Transgenerational effects of early experience on behavioral, hormonal and gene expression responses to acute stress in the precocial chicken

Hormones and Behavior 61 (2012) 711–718

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Transgenerational effects of early experience on behavioral, hormonal and gene expression responses to acute stress in the precocial chicken Vivian C. Goerlich 1, Daniel Nätt 1, Magnus Elfwing, Barry Macdonald, Per Jensen ⁎ IFM Biology, Division of Zoology, Avian Behavioural Genomics and Physiology Group, Linköping University, Sweden

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Article history: Received 5 December 2011 Revised 13 March 2012 Accepted 15 March 2012 Available online 23 March 2012 Keywords: Early growth response Corticotropin releasing hormone receptor Postnatal stress Transgenerational effects Steroid hormones Gene expression

a b s t r a c t Stress during early life can profoundly influence an individual's phenotype. Effects can manifest in the shortterm as well as later in life and even in subsequent generations. Transgenerational effects of stress are potentially mediated via modulation of the hypothalamic–pituitary–adrenal axis (HPA) as well as epigenetic mechanisms causing heritable changes in gene expression. To investigate these pathways we subjected domestic chicken (Gallus gallus) to intermittent social isolation for the first three weeks of life. The early life stress resulted in a dampened corticosterone response to restraint stress in affected birds and in their male offspring. Stress-specific genes, such as early growth response 1 (EGR1) and corticotropin releasing hormone receptor 1 (CRHR1), were upregulated immediately after restraint stress, but not under baseline conditions. Treatment differences in gene expression were also correlated across generations which indicate transgenerational epigenetic inheritance. In an associative learning test early stressed birds made more correct choices suggesting a higher coping ability in stressful situations. This study is the first to show transgenerational effects of early life stress in a precocial species by combining behavioral, endocrinological, and transcriptomic measurements. © 2012 Elsevier Inc. All rights reserved.

Introduction The perinatal environment can have profound long-lasting impact on traits such as personality, reproduction, fecundity, behavior, and physiology. Unfavorable early experiences can result in long-term phenotypic changes, potentially via programming of the hypothalamic–pituitary–adrenal (HPA) axis which regulates stress physiology (reviewed by Lindström, 1999; Romero, 2004). Prolonged stress can even induce heritable changes in gene expression patterns, through for example methylation of the DNA or histone tail modifications (reviewed by Joëls et al., 2007; Kim et al., 2009). Recently, evidence is accumulating that the programming effects of conditions during early development can be transmitted to the offspring (Champagne and Rissman, 2011). Steroid hormones are potential candidates in mediating variation in the epigenome since they are influenced by the surrounding environment and orchestrate physiology and behavior partly by acting as transcription factors regulating gene activity levels (Zhang and Ho, 2011). The majority of avian studies investigating effects of early environment have focused on the effects of nutritional stress, caused either directly by manipulation of food quality (Krause et al., 2009), or indirectly by manipulating brood size, thereby enhancing nestling competition (Naguib and Gil, 2005). This approach cannot rule out ⁎ Corresponding author. E-mail address: [email protected] (P. Jensen). 1 Equal contribution. 0018-506X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2012.03.006

the potentially confounding factors of parental care (Banerjee et al., 2011) and compensatory growth (Metcalfe and Monaghan, 2001). On the other hand, social isolation, an often used paradigm in mammals (Pryce et al., 2002), represents a relevant stressor in group living avian species as well, and results in increased plasma corticosterone levels (Yanagita et al., 2011) while minimizing nutritional effects. Under commercial conditions, birds such as ducks, quail, and chickens, are exposed to a myriad of stressors during early development such as hatching without maternal contact, transportation, heat and cold stress, and separation from social mates (Frazer and Broom, 1990). Identifying the impact of such early stressors on behavior and physiology is important both for animal welfare and for understanding the importance of the early environment for the evolution of phenotypic variation. Our study aimed at identifying the short- and long-term effects of a limited period of stress during the postnatal period. We repeatedly applied short term isolation stress to avoid effects due to nutritional deficits and used the precocial domestic chicken (Gallus gallus) to rule out confounding parental care. After the stress period we took several behavioral and physiological measurements in both parents and the subsequent offspring generation to explore the short- and long-term effects of the experimental treatment. We measured the corticosterone response to restraint stress, expecting that early stressed birds would show lower baseline and dampened peak levels (Rich and Romero, 2005). Chronic stress has shown to diminish fecundity and delay sexual maturation (Lindström, 1999). Sexual maturation is characterized by a marked increase in gonadal steroids

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(Eitan et al., 1998; Vanmontfort et al., 1995) therefore we repeatedly measured plasma testosterone (males) and estradiol (females) prior to sexual maturity, expecting early stressed birds to show a delayed increase. Moreover we scored the performance in an associative learning test, a trait which has been shown to be affected both negatively and positively by chronic stress (Schwabe et al., 2011). Finally we quantified hypothalamic gene expression patterns to explore regulatory changes due to early experiences (Lindqvist et al., 2007). This comprehensive approach allowed us to investigate mechanisms related both to steroid hormones and gene expression, potentially underlying long-term and transgenerational epigenetic effects of early life stress. Materials and methods Study animals and housing The experiment and all its procedures were approved by a Swedish regional ethical committee. The time line of the experiment is

shown in Fig. 1A. We purchased fertilized HyLine eggs from a commercial breeder (Swedegg, Mantorp, Sweden) which then were incubated and hatched according to standards at the hatching facility of Linköping University. At day two after hatching, chicks were assigned to either the control (C) or early stress (ES) treatment, balanced for sex. Birds from both groups (94 ES and 92 C) were equally distributed over three mixed-sex pens per group, all housed within one room under the same conditions (12L:12D, 28 °C) with free access to feed and water. Between days 4 and 26 the birds in the ES group were exposed to the stress treatment at random times once per day. During treatment, the birds were placed individually in a metal mesh box (25 × 38 × 18 cm) where they had vocal contact, but limited sight and no physical contact with other chicks. Hence, the birds were exposed to a combination of handling, social isolation, feed and water deprivation, and temperature stress, since the ambient temperature was 10° lower in the cages than in the home pens. Time in the stress box was gradually increased from 1 h during the first, to 2 h during the second, and 3 h during the third week. The C group was kept

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Fig. 1. The timeline of the experiment (A) and the design of the microarray analysis (B). Time line for the experiment (A) shows each separate group (test, naive and offspring birds), with an equal number of ES, C, females and males present. Numbers in brackets indicate days after hatch. ES = Early stress treatment (filled box, only ES birds); NS = Naive separated from test birds; B1,2 = Brain sampling; RS1,2 = Restraint stress test; AL1,2 = Associative learning test; SM = Blood sampling for sexual maturation; OE = Egg collection for offspring generation; HE = Egg collection for hormonal analysis; OF = Open field; SR = Social reinstatement in open field; TI = Tonic immobility. Different comparisons in the microarray analysis (B) are indicated by Greek letters. (α) = Parental controls (C) versus early stressed (ES) treated groups. (β) = Parental baseline versus restrained groups. (γ) = Restrained male offspring of control parents (CPo) versus restrained male offspring of early stressed parents (ESPo). (δ) = Transgenerational correlation in the difference of (α) and (γ). Each black circle represents microarray duplicates of in total six individuals per group (hybridized in pools of three). The whole experiment contained 20 microarrays divided on 60 birds.

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unexposed to physical stressors except for weekly handling to obtain body measurements. After the three weeks of stress treatment, 48 birds (equally balanced for sex, treatment, and home pen) were culled for brain tissue sampling. The remaining surviving birds were split into a test group (n = 78) and a naïve group (n = 57) (also equally balanced). The test birds were used for behavioral testing and physiological sampling while the naïve birds were only handled for regular weighing throughout life. At day 55 all birds were moved to an adult chicken facility and housed, separated for sex, under a 12L:12D light cycle at 22 °C in pens measuring 3 × 3 × 3 m with perches, nests and free access to water and feed. At days 152–153, as well as day 166 after hatch, the surviving naïve birds (ES female n = 9, ES male n = 19, C female n = 7, C male n = 17) were mated within group. Each female was mated with multiple males, but as far as possible (due some cases of sterility) each male was only mated with one female. In total, 44 offspring of ES parents (ESPo) and 53 offspring of C parents (CPo) were hatched. Incubation, hatching and physical environment of the offspring were the same as for the C parents. Offspring groups were neither exposed to early stress treatment (like ES parents) nor the potential stress of daily bird catching in neighboring ES pens (like C parents). Sample collection Restraint stress At the age of 29 days parental chicks were subjected to restraint stress to measure the sensitivity of the HPA axis. Birds were removed from their home pens and blood sampled within 3 min to determine baseline corticosterone (CORT) levels (Romero and Reed, 2005). Sampling was balanced for treatment and sex. After the baseline sample, each bird was restrained in a tight cloth bag made of netting. At 10 and 30 min after initial disturbance birds were removed from the bags and blood sampled again. Blood was collected in heparinized glass capillaries after venipuncture of the brachial vein. The same protocol was followed for the offspring chicks. Sexual maturation The domestic White leghorn becomes sexually mature around 110 days of age, indicated by a marked increase in plasma 17-βestradiol (E2, females) and testosterone (T, males) peak (Eitan et al., 1998; Vanmontfort et al., 1995). We collected blood samples of the parental birds between the age of 91 and 119 days to measure E2 and T. All females were sampled weekly while males were split into two groups which each was sampled every second week. Brain tissue sampling We collected brain samples enriched of the thalamus/hypothalamus at baseline and after exposure to 30 min restraint stress (as described above). In the parental generation we sampled both sexes (6/treatment, n = 48) while in the offspring generation we analyzed only samples from male offspring that had undergone the restraint period (6/treatment, n = 12). Samples were immediately frozen in liquid nitrogen. Body mass and egg collection Both parents and offspring were weighed once a week and tarsus length was measured using a digital caliper (to the nearest 0.01 mm). From the age of 175 days we collected fresh eggs from the naïve birds on a daily basis, and weighed and froze the eggs at −20 for later yolk E2 and T analysis. Since parental females were group housed during egg collection we could not determine which bird had laid a particular egg, but since chicken normally do not lay more than one egg per day, we used independent t-tests per day for the analysis of yolk and egg weights.

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Behavioral measurements Associative learning An associative learning test was carried out at days 50–51 after hatch in both parents and offspring. Following water deprivation for 2 h 30 min +/− 30 min the birds were placed in the test arena, which contained two nipple bottles, one blue and one yellow. One bottle contained tap water, while the other contained 3% quinine solution, which is aversive to birds (Yeomans and Savory, 1989). During 20 min training sessions, the chicks had free access to both bottles. The color (blue or yellow) and spatial location (right or left) associated with quinine were balanced across birds. Approximately 2 h after training, the latency to first step, latency to first choice, and the color of first choice were recorded during a 5 min test. Offspring fear related behaviors We conducted three behavioral tests for fear and explorative behavior in the offspring: the time spent in induced tonic immobility (age 46/47 days), where a longer time to emerge from immobility indicates higher fearfulness (Natt et al., 2007) activity in an open field (age 33/34 days), where less fearful animals usually are more active (Heiblum et al., 1998), and latency to reinstate with social companions in an open field arena (age 39/40 days), where more fearful animals are expected to spend more time closer to social companions (Karlsson et al., 2010). Hormone measurements Extraction The extraction procedure was based on Kozlowski et al. (2009). Plasma and yolk samples were weighed (to the nearest 0.001 g) in 2 ml Eppendorf tubes and 200 μl phosphate buffered saline with gelatin (PBSG) was added to plasma samples. All samples were spiked with approximately 4000 cpm of radioactive labeled hormone (T, E2, or CORT) to correct for extraction losses. After vortexing, samples were incubated in a 37 °C waterbath for 45 min. Then 500 μl 100% EtOH was added and samples were vortexed for 15 min. Following vortexing, samples were centrifuged at 13,000 rpm, 4 °C, for 10 min. The supernatant was decanted into fresh tubes and frozen at − 80 °C overnight. The next day samples were centrifuged at 4000 rpm, 4 °C, for 10 min. Again the supernatant was decanted into fresh tubes and completely dried at 37 °C in a vacuum concentrator. The pellet was re-dissolved in PBSG. Average recoveries (± SDEV) were 81.4 ± 3.2% for plasma T, 79.3 ± 6% for yolk T, 76.1 ± 4.9% for plasma E2, 75 ± 7.3% for yolk E2, and 80.1 ± 3.9% for plasma CORT. Radioimmunoassay Steroid concentrations were determined with commercially available kits: Coat-a-Count® total testosterone (Siemens Medical Solutions Diagnostics, Sweden), Spectria® Estradiol Sensitive RIA (Orion Diagnostica AB, Sweden), and ImmunoChemTM Double Antibody Corticosterone (MP Biomedical via Fisher Scientific, Sweden). Each kit was tested for parallelism between serial dilutions of plasma/ yolk samples and a PBSG standard curve. Plasma and yolk E2 and T were determined in 4 RIAs, parental chick CORT in 2 RIAs (individual sample series within one RIA and both RIAs balanced for treatment), and offspring CORT in one RIA. The intra-assay coefficients of variation (CV) were: T: 1.75% (plasma), 1.24% (yolk); E2: 2.14% (plasma), 3.69% (yolk); CORT: 1.86, 1.25, and 5.04%; inter-assay CV for CORT was 4.07%. The RIA results were corrected for recovery and initial sample mass. Statistical analyses Plasma CORT levels of parents and offspring chicks were log transformed to meet a normal distribution. The corticosterone

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response to restraint test was analyzed using a repeated measures ANOVA and LSD corrected post hoc comparisons. We used sex and treatment as categorical predictors. Prior to analyses we tested for effects of body condition (un-standardized residuals of a linear regression of body mass on tarsus length) and time until sample retrieval with a multivariate GLM. Non-significant terms were removed from the model in a backwards stepwise manner. Growth was analyzed using repeated measures ANOVA with sex, treatment and their interaction. Due to the unbalanced design of the sexual maturation blood sampling we fitted linear mixed models which can handle repeated measures with missing data. The individual is accounted for as random factor, while fixed effects (treatment and sex) as well as covariates (sampling time and residual body condition) can be incorporated. Microarray analysis RNA-extraction, labeling and hybridization Brain tissue samples were homogenized in Lyzing matrix D tubes with the FastPrep®-24 (MP Biomedicals) and RNA was extracted with the same method as has been described previously (Nätt et al., 2009). The experimental design underlying the microarray analyses is shown in Fig. 1B. Only male restraint samples were analyzed in the offspring generation. Microarray hybridization has been described elsewhere (Natt et al., 2012). Before hybridization, samples were mixed in pools of three birds each. Labeling and hybridization were performed at Uppsala Array Platform at Uppsala University, Sweden (www.medsci.uu.se/klinfarm/arrayplatform). Biotinylated fragmented RNA was prepared for each pool using standard procedures in GeneChip ® 3′ IVT Express Kit Users manual (Affymetrix Inc., Rev. 1, 2008). Each pool was then hybridized to GeneChip Chicken Genome Arrays (Affymetrix Inc.) for 16 h in 45 °C under constant rotation. Washing and staining were performed in a Fluidics Station 450 and scanned using the GeneChip Scanner 3000 7G (Affymetrix Inc.).

8 of 8 versus 8 of 10, χ 2 = 1.8, p = 0.2). Correct choice showed no difference in the offspring (p > 0.1).

Fear related behavior in offspring We found no differences between ESPo and CPo offspring in time spent in tonic immobility (females: χ 21 = 1.7, p = 0.2; males: χ 21 = 1.1, p = 0.3), total distance moved in the open field (males: t17 = 0.7, p = 0.4, females: t25 = −1.1, p = 0.3) or latency to approach social mates (male: t17 = 0.6, p = 0.6, female: t25 = 0.2, p = 0.9).

Glucocorticoid stress response to restraint stress Stress protocol parental chicks (ng/ml CORT) Neither time until sample retrieval nor body condition reached significance (all p > 0.1), therefore these covariates were not included in further models. The repeated measures ANOVA on log CORT revealed significant differences in the stress response between the treatment groups (F1,29 = 4.7, p = 0.04; Fig. 2) and sexes (F1,29 = 10.9, p = 0.003; Fig. 2). Separate analyses of the sexes showed a significant treatment effect on the CORT response in females (F1,16 = 0.047) which was absent in males (F1,12 = 0.6, p = 0.44). Post hoc tests revealed that 10 min CORT concentrations were significantly higher in ES females compared to the C females (p = 0.043) while baseline and 30 min concentrations did not differ (all p > 0.06). Similarly, ES males had higher 10 min CORT levels compared to C males (p = 0.047) while baseline and 30 min concentrations did not differ (all p > 0.6).

parents

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Associative learning More ES (parental chicks) than C birds made a choice by pecking at one of the two drinkers in the associative learning test (sexes pooled: 32 of 39 versus 20 of 39 C birds, χ 2 = 8.3, p b 0.01; females: 14 of 17 versus 6 of 14, χ 2 = 5.2, p b 0.05; males: 18 of 22 versus 14 of 25, χ 2 = 3.6, p = 0.06). Among those that made a choice, ES females made more correct choices (sexes pooled: 21 of 32 birds versus 7 of 20 birds, χ 2 = 4.6, p b 0.05; females: 9 of 14 versus 1 of 6, χ 2 = 3.8, p = 0.06; males: 12 of 18 versus 6 of 14, χ 2 = 1.8, p = n.s.). Similarly, a higher number of the ESPo than CPo offspring made a choice (19 of 19 versus 20 of 26, χ 2 = 5.1, p b 0.05), however, not if analyzed by sex (females: 11 of 11 versus 12 of 16, χ 2 = 3.2, p = 0.07; males:

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Gene expression data analysis Gene expression was analyzed in R/Bioconductor software environment (www.bioconductor.org) as described before (Natt et al., 2012), except for using nsFilter function in the geneFilter package to exclude Affymetrix control probes, non-variable and the least variable probesets (IQR, cut off at 0.5) (Bourgon et al., 2010). Cross generational correlation analysis was done on the overlapping 1000 top genes (ranked by log2 fold change) in the parents and offspring respectively. Hierarchical average linkage cluster analysis was performed with the Genesis software v 1.7.5 (Sturn et al., 2002), while gene ontology analysis was performed using DAVID (Huang et al., 2008). Microarray data has been uploaded to the ArrayExpress database (http://www.ebi.ac.uk/miamexpress/) with the E-MTAB-924 (parents) and E-MTAB-925 (offspring) accession numbers.

mean plasma corticosterone (ng/ml)

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time (min) Fig. 2. Blood plasma corticosterone levels (ng/ml) after 0 (baseline), 10, and 30 min of physical restraint. The upper panel represents female parents and offspring, the lower male parents and offspring. C parents and CPo offspring are represented by dashed lines, ES parents and ESPo offspring by solid lines. * p b 0.05 in LSD post hoc test following repeated measures ANOVA.

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Sex steroid levels during the course of sexual maturation Parental females plasma 17-β-estradiol (pg/ml E2) As expected, plasma E2 concentrations rose steadily in the weeks before first eggs were laid (effect of sampling date all p b 0.05). However, the early stress treatment had no effect on female plasma E2 levels (F1, 35.3 = 1.2, p = 0.28).

EGR1 up

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We chose eggs for hormone analyses from the three days on which egg counts were highest and found yolk E2 concentrations to be slightly higher in C yolks on day one (mean ± SD: C: 1.17 ± 0.12, ES: 1.02 ± 0.14, t11 = 1.98, p = 0.07), but significantly higher in ES yolks on day two (C: 0.95 ± 0.11, ES: 1.12 ± 0.10, t11 = −2.92, p b 0.05) while there was no difference in E2 on day three (C: 1.07 ± 0.23, ES: 1.12 ± 0.12, t10 = − 0.45, p = 0.66). T concentrations did not differ except for day two when, in line with E2 concentrations, ES yolks contained higher T concentrations than C yolks (day 1: C: 22.62 ± 4.7, ES: 22.6 ± 3.79, t11 = 0.01, p = 0.99; day 2: C: 19.5 ± 2.27, ES: 22.67 ± 2.38, t11 = −2.45, p b 0.05; day 3: C: 25.18 ± 2.65, ES: 23.84 ± 3.54, t10 = 0.71, p = 0.49). During the egg collection for the offspring generation, eggs from ES females were numerically heavier at 11 out of 12 days and this difference was significant on 3 out of 12 days (independent t-test, p b 0.05).

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Parental males plasma testosterone (ng/ml T) Plasma T increased prior to expected sexual maturation (effect of sampling date all p b 0.05), though ES and C males did not differ significantly in their plasma T levels (treatment: F1, 67.32 = 0.6, p = 0.45). Parental yolk hormones and egg weights

CRHR1

(GgaAffx.11738.1.S1_s_at) (Gga.14391.1.S1_at)

gene regulation (log2 FC)

Stress protocol offspring chicks (ng/ml CORT) Again, time until sample retrieval and body condition did not significantly predict variation in the CORT levels (all p > 0.3). In the repeated measures ANOVA none of the predictors reached significance (all p > 0.15). Also the sex separate analyses did not find differences between CPo and ESPo females (p = 0.9, post hoc all p > 0.4). In males, however, the repeated measures showed a trend (F1,14 = 4.3, p = 0.057; Fig. 2), and post hoc tests revealed that CPo males had significantly higher CORT levels at 10 min (p = 0.02) and 30 min (p = 0.03) compared to ESPo males (Fig. 2).

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Fig. 3. Brain gene expression changes from baseline to 30 min of restraint for transcripts of the EGR1 and CRHR1 genes. Significant bars represent up-regulation in restrained birds. Brackets show array probeset IDs. *** Denote p b 0.001, ** p b 0.01 respectively (adjusted for FDR). ¤ Indicate p b 0.001 without FDR, but was ranked 3 out of 12,968 probesets analyzed for differential expression.

correlation between DE female parents and male offspring was stronger than between male parents and male offspring (δ in Fig. 1B) (Fig. 4). All genes which were significant in both male parents and offspring were DE in the same direction, while three out of four were so when comparing female parents and male offspring (Table 1). Two genes, Uridine monophosphate synthetase (UMPS) and Succinate-CoA ligase, GDP-forming, beta subunit (SUCLG2) were significant in both male and female parents, as well as in the offspring.

Gene expression Treatment effects (ES versus C) Focusing on treatment effects in the parents (α in Fig. 1B), no genes were significantly differentially expressed (DE) between ES and C males, while six genes were DE in the females. In the parental restrained birds, 27 genes were significantly DE in the males, and six in the females (Supplementary Table 1). Within treatment effects (baseline versus restraint) Comparing baseline and restraint (β in Fig. 1B), ES males had more significantly DE genes than the controls (26 DE genes versus 1), which was not the case for females (9 versus 8) (Supplementary Table 2). All groups responded to the restraint by up-regulating early growth factor 1 (EGR1), but only the parental male ES birds responded by significantly up-regulating corticotropin releasing hormone receptor 1 (CRHR1) (Fig. 3). Hierarchical clustering with the 26 significant DE genes in ES males showed that EGR1, CRHR1 and two other genes, pyruvate dehydrogenase kinase 4 (PDK4) and phosphodiesterase 5A (PDE5A), shared the same clade. Transgenerational effects (restrained male offspring only) Comparing male ESPo and CPo restrained offspring (γ in Fig. 1B) 44 genes were significantly DE (Supplementary Table 3). The

Analysis of gene function By pooling the significant genes between ES and C in the restrained male parents with the significant genes between ESPo and CPo in the restrained male offspring, (p b 0.05), the top three significant (p b 0.05) GO terms were related to endocrine responses: response to hormone stimulus (GO:0009725, 10% of the genes, p b 0.01); response to organic substance (GO:0010033, 14%, p b 0.01); response to endogenous stimulus (GO:0009719, 10%, p b 0.05). When looking at DE caused by restraint, the ES males had three immune response related terms as the top ranked: antigen processing and presentation (GO:0019882, 16%, p b 0.01); immune response (GO:0006955, 26%, p b 0.01); antigen processing and presentation of peptide antigen via MHC class I (GO:0002474, 11%, p b 0.05). Growth Parental generation Repeated measures showed a significant interaction between sex and treatment (F1,4 = 5.0, p b 0.03; C female n = 8, C male n = 14, ES female n = 11, ES male n = 14) which was due to a significant difference between ES and C females (F4,17 = 4.8, p b 0.04). No treatment effect was apparent in males (F1,26 = 0.5, p = 0.47). The 3 week

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A

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Fig. 4. Gene expression inheritance in thalamus/hypothalamus of birds that undergone 30 min of restraint. Scatterplots show the correlations of the differences in gene expression (FC = log2 fold change) in each generation. In A) the male parental ES and C difference is plotted against the male offspring ESPo and CPo difference. In B) the comparison is shown for female parents and the same male offspring. Gene subsets were chosen on the overlap of the 1000 topmost differentially expressed genes in each generation (Venn-diagrams). Arrows highlight two genes that were significant in all three datasets.

isolation period had no direct effect on body mass at age 26 in females (t17 = − 1.05, p = 0.31) or males (t26 = 0.01, p = 0.99). ES females started to become heavier than C females only at the age of 55 days (p = 0.07), 75 days (p = 0.09) and were significantly heavier at the age of 152 days (t17 = −2.39, p = 0.03). Offspring generation We found a significant interaction between parental treatment and sex (F1,41 = 4.2, p b 0.05; CPo female n = 16, CPo male n = 16, ESPo female n = 10, ESPo male n = 8). Analyses split for sex showed no treatment effect in female offspring (F1,24 = 0.9, p = 0.37) but a significant difference between CPo and ESPo male offspring (F1,17 = 7.8, p b 0.05). ESPo males were heavier than CPo males at 56 days (t17 = − 2.5, p b 0.05) and 74 days (t17 = −3.97, p b 0.001). Despite the overall similarity in body mass in female offspring, CPo females were heavier than ESPo females at the age of 2 days (t24 = 2.48, p b 0.05) while at 74 days (t25 = −2.3, p b 0.05) ESPo females were heavier than CPo females. Discussion In chickens reared without parental care a period of isolation stress during early life resulted in sex-specific effects, within and across generations, on glucocorticoid release, brain gene expression, and growth. Transgenerational effects on behavior were weak but discernible. Our study suggests that early experiences may shape phenotypes of chickens in a long-term way. In the parents, the three week isolation treatment did not affect baseline levels of circulating corticosterone but resulted in a suppression of the HPA axis sensitivity shortly after the treatment. Since the restraint stress protocol differed markedly from the daily isolation it is not likely that the chicks acclimated to the stressor (Romero, 2004). Rather the desensitization of the endocrine stress response occurred on a physiological level, a mechanism thought to protect the individual from negative effects induced by chronically elevated glucocorticoids due to repeated stressful events (Rich and Romero, 2005; Romero, 2004). Stress recovery was similar between treatment groups in the parental chicks and the female offspring, while the exaggerated corticosterone peak levels at 10 min and the slower recovery suggest that CPo males were more susceptible to stress or the response was suppressed in ESPo males. Intriguingly, the corticosterone release patterns of the ESPo males are comparable with the male C parents. We can only speculate on the underlying reasons, possibly the commercial (stressful) hatchery background resulted in suppressed stress responses in the parental generation which was then further altered by the early isolation period. In turn, the absence of severe stressors in the C parents might have led to a more sensitive HPA axis in the CPo males. Baseline corticosterone levels were higher in all offspring which might again be due to the different backgrounds

of the grandparents raised in a commercial hatchery or small changes in rearing conditions in the offspring generation due to different times of the year or the decrease in human presence when no early stress treatment was performed. Regarding the male specific transgenerational effect, sex specific sensitivity to stress has been reported in previous studies on chicken in which males have been shown to be more responsive to stress than females (Chaturvedi et al., 2000; Madison et al., 2008; Schmeling and Nockels, 1978), a pattern corroborated by our findings in the parental and offspring chicks. A stronger effect in male offspring might indicate parental imprinting of certain genes (Curley et al., 2011) since we found greater differences in corticosterone response the parental males as well and a significant correlation between differential gene expression in the male parents and the offspring. Gene ontology analysis showed that genes affected by the ES treatment in the parental birds and the restrained male offspring were significantly enriched with genes associated with hormonal responses. Most interesting, in samples taken after the restraint period the corticotropin releasing hormone receptor 1 (CRHR1) was up-regulated in the parental ES males, but not in the parental females or male offspring. CRHR1 is one of the main controllers of the HPA-axis, with distributions in multiple nuclei of avian thalamus/hypothalamus (Richard et al., 2004). Restraining the birds also induced an upregulation of early growth response 1 (EGR1, also known as zif268, NGFI-A) in all groups investigated. EGR1 is an immediate early gene coding for a transcription factor that initiates the genomic response for example under stressful events (Malkani et al., 2004). Since our clustering analysis showed that CRHR1 was co-regulated with EGR1 in response to restraint in parental ES males, it may play a significant role in the sex differences observed, especially when both CRHR1 and EGR1 have previously been reported to have sex dependent brain distributions and effects (Bangasser et al., 2009; Stack et al., 2009). Finally, SUCLG2 and UMPS, both highly differentially expressed in male and female parents and the male restraint offspring, are likely to be involved in different parts of the stress response possibly through mitochondrion regulation (Hazard et al., 2008; Naviaux and McGowan, 2000). Early life stress is not necessarily detrimental but can result in phenotypic changes which are thought to be beneficial, such as reduced susceptibility to external stressors later in life (Macrì et al., 2007; Salvatierra et al., 2009). Accordingly, ES parental birds performed better in the associative learning task, which was also the case for their ESPo offspring. However, no differences between ESPo and CPo offspring were apparent in any of the fear-related tests, suggesting either that the transgenerational effects on the HPA axis and gene expression do not manifest in the behaviors studied, or they develop later in life. Hypothetically, transgenerational behavioral effects could also become apparent only in the presence of an early life stressor.

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Table 1 Significantly differentially expressed genes in the thalamus/hypothalamus of restraint treated birds in both generations. Affymetrix ID

Description

Gene

Offspring

Parents

Males

Males

FC GgaAffx.11425.1.S1_at Gga.17147.1.S1_s_at GgaAffx.20374.1.S1_at Gga.2148.1.S1_at GgaAffx.26461.1.S1_at Gga.1849.1.S1_at a b c

Uridine monophosphate synthetase Coiled-coil domain containing 127 Succinate-CoA ligase, GDP-forming, beta subunit Solute carrier family 31 (copper transporters) CDC14 cell division cycle 14 homolog A Choline acetyltransferase

UMPS CCDC127 SUCLG2 SLC31A1 CDC14A CHAT

a

− 0.72 − 1.14 3.67 0.63 − 0.80 − 1.19

Adj. p

b

b0.01 b0.001 b0.001 b0.05 b0.001 b0.001

FC

c

− 1.23 − 1.21 2.47 0.81

Females Adj. p

b

b 0.001 b 0.001 b 0.01 b 0.05

FCc

Adj. pb

− 1.77

b0.001

2.45

(0.052)

1.06 − 0.82

b0.01 b0.05

Positive value means up-regulation (log2 fold change) of ESPo male offspring in relation to CPo male offspring. Linear model p-value adjusted for false discovery rate. Positive value means up-regulation (log2 fold change) of ES in relation to C birds.

The early isolation treatment enhanced growth in both the parental and offspring generations while body mass differences emerged only at later age, consistent with earlier studies (Nätt et al., 2009). Weight gain during early life has been shown to be regulated by a different set of genes than during adolescence (Kerje et al., 2003), which may be related to the present findings. Corticosterone is an important metabolic steroid regulating muscle growth and lipid metabolism (Landys et al., 2006) and the repeated early stress might have lead to changes in metabolism later in life. Though we did not find differences in baseline corticosterone concentrations changes might have occurred at the steroid receptor or carrier protein levels. By using a precocial species we avoided confounding factors of parental care in our experiment and restricted the occurrence of transgenerational effects to mechanisms acting on the molecular level. A potential pathway for transgenerational effects in birds is the egg (yolk) which contains substantial amounts of steroid hormones from maternal origin which profoundly influence offspring phenotype (Groothuis et al., 2005). We have previously reported increased testosterone and estradiol levels in eggs of chronically stressed chickens (Nätt et al., 2009) and found similar trends in the current study. However, since we could not determine individual origin of the eggs and the increase in T and E2 was apparent on only one of three sampling days, our data do not allow us to validate this pathway. Moreover, several other egg components, such as antibodies (Naguib et al., 2004), carotenoids (Saino et al., 2003), lipids and proteins (Bonisoli-Alquati et al., 2008) have been shown to affect offspring development as well. The eggs from ES females were heavier than the eggs from C females, which may indicate that other maternal mediators underlie the transgenerational effects in our study.

Conclusion We showed that in chickens stressful experiences early in life affect the corticosteroid response to acute stress and related genes. These effects were transferred to the offspring, probably through epigenetic mechanisms, since parental care was absent. Although offspring of early stressed parents was raised in a mis-matching control environment they had a higher growth and performed better than control offspring in a potentially stressful learning test. The reduced corticosterone response possibly indicates a higher coping ability; however, there might be a potential trade-off yet to be identified. Across all variables we found clear sex-specific treatment effects which might be due to differential gene expression and certainly are worth further exploration. Intriguingly, the largest treatment effects were seen in male birds subjected to restraint stress, both on corticosterone release and gene regulatory changes. We therefore emphasize the importance of studying gene expression under conditions more relevant to the stress treatment and from the perspective of both sexes.

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