipara S. Meylana,*, A.M. Duftyb, J. Cloberta a
b
Laboratoire d’Ecologie, CNRS-UMR 7625, Universite´ P. et M. Curie, Paris, France Biology Department, Boise State University, 1910 University Drive, Boise, ID 83725, USA
Received 5 June 2002; received in revised form 2 September 2002; accepted 10 November 2002
Abstract Relationships between hormones and behaviour can be explored by altering endogenous hormone levels, often through implantation of silastic tubing or osmotic pumps filled with a hormone or its agonists or antagonists. However, organisms in sensitive life-history stages (such as pregnancy) may be adversely affected by the surgical procedures associated with these manipulations, necessitating use of non-invasive techniques. We demonstrate that the application of a sesame oil– corticosterone mixture to the skin of pregnant female common lizards (Lacerta vivipara) elevates plasma levels of the hormone. Pregnant female L. vivipara were captured and treated daily for 1–20 days with the sesame oil–corticosterone mixture (experimental group) or with vehicle only (control group). Blood samples were collected and analyzed for corticosterone by radioimmunoassay. Baseline plasma corticosterone levels were elevated within 1 h in the experimental group. Similar levels (f145 ngyml) were found over the subsequent 2 days, and by day 5 had risen significantly higher (f281.9 ngyml), where they remained for the duration of the experiment. These increases are comparable to those found in other species using related techniques. No significant changes in plasma corticosterone levels occurred in the control group. Finally, corticosterone levels also were determined for untreated females that were captured, held overnight, sampled, and released to access to the natural range of basal corticosterone levels. Basal plasma levels of corticosterone in pregnant females varied among individuals independently of female body size or corpulence. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Corticosterone; Maternal hormones; Transdermal application; Non-invasive method; Radioimmunoassays; Lacerta vivipara; Common lizard
1. Introduction Corticosteroid hormones are secreted by the adrenal glands as an adaptive response to stressful stimuli and, in reptiles, corticosterone is the major adrenal corticosteroid (Greenberg and Wingfield, 1987). Adrenocorticosteroid hormones are involved in the regulation of metabolism, the *Corresponding author. Tel.: q1-44-273-145; fax: q1-44273-516. E-mail address: [email protected] (S. Meylan).
immune system, and behaviour, and manipulating plasma corticosterone levels is fundamental to experimental studies of behavioural endocrinology. Steroid hormone supplementation generally is achieved through injections (Summers et al., 2000) or through chronic implantation of hormone-filled silastic tubing (DeNardo and Sinervo, 1994a,b) or hormone pellets (Tokarz, 1987). This can raise circulating levels of corticosterone for a period of days to weeks, and is convenient because implants can be given to free-living animals. However, the associated surgical procedure may be stressful
1095-6433/03/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 2 . 0 0 3 4 3 - 4
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S. Meylan et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 497–503
itself (Knapp and Moore, 1997; Breuner et al., 1998), especially during life-history stages in which organisms are sensitive to stressors, such as pregnancy. Our research focuses on the effects of maternal corticosterone levels during gestation on the morphology and behaviour of juvenile phenotypes. We use the common lizard (Lacerta vivipara) as our model system because previous studies found that offspring phenotype is influenced by the maternal environment (Sorci et al., 1994; Massot and Clobert, 1995; Lorenzon et al., 1999), maternal age ´ (Ronce et al., 1998) and maternal condition (Lena and de Fraipont, 1998; de Fraipont et al., 2000; Meylan et al., 2002). Corticosterone can act as a cue for developing offspring, providing information regarding both maternal condition and the external environment (Dufty et al., 2002). Thus, to tease apart the relationship between the maternal environment and offspring phenotype, experimental manipulation of corticosterone is important. However, in L. vivipara such work requires a noninvasive method to increase corticosterone levels because an exploratory experiment that manipulated the corticosterone levels of pregnant females (de Fraipont et al., 2000) showed that inserting subcutaneous silastic implants caused increased mortality in embryos and adults and may have affected the behaviour of the surviving individuals. Reptilian skin and many of its secretions contain lipids (Mason, 1992) and lipophilic molecules, such as steroids, can cross lizard skin readily. Consequently, we applied a mixture of sesame oil and corticosterone nightly to the skin of pregnant female common lizards. We modified the method of Knapp and Moore (1997), who delivered corticosterone transdermaly using a dermal patch fastened to the skin of tree lizards (Urosaurus ornatus). We did not use dermal adhesive bandages. The aim of the present study was to determine the effects of the transdermal application of corticosterone on circulating corticosterone levels of the pregnant females in the common lizard. It is important to know the consequences of such manipulations on circulating levels of the hormone because pharmacological doses could produce misleading results by disrupting cell processes, inducing neuronal death, or increasing susceptibility to disease (Axelrod and Reisine, 1984; Greenberg and Wingfield, 1987). Specifically, we sought to verify that the daily application of corticosterone
to the skin of females elevates and maintains their plasma corticosterone levels. 2. Materials and methods 2.1. The species The common lizard is a small lacertid (adult’s snout-vent lengths50–60 mm, juvenile’s snoutvent lengths18 mm on average), widely distributed across Europe and Asia. We studied ` (1500 m, Massif populations on Mont–Lozere Central, South-eastern France, 448009N, 38459E), where males emerge from hibernation in midApril, followed by yearlings and females in midMay. Mating takes place as soon as females emerge from hibernation (Pilorge et al., 1987). After 2 months of gestation, females lay a clutch, on average, of five soft-shelled eggs. Offspring hatch within 1 h of oviposition. On 20 June 2001, we captured 61 pregnant females and kept them in the laboratory until parturition (usually at the beginning of August). Pregnant females are easily discernible by visual inspection or gentle palpation of their abdomen. Each lizard was housed in individual plastic terrarium (18=12=12 cm3) with 1 cm of damp soil. A basking site and a shelter permitted thermoregulation. Terraria were stored in a room at ambient temperature (22–30 8C) and exposed to natural daylight. Terraria were also heated 6 h per day with an incandescent light bulb (25 W) at 20 cm above the terrarium (peak temperature of 30 8C). We also provided water ad libidum and offered food (Pyralis farinalis larvae) once per week. Housing conditions were in accord with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The females were measured (snout-vent length) and weighed weekly. Corpulence was calculated as the residual of the regression of body mass against snout-vent length. There was no mortality and all females laid clutches. Females were treated either with exogenous corticosterone or with vehicle alone (see below). After the experiment, mothers and offspring were returned to their original population at the capture point of the mother. Inter-individual variation in basal corticosterone levels also was determined. We captured 34 additional pregnant females on the 25 June, 2001. These individuals were kept overnight in the laboratory and blood samples were taken the next
S. Meylan et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 497–503
afternoon, after which the females were released at their site of capture. Dauphin-Villemant and Xavier (1987) noted that 1 h of confinement induced elevated corticosterone levels in nonreproductive L. vivipara. After 18 h of confinement corticosterone levels were lower, but were still more variable than those of control animals and had not quite returned to baseline. Thus, by sampling blood after 24 h of captivity, corticosterone levels in control animals may still have been slightly more variable and slightly higher than those found in free-living lizards. 2.2. Hormonal treatment We manipulated circulating levels of corticosterone using a non-invasive method modified from Knapp and Moore (1997). Corticosterone was delivered transdermally to the lizards in a mixture of the steroid hormone and sesame oil. We did not use dermal adhesive bandages as done by Knapp and Moore (1997) to decrease the risk of stress associated with the application, presence, and removal of a patch. Lacerta vivipara is very similar in size and weight to the tree lizards used by Knapp and Moore (1997), so we used the same corticosterone concentration. We diluted corticosterone (Sigma C2505) in commercial pure sesame oil (3 mg corticosteroney1 ml sesame oil). We applied the hormone solution nightly (4.5 ml with a pipette) to the backs (between the two shoulders) of the treatment group (ns36) for 1–20 days, after which a blood sample was taken. Control females (ns25) were treated similarly, but with the oil vehicle only. Both the oil and the oily hormone mixture were completely absorbed within 2–3 h of application. We treated the animals during the night for two reasons. First, the temperature in the terraria decreased during the night so the females were less active and more easily handled. Second, lower temperature reduced the risk of evaporation of the solution before it crossed the skin. 2.3. Blood sampling Blood samples (40–80 ml whole blood) were collected from the post-orbital sinus using 2–3 50 ml microhematocrit tubes. Blood sampling was conducted between 25 June and 15 July 2001. All samples were collected within 3 min of removal of an animal from its home cage to avoid a
499
Table 1 The sample size used in each of the experimental treatment groups Blood sample periods
1h Day Day Day Day Day Day
1 2 5 10 15 20
Sample size Placebo
Corticosterone
4 4 3 3 3 4 4
4 5 5 5 5 5 7
handling-induced increase in plasma corticosterone levels (Dauphin-Villemant and Xavier, 1987). To reduce variability in corticosterone levels due to diurnal rhythms in its secretion, we collected all blood samples between 16:00 and 17:30 h. To determine the effect of the transdermal corticosterone application on circulating corticosterone levels we took blood from 3 to 5 lizards of each group (placebo and corticosterone; Table 1) at the following times: 1 h after the first treatment, and after 1, 2, 5, 10, 15 and 20 days of treatment. Each individual was sampled only once. Blood samples were centrifuged and the plasma harvested and stored at y20 8C until transported on dry ice (4 kg, y80 8C) to Paris, where they were maintained at y80 8C. The samples subsequently were transported to the radioimmunoassay laboratory, also on dry ice, where they were kept at y20 8C until assayed. 2.4. Hormone assays Corticosterone values were determined in a single radioimmunoassay following procedures described by Wingfield et al. (1992). In brief, a small amount of labelled corticosterone was added to all sample test tubes and to each of two test tubes containing a standard amount of corticosterone to determine loss of sample during the extraction process. Corticosterone was extracted from 10 to 30 ml of plasma in 4 ml of freshly distilled dichloromethane, dried under nitrogen gas, and resuspended with 550 ml of buffer. We placed 200 ml of this solution into duplicate assay tubes and placed another 100 ml into a vial with scintillant. The latter vials provided an estimate of the percentage of steroid recovered after extraction. Mean recovery was 87%. A standard curve, consisting
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varied among individuals (Fig. 1). The mean corticosterone level was 21.64"4.52 ngyml (ns 34). The maximum baseline corticosterone level in these females was 101.97 ngyml. However, most levels were well below this, as the median level was 10.58. There was neither an effect of female body size (F1,33s0.03, Ps0.87) nor of female corpulence (F1,33s0.001, Ps0.98) on circulating basal corticosterone levels. Fig. 1. Natural variation in circulating corticosterone levels among individual female Lacerta vivipara.
of nine serially-diluted points (starting at 2000 pgy100 ml) was prepared in duplicate assay tubes. Each sample and standard curve tube received 100 ml of labeled corticosterone and 100 ml of corticosterone antibody (Endocrine Sciences) and was allowed to incubate overnight at 4 8C. After treatment with dextran-coated charcoal for 10 min to remove the unbound fraction, tubes were centrifuged at 2000 rpm for an additional 10 min whereupon the supernatant was decanted into scintillation vials, scintillant added, and the vials were counted on a Beckman LS-6500 scintillation counter the following day. Non-specific binding of the antibody was less than 5%. Two water blanks measured undetectable levels of corticosterone. Two corticosterone standards, each of 1000 pg, produced values that averaged 1120 pg (S.E.s 43.84).
3.2. Circulating treatment
corticosterone
levels
after
There was a significant effect of transdermal corticosterone application on plasma corticosterone levels (F1,55s25.8, P-0.0001). Circulating corticosterone levels were higher in corticosteronetreated females than in control females (Fig. 2), and the difference was seen within 1 h of treatment. In addition, treatment duration affected the hormone levels (F1,34s4.81, Ps0.035). Elevated corticosterone levels occurred within 1 h of the first transdermal application and were maintained during the following 2 days of treatment. However, beginning at day 3 and continuing until day 20 (when treatment ended), circulating corticosterone levels were significantly higher than those measured during the first 2 days of treatment, averaging 281.9 ngyml (Fig. 2). Corticosterone levels of control females remained low and did not increase
2.5. Statistics Statistical analyses were performed using SAS software (SAS Institute, 1996). Plasma concentrations of corticosterone were compared according to the treatment and the duration of the treatment. We also introduced individual covariables into the statistical model, using GLM procedure (ANOVA). We used type III sum of squares (nonsequential decomposition). We started with a general model including all the potential effects and their interactions. We then dropped the nonsignificant effects, starting with the most complex interaction terms. Only the results of the final model are reported. 3. Results 3.1. Natural variation in corticosterone levels among individuals Basal plasma levels of corticosterone in pregnant female L. vivipara captured and held overnight
Fig. 2. Effect of transdermal corticosterone application on the circulating level of corticosterone in pregnant female lizards. B, experimental (corticosterone) treatment; P, placebo treatment. There was a significant effect of the treatment on corticosterone levels (ns60, Fs25.8, P-0.0001). For corticosterone-treated females, there was a significant effect of the duration of the hormonal treatment on circulating corticosterone levels in pregnant females (ns35, Fs4.81, Ps0.035).
S. Meylan et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 497–503
over the course of the treatment (F1,24s3.39, Ps 0.078), although there was a trend toward increased corticosterone levels. As was the case with females held overnight, there was considerable variation in corticosterone levels among corticosterone-treated females. We investigated whether these differences were due to differences in individual morphological characteristics by including body size and corpulence (i.e. mass corrected for size) in the statistical model. There was neither an effect of female body size (F1,55s0.24, Ps0.62) nor of female corpulence (F1,55s0.31, Ps0.58) on circulating corticosterone levels. 4. Discussion Our results demonstrated that the transdermal application of corticosterone significantly increases circulating corticosterone levels within 24 h. We used this non-invasive technique because of the small size of our study organism and because previous experience suggested that such treatments would be stressful. This technique does not require surgery and limits the undesirable impacts of hormone manipulation on the females and their offspring. Pregnant females treated with corticosterone experienced no mortality and no abortion during the experiment, although such losses had occurred during earlier experiments using more invasive techniques (de Fraipont et al., 2000). Mean corticosterone levels in the untreated pregnant females that were captured and held overnight prior to sampling were only slightly higher and slightly more variable than those of non-reproductive females housed by Dauphin-Villemant and Xavier (1987) in large terraria designed to mimic field conditions (21.64"4.52 ngyml vs. 14.4"2.3 ngyml, respectively). Since corticosterone levels in pregnant females tends to be higher than those in non-reproductive females (Dauphin-Villemant et al., 1990), this sample of pregnant females may represent baseline values, although this must be verified by sampling free-living lizards immediately after capture. Corticosterone levels increased within 1 h of the first corticosterone application and remained at similar levels (f145"32 ngyml) over the subsequent 2 days of treatment. By day 5 of the treatment corticosterone concentrations rose to 281.9 ngyml, where they remained for the remainder of the experiment. These values represent approximately a 12-fold increase over mean base-
501
line levels and are 2.8 times the maximum baseline value measured in controls. However, the difference in corticosterone levels between the placeboand corticosterone-treated females in our study may have been affected by diurnal factors. That is, Dauphin-Villemant and Xavier (1987) found that basal corticosterone levels in captive, active Lacerta females (i.e. those sampled during the day when a heating light was turned on) were approximately 50 ngyml. This is 3-fold higher than the levels they found in quiescent females sampled in the late afternoon when the heating lights were turned off. We sampled all our captive Lacerta females in the late afternoon, and the corticosterone values we measured in placebo-females were similar to those obtained at a comparable time by Dauphin-Villemant and Xavier (1987). Thus, by sampling females during their inactive period, we may have maximized the differences in corticosterone levels measured in the two treatment groups. Similarly-sized induced increases in baseline plasma corticosterone levels also have been found in other reptile species. For example, Knapp and Moore (1997) increased plasma corticosterone concentrations by approximately 13-fold with transdermal corticosterone application, and Tyrrell and Cree (1998) observed that 3 h of confinement increased corticosterone levels more than 10-fold in Sphenodon punctatus. Although these results are consistent with the increases we induced in L. vivipara by transdermal application of corticosterone, it remains to be seen if similar increases are produced by natural stressors. Increases in baseline plasma corticosterone levels in L. vivipara may be a normal part of their reproductive cycle, for the increased energy demands of parturition may require elevated corticosterone levels to help mobilize energy stores, and elevated baseline corticosterone levels may help to regulate the timing of parturition. For example, Dauphin-Villemant et al. (1990) observed endogenous increases in plasma corticosterone levels in untreated, captive females over the course of gestation, with corticosterone levels reaching 80 ngyml just prior to parturition. We did not find such increases in basal corticosterone levels in our control females, although we may have stopped sampling the animals before the changes became evident. Furthermore, Lacerta adrenal glands remain sensitive to stimulation by ACTH during the reproductive period (Dauphin-
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Villemant et al., 1990), demonstrating that additional increases in corticosterone secretion above baseline levels can occur, such as those generated by stressors. Our results show that transdermal application of corticosterone can produce such increases in pregnant females without disrupting gestation. The sensitivity of the hypothalamo-pituitaryadrenal axis varies among individuals. For example, corticosterone level can fluctuate with intrinsic body condition (Wilson and Wingfield, 1992; Dauphin-Villemant and Xavier, 1987) and with age (Wilson and Wingfield, 1992). However, in our case, circulating corticosterone levels were related neither to female size nor to female corpulence. This is similar to the findings of Cash et al. (1997), who found no correlation between basal plasma corticosterone levels and energetic condition. Furthermore, in L. vivipara, size increases with age until the former reaches a plateau (Ronce et al., 1998, 90% of size variation is explained by age: Massot, unpublished data). Thus, within limits, our data also suggest that corticosterone levels do not vary systematically with age in this species. Therefore, other explanations must be sought to explain differences in baseline corticosterone levels among individual L. vivipara, such as differences in genotype, in previous experience, or a lingering effect of captivity. Our laboratory is using the transdermal application of corticosterone to pregnant L. vivipara to explore the consequences of stress during gestation on females and on offspring phenotype. We have already demonstrated, in a previous corticosterone manipulation using silastic implants (de Fraipont et al., 2000), that the elevation of corticosterone in pregnant females affects their behaviour, with corticosterone-treated females more active than placebo females. We currently are investigating whether maternal stress is perceived by developing young and, if so, how this information affects their behaviour and life-history traits (Meylan et al., 2002). Acknowledgments ´ We are grateful to the ‘Parc national des Cevˆ ennes’ and the ‘Office National des Forets’ for providing facilities during our field season, a PhD grant from the French Ministry of Research and Education, a grant from the Idaho State Board of
Education, and a Faculty Research Grant from Boise State University. References Axelrod, J., Reisine, T.D., 1984. Stress hormones: their interaction and regulation. Science 224, 452–459. Breuner, C.W., Greenberg, A.L., Wingfield, J.C., 1998. Noninvasive corticosterone treatment rapidly increases activity in Gambel’s white-crowned sparrows. Gen. Comp. Endocrinol. 111, 386–394. Cash, W.B., Holberton, R.L., Knight, S.S., 1997. Corticosterone secretion in response to capture and handling in freeliving red-eared slider turtles. Gen. Comp. Endocrinol. 108, 427–433. Dauphin-Villemant, C., Xavier, F., 1987. Nychthemeral variations of plasma corticosteroids in captive female Lacerta vivipara Jacquin: influence of stress and reproductive state. Gen. Comp. Endocrinol. 67, 292–302. Dauphin-Villemant, C., Leboulenger, F., Vaudry, H., 1990. Adrenal activity in the female lizard Lacerta vivipara Jacquin during artificial hibernation. Gen. Comp. Endocrinol. 79, 201–214. de Fraipont, M., Clobert, J., John-Alder, H., Meylan, S., 2000. Pre-natal maternal stress increases philopatry of offspring in common lizard (Lacerta vivipara). J. Anim. Ecol. 69, 404–413. DeNardo, D.F., Sinervo, B., 1994a. Effects of corticosterone on activity and home range size of free-living male lizards. Horm. Behav. 28, 53–62. DeNardo, D.F., Sinervo, B., 1994b. Effects of steroids hormone interaction on activity and home range size of free-living male lizards. Horm. Behav. 28, 273–287. Dufty Jr., A.M., Clobert, M., Møller, A.P., 2002. Hormones, developmental plasticity, and adaptation. Trends Ecol. Evol. 17, 190–196. Greenberg, N., Wingfield, J.C., 1987. Stress and reproduction: reciprocal relationships. In: Norris, D.O., Jones, R.E. (Eds.), Hormones and Reproduction in Fishes, Amphibians and Reptiles. Plenum Press, New York. Knapp, R., Moore, M.C., 1997. A non-invasive method for sustained elevation of steroid hormone levels in reptiles. Herpetol. Rev. 28, 33–36. ´ Lena, J.P., de Fraipont, M., 1998. Kin recognition in the common lizard. Behav. Ecol. Sociobiol. 42, 341–347. Lorenzon, P., Clobert, J., Oppliger, A., John-Alder, H., 1999. Effect of water constraint on growth rate, activity and body temperature of yearling common lizard (Lacerta vivipara). Oecologia 118, 423–430. Mason, R.T., 1992. Reptilian pheromones. In: Gans, C., Crews, D. (Eds.), Biology of the Reptilia, Vol. 18. University of Chicago Press, Chicago, IL, pp. 114–228. Massot, M., Clobert, J., 1995. Influence of maternal food availability on offspring dispersal. Behav. Ecol. Sociobiol. 37, 413–418. Meylan, S., Belliure, J., Clobert, J., de Fraipont, M., 2002. Stress and body condition as prenatal and postnatal determinants of dispersal in the common lizard (Lacerta vivipara). Horm. Behav. 42, 319–326.
S. Meylan et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 497–503 Pilorge, T., Clobert, J., Massot, M., 1987. Life history variations according to sex and age in Lacerta vivipara. In: van, G.J.J., Strijbosh, H., Bergers, P.M.J. (Eds.), Proceedings of the Fourth Ordinary General Meeting of the Societas Europe Herpetological. Faculty of Sciences, Nijmegen, pp. 32–39. Ronce, O., Clobert, J., Massot, M., 1998. Natal dispersal and senescence. Proceedings of the National Academy of Sciences USA, 95, pp. 600–605. SAS, 1996. SAS ySTAT software changes and enhancements through release 6.11. SAS Institute, Cary, North Carolina. Sorci, G., Massot, M., Clobert, J., 1994. Maternal parasite load increases sprint speed and philopatry in female offspring of the common lizard. Am. Nat. 144, 153–164. Summers, C.H., Larson, E.T., Ronan, P.J., Hofmann, P.M., Emerson, A.J., Renner, K.J., 2000. Serotonergic responses
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to corticosterone and testosterone in the limbic system. Gen. Comp. Endocrinol. 117, 151–159. Tokarz, R.R., 1987. Effects of corticosterone treatment on male aggressive behaviour in a lizard (Anolis sagrei). Horm. Behav. 21, 358–370. Tyrrell, C., Cree, A., 1998. Relationships between corticosterone concentration and season, time of day and confinement in a wild reptile (Tuatara, Sphenodon punctatus). Gen. Comp. Endocrinol. 110, 97–108. Wilson, B.S., Wingfield, J.C., 1992. Correlation between female reproductive condition and plasma corticosterone in the lizard Uta stansburiana. Copei 1992, 691–697. Wingfield, J.C., Vleck, C.M., Moore, M.C., 1992. Seasonal changes of adrenocortical response to stress in birds of sonoran desert. J. Exp. Zool. 264, 419–428.
The effect of transdermal corticosterone application on plasma corticosterone levels in pregnant Lacerta vivipara S. Meylana,*, A.M. Duftyb, J. Cloberta a
b
Laboratoire d’Ecologie, CNRS-UMR 7625, Universite´ P. et M. Curie, Paris, France Biology Department, Boise State University, 1910 University Drive, Boise, ID 83725, USA
Received 5 June 2002; received in revised form 2 September 2002; accepted 10 November 2002
Abstract Relationships between hormones and behaviour can be explored by altering endogenous hormone levels, often through implantation of silastic tubing or osmotic pumps filled with a hormone or its agonists or antagonists. However, organisms in sensitive life-history stages (such as pregnancy) may be adversely affected by the surgical procedures associated with these manipulations, necessitating use of non-invasive techniques. We demonstrate that the application of a sesame oil– corticosterone mixture to the skin of pregnant female common lizards (Lacerta vivipara) elevates plasma levels of the hormone. Pregnant female L. vivipara were captured and treated daily for 1–20 days with the sesame oil–corticosterone mixture (experimental group) or with vehicle only (control group). Blood samples were collected and analyzed for corticosterone by radioimmunoassay. Baseline plasma corticosterone levels were elevated within 1 h in the experimental group. Similar levels (f145 ngyml) were found over the subsequent 2 days, and by day 5 had risen significantly higher (f281.9 ngyml), where they remained for the duration of the experiment. These increases are comparable to those found in other species using related techniques. No significant changes in plasma corticosterone levels occurred in the control group. Finally, corticosterone levels also were determined for untreated females that were captured, held overnight, sampled, and released to access to the natural range of basal corticosterone levels. Basal plasma levels of corticosterone in pregnant females varied among individuals independently of female body size or corpulence. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: Corticosterone; Maternal hormones; Transdermal application; Non-invasive method; Radioimmunoassays; Lacerta vivipara; Common lizard
1. Introduction Corticosteroid hormones are secreted by the adrenal glands as an adaptive response to stressful stimuli and, in reptiles, corticosterone is the major adrenal corticosteroid (Greenberg and Wingfield, 1987). Adrenocorticosteroid hormones are involved in the regulation of metabolism, the *Corresponding author. Tel.: q1-44-273-145; fax: q1-44273-516. E-mail address: [email protected] (S. Meylan).
immune system, and behaviour, and manipulating plasma corticosterone levels is fundamental to experimental studies of behavioural endocrinology. Steroid hormone supplementation generally is achieved through injections (Summers et al., 2000) or through chronic implantation of hormone-filled silastic tubing (DeNardo and Sinervo, 1994a,b) or hormone pellets (Tokarz, 1987). This can raise circulating levels of corticosterone for a period of days to weeks, and is convenient because implants can be given to free-living animals. However, the associated surgical procedure may be stressful
1095-6433/03/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 5 - 6 4 3 3 Ž 0 2 . 0 0 3 4 3 - 4
498
S. Meylan et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 497–503
itself (Knapp and Moore, 1997; Breuner et al., 1998), especially during life-history stages in which organisms are sensitive to stressors, such as pregnancy. Our research focuses on the effects of maternal corticosterone levels during gestation on the morphology and behaviour of juvenile phenotypes. We use the common lizard (Lacerta vivipara) as our model system because previous studies found that offspring phenotype is influenced by the maternal environment (Sorci et al., 1994; Massot and Clobert, 1995; Lorenzon et al., 1999), maternal age ´ (Ronce et al., 1998) and maternal condition (Lena and de Fraipont, 1998; de Fraipont et al., 2000; Meylan et al., 2002). Corticosterone can act as a cue for developing offspring, providing information regarding both maternal condition and the external environment (Dufty et al., 2002). Thus, to tease apart the relationship between the maternal environment and offspring phenotype, experimental manipulation of corticosterone is important. However, in L. vivipara such work requires a noninvasive method to increase corticosterone levels because an exploratory experiment that manipulated the corticosterone levels of pregnant females (de Fraipont et al., 2000) showed that inserting subcutaneous silastic implants caused increased mortality in embryos and adults and may have affected the behaviour of the surviving individuals. Reptilian skin and many of its secretions contain lipids (Mason, 1992) and lipophilic molecules, such as steroids, can cross lizard skin readily. Consequently, we applied a mixture of sesame oil and corticosterone nightly to the skin of pregnant female common lizards. We modified the method of Knapp and Moore (1997), who delivered corticosterone transdermaly using a dermal patch fastened to the skin of tree lizards (Urosaurus ornatus). We did not use dermal adhesive bandages. The aim of the present study was to determine the effects of the transdermal application of corticosterone on circulating corticosterone levels of the pregnant females in the common lizard. It is important to know the consequences of such manipulations on circulating levels of the hormone because pharmacological doses could produce misleading results by disrupting cell processes, inducing neuronal death, or increasing susceptibility to disease (Axelrod and Reisine, 1984; Greenberg and Wingfield, 1987). Specifically, we sought to verify that the daily application of corticosterone
to the skin of females elevates and maintains their plasma corticosterone levels. 2. Materials and methods 2.1. The species The common lizard is a small lacertid (adult’s snout-vent lengths50–60 mm, juvenile’s snoutvent lengths18 mm on average), widely distributed across Europe and Asia. We studied ` (1500 m, Massif populations on Mont–Lozere Central, South-eastern France, 448009N, 38459E), where males emerge from hibernation in midApril, followed by yearlings and females in midMay. Mating takes place as soon as females emerge from hibernation (Pilorge et al., 1987). After 2 months of gestation, females lay a clutch, on average, of five soft-shelled eggs. Offspring hatch within 1 h of oviposition. On 20 June 2001, we captured 61 pregnant females and kept them in the laboratory until parturition (usually at the beginning of August). Pregnant females are easily discernible by visual inspection or gentle palpation of their abdomen. Each lizard was housed in individual plastic terrarium (18=12=12 cm3) with 1 cm of damp soil. A basking site and a shelter permitted thermoregulation. Terraria were stored in a room at ambient temperature (22–30 8C) and exposed to natural daylight. Terraria were also heated 6 h per day with an incandescent light bulb (25 W) at 20 cm above the terrarium (peak temperature of 30 8C). We also provided water ad libidum and offered food (Pyralis farinalis larvae) once per week. Housing conditions were in accord with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The females were measured (snout-vent length) and weighed weekly. Corpulence was calculated as the residual of the regression of body mass against snout-vent length. There was no mortality and all females laid clutches. Females were treated either with exogenous corticosterone or with vehicle alone (see below). After the experiment, mothers and offspring were returned to their original population at the capture point of the mother. Inter-individual variation in basal corticosterone levels also was determined. We captured 34 additional pregnant females on the 25 June, 2001. These individuals were kept overnight in the laboratory and blood samples were taken the next
S. Meylan et al. / Comparative Biochemistry and Physiology Part A 134 (2003) 497–503
afternoon, after which the females were released at their site of capture. Dauphin-Villemant and Xavier (1987) noted that 1 h of confinement induced elevated corticosterone levels in nonreproductive L. vivipara. After 18 h of confinement corticosterone levels were lower, but were still more variable than those of control animals and had not quite returned to baseline. Thus, by sampling blood after 24 h of captivity, corticosterone levels in control animals may still have been slightly more variable and slightly higher than those found in free-living lizards. 2.2. Hormonal treatment We manipulated circulating levels of corticosterone using a non-invasive method modified from Knapp and Moore (1997). Corticosterone was delivered transdermally to the lizards in a mixture of the steroid hormone and sesame oil. We did not use dermal adhesive bandages as done by Knapp and Moore (1997) to decrease the risk of stress associated with the application, presence, and removal of a patch. Lacerta vivipara is very similar in size and weight to the tree lizards used by Knapp and Moore (1997), so we used the same corticosterone concentration. We diluted corticosterone (Sigma C2505) in commercial pure sesame oil (3 mg corticosteroney1 ml sesame oil). We applied the hormone solution nightly (4.5 ml with a pipette) to the backs (between the two shoulders) of the treatment group (ns36) for 1–20 days, after which a blood sample was taken. Control females (ns25) were treated similarly, but with the oil vehicle only. Both the oil and the oily hormone mixture were completely absorbed within 2–3 h of application. We treated the animals during the night for two reasons. First, the temperature in the terraria decreased during the night so the females were less active and more easily handled. Second, lower temperature reduced the risk of evaporation of the solution before it crossed the skin. 2.3. Blood sampling Blood samples (40–80 ml whole blood) were collected from the post-orbital sinus using 2–3 50 ml microhematocrit tubes. Blood sampling was conducted between 25 June and 15 July 2001. All samples were collected within 3 min of removal of an animal from its home cage to avoid a
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Table 1 The sample size used in each of the experimental treatment groups Blood sample periods
1h Day Day Day Day Day Day
1 2 5 10 15 20
Sample size Placebo
Corticosterone
4 4 3 3 3 4 4
4 5 5 5 5 5 7
handling-induced increase in plasma corticosterone levels (Dauphin-Villemant and Xavier, 1987). To reduce variability in corticosterone levels due to diurnal rhythms in its secretion, we collected all blood samples between 16:00 and 17:30 h. To determine the effect of the transdermal corticosterone application on circulating corticosterone levels we took blood from 3 to 5 lizards of each group (placebo and corticosterone; Table 1) at the following times: 1 h after the first treatment, and after 1, 2, 5, 10, 15 and 20 days of treatment. Each individual was sampled only once. Blood samples were centrifuged and the plasma harvested and stored at y20 8C until transported on dry ice (4 kg, y80 8C) to Paris, where they were maintained at y80 8C. The samples subsequently were transported to the radioimmunoassay laboratory, also on dry ice, where they were kept at y20 8C until assayed. 2.4. Hormone assays Corticosterone values were determined in a single radioimmunoassay following procedures described by Wingfield et al. (1992). In brief, a small amount of labelled corticosterone was added to all sample test tubes and to each of two test tubes containing a standard amount of corticosterone to determine loss of sample during the extraction process. Corticosterone was extracted from 10 to 30 ml of plasma in 4 ml of freshly distilled dichloromethane, dried under nitrogen gas, and resuspended with 550 ml of buffer. We placed 200 ml of this solution into duplicate assay tubes and placed another 100 ml into a vial with scintillant. The latter vials provided an estimate of the percentage of steroid recovered after extraction. Mean recovery was 87%. A standard curve, consisting
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varied among individuals (Fig. 1). The mean corticosterone level was 21.64"4.52 ngyml (ns 34). The maximum baseline corticosterone level in these females was 101.97 ngyml. However, most levels were well below this, as the median level was 10.58. There was neither an effect of female body size (F1,33s0.03, Ps0.87) nor of female corpulence (F1,33s0.001, Ps0.98) on circulating basal corticosterone levels. Fig. 1. Natural variation in circulating corticosterone levels among individual female Lacerta vivipara.
of nine serially-diluted points (starting at 2000 pgy100 ml) was prepared in duplicate assay tubes. Each sample and standard curve tube received 100 ml of labeled corticosterone and 100 ml of corticosterone antibody (Endocrine Sciences) and was allowed to incubate overnight at 4 8C. After treatment with dextran-coated charcoal for 10 min to remove the unbound fraction, tubes were centrifuged at 2000 rpm for an additional 10 min whereupon the supernatant was decanted into scintillation vials, scintillant added, and the vials were counted on a Beckman LS-6500 scintillation counter the following day. Non-specific binding of the antibody was less than 5%. Two water blanks measured undetectable levels of corticosterone. Two corticosterone standards, each of 1000 pg, produced values that averaged 1120 pg (S.E.s 43.84).
3.2. Circulating treatment
corticosterone
levels
after
There was a significant effect of transdermal corticosterone application on plasma corticosterone levels (F1,55s25.8, P-0.0001). Circulating corticosterone levels were higher in corticosteronetreated females than in control females (Fig. 2), and the difference was seen within 1 h of treatment. In addition, treatment duration affected the hormone levels (F1,34s4.81, Ps0.035). Elevated corticosterone levels occurred within 1 h of the first transdermal application and were maintained during the following 2 days of treatment. However, beginning at day 3 and continuing until day 20 (when treatment ended), circulating corticosterone levels were significantly higher than those measured during the first 2 days of treatment, averaging 281.9 ngyml (Fig. 2). Corticosterone levels of control females remained low and did not increase
2.5. Statistics Statistical analyses were performed using SAS software (SAS Institute, 1996). Plasma concentrations of corticosterone were compared according to the treatment and the duration of the treatment. We also introduced individual covariables into the statistical model, using GLM procedure (ANOVA). We used type III sum of squares (nonsequential decomposition). We started with a general model including all the potential effects and their interactions. We then dropped the nonsignificant effects, starting with the most complex interaction terms. Only the results of the final model are reported. 3. Results 3.1. Natural variation in corticosterone levels among individuals Basal plasma levels of corticosterone in pregnant female L. vivipara captured and held overnight
Fig. 2. Effect of transdermal corticosterone application on the circulating level of corticosterone in pregnant female lizards. B, experimental (corticosterone) treatment; P, placebo treatment. There was a significant effect of the treatment on corticosterone levels (ns60, Fs25.8, P-0.0001). For corticosterone-treated females, there was a significant effect of the duration of the hormonal treatment on circulating corticosterone levels in pregnant females (ns35, Fs4.81, Ps0.035).
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over the course of the treatment (F1,24s3.39, Ps 0.078), although there was a trend toward increased corticosterone levels. As was the case with females held overnight, there was considerable variation in corticosterone levels among corticosterone-treated females. We investigated whether these differences were due to differences in individual morphological characteristics by including body size and corpulence (i.e. mass corrected for size) in the statistical model. There was neither an effect of female body size (F1,55s0.24, Ps0.62) nor of female corpulence (F1,55s0.31, Ps0.58) on circulating corticosterone levels. 4. Discussion Our results demonstrated that the transdermal application of corticosterone significantly increases circulating corticosterone levels within 24 h. We used this non-invasive technique because of the small size of our study organism and because previous experience suggested that such treatments would be stressful. This technique does not require surgery and limits the undesirable impacts of hormone manipulation on the females and their offspring. Pregnant females treated with corticosterone experienced no mortality and no abortion during the experiment, although such losses had occurred during earlier experiments using more invasive techniques (de Fraipont et al., 2000). Mean corticosterone levels in the untreated pregnant females that were captured and held overnight prior to sampling were only slightly higher and slightly more variable than those of non-reproductive females housed by Dauphin-Villemant and Xavier (1987) in large terraria designed to mimic field conditions (21.64"4.52 ngyml vs. 14.4"2.3 ngyml, respectively). Since corticosterone levels in pregnant females tends to be higher than those in non-reproductive females (Dauphin-Villemant et al., 1990), this sample of pregnant females may represent baseline values, although this must be verified by sampling free-living lizards immediately after capture. Corticosterone levels increased within 1 h of the first corticosterone application and remained at similar levels (f145"32 ngyml) over the subsequent 2 days of treatment. By day 5 of the treatment corticosterone concentrations rose to 281.9 ngyml, where they remained for the remainder of the experiment. These values represent approximately a 12-fold increase over mean base-
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line levels and are 2.8 times the maximum baseline value measured in controls. However, the difference in corticosterone levels between the placeboand corticosterone-treated females in our study may have been affected by diurnal factors. That is, Dauphin-Villemant and Xavier (1987) found that basal corticosterone levels in captive, active Lacerta females (i.e. those sampled during the day when a heating light was turned on) were approximately 50 ngyml. This is 3-fold higher than the levels they found in quiescent females sampled in the late afternoon when the heating lights were turned off. We sampled all our captive Lacerta females in the late afternoon, and the corticosterone values we measured in placebo-females were similar to those obtained at a comparable time by Dauphin-Villemant and Xavier (1987). Thus, by sampling females during their inactive period, we may have maximized the differences in corticosterone levels measured in the two treatment groups. Similarly-sized induced increases in baseline plasma corticosterone levels also have been found in other reptile species. For example, Knapp and Moore (1997) increased plasma corticosterone concentrations by approximately 13-fold with transdermal corticosterone application, and Tyrrell and Cree (1998) observed that 3 h of confinement increased corticosterone levels more than 10-fold in Sphenodon punctatus. Although these results are consistent with the increases we induced in L. vivipara by transdermal application of corticosterone, it remains to be seen if similar increases are produced by natural stressors. Increases in baseline plasma corticosterone levels in L. vivipara may be a normal part of their reproductive cycle, for the increased energy demands of parturition may require elevated corticosterone levels to help mobilize energy stores, and elevated baseline corticosterone levels may help to regulate the timing of parturition. For example, Dauphin-Villemant et al. (1990) observed endogenous increases in plasma corticosterone levels in untreated, captive females over the course of gestation, with corticosterone levels reaching 80 ngyml just prior to parturition. We did not find such increases in basal corticosterone levels in our control females, although we may have stopped sampling the animals before the changes became evident. Furthermore, Lacerta adrenal glands remain sensitive to stimulation by ACTH during the reproductive period (Dauphin-
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Villemant et al., 1990), demonstrating that additional increases in corticosterone secretion above baseline levels can occur, such as those generated by stressors. Our results show that transdermal application of corticosterone can produce such increases in pregnant females without disrupting gestation. The sensitivity of the hypothalamo-pituitaryadrenal axis varies among individuals. For example, corticosterone level can fluctuate with intrinsic body condition (Wilson and Wingfield, 1992; Dauphin-Villemant and Xavier, 1987) and with age (Wilson and Wingfield, 1992). However, in our case, circulating corticosterone levels were related neither to female size nor to female corpulence. This is similar to the findings of Cash et al. (1997), who found no correlation between basal plasma corticosterone levels and energetic condition. Furthermore, in L. vivipara, size increases with age until the former reaches a plateau (Ronce et al., 1998, 90% of size variation is explained by age: Massot, unpublished data). Thus, within limits, our data also suggest that corticosterone levels do not vary systematically with age in this species. Therefore, other explanations must be sought to explain differences in baseline corticosterone levels among individual L. vivipara, such as differences in genotype, in previous experience, or a lingering effect of captivity. Our laboratory is using the transdermal application of corticosterone to pregnant L. vivipara to explore the consequences of stress during gestation on females and on offspring phenotype. We have already demonstrated, in a previous corticosterone manipulation using silastic implants (de Fraipont et al., 2000), that the elevation of corticosterone in pregnant females affects their behaviour, with corticosterone-treated females more active than placebo females. We currently are investigating whether maternal stress is perceived by developing young and, if so, how this information affects their behaviour and life-history traits (Meylan et al., 2002). Acknowledgments ´ We are grateful to the ‘Parc national des Cevˆ ennes’ and the ‘Office National des Forets’ for providing facilities during our field season, a PhD grant from the French Ministry of Research and Education, a grant from the Idaho State Board of
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