Transgenerational Effects of Prenatal Synthetic Glucocorticoids on ...

GLUCOCORTICOIDS-CRH-ACTH-ADRENAL

Transgenerational Effects of Prenatal Synthetic Glucocorticoids on Hypothalamic-Pituitary-Adrenal Function Majid Iqbal,* Vasilis G. Moisiadis,* Alisa Kostaki, and Stephen G. Matthews Departments of Physiology (M.I., V.G.M., A.K., S.G.M.), Obstetrics and Gynecology (S.G.M., and Medicine (S.G.M.), Faculty of Medicine; University of Toronto, Toronto, Ontario, Canada M5S 1A8

Approximately 10% of pregnant women are at risk of preterm delivery and receive synthetic glucocorticoids (sGC) to promote fetal lung development. Studies have indicated that prenatal sGC therapy modifies hypothalamic-pituitary-adrenal (HPA) function in first-generation (F1) offspring. The objective of this study was to determine whether differences in HPA function and behavior are evident in the subsequent (F2) generation. Pregnant guinea pigs (F0) received betamethasone (BETA; 1 mg/kg) or saline on gestational d 40/41, 50/51, and 60/61. F1 females were mated with control males to create F2 offspring. HPA function was assessed in juvenile and adult F2 offspring. Locomotor activity was assessed in juvenile offspring. Analysis of HPA-related gene expression was undertaken in adult hippocampi, hypothalami, and pituitaries. Locomotor activity was reduced in F2 BETA males (P ⬍ 0.05). F2 BETA offspring displayed blunted cortisol response to swim stress (P ⬍ 0.05). After dexamethasone challenge, F2 BETA males and females displayed increased and decreased negative feedback, respectively. F2 BETA females had reduced pituitary levels of proopiomelanocortin (and adrenocorticotropic hormone), and corticotropin-releasing hormone receptor mRNA and protein (P ⬍ 0.05). F2 BETA males displayed increased hippocampal glucocorticoid receptor (P ⬍ 0.001), whereas in BETA females, hippocampal glucocorticoid receptor and mineralocorticoid receptor mRNA were decreased (P ⬍ 0.05). In conclusion, prenatal BETA treatment affects HPA function and behavior in F2 offspring. In F2 BETA females, pituitary function appears to be primarily affected, whereas hippocampal glucocorticoid feedback systems appear altered in both F2 BETA males and females. These data have clinical implication given the widespread use of repeat course glucocorticoid therapy in the management of preterm labour. (Endocrinology 153: 3295–3307, 2012)

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reterm birth occurs in more than 10% of pregnancies and can result in a number of negative health outcomes for a newborn child, such as neurodevelopmental complications, lung immaturity, sepsis, and increased neonatal morbidity and mortality (1, 2). Synthetic glucocorticoids (sGC), such as betamethasone (BETA), are administered to pregnant women at risk of preterm delivery to promote the development of the fetal lungs and reduce the occurrence of respiratory distress syndrome and neonatal death (3–5). However, various animal studies have shown that prenatal sGC exposure can lead to alterations in the de-

velopment of a number of organ systems leading to modified function throughout life (6, 7). The hypothalamopituitary-adrenal (HPA) axis and stress-related behaviors have been shown to be particularly sensitive to the effects of prenatal sGC exposure (8 –10). Altered regulation of HPA function has been linked to the premature development of adult-onset pathologies, such as insulin resistance and hypertension in rats and humans (11–13). We have shown that repeated fetal exposure to sGC during late gestation results in reduced basal HPA function in the first-generation (F1) adult male guinea pig offspring (14).

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2012 by The Endocrine Society doi: 10.1210/en.2012-1054 Received January 16, 2012. Accepted April 12, 2012. First Published Online May 7, 2012

* M.I. and V.G.M. share joint first-authorship. Abbreviations: ACTH, Adrenocorticotropic hormone; AUC, area under the curve; AVP, arginine vasopressin; AVPR1, AVP receptor type 1; BETA, betamethasone; CRHR1, CRH type 1 receptor; GR, glucocorticoid receptor; HPA, hypothalamo-pituitary-adrenal; ISH, in situ hybridization; MR, mineralocorticoid receptor; POMC, proopiomelanocortin; sGC, synthetic glucocorticoids; PND25, postnatal d 25; PVN, paraventricular nucleus; ROD, relative optical density.

Endocrinology, July 2012, 153(7):3295–3307

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Transgenerational Effects of Prenatal sGC

We have also demonstrated that adult female F1 offspring that had been prenatally exposed to sGC exhibit reduced basal HPA activity during the luteal phase of the reproductive cycle (14, 15). These alterations in HPA function involve changes in the expression of HPA regulatory genes at the level of the hippocampus, hypothalamus, and pituitary (9, 14 –16). The current study investigated whether the effects of prenatal sGC exposure on HPA function and behaviors are present in second-generation (F2) offspring. We hypothesized that one F2 offspring from F1 mothers that had been prenatally exposed to sGC but had gone through an undisturbed pregnancy would exhibit reduced HPA axis function due to altered gene expression in key regulatory regions of the HPA axis and modified open-field behavior and 2) these effects would be sex-specific.

Materials and Methods Animals and treatments Nulliparous female Dunkin-Hartley strain guinea pigs (Charles River Canada, St. Constant, Quebec, Canada) were mated as previously described (17). Guinea pigs were chosen as the model organism because they have similar placentation to humans and give birth to neuroanatomically mature young that undergo a pattern of brain development that more closely resembles primate and human fetal development than do the rat or mouse (18). Food and water were available ad libitum, and a 12-h light, 12-h dark cycle was maintained. Females were housed separately during pregnancy and the perinatal period, but remained in visual, olfactory, and auditory contact with other animals at all times. On postnatal d 25 (PND25), offspring were weaned and pair-housed in clear, polycarbonate cages. All studies were performed according to protocols approved by the Animal Care Committee at the University of Toronto, in accordance with the Canadian Council on Animal Care. Pregnant guinea pigs (F0) were subcutaneously injected with three courses (each course consisted of two injections, 24 h apart) of either BETA (1 mg/kg Betaject; Sabex, Boucherville, Quebec, Canada; n ⫽ 8) or vehicle (control, 0.166 ml/kg saline; n ⫽ 8) on gestational d 40 and 41 (period of rapid neurogenesis), 50 and 51 (period of rapid brain growth), and 60 and 61 (period of rapid myelination) (19). The dose of BETA used in this study is comparable to the dose used in pregnant women (approximately 0.25 mg/kg) because the guinea pig glucocorticoid receptor (GR) has a 4-fold lower affinity for sGC (20). We have previously shown that maternal subcutaneous saline injection into the intrascapular region does not cause activation of the maternal HPA axis in late gestation (Kapoor, A., and S. G. Matthews, unpublished observation). Animals were allowed to deliver undisturbed. There was no effect of repeated BETA treatment on gestation length (control 69.4 ⫾ 0.3 d; BETA 69.6 ⫾ 0.3 d) or birth weight (males: control, 102.2 ⫾ 1.9 g, and BETA, 107.6 ⫾ 3.1 g; females: control, 99.3 ⫾ 3.3 g, and BETA 110.2 ⫾ 6.2 g) (15). Offspring (F1 generation) were weaned on PND25.


F1 pregnancy and F2 offspring

The reproductive status of F1 females (control, n ⫽ 11; BETA, n ⫽ 8) was monitored beginning on PND25, as previously described (21). Briefly, the reproductive cycle is 16 d, with the beginning (estrus) marked by an opening of the vaginal mucous membrane. After a series of behavioral and endocrine tests (see Dunn et al., Ref. 15), adult F1 female offspring (PND157–196) were bred with untreated control males. F1 females were left undisturbed during pregnancy and delivery, apart from routine cage maintenance. F2 offspring were weighed every 10 d from birth and at the time of euthanasia. Measurements of body length and abdominal circumference were taken on PND0 and 25 (at weaning). Male and female F2 offspring (control: males n ⫽ 8, females n ⫽ 11; BETA: males n ⫽ 6, females n ⫽ 8) underwent a series of behavioral and endocrine tests during juvenile and adult life (described below). F2 animals were euthanized at 1200 h. Females were euthanized during the midluteal phase of their reproductive cycle (d 11) after behavioral/endocrine testing (PND158 ⫾ 4 d, one complete cycle after behavioral/endocrine testing), and males on approximately PND103 (⫾ 1 d). Trunk blood was collected in heparinized tubes, centrifuged (10,000 ⫻ g for 30 sec at 4 C) and plasma separated and stored at ⫺20 C. Brains from each animal were hemisected on an angle such that the left hemisphere was larger and contained the entire paraventricular nucleus (PVN) of the hypothalamus. The hippocampus was dissected from the right hemisphere, and the pituitary gland was removed from the floor of the skull. All tissues were immediately frozen on dry ice and stored at ⫺81 C for molecular analyses.

Juvenile F2 animals (PND25–35) Total activity and time spent in the outer zone were monitored in a novel, open-field environment (42 ⫻ 42 cm; 30 min) by an Opto-Max animal activity meter (Columbus Instruments, Columbus, OH), as previously described (22, 23). Computer software was used to divide the testing chamber into inner (a 14 ⫻ 14 cm center-square) and outer (within 14 cm of the perimeter) zones (22). Individual animals were tested on PND25 between 0800 and 1200 h in a quiet testing room. Saliva samples were taken at 0 (basal; before open-field testing, in the home cage), 30 (conclusion of test), 60 and 120 min (in the weaning cage) for salivary cortisol measurement. Saliva collection in the guinea pig is noninvasive, does not involve restraint (other than normal handling), and does not result in activation of the HPA axis (24). On PND35, F2 offspring underwent swim tasks at 0900 h, as part of the Morris water maze test (not presented). Saliva samples were taken before and 10 min after the first swim sessions to assess basal and activated HPA function via salivary cortisol measurement.

Adult F2 animals On approximately PND70 (and during the luteal phase of the second full cycle after PND70 in females), animals underwent another series of swim tasks at 0900 h, and salivary samples were collected as described above (before and 10 min after swim task). On PND93 (⫾ 1 d) in males and in females on d 8 of the reproductive cycle (luteal phase of the second full cycle after water maze testing; PND138 ⫾ 4 d), salivary samples were collected from animals in their home cage at 0800, 1200, and 1600 h to assess basal cortisol levels in the morning, midday, and after-


noon, respectively. On PND94 (⫾ 1 d) in male F2 offspring and during the luteal phase (d 9; PND139 ⫾ 4 d) of the reproductive cycle in female offspring, animals were exposed to a high-frequency strobe light at 1300 h (30 min, Micro Strobelite; ProGear Warehouse, Springfield, OH), a modest psychological stressor (22). Saliva samples were collected before testing and at 30, 60, and 120 min after initiation of the stressor. On PND96 (⫾ 1 d; males) and on d 11 of the female reproductive cycle (PND141 ⫾ 4 d), animals were injected with dexamethasone (1 mg/kg; dexamethasone challenge) (25) at 0800 h to assess suppressed HPA function and negative feedback regulation of the HPA axis. Salivary samples were collected at 0 (0800 h), ⫹8 (1600 h), and ⫹24 (0800 h the next day to demonstrate that the HPA axis had returned to normal) hours after dexamethasone administration.

Saliva and plasma hormone measurements Salivary cortisol (Salimetrics LLC, State College, PA) and plasma estradiol (MP Biomedicals, Orangeburg, NY) were measured using ELISA as previously described (21, 26). Plasma levels


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of cortisol, adrenocorticotropic hormone (ACTH) (ICN Pharmaceuticals Inc., Medicorp, Montreal, Quebec, Canada) and progesterone (MP Biomedical Inc., Costa Mesa, CA) were measured by RIA as we have previously described (15, 21). For all ELISA and RIA, samples were run in the same assay to negate interassay bias.

In situ hybridization (ISH) The method of ISH has been described in detail previously (27, 28). Briefly, 10-␮m coronal sections of brain and pituitary were mounted on poly-L-lysine-coated slides, dried, and postfixed with 4% paraformaldehyde. The use of antisense oligonucleotide probes for the mineralocorticoid receptor (MR), GR, CRH, CRH type 1 receptor (CRHR1), arginine vasopressin (AVP), and proopiomelanocortin (POMC) has been previously described (17, 26, 29, 30). Probes were end-labeled with [35S]deoxyadenosine-5⬘-␣-thiophosphate (1300 Ci/mmol; PerkinElmer, Woodbridge, Canada) using terminal deoxynucleotidyl transferase (15 U/␮l; Invitrogen Canada Inc., Burlington, Can-

FIG. 1. Male and female locomotor activity in an open field (A and B) and salivary cortisol (C and D) in F2 control (CTRL) (males, n ⫽ 7; females, n ⫽ 9; —䡺—, white bars) and BETA (males, n ⫽ 8; females, n ⫽ 6; - - -f- - -, black bars) offspring at PND25. Inset, total activity. The level of activity was assessed over the 30-min test (arbitrary activity units), whereas salivary cortisol was measured before and 30, 60, and 120 min after exposure to the open field on PND25 using ELISA. Data are presented as mean ⫾ SEM. A significant difference between control and BETA groups is represented as follows: *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001.

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ada) to a specific activity of 1.0 ⫻ 109 cpm/␮g. Slides hybridized with AVP probes were exposed to autoradiographic film (Biomax; Kodak, Rochester, NY) for 2 d, whereas slides for all other probes were exposed for variable periods of time as previously described (26, 31). A sense oligonucleotide probe was run in each ISH to demonstrate the level of nonspecific binding. Film analysis was carried out on an average of 9, 12 and 12 sections per gene or per animal for hippocampus or hypothalamus and pituitary, respectively, using the MCID software package (Imaging Research, St. Catharines, Canada). Care was taken to ensure that analysis was undertaken at the same neuroanatomical level in each animal. The relative optical density (ROD) was quantified after subtraction of background values. Standard slides (14C; American Radiochemical, St. Louis, MO) were incorporated in each run to ensure that densitometric analysis was undertaken within the linear range of the autoradiographic film. GR mRNA was measured in the hippocampus (CA1/2, CA3, and CA4 regions), dentate gyrus, PVN of the hypothalamus, and anterior lobe of the pituitary. MR mRNA levels were determined in the hippocampus (as for GR) and the dentate gyrus. CRH and AVP mRNA were measured in the PVN. POMC, GR, and CRHR1 mRNA levels were determined in the anterior lobe of the pituitary.

Western blot analysis GR, CRHR1, AVP receptor type 1 (AVPR1), and ACTH protein levels in anterior pituitary homogenate were assessed as we have described previously (15). Briefly, samples were homogenized in RIPA lysis buffer, and total protein concentration was


measured by the Bradford method. Denatured samples were separated by SDS-PAGE (10% gel for ACTH, 8% for other proteins) and transferred to a nitrocellulose membrane using the iBLOT system (Invitrogen Canada). Membranes were blocked overnight (4 C) in 5% skim milk with PBS with Tween 20 (blocking solution). They were then incubated with primary antibodies for GR (H-300, 1:500, rabbit polyclonal; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), CRHR1 (V-14, 1:500, goat polyclonal; Santa Cruz), AVPR1 (H-90, 1:1000, rabbit polyclonal; Santa Cruz), ACTH (B427; 1:5000, mouse monoclonal; Santa Cruz), and ␤actin (1:5000, rabbit polyclonal; Sigma) in blocking solution (1 h at 23 C). Membranes were then washed in PBS with Tween 20 and incubated with the horseradish peroxidase-conjugated secondary antibody, raised against the appropriate species (1:5000 dilution, 2 h at 23 C; PerkinElmer) followed by Western Lightning Chemiluminescence Reagent Plus (PerkinElmer). Bands were visualized by exposure to Kodak Blue X-OMAT film (PerkinElmer). The ROD of the bands was measured using computerized image analysis and was standardized against the actin signal (MCID Core version 7.0; Imaging Research Inc., Interfocus Imaging Ltd., Cambridge, UK).

Statistical analysis Analysis was conducted using GraphPad Prism version 4 (GraphPad Software, Inc., San Diego, CA). All data are expressed as means ⫾ SEM; significance is set at P ⬍ 0.05. If more than one male or female was sampled from a litter, data from same-sex littermates were averaged to eliminate litter bias. All analyses were carried out separately for males and females be-

FIG. 2. F2 male and female salivary cortisol before and after a swim stress on PND35 (A and B) and PND70 (C and D) in control (males, n ⫽ 8; females, n ⫽ 9; —䡺—) and BETA (males n ⫽ 8, females n ⫽ 5; - - -f- - -) offspring. The level of salivary cortisol was measured before and after exposure to a swim stress using ELISA. Data are presented as mean ⫾ SEM. A significant difference between control and BETA groups is represented as follows: *, P ⬍ 0.05; **, P ⬍ 0.01.


cause we initially observed profound sex differences in the effects of prenatal BETA treatment (F0 pregnancy) on body conformation and behavior in young F2 offspring. Litter size, mRNA, protein & plasma hormone levels, and time spent in the outer zone of the open field were analyzed by one-way ANOVA. Twoway ANOVA with repeated measures (and Bonferroni post hoc analyses) was used to analyze activity in the open field, salivary cortisol (treatment ⫻ time), body measures and weight gain (treatment ⫻ age). Area under the curve (AUC) was calculated for salivary cortisol samples. Total and net AUC were calculated using the trapezoid rule (32), with net AUC combining both positive and negative peaks. The baseline for total AUC was set to y ⫽ 0, whereas for net AUC it was the value of the first (or control, pretest) sample. Total AUC was calculated for basal salivary cortisol to provide an index of levels across the day, whereas net AUC was calculated for open-field, and flashing light tests to determine response to the stressor. Net area above the curve was used for the dexamethasone challenge to assess salivary cortisol suppression. One-way ANOVA was used to determine treatment-specific effects of time in salivary cortisol samples.

Results F2 litter size, body weights, and measurements There were no differences in litter sizes, number of males/females per litter, or male and female birth weight between treatment groups, as previously reported (15). However, F2 BETA males were found to have significantly increased body length compared with control animals at PND0 (control, 15.1 ⫾ 0.2 cm; BETA, 16.2 ⫾ 0.3 cm; P ⬍ 0.01) and PND25 (control, 21.2 ⫾ 0.5 cm; BETA, 23.7 ⫾ 0.6 cm; P ⬍ 0.001). There were no differences in growth (increasing body weight) from birth to adulthood (70 d) between the F2 control and BETA offspring (data not shown). There were also no differences in body weight at the time of euthanasia (103 d in males, ⬃158 d in females). Open-field activity Repeated-measures ANOVA revealed that total activity in BETA F2 juvenile males (PND 25) was significantly reduced (Fig. 1A; P ⬍ 0.05) compared with control animals over the 30-min testing period. Post hoc analysis identified reductions in BETA F2 male offspring activity during several 5-min periods of the test, particularly during the early phase of open-field exposure (0 –5 min, P ⬍ 0.001; 5–10 min, P ⬍ 0.01; 15–20 min, P ⬍ 0.05). There was also a significant decrease in activity over the 30-min test in F2 control animals (Fig. 1B; P ⬍ 0.01) but not in the BETA F2 offspring. There were no significant differences in the open-field activity between female F2 control and BETA offspring, although there was a trend for increased activity in the BETA F2 offspring. Separate analyses of the total time spent in the inner and outer zone of the open-


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field chamber revealed that animals from both sexes spent significantly more time in the outer vs. inner zone (data not shown; P ⬍ 0.001), with no effect of prenatal sGC treatment. However, F2 BETA males spent less time in the inner zone during the last 5 min compared with the first 5 min (P ⬍ 0.01); there was no difference in control males. Basal, stress-activated, and dexamethasonesuppressed HPA function Juvenile F2 offspring (PND25–35) There was no effect of prenatal sGC treatment on basal salivary cortisol concentrations in juvenile (PND25) male and female F2 offspring measured before exposure to the open field (Fig. 1, C and D). There was also no significant effect of prenatal sGC treatment on the activation (0 – 60 min), recovery (60 –120 min), or overall (0 –120 min) salivary cortisol response to exposure to the novel open-field environment (Fig. 1, C and D). On PND35, juvenile animals were exposed to a swim stress. There were no differences in basal (preswim) cortisol levels between the groups (Fig. 2, A and B). Swim exposure resulted in significant (P ⬍ 0.01) increases in salivary cortisol levels in all groups except F2 BETA females (no cortisol response to swim stress). However, in F2 BETA male and female offspring the magnitude of the response was significantly

FIG. 3. F2 male (PND93) and female (⬃PND138) salivary cortisol over an 8-h period (A and B) in control (males, n ⫽ 5; females, n ⫽ 8; — 䡺—) and BETA (males, n ⫽ 7; females, n ⫽ 5; - - -f- - -) offspring. The level of basal salivary cortisol was measured at 0800, 1200, and 1600 h using ELISA. Data are presented as mean ⫾ SEM.

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reduced compared with controls (male, P ⬍ 0.01; female, P ⬍ 0.05) (Fig. 2, A and B). Adult F2 offspring On approximately PND70, adult male and female (during the luteal phase) animals were exposed to a second swim stress. There were no differences in basal (preswim) cortisol levels between the groups (Fig. 2, C and D); however, there was a strong trend toward reduced basal cortisol levels in female BETA F2 offspring (P ⫽ 0.08). Swim exposure resulted in significant increases in salivary cortisol levels in males (control, P ⬍ 0.001; BETA, P ⬍ 0.01) and BETA females (P ⬍ 0.01); there was a trend toward a significant rise in cortisol in control females (P ⫽ 0.079). In F2 BETA male offspring, there was a tendency for a reduced cortisol response; however, this failed to attain significance (Fig. 2C; P ⬎ 0.05). In adult female F2 offspring, the magnitude of the response was similar between the groups albeit from different baselines (Fig. 2D). On approximately PND93 (males) and on d 8 of the reproductive cycle (females, midluteal, ⬃PND138), salivary cortisol concentrations were measured at 0800, 1200, and 1600 h (Fig. 3, A and B) to assess basal HPA function throughout the subjective day. There were no significant differences in cortisol values between the treatments, but there were significant effects of time of day


(elevated basal salivary cortisol at 0800 h compared with 1200 and 1600 h) only in control animals (P ⬍ 0.05). There was also a strong trend toward reduced basal salivary cortisol in the female BETA F2 offspring (P ⫽ 0.08). Exposure to the psychological stress of an inescapable strobe light resulted in a significant increase (P ⬍ 0.05) in salivary cortisol in control females (Fig. 4C). In contrast, male control and BETA F2 offspring failed to mount any cortisol response to the psychological stress (Fig. 4A). This is further illustrated by analysis of the of the Net AUC response to the strobe light (Fig. 4, B and D). Indeed, F2 BETA females displayed a significant decrease in Net AUC of cortisol (P ⬍ 0.05) compared with control females. On approximately PND96 (males) and on d 11 of the reproductive cycle (females, ⬃PND141), animals underwent a dexamethasone-suppression test to investigate glucocorticoid feedback sensitivity. F2 BETA males and control females exhibited significant suppression of salivary cortisol levels (P ⬍ 0.01 and P ⬍ 0.05, respectively) 8 h after dexamethasone administration (1600 h; Fig. 5, A and B). F2 BETA male offspring also displayed a significant recovery of cortisol levels (P ⬍ 0.05) over the next 16 h (1600 – 0800 h the next morning), and there was a strong trend for recovery in female controls (P ⫽ 0.055). In contrast, female F2 BETA and male control offspring displayed no suppression of salivary cortisol to the administration of dexamethasone. Plasma hormone concentrations Analysis of plasma hormone levels at the time of euthanasia revealed that there were no significant differences in plasma ACTH (males: control, 83.1 ⫾ 18.3 pg/ml; BETA, 56.6 ⫾ 10.5 pg/ml; females: control, 32.6 ⫾ 3.8 pg/ml; BETA, 37.9 ⫾ 5.0 pg/ml) or cortisol (males: control, 20.8 ⫾ 8.0 ng/ml; BETA, 21.0 ⫾ 5.6 ng/ml; females: control, 51.6 ⫾ 7.4 ng/ml; BETA, 34.4 ⫾ 10.7 ng/ml) levels between treatment groups for either sex. Similarly, plasma levels of estradiol (control, 26.9 ⫾ 1.3 pg/ml; BETA, 23.0 ⫾ 2.4 pg/ml) and progesterone (control, 1.9 ⫾ 0.3 ng/ml; BETA, 1.4 ⫾ 0.2 ng/ml) were not significantly different between control and BETA females.

FIG. 4. F2 male (PND94) and female (⬃PND139) salivary cortisol in response to a 30-min flashing light stressor (A and C) AUC (B and D) in control (CTRL) (males, n ⫽ 7; females, n ⫽ 8; —䡺—, white bars) and BETA (males, n ⫽ 6; females, n ⫽ 6; - - -f- - -, black bars) offspring. The level of salivary cortisol was measured before and 30 and 60 min after exposure to strobe light using ELISA. Data are presented as mean ⫾ SEM. A significant difference between control and BETA groups is represented as follows: *, P ⬍ 0.05.

Expression of HPA-related genes Hippocampus Two-way ANOVA revealed a significant (P ⬍ 0.001) increase in hippocam-



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0.05); however, there were no corresponding changes in protein. AVPR1 protein levels were not different between treatment groups for either males or females (Fig. 7G).

Discussion

FIG. 5. F2 male (PND96) and female (⬃PND141) salivary cortisol in response to an injection of dexamethasone (1 mg/kg; A and B) in control (males n ⫽ 7, females n ⫽ 6; —䡺—) and BETA (males n ⫽ 6, females n ⫽ 7; - - -f- - -) offspring. The level of salivary cortisol was measured before, 8 and 24 h after receiving dexamethasone using ELISA. Data are presented as mean ⫾ SEM. A significant difference between time 0 and 8 h is represented as follows: ‡, P ⬍ 0.05; ‡‡, P ⬍ 0.01.

pal GR mRNA expression in F2 BETA males compared with controls. Post hoc analysis revealed that this effect was most prevalent in the CA1/2 region (P ⬍ 0.001; Fig. 6A). In contrast, two-way analysis identified significant reductions in GR (Fig. 6B; P ⬍ 0.05) and MR (Fig. 6D; P ⬍ 0.05) mRNA levels in the hippocampus of F2 BETA females. However, subsequent post hoc analyses did not reveal these changes to be attributable to any particular region of the hippocampus. Hypothalamic PVN There were no significant effects of antenatal sGC treatment on the expression of AVP, CRH, and GR mRNA in the PVN in male or female F2 offspring (Table 1). Anterior pituitary Levels of POMC, CRHR1, and GR mRNA were significantly reduced in the anterior pituitary of F2 BETA females compared with controls (Fig. 7, A–C; P ⬍ 0.05). These decreases corresponded to reductions in ACTH, CRHR1, and GR protein in the anterior pituitary (Fig. 7, D–F; P ⬍ 0.05). F2 BETA males also exhibited reduced POMC and CRHR1 mRNA expression in the anterior pituitary compared with controls (Fig. 7, A and B; P ⬍

This is the first study to demonstrate transgenerational effects (via maternal transmission) of prenatal synthetic glucocorticoid exposure on HPA function and open-field behavior. These effects occurred despite no manipulation of the F1 pregnancy. We have demonstrated that both male and female F2 offspring, whose grandmothers received sGC during pregnancy, exhibit reductions in stress responsiveness and basal cortisol regulation that correspond to alterations in molecular regulation of the HPA axis at the level of the hippocampus and anterior pituitary. In addition, juvenile F2 BETA male offspring exhibited profoundly decreased locomotor activity in a novel environment. Previous studies from our laboratory (33) and other laboratories (34 –36) have identified potent transgenerational effects of prenatal manipulation. Bertram et al. (33) demonstrated that maternal undernutrition during late F0 pregnancy (GD36 –70) in the guinea pig resulted in elevated basal plasma cortisol levels and altered HPA response to dexamethasone and CRH challenge in both F1 and F2 adult male offspring. Drake et al. (35) reported that prenatal dexamethasone exposure in rats led to reduced birth weight, glucose intolerance, and altered glucose metabolism (in the liver) in F2 offspring, although this phenotype was not transmitted to the F3 generation. Maternal high-fat diet during pregnancy in rats has been shown to result in increased F1 and F2 body length and insulin insensitivity (34). In the present study, we found significant differences between the daily profiles of basal cortisol in adult F2 BETA and control animals. BETA offspring also exhibited a reduced HPA response to different stressors as well as sex-specific alterations in HPA response to dexamethasone challenge. There was no significant effect of antenatal BETA on basal cortisol levels in F2 offspring; however, BETA exposure abolished the diurnal changes in salivary cortisol that were evident in adult control animals. Additional studies are required to determine whether this lack of diurnal HPA regulation extends through the night as well as the mechanisms involved. It is important to note that the behavioral and endocrine phenotypes observed in F1 mothers that were themselves exposed to BETA in utero are not simply replicated in their F2 offspring. Adult F1 BETA females were hypocortisolemic during the luteal (d 8) phase of their reproductive cycle (15).

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FIG. 6. ROD of GR (A and C) and MR (B and D) mRNA in the hippocampus in adult F2 male (A and B; PND103) and female (C and D; PND158) guinea pig offspring whose grandmothers had been injected with control (males, n ⫽ 6 – 8; females, n ⫽ 6 – 8; white bars) or BETA (males, n ⫽ 8; females, n ⫽ 6 –7; black bars). E and F, Representative images of GR mRNA expression in male control (E) and BETA (F) hippocampi. Ctrl, Control. Data are presented as mean ⫾ SEM. A significant difference between control and BETA groups is represented as follows: ***, P ⬍ 0.001.

The lower basal salivary cortisol levels correlated with lower plasma estradiol levels (during the luteal phase), suggesting a role of sex hormones in regulating basal cortisol levels (37). In the present study, F2 BETA females displayed a trend toward reduced basal salivary cortisol levels during the luteal phase but with no accompanying difference in sex hormone levels. The effects of prenatal BETA have not been documented in adult F1 male offspring, although F1 adult male guinea pig offspring that were prenatally exposed to dexameth-

asone display a hypocortisolemic phenotype (14, 26). It is difficult to directly compare these latter results with those in the present study because the effects of prenatal dexamethasone and BETA on HPA function may differ due to these compounds having different pharmacokinetics and genomic efficacies (38, 39). In the present study, there was no difference in the HPA response of F2 BETA females or males to a novel environment compared with controls. However, BETA females failed to mount an HPA response to a flashing strobe light



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TABLE 1. ROD of CRH, AVP, and GR mRNA in the PVN of adult F2 male and female control (males, n ⫽ 7; females, n ⫽ 4 –7) and BETA (males, n ⫽ 6 – 8; females, n ⫽ 4 – 6) offspring Male

Female

Gene

Control

BETA

Control

BETA

CRH AVP GR

100.0 ⫾ 14.4 100.0 ⫾ 10.1 100.0 ⫾ 7.5

73.8 ⫾ 7.8 117.5 ⫾ 10.8 101.3 ⫾ 8.2

100.0 ⫾ 11.5 100.0 ⫾ 16.5 100.0 ⫾ 1.0

95.2 ⫾ 23.9 113.4 ⫾ 20.5 85.2 ⫾ 8.6

Data are expressed as percentage of control and presented as mean ⫾ SEM.

compared with F2 control females. As juveniles, both male and female F2 BETA offspring also elicited reduced HPA responses to swim stress. However, although there was a trend toward reduced responsiveness to swim stress in the adult F2 BETA male offspring, this effect was not significant. To our knowledge, no studies have demonstrated habituation of the HPA response to swim stress, and because adult animals had not been exposed to swim stress for 35–50 d since their juvenile swim experience, we believe it unlikely that the results we observed were due to habituation but rather due to an effect of age. Notwithstanding, we have observed clear sex- and age-specific changes in HPA response to both psychological and strong somatic stressors after grandmaternal sGC treatment. Future studies are required to allow direct comparison of HPA phenotype across multiple generations. Dexamethasone challenge revealed increased negative feedback sensitivity (i.e. lower salivary cortisol levels) in F2 BETA males compared with controls, whereas there was reduced feedback sensitivity in F2 BETA females. In addition, juvenile F2 BETA males were less active in a novel environment compared with controls with no significant differences in females. Sex-specific transgenerational effects have been identified in other studies. In mice, maternal high-fat diet led to increased GH secretagogue receptor expression in F2 female hypothalami compared with control animals, whereas F2 males were no different from controls (34). In another study, anxiety levels in F3 offspring descended from F0 mothers that had received an endocrine disruptor, vinclozolin, during pregnancy were reduced in males but elevated in females (40). The mechanisms by which sexspecific effects occur in F2 offspring are not known. However, F1 BETA females have been shown to exhibit reduced plasma estradiol levels (15). It is possible that altered maternal sex-hormone balance leads to sex-specific effects in F2 offspring. The decrease in the expression of POMC mRNA and ACTH protein in the anterior pituitary of female F2 BETA offspring clearly corresponds to a reduction in corticotroph ACTH content, suggesting a reduction in overall corticotroph activity. There was also a reduction in cor-

ticotroph sensitivity as indicated by the reduction in CRHR1 mRNA and protein expression. These changes corresponded to the decreased cortisol release that was observed in PND35 F2 BETA females after the swim-stress task and reduced stress responsiveness in adult offspring. F2 BETA females also exhibited reduced levels of GR mRNA and protein in the anterior pituitary as well as reduced GR and MR in the hippocampus, suggesting decreased glucocorticoid negative feedback inhibition of the stress response. Indeed, this was demonstrated after dexamethasone challenge. Interestingly, adult F2 BETA females displayed reduced cortisol levels before the swim task (compared with female controls) yet were able to mount a cortisol response to stress of similar magnitude to controls. This suggests a complex interplay between reduced corticotroph activity/sensitivity and decreased glucocorticoid negative feedback. Like females, F2 BETA males exhibited a reduced cortisol response to a swim stress. This may be attributed to a decreased ability of hypothalamic CRH to stimulate ACTH release from the anterior pituitary (i.e. reduction in CRHR1 and POMC mRNA). Surprisingly, CRHR1 and ACTH protein were not significantly different from control males. The reason for the inconsistency between mRNA and translated protein is unclear. The blunted stress response in F2 BETA males may also be due to their significantly elevated levels of GR in the hippocampus. Elevated GR levels would suggest increased glucocorticoid negative feedback inhibition. Indeed, this was evident after dexamethasone challenge. Juvenile F2 BETA males also exhibited reduced locomotor activity (total activity and distance traveled) in a novel environment when compared with controls. Although F2 BETA and control males spent the same amount of time in the inner zone of the testing chamber, F2 BETA males spent significantly less time in the inner zone during the final 5 min of testing when compared with the first 5 min. Together, these data indicate that F2 BETA males are less active and show a greater aversion to the center of the open field (at a time when they should be more familiar with their environment). The latter may suggest an increase in anxiety-like behavior in response to a novel environment. Previous

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FIG. 7. Anterior pituitary expression of POMC mRNA (A), CRHR1 mRNA (B), GR mRNA (C), ACTH protein (D), CRHR1 protein (E), GR protein (F), and AVPR protein (G) in adult F2 male (PND103) and female (PND158) control (males, n ⫽ 5– 6; females, n ⫽ 6 –10; white bars) and BETA (males, n ⫽ 4 –5; females, n ⫽ 4 – 6; black bars) offspring. H and I, Representative images of POMC mRNA expression in female control (H) and BETA (I) pituitaries. AP, Anterior (lobe) pituitary; IP, intermediate (lobe) pituitary. Data are expressed as percentage of control (CTRL) and presented as mean ⫾ SEM. A significant difference between control and BETA groups is represented as follows: *, P ⬍ 0.05.


studies have identified a relationship between HPA regulation and anxiety behavior, linking increased hippocampal expression of GR with anxiety (41). In our current study, we found increased expression of GR mRNA in the hippocampi of F2 BETA males, supporting this hypothesis. However, additional behavioral testing is required to confirm changes in anxiety behavior. The mechanisms by which transgenerational effects of prenatal manipulations are transmitted represents an area of increasing research focus. In the present study, we propose that exposure to excess prenatal synthetic glucocorticoids results in maternal adaptations to pregnancy, altered epigenetic regulation of gene expression, or a combination of both. It is possible that the altered HPA and cardiovascular phenotypes in F1 females, described previously in a number of studies (14, 15, 35), result in adaptive changes in metabolic and endocrine function. These changes to the females’ internal environment could lead to alterations in pregnancy parameters (hormone levels, placental function, blood flow) (15, 42, 43) and consequently affect development of the F2 offspring, contributing to the changes we have identified in F2 BETA offspring. With respect to the present study, it is important to note that the BETA F2 generation developed from germline cells (in this case, ova) that were exposed in utero to BETA (i.e. F1 female primordial germ cells were exposed during gestation). This exposure may, in part, have programmed the development of BETA F2 offspring. In addition, the exposure of developing F1 ovarian germ cells to synthetic glucocorticoids could potentially contribute to the sex-specific alterations exhibited by the F2 BETA offspring. Recent studies have found that changes to fetal and early life environments can dramatically alter DNA methylation and histone acetylation of specific gene promoters (44 – 46) and that the effects can be transferred to the subsequent generations (47). Moreover, recent studies have demonstrated that epigenetic silencing of alleles that occurs during meiosis does not completely erase all epigenetic modification as previously thought (36, 48). In the context of the present study, prenatal exposure to synthetic glucocorticoids during critical phases of fetal development may change the epigenetic regulation of gene promoters leading to the programming of HPA function and behavior that we observed. It is possible that these effects could then be transmitted across multiple generations via the oocyte or sperm. We have recently demonstrated that prenatal exposure to synthetic glucocorticoids leads to acute and long-term changes in global methylation in the brain, liver, kidney, and adrenal that this is associated with long-term changes in the expression of the cellular machinery responsible for maintenance of DNA methylation


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and that similar effects are seen in F2 offspring (24). Clearly, additional studies are required to investigate a potential epigenetic basis for the transgenerational effects of synthetic glucocorticoids on HPA function, behaviors, and related changes in gene expression. In conclusion, prenatal exposure to multiple courses of synthetic glucocorticoids has profound effects on HPA function and behavior in F2-generation offspring. The fact that BETA exposure exhibits transgenerational effects is clinically significant. BETA is the treatment of choice for mothers at risk of preterm delivery (15). Administration of multiple courses of prenatal sGC became common practice at many health centers despite limited knowledge on the potential for effects on long-term health outcomes (49). Furthermore, the mechanisms by which early-life events lead to transgenerational memory of endocrine function and behaviors need to be further elaborated. It is likely that they involve altered maternal adaptations to pregnancy and altered maternal behaviors as well as epigenetic processes.

Acknowledgments Address all correspondence and requests for reprints to: Dr. S. G. Matthews, Department of Physiology, Faculty of Medicine, University of Toronto, Medical Sciences Building Room 3302, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8. Email: [email protected]. This work was supported by the Canadian Institutes for Health Research (FRN-97736 to to S.G.M. and Doctoral Research Award to M.I.). Different elements of this work were presented by S.G.M and A.K. at the 2008 Society for Gynecologic Investigation Meeting, by M.I. at the 2010 Developmental Origins of Health and Disease Meeting, and by V.G.M at the 2010 Society for Neuroscience Meeting. Disclosure Summary: M.I., V.G.M., A.K., and S.G.M. have nothing to disclose.

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