Attenuated neuroendocrine responses to emotional and physical stressors in pregnant rats involve adenohypophysial changes

Authors


Corresponding author I. D. Neumann: Max Planck Institute of Psychiatry, Kraepelinstrasse 2, D 80804 Munich, Germany. Email: INEU@MPIPSYKL.MPG.DE

Abstract

  • 1The responsiveness of the rat hypothalamo-pituitary-adrenal (HPA) axis and hypothalamo-neurohypophysial system (HNS) to emotional (elevated plus-maze) and physical (forced swimming) stressors and to administration of synthetic corticotrophin-releasing hormone (CRH) was investigated during pregnancy and lactation. In addition to pregnancy-related adaptations at the adenohypophysial level, behavioural responses accompanying the neuroendocrine changes were studied.
  • 2Whereas basal (a.m.) plasma corticosterone, but not corticotrophin (adrenocorticotrophic hormone; ACTH), levels were increased on the last day (i.e. on day 22) of pregnancy, the stress-induced rise in both plasma hormone concentrations was increasingly attenuated with the progression of pregnancy beginning on day 15 and reaching a minimum on day 21 compared with virgin control rats. A similar attenuation of responses to both emotional and physical stressors was found in lactating rats.
  • 3Although the basal plasma oxytocin concentration was elevated in late pregnancy, the stress-induced rise in oxytocin secretion was slightly lower in day 21 pregnant rats. In contrast to vasopressin, oxytocin secretion was increased by forced swimming in virgin and early pregnant rats indicating a differential stress response of these neurohypophysial hormones.
  • 4The blunted HPA response to stressful stimuli is partly due to alterations at the level of corticotrophs in the adenohypophysis, as ACTH secretion in response to CRH in vivo (40 ng kg−1, i.v.) was reduced with the progression of pregnancy and during lactation. In vitro measurement of cAMP levels in pituitary segments demonstrated reduced basal levels of cAMP and a lower increase after CRH stimulation (10 nM, 10 min) in day 21 pregnant compared with virgin rats, further indicating reduced corticotroph responsiveness to CRH in pregnancy.
  • 5The reduced pituitary response to CRH in late pregnancy is likely to be a consequence of a reduction in CRH receptor binding as revealed by receptor autoradiography. [125I] CRH binding in the anterior pituitary was significantly reduced in day 11, 17 and 22 pregnant rats compared with virgin controls.
  • 6Anxiety-related behaviour of the animals as revealed by the time on and entries into the open arms of the elevated plus-maze was different between virgin and pregnant rats with decreased number of entries indicating increased anxiety with the progression of pregnancy (except on pregnancy day 18). The emotional behaviour, however, was not correlated with the neuroendocrine responses.
  • 7The results indicate that the reduced response of the HPA axis to stressors described previously during lactation is already manifested around day 15 of pregnancy in the rat and involves physiological adaptations at the adenohypophysial level. However, alterations in stressor perception at higher brain levels with the progression of pregnancy may also be involved.

In addition to its normal circadian variation, the hypothalamo-pituitary-adrenal (HPA) axis activity is altered by physiological or pathophysiological states that challenge the internal homeostasis of the organism. In addition to chronic stress (Fuchs & Flügge, 1994), psychiatric diseases (Holsboer & Barden, 1996) or ageing (Hatzinger, Reul, Landgraf, Holsboer & Neumann, 1996), pregnancy and lactation might represent a similar challenge due to the profound physiological adaptations occurring in the mother. Thus, in pregnancy there is increased sensitivity of the adrenal gland to corticotrophin (ACTH) and increased glucocorticoid secretion in several species (Dupouy, Coffigny & Magre, 1975; Carr, Parker, Madden, MacDonald & Porter, 1981; Waddell & Atkinson, 1994; Keller-Wood, 1996). However, there are no detailed studies regarding the responsiveness to stressors of the HPA axis reflected primarily by plasma ACTH and corticosterone concentrations, or of underlying mechanisms of possible pregnancy-related changes in HPA axis activity, or of associated behavioural alterations. The responsiveness of the HPA axis is reduced during lactation (see below), and we have now studied whether such altered responsiveness is established in pregnancy. Reduced reactivity of the maternal HPA axis may protect both the pregnant rat and her offspring from harmful excessive levels of glucocorticoids (for review, see Weinstock, 1997).

During lactation, the activity and regulation of the HPA axis are altered in ways that are dependent on the presence or suckling of the young (Walker, Lightman, Steele & Dallman, 1992; Windle et al. 1997). Thus, an increase in glucocorticoid secretion and a flattening of the diurnal rhythm in glucocorticoid secretion have been described (Stern, Goldman & Levine, 1973; Walker et al. 1992; Fischer, Patchev, Hellbach, Hassan & Almeida, 1995). In addition, secretion of ACTH from the anterior pituitary and, consequently, of cortisol (humans) and corticosterone (rats) from the adrenal glands is reduced during physical (Altemus, Deuster, Galliven, Carter & Gold, 1995; Walker, Trottier, Rochford & Lavallée, 1995), ether (Lightman & Young, 1989; Walker et al. 1992) and noise stress (Windle et al. 1997), and in response to cardiovascular stimuli (Keller-Wood, 1996). Furthermore, there are indications of altered emotional behaviour in lactating rats (Hard & Hansen, 1984; Walker et al. 1995).

Besides the well-described stress responses of the HPA axis there is also activation of the hypothalamo-neurohypophysial system (HNS), releasing either oxytocin or vasopressin or both from the neurohypophysis into blood, in response to various stressors (Lang, Heil, Ganten, Hermann, Unger & Rascher, 1983; Kasting, 1988; Wotjak, Kubota, Ganster, Liebsch, Neumann & Landgraf, 1996). During lactation, the stimulated oxytocin secretion is reduced in response to physical stress (Carter & Lightman, 1987; Lightman, 1992; Neumann, Pittman & Landgraf, 1995b; Walker et al. 1995) and hyperosmotic or pharmacological stimulation (Patel, Chowdrey & Lightman, 1991; Koehler, McLemore, Tang & Summy-Long, 1993; Neumann, Landgraf, Bauce & Pittman, 1995a). However, the reactivity of the oxytocinergic system during pregnancy has not been studied.

The present study was designed to test the secretory responses of the HPA axis and the HNS to the emotional and physical stressors of exposure to the elevated plus-maze and forced swimming, respectively, during pregnancy. We then studied the responsiveness to exogenous corticotrophin-releasing hormone (CRH) in the pregnant and virgin rats to seek changes at the level of the adenohypophysis in mechanisms regulating ACTH secretion from corticotrophs. We further sought modifications in CRH receptors or their coupling in corticotrophs by measuring cAMP production by anterior pituitaries from pregnant and virgin rats in vitro in response to CRH, since cAMP is a second messenger that is positively coupled to the CRH receptor in the pituitary (Antoni, 1986; King & Baertschi, 1990); we measured [125I] CRH binding-site density in the pituitary to examine the possibility of CRH receptor downregulation. Finally, to see if there is an alteration in emotionality accompanying the attenuated HPA axis and HNS responses to stressors, the anxiety-related behaviour of pregnant and virgin rats on the elevated plus-maze (Pellow, Chopin, File & Briley, 1985) was monitored.

Preliminary results have been presented (Neumann, Johnstone, Landgraf, Russell & Douglas, 1996; Douglas, Johnstone, Hatzinger, Neumann, Landgraf & Russell, 1996).

METHODS

Animals

In experiment 1, virgin female Wistar rats (260-290 g) were mated overnight with sexually experienced males, and pregnancy was confirmed by the presence of a vaginal plug of semen in the mating cages the following morning (day 1 of pregnancy). Rats were housed in groups of four to six under standard laboratory conditions at the Max Planck Institute of Psychiatry (12 : 12 h light-dark cycle, lights on at 07.00 h, 22°C, 60 % humidity, food and water ad libitum) for at least 5 days before surgery and after delivery from the supplier (Charles River, Sulzfeld, Germany). Another group of virgin Wistar rats was mated overnight and housed singly 3 days prior to parturition; surgery was performed on day 4 to 6 of lactation. Lactating rats were kept with their litters until just before the experiments.

For experiments 2 and 3, virgin and pregnant Sprague-Dawley rats (Bantin and Kingman, UK), mated as above, were housed singly (12 : 12 h light-dark cycle, lights on at 07.00 h, 21°C, food and water ad libitum) at the University of Edinburgh for at least 5 days prior to the experiment.

Experiment 1: ACTH, corticosterone, oxytocin and vasopressin secretory responses to emotional and physical stressors and to CRH in virgin, pregnant and lactating rats

Surgery for blood sampling

Under halothane (2-3 %) anaesthesia and using sterile procedures, rats were implanted with chronic jugular vein catheters 5 days before the start of the experiments. The jugular vein was exposed and a silicone tubing catheter (4 cm; Dow Corning) connected to a PE-50 polyethylene tubing was inserted approximately 3 cm into the vessel until the tip reached the right atrium; the catheter was exteriorized dorsally in the cervical region. The catheter was filled with sterile saline (0.9 %) containing gentamicin (30 000 i.u. ml−1; Centravet, Germany) and was flushed with the same solution after 3 days. Following surgery, rats were housed singly and handled carefully each day to familiarize them with the blood sampling procedure and to reduce non-specific stress responses during the experiments.

Behavioural testing and stress procedures

Elevated plus-maze.

The elevated plus-maze has been validated for the detection of emotional responses to anxiogenic and anxiolytic substances (Pellow et al. 1985) and to stressful external stimuli (Heinrichs, Pich, Miczek, Britton & Koob, 1992; Liebsch et al. 1995). The plus-maze was used to assess the emotional state and as a mild emotional stressor (novel environment) in our experiment. The test is based on creating a conflict between the exploratory drive of the rat and its innate fear of open and exposed areas. Thus, increased open-arm exploration indicates reduced anxiety-related behaviour. As described in detail by Liebsch et al. (1995), the apparatus consists of a plus-shaped platform elevated 70 cm from the floor. Two of the opposing arms (50 cm × 10 cm) are closed by 40 cm-high side and end walls (closed arms), whereas the other two arms have no walls (open arms). At the beginning of the test, the rat was placed onto the central area (10 cm × 10 cm) of the maze. The following parameters were recorded by means of a video camera-computer set-up during the 5 min exposure: (1) entries into open arms (ratio of open-arm entries to total number of entries into all arms), (2) time spent on the open arms (ratio of time spent on open arms to total time spent on all arms), and (3) overall activity (total number of entries into closed arms).

Forced swimming.

Forced swimming represents an ethologically relevant complex physical and emotional stressor for rats (Abel, 1994). With the extension tubing of the venous catheter still attached, rats were forced to swim for 90 s in a black plastic cylinder (40 cm in diameter and 50 cm in height) filled with tap water (19°C) to a depth of ca 40 cm. After the swim, the rats were gently dried using towels for 10 s and returned to their home cages.

Experimental protocols

Stressors in virgin, pregnant and lactating rats (day 1).

Five days after surgery, the responses of the HPA axis and the HNS to emotional and physical stressors were tested in virgin rats (289 ± 4.20 g; mean ±s.e.m.; n= 19), in rats on day 10 (327 ± 4.40 g; n= 6), day 15 (355 ± 7.30 g; n= 7), day 18 (374 ± 10.5 g; n= 7) and day 21 (430 ± 4.30 g; n= 7) of pregnancy and, in a separate experiment, in lactating rats (days 9-12 of lactation, 345 ± 5.80 g; n= 7) and their virgin controls (295 ± 5.90 g; n= 5).

At 08.00 h the catheters were attached to an extension tubing connected to a 1 ml plastic syringe filled with sterile heparinized saline (30 i.u. ml−1), and the rats were left undisturbed for 90 min. Either 0.2 ml (for detection of ACTH and corticosterone) or 0.6 ml (for ACTH, corticosterone, oxytocin and vasopressin, and lactate as an indicator of muscle activity) blood samples, replaced immediately by sterile 0.9 % saline, were taken under basal conditions at 09.30 and 10.00 h and after the respective stress exposure. For transfer to the elevated plus-maze, the catheter was disconnected and closed. Rats were then placed on the elevated plus-maze for 5 min, returned to their home cage and the catheter was reattached to the syringe. Ten minutes later, a blood sample was taken and immediately afterwards rats were exposed to the forced swim stress (90 s). Further blood samples were taken 5, 15 and 25 min after forced swimming.

At the end of the experiment, catheters were gently flushed with 0.3 ml gentamicin solution and closed.

Stimulation with i.v. CRH in virgin, pregnant and lactating rats (day 2).

On the following day at 08.00 h, the rats were weighed, then the catheter was reconnected to a syringe filled with heparinized saline as described above, and at 09.30 and 10.00 h basal blood samples (0.2 ml for ACTH and corticosterone) were taken. Further blood samples replaced by sterile saline were collected 10, 30 and 50 min after i.v. bolus injection of human CRH (40 ng kg−1, 80 ng ml−1; Bissendorf Peptide, Wedemark, Germany).

At the end of the experiment, the rats were killed by an overdose of halothane and pregnancy state was carefully checked in all rats post mortem.

Treatment of blood samples

All blood samples were collected on ice in EDTA-coated tubes containing 10 μl aprotinin (Trasylol; Bayer AG) and centrifuged at 4°C (5000 r.p.m., 5 min). Plasma samples were stored at -20°C (80 μl for ACTH, 200 μl for oxytocin and vasopressin, 50 μl for lactate) or -80°C (30 μl for corticosterone) until assay.

Radioimmunoassays (RIAs) for ACTH, corticosterone, oxytocin, vasopressin and plasma lactate measurement

Plasma ACTH and corticosterone concentrations were measured using commercially available kits (ICN) according to the respective protocols. The intra- and inter-assay coefficients of variation were below 7 and 10 %, respectively. Plasma ACTH and corticosterone concentrations from virgin and pregnant, and virgin and lactating rats, respectively, were estimated in different assays.

Oxytocin and vasopressin concentrations were estimated in extracted plasma samples by highly sensitive and selective RIAs (detection limit, 0.1 pg sample−1; cross-reactivity of the antisera with other related peptides, including vasopressin and oxytocin, was < 0.7 %) (for a detailed description see Landgraf, 1981).

Plasma lactate concentrations were measured enzymatically (MPR1 Lactat; Boehringer Mannheim).

Experiment 2: in vitro basal and CRH-stimulated cAMP levels in pituitary segments from virgin and pregnant rats

Conscious virgin (n= 16) and day 11 (n= 6), day 17 (n= 6) and day 21 (n= 8) pregnant Sprague-Dawley rats caged separately were transferred individually to the experimental room immediately before decapitation between 09.00 and 11.00 h to minimize stress. Pituitaries were rapidly removed and washed in 0.5 ml Dulbecco's modified Eagle's medium (Gibco BRL), buffered with 25 mM Hepes to pH 7.4 and containing 0.25 % bovine serum albumin (BSA; Sigma, RIA grade V) (thereafter solution referred to as DMEM). The posterior lobe was removed, anterior pituitaries weighed, and cut free-hand under microscopic control into eight similar segments. Pituitary segments were incubated for at least 1 h at 37°C in 1 ml of DMEM in 24-well cluster plates (Costar). Subsequently, each segment was transferred into fresh DMEM (250 μl) containing 0.5 mM of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). After incubation for 15 min at 37°C, segments were challenged for a further 10 min with either 250 μl of 20 nM CRH (final concentration 10 nM; Peninsula, St Helens, UK) or vehicle (DMEM). The reaction was terminated by the addition of 0.5 ml ice-cold 0.2 n HCl, and culture plates were sealed with parafilm and stored at -70°C until trituration and cAMP determination.

Determination of cAMP

After two rounds of freeze-thawing (-70 to 0°C), pituitary segments were homogenized by trituration through a gauge 26 needle. cAMP accumulation in duplicate samples of the acidic extract (pituitary tissue + extracellular medium) was determined using a specific double-antibody precipitation RIA as previously described (Woods, Shipston, Mullens & Antoni, 1992). Intra- and inter-assay variability was less than 5 and 10 %, respectively.

Experiment 3: measurement of [125I] CRH binding-site density in the pituitary gland

Virgin rats and rats on pregnancy day 11, 17 and 22 (n= 6 in each group) were housed singly for at least 1 week prior to tissue collection. Between 10.00 and 11.00 h the rats were decapitated and the whole pituitary carefully removed. Each pituitary was placed on a piece of aluminium foil on dry ice, covered in powdered dry ice and stored at -70°C.

Horizontal pituitary cryostat sections (20 μm) were thaw-mounted onto poly-L-lysine-coated slides, desiccated overnight at 4°C and stored at -20°C in sealed boxes until use.

For receptor autoradiography, sections were preincubated for 20 min at 21°C in 50 mM Tris-HCl (pH 7.4) containing 10 mM MgCl2, 2 mM EGTA, 0.1 % BSA (RIA grade V), 100 KIU ml−1 aprotinin (Trasylol), and 0.1 mM bacitracin (Sigma). Incubation was carried out subsequently in fresh buffer for 60 min with 0.2 nM [125 I] CRH (ovine; specific activity, 2200 Ci mmol−1; DuPont). Non-specific binding was determined in the presence of 10−6 M unlabelled ovine CRH (Sigma). The sections were washed three times in BSA-free Tris-HCl buffer at 4°C for 2 min, dipped in distilled water and rapidly dried in a stream of cold air. Dried sections were exposed to Agfascopix Video 5B films for 22 days.

Quantification of autoradiographs was performed microscopically (× 10 objective lens; × 1.6 additional magnification) with a computer-assisted image-analysis system (Joyce-Loebl MicroMagiscan; Vickers, Gateshead, UK). Silver grain area per unit area (silver grain density) was measured within a 100 μm × 100 μm frame over each section. Total binding (silver grain density) was measured over two different areas per anterior pituitary section, one area for pars intermedia and one area for the posterior pituitary gland; for each animal such measurements were made on three pituitary sections. Similar measurements were made on two adjacent background areas off the tissue for each pituitary. Background silver grain density was subtracted from tissue grain density. For tissue incubated with excess cold CRH, three measurements per section of pituitary grain density were similarly made (pituitary lobes could not be distinguished). Mean density values were calculated for each rat per pituitary lobe, and the mean non-specific binding value subtracted from the total. Group means were then calculated.

Statistical analysis

Data are presented as group means ±s.e.m. To estimate total amount of hormone release in response to a given stimulus, the area under the curve corrected for baseline (AUC) was calculated using trapezoidal integration (Forsythe, Keenan, Organick & Stenberg, 1969). In addition, increments (δ) were calculated as the maximal poststimulation increase minus baseline values.

Statistical analysis was performed by means of statistical software (GB-Stat V5.4, Dynamic Microsystems, USA, and SigmaStat, Jandel Scientific). The tests used were two-way (factors: pregnancy state × time) and one-way (factor: time) analyses of variance (ANOVA) for repeated measures followed by Newman-Keuls test (ACTH, corticosterone, oxytocin, vasopressin, lactate), or completely randomized one-way ANOVA (AUC, ratio, δ, plus-maze parameters during various pregnancy states, cAMP levels, CRH receptor autoradiography data) followed by Newman-Keuls test. P < 0.05 was considered statistically significant.

RESULTS

Experiment 1: neuroendocrine responses to emotional and physical stressors and to CRH in virgin, pregnant and lactating rats

Emotional and physical stressors (day 1)

ACTH and corticosterone.

Basal plasma levels of ACTH and corticosterone at 09.30 or 10.00 h did not differ significantly between virgin rats and rats on day 10, 15, 18 or 21 of pregnancy (P= 0.77), although basal corticosterone levels in day 21 pregnant rats tended to be higher compared with all other groups (Fig. 1).

Figure 1.

ACTH (A) and corresponding corticosterone (B) concentrations in plasma collected from the jugular vein of conscious virgin and day 10, 15, 18 and 21 pregnant rats on experimental day 1 under basal conditions (at 09.30 and 10.00 h) and 10 min after exposure to the elevated plus-maze (EPM; 5 min), as well as 5, 15 and 25 min after forced swimming (FS; 90 s, 19 °C). Five days before the experiment, rats were fitted with a jugular vein catheter under halothane anaesthesia and caged singly afterwards. Data are presented as means ±s.e.m.; numbers in parentheses indicate group size. Two-way ANOVA for repeated measures for ACTH (corticosterone); factor: time × group, P < 0.0001 (P < 0.0083). *P < 0.01, virgin and day 10 vs. day 18 and 21; †P < 0.01, day 15 vs. day 21; ²P < 0.01, day 10 vs. day 18 and 21.

Mild emotional stress (5 min exposure to the elevated plus-maze) followed by a complex physical-emotional stressor (90 s forced swimming) significantly increased the secretion of ACTH and, consequently, of corticosterone in all groups (P < 0.0001; Fig. 1). The maximum rise in circulating hormone levels was reached 5 min (ACTH) and 15 min (corticosterone) after forced swimming. There was an inverse relationship between the stage of pregnancy and the magnitude of ACTH secretion in response to the elevated plus-maze with a ca 6.0- and 7.2-fold increase in virgin and day 10 pregnant rats, and a 3.1- and 1.9-fold increase in day 18 and day 21 pregnant rats, respectively. Similarly, subsequent forced swimming caused a 15- and 16-fold increase in plasma ACTH concentration in virgin and day 10 and only a 9.5- and 8.3-fold increase in day 18 and day 21 pregnant rats, respectively, compared with basal values (P < 0.0001; Fig. 1). Secretion of corticosterone in response to both the elevated plus-maze and forced swimming was also found to be significantly lower at the end of pregnancy (P < 0.005) with significant differences between virgin and day 10 pregnant rats, and day 18 and day 21 pregnant rats (P < 0.01; Fig. 1). The increasingly blunted neuroendocrine response to the emotional and physical stressors with the progression of pregnancy was also reflected by significantly reduced AUC values for ACTH (virgin, 17.1 ± 1.08 arbitrary units (a.u.); day 10, 18.8 ± 1.14 a.u.; day 15, 9.90 ± 1.45 a.u.; day 18, 9.50 ± 1.48 a.u.; day 21, 8.20 ± 3.90 a.u.; P < 0.01 days 15, 18 and 21 vs. day 10 and virgin); corresponding AUC values for corticosterone were not different between groups.

When the responses to exposure to emotional and physical stressors, respectively, throughout pregnancy were compared, as reflected by respective ACTH increments, the pregnancy-related difference in responsiveness was more pronounced during exposure to the elevated plus-maze, as there was a 6.2-fold difference in the increments between virgins and day 21 pregnant rats compared with a 1.7-fold difference after the forced swimming between these two groups (P < 0.05). In a further series of experiments on Sprague- Dawley rats in Edinburgh, similar attenuation of ACTH and corticosterone secretion in response to swim stress alone was found in late pregnant rats (A. J. Douglas, H. Johnstone, A. Wigger, R. Landgraf, J. A. Russell & I. D. Neumann, unpublished data).

In lactating rats, the ACTH and corticosterone responses to both exposure to the elevated plus-maze and forced swimming were significantly less than those in virgin rats (P < 0.0001; Fig. 2). Basal ACTH and corticosterone concentrations, respectively, were similar in both groups.

Figure 2.

Plasma ACTH (A) and corticosterone (B) concentrations in virgin rats and between days 9 and 12 of lactation under basal conditions and in response to exposure to the elevated plus-maze (EPM) followed by forced swimming (FS) according to the protocol described in the legend to Fig. 1. Data are means ±s.e.m.; numbers in parentheses indicate group size. Two-way ANOVA for repeated measures for ACTH and corticosterone; factor: time × group, P < 0.0001; *P < 0.01, vs. lactating rats. Except for the ACTH level 10 min after EPM in lactating rats, ACTH and corticosterone concentrations were significantly increased at each time point after stimulation (P < 0.01).

Oxytocin and vasopressin.

Basal plasma levels of oxytocin differed significantly among virgin and pregnant rats (one-way ANOVA, P < 0.006; Fig. 3), with increased basal oxytocin levels on days 18 and 21 compared with virgin and day 10 pregnant rats (P < 0.01). The blood sample taken 15 min after forced swimming was used for the oxytocin and vasopressin assay. However, including pre- and poststimulation values of all groups, two-way ANOVA for repeated measurements did not reveal statistical differences among groups (factor: time × group; P= 0.532). When pre- and poststimulation values were compared (factor: time; P < 0.0001), forced swimming significantly increased the oxytocin concentration in the plasma only in virgin and day 10 and day 15 pregnant rats (P < 0.05), but not in day 18 and 21 pregnant rats (Fig. 3). The reduced response to this kind of a combined emotional and physical stressor in late pregnancy is also reflected in a significantly lower ratio between poststimulation and prestimulation levels on day 21 compared with day 10 pregnant rats (P < 0.05; Fig. 3).

Figure 3.

Plasma oxytocin concentration in conscious virgin and pregnant rats under basal conditions and 15 min after exposure to forced swimming (FS + 15 min) (A) as described in the legend to Fig. 1 and the calculated ratio between stimulated and basal neuropeptide concentrations for both oxytocin and vasopressin (B). Data are means ±s.e.m.; numbers in parentheses indicate group size. Two-way ANOVA for repeated measures; factor: time, P < 0.0001; factor: group, P < 0.503, factor: time × group, P < 0.7134. *P < 0.05, vs. corresponding basal values; †P < 0.045, vs. virgin, day 10 (Kruskal-Wallis test, P < 0.0027).

In contrast to oxytocin, forced swim stress did not provoke an increase in vasopressin release into the blood, in either virgin or in pregnant rats (factor: time; P= 0.203) as reflected by the respective ratio values (Fig. 3).

In lactating rats between days 9 and 12 of lactation, basal oxytocin and vasopressin levels did not differ in comparison with virgin rats (oxytocin: 7.30 ± 0.50 vs. 8.05 ± 0.90 pg ml−1, P= 0.45; vasopressin: 11.1 ± 2.85 vs. 12.0 ± 4.15 pg ml−1). The stress-induced increase in oxytocin secretion was significantly reduced (lactating: 1.3-fold, virgin: 2.7-fold increase; P < 0.0002) and did not reach statistical significance in the lactating group at any time point, i.e. 5, 15 or 50 min after the swim stress.

Again, plasma vasopressin levels remained unchanged in response to forced swimming in both virgin and lactating rats.

Lactate.

In a separate follow-up study, when plasma lactate levels in day 21 pregnant and virgin rats both under basal conditions and in response to forced swimming were compared, plasma concentrations after stress were found to be significantly higher in late pregnant rats (from 8.13 ± 0.74 to 22.7 ± 3.50 mmol l−1; virgin: from 6.42 ± 0.82 to 14.7 ± 2.29 mmol l−1; n= 7 each; P < 0.01) with no significant differences in basal levels.

Anxiety-related behaviour on the elevated plus-maze.

With respect to their anxiety-related behaviour, virgin rats and rats at different stages of pregnancy differed significantly (P < 0.021; Fig. 4). In general, pregnant rats evidently became more anxious with the progression of pregnancy, i.e. on days 15 and 21, compared with virgin and day 10 pregnant animals, as indicated by a reduced percentage of entries into the open arms (P < 0.05; Fig. 4) as well as the tendency for a reduced percentage of time spent on the open arms during the 5 min exposure on the plus-maze. On day 18 only, pregnant rats spent significantly more time on the open arms and thus seemed, by this measure, to be less anxious compared with day 15 and day 21 pregnant animals (P < 0.01; Fig. 4). The locomotor activity, reflected by the total number of entries into the closed arms, did not differ among groups (P= 0.57).

Figure 4.

Anxiety-related behaviour of virgin and day 10, 15, 18 and 21 pregnant rats on the elevated plus-maze on day 1 of the experiment as indicated by the percentage of time spent on the open arms (□) and by the percentage of entries into the open arms (▪). For details see legend to Fig. 1. One-way ANOVA; percentage time: P < 0.021; †P < 0.01, vs. day 15 and 21. One-way ANOVA; percentage entries: P < 0.027; ²P < 0.05, vs. day 10, *P < 0.01, vs. virgin, day 10 and day 18. Data are means ±s.e.m.

Lactating and virgin rats did not differ in their anxiety-related behaviour (16.3 ± 3.17 vs. 13.8 ± 2.29 % of total time spent in open arms, respectively).

Intravenous administration of CRH (day 2)

ACTH and corticosterone.

On day 2 of the experiment, basal levels of circulating corticosterone, but not ACTH, between 09.00 and 10.00 h differed significantly among groups (P < 0.0001), with elevated corticosterone levels on day 22 of pregnancy compared with virgins and rats on days 11, 16 and 19 of pregnancy (P < 0.01; Fig. 5).

Figure 5.

ACTH (A) and corresponding corticosterone (B) concentrations in plasma collected from the jugular vein of conscious virgin and day 11, 16, 19 and 22 pregnant rats on experimental day 2 in response to i.v. CRH (40 ng kg−1, arrow). For details see legend to Fig. 1. Insets show the area under the curve corrected for baseline (AUC; in arbitrary units (a.u.)). Data are means ±s.e.m.; numbers in parentheses indicate group size. *P < 0.01, virgin and pregnancy day 11 vs. days 19 and 22; †P < 0.05, pregnancy day 11 vs. days 16 and 19; ²P < 0.01, pregnancy day 22 vs. all other groups; ³P < 0.01, **P < 0.05, vs. virgin and day 11.

Intravenous administration of CRH (40 ng kg−1) increased ACTH and, consequently, corticosterone secretion in all groups studied (factor: time; P < 0.0001 for both). Maximum hormone levels were reached 10 min (ACTH) and 30 min (corticosterone) after administration of CRH. CRH was less effective with the progression of pregnancy (factor: time × group; P < 0.05 for both) with significant differences in ACTH secretion at 10 min among virgin (4.4-fold increase), day 11 (4.0-fold) and day 16 (2.6-fold), day 19 (1.6-fold) and day 22 (1.5-fold) pregnant rats. This was also reflected by reduced AUC values for both ACTH and corticosterone on days 16, 19 and 22 of pregnancy (Fig. 5).

In lactating rats, basal ACTH levels tended to be higher compared with virgin rats (18.2 ± 3.71 vs. 8.76 ± 1.78 pg ml−1, n.s.). There was a significant difference between lactating and virgin rats in their response to i.v. CRH (P < 0.001) with no significant response in lactating rats and a 2.4-fold increase (P < 0.01) in virgin rats at 10 min.

Experiment 2: in vitro basal and CRH-stimulated cAMP levels in pituitary segments from virgin and pregnant rats

In the presence of the phosphodiesterase inhibitor IBMX, basal as well as CRH-stimulated cAMP accumulation was significantly lower in pituitary segments isolated from day 17 and day 21 pregnant rats compared with the respective virgin groups (Table 1), whereas no such difference was found in pituitary segments from day 11 pregnant rats compared with their virgin controls. Importantly, the CRH-induced increment (CRH-stimulated minus basal) in cAMP accumulation was significantly attenuated in late pregnant rats on day 21 of pregnancy (P < 0.05; Table 1) indicating a reduced efficacy of CRH-stimulated accumulation compared with virgin controls.

Table 1. Total cyclic AMP content in segments of the adenohypophysis of day 11, day 17 and day 21 pregnant rats and their respective virgin controls incubated either in vehicle (Basal) or CRH (10 nM) at 37 °C for 10 min, and the respective increments (δ; CRH-stimulated minus vehicle)
 [cAMP] (nmol)
 BasalCRH-stimulatedδ
  1. Data are means ±s.e.m.; numbers in parentheses indicate group size. **P < 0.01, *P < 0.05, vs. respective virgin group.

Day 11 (6)7.06 ± 0.7613.4 ± 1.286.92 ± 1.15
Virgin (4)6.90 ± 1.6014.3 ± 3.487.72 ± 2.04
Day 17 (6)6.10 ± 0.78 *15.3 ± 0.83 *9.22 ± 0.68
Virgin (4)10.4 ± 1.3118.8 ± 1.818.38 ± 1.42
Day 21 (8)5.20 ± 0.27 *11.5 ± 0.70 *6.30 ± 0.68 **
Virgin (8)7.06 ± 0.8916.3 ± 1.389.26 ± 0.86

There was no difference (ANOVA, P < 0.066) in anterior pituitary weights between virgin (11.1 ± 0.2 mg), day 11 (11.1 ± 0.14 mg), day 17 (11.6 ± 0.37 mg) and day 21 (12.1 ± 0.35 mg) pregnant rats.

Experiment 3: measurement of [125I] CRH binding-site density in the pituitary gland

[125I] CRH binding-site density as reflected by silver grain densities was estimated in the anterior, posterior and intermediate pituitary lobes of virgin and day 11, 17 and 22 pregnant rats. Compared with virgin rats, there was a significantly reduced [125I] CRH binding in the anterior pituitary of all pregnancy groups studied (P < 0.01 vs. virgin; Fig. 6) and a further reduction in specific [125I] CRH binding on day 22 compared with days 11 and 17 of pregnancy (P < 0.05; Fig. 6). [125I] CRH binding in the intermediate lobe of the pituitary was detectable and was found to be reduced in day 22 pregnant rats (P < 0.05 vs. virgin). In the posterior pituitary, virtually no specific [125I] CRH binding was detectable.

Figure 6.

Specific [125I] CRH binding in the anterior and intermediate lobe of pituitaries collected from virgin, day 11, day 17 and day 22 pregnant rats (n= 6 each). The film was quantified by image analysis to measure silver grain density (arbitrary units; a.u.). Data are means ±s.e.m.*P < 0.05 and **P < 0.01, vs. virgin; †P < 0.05, vs. day 11 and day 17 pregnant groups.

DISCUSSION

The present results demonstrate that, in rats, the attenuated HPA axis responsiveness to stressors observed during lactation is already manifested in mid-gestation. Specifically, we showed a significant hyporesponsiveness to both emotional (elevated plus-maze) and physical (forced swimming) stressors from day 15 until day 21 of pregnancy as well as during lactation, reflected in a reduced stress-induced secretion of ACTH from the adenohypophysis and reduced corticosterone secretion from the adrenal gland. Similarly, the oxytocin secretory response to forced swimming tended to be reduced on the last days of pregnancy. The attenuated response of the HPA axis is likely to be at least partly due to a reduced reactivity of corticotrophs to CRH as demonstrated in vivo by reduced CRH-stimulated ACTH secretion from the adenohypophysis into blood and in vitro by lower stimulation by CRH of cAMP production in corticotrophs of the adenohypophysis. Moreover, receptor autoradiography revealed a significant reduction in [125I] CRH binding-site density in the anterior pituitary of pregnant (days 11, 17 and 22) compared with virgin rats. Independent of the reproductive state, the neuroendocrine responses to the emotional stressor did not correlate with the anxiety-related behaviour of the animals on the elevated plus-maze.

Basal and stress-induced activity of the HPA axis and HNS during pregnancy

In our study, basal (a.m.) levels of ACTH remained stable throughout pregnancy, including on day 22; these findings are comparable with the trough ACTH values in pregnant rats described by Atkinson & Waddell (1995). In human and ovine pregnancy, increased basal ACTH concentrations may be partly placental in origin (for review, see Keller-Wood, 1994). In the rat, the placenta may be a source of circulating ACTH (Chen, Chang, Krieger & Bardin, 1986), although this is uncertain (Waddell, 1993), but this is not released in response to maternal stress (Ohkawa, Takeshita, Murase, Kambegawa, Okinaga & Arai, 1991). Similarly, basal corticosterone concentrations did not differ between virgin and pregnant rats, except for an increase on day 22 of pregnancy, the first expected day of parturition. Previously, decreased plasma corticosterone concentration has been found early in pregnancy (Ogle & Kitay, 1977; Atkinson & Waddell, 1995) followed by a return to prepregnancy levels and a further increase on the last day(s) of pregnancy as we found. This late increase in plasma corticosterone concentration may reflect increased sensitivity of the adrenal glands to ACTH, reduced metabolic clearance rate (Waddell & Atkinson, 1994), stimulation by increased ovarian oestrogen secretion or a contribution by the fetal adrenal glands (Dupont, Rheaume, Simard, Luu-The, Labrie & Pelletier, 1991).

Our novel finding is that the response of the HPA axis to both emotional and physical stressors is significantly blunted in the pregnant rat, beginning around day 15. These adaptations of the HPA axis manifest in mid-gestation seem to persist throughout lactation thus confirming and extending recent results (Altemus et al. 1995; Walker et al. 1995; Neumann et al. 1995b; da Costa, Wood, Ingram & Lightman, 1996; Windle et al. 1997). However, the mechanisms of adaptation may not be the same in pregnancy and lactation. Diminished ACTH secretion in response to stimuli including CRH in pregnancy has been described also in sheep (Keller-Wood, 1994), baboons (Goland, Wardlaw, MacCarter, Warren & Stark, 1991) and humans (Magiakou, Mastorakos, Rabin, Dubbert, Gold & Chroussos, 1996).

The pregnancy-related adaptations of the stress response include the HNS with oxytocin being released in response to a variety of stressors in the rat (Lang et al. 1983; Kasting, 1988; Wotjak et al. 1996). There are increased stores of oxytocin in the neural lobe (Douglas, Dye, Leng, Russell & Bicknell, 1993) and basal oxytocin secretion increases toward the end of pregnancy as confirmed in the present study (Fig. 3). However, the stress-induced rise in oxytocin release in late pregnancy was slightly attenuated and did not reach statistical significance in day 18 and 21 pregnant rats; restraint by endogenous opioid peptides may be important (Douglas et al. 1995). Similarly there is an increased basal and attenuated stress-induced local release of oxytocin within the hypothalamic paraventricular nucleus (PVN) at the end of pregnancy (Neumann et al. 1997). The altered oxytocin responses in pregnant rats differ from the pattern of attenuated responses of the HPA axis, which were markedly reduced from day 15 of pregnancy onward, through lactation (Figs 1 and 2). In lactation, the oxytocin response was essentially abolished, consistent with previous reports of reduced responses to non-suckling-related stimuli (Carter & Lightman, 1987; Lightman & Young, 1989; Patel et al. 1991; Koehler et al. 1993; Neumann et al. 1995a, b).

Mechanisms of the attenuated HPA axis activity during pregnancy

The physiological adaptations of the HPA axis during gestation occur at several levels including limbic feedback systems (Johnstone, Douglas, Seckl & Russell, 1997), CRH/ vasopressin neurons within the hypothalamus (Douglas & Russell, 1994), corticotrophs of the adenohypophysis as shown in the present study, and cortical cells of the adrenal gland (Carr et al. 1981; Waddell & Atkinson, 1994). An enhanced glucocorticoid feedback especially at hippocampal, but also hypothalamic, brain areas would negatively control CRH/vasopressin neurons and, thus, attenuate ACTH secretion. With respect to glucocorticoid feedback control during pregnancy, various species-dependent results have been published with unchanged (Keller-Wood, 1996) or reduced feedback sensitivity (Owens et al. 1987). Changes in glucocorticoid receptor binding capacity within the hippocampus as observed in lactation (Meaney, Viau, Aitken & Bhatnagar, 1989) are being studied in our laboratories also during pregnancy (Johnstone et al. 1997). However, the stress response is also reduced in adrenalectomized lactating rats (Walker et al. 1992), so altered feedforward control mechanisms of the HPA axis could be involved. The pregnancy-related alterations in plasma levels of the sex steroids, oestrogens and progesterone, could alter hippocampal mineralo- (Carey, Deterd, de Koning, Helmerhorst & de Kloet, 1995) and glucocorticoid (Burgess & Handa, 1992) receptor binding, hypothalamic CRH expression (Douglas & Russell, 1994; Grino, Héry, Paulmyer-Lacroix & Anglade, 1995) and ACTH and corticosterone secretion (Viau & Meaney, 1991; Burgess & Handa, 1992). However, it is uncertain whether the secretion of hypothalamic corticotrophin-releasing factors is reduced during pregnancy (Plotsky, 1986).

In the present study several lines of evidence indicate pregnancy-related alterations at corticotrophs of the pituitary synthesizing and releasing ACTH. In rats between days 16 and 22 of pregnancy, these cells showed a reduced response to CRH in vivo as reflected in a reduced ACTH secretion (Fig. 5). Although receptor autoradiography revealed a reduction in CRH receptor density in pregnant rats beginning on day 11 no significant reduction of CRH-stimulated cAMP accumulation was observed until day 17 of pregnancy. The normal CRH response, in terms of cAMP accumulation in vitro, observed between days 10 and 16 in the face of a marked reduction in CRH receptor density is most probably a result of the large CRH receptor capacity reported in corticotrophs (Antoni, 1986; King & Baertschi, 1990). This suggests that the reduced CRH-stimulated ACTH response observed in vivo at days 11-17 of pregnancy is not a consequence of alterations in CRH receptor coupling to the cAMP pathway but results from alterations either downstream of cAMP accumulation in corticotrophs or as a result of pregnancy-induced changes at higher levels of the HPA axis. The reduced CRH-stimulated cAMP accumulation at day 21 of pregnancy, perhaps resulting from a further decrease in CRH receptor density, may underly the mechanisms of attenuated ACTH responses in vivo at this time. However, it should be noted that, in the rat, the placenta does not synthesize CRH as found in several other mammalian species (Jones, Gu & Parer, 1989; Robinson, Arbiser, Emanuel & Majzoub, 1989; Goland et al. 1991); thus, the downregulation of pituitary receptors is not likely to be due to an increased level of circulating CRH of peripheral origin.

We can exclude the possibility that reduced locomotor activity during exposure to the elevated plus-maze or forced swimming led to reduced HPA axis responses in pregnant rats, since (i) the total number of entries into the closed arms of the plus-maze, and (ii) the amount of time that animals spent struggling, swimming or floating during forced swimming were similar among groups. The increase in plasma lactate concentration, indicative of muscle activity during the swim stress (Abel, 1994), was even higher in late pregnant rats. Alternatively, reduced stressor perception, resulting from lowered stimulatory inputs from suprahypothalamic, e.g. limbic and cortical, brain areas and the brainstem to the hypothalamic PVN containing CRH/ vasopressin neurons could be postulated; consistent with this is reduced stress-induced c-fos mRNA expression within the parvocellular part of the PVN in late pregnant and lactating rats (da Costa et al. 1996). Although our experimental design did not allow a clear distinction between emotional and physical components, our observations suggest that the pregnancy-related reduction in ACTH secretion is at least as pronounced in response to a mild emotional as it is to a predominantly physical stressor (Fig. 1), thus supporting the notion of reduced stress perception.

Significance of reduced HPA axis and HNS responsiveness during pregnancy

Negative effects of exogenous ACTH on implantation and the progress of gestation were described and interpreted as an excessive stimulation of secretion of steroids other than corticosteroids from the adrenal gland (Chatterjee & Harper, 1970). Daily treatment with ACTH during the last third of gestation causes abnormal development of the young, prolonged pregnancy and impairs the onset of maternal behaviour (Fameli, Kitraki & Stylianopoulou, 1993). Similarly, increased HPA axis activity during pregnancy triggered by excessive stress adversely affects the behavioural (Fameli, Kitraki & Stylianopoulou, 1994) and endocrine development of the offspring (for review see Weinstock, 1997). Comparable findings were observed in the offspring of aged pregnant females with impaired glucocorticoid feedback and thus elevated corticosteroid levels during pregnancy (Erisman, Carnes, Takahashi & Lent, 1990). Thus, the dampened stress response of the maternal HPA axis during normal pregnancy described in the present study may provide a protective mechanism against excessive levels of circulating ACTH/corticosteroids during the sensitive period of fetal HPA axis development and maturation.

In contrast, the attenuated stress-induced release of oxytocin at the last day(s) of pregnancy may serve the purpose of storing neurohypophysial oxytocin prior to delivery when demands for neurohypophysial oxytocin are high.

Dissociation between neuroendocrine and behavioural responses throughout pregnancy

The neuroendocrine responses to the emotional stressor were not correlated with the anxiety-related behaviour of virgin, pregnant or lactating rats. Thus, despite a continuous reduction in the HPA axis responsiveness with the progression of pregnancy, pregnant rats (except on day 18 of pregnancy) were evidently more anxious on the elevated plus-maze compared with virgin controls. Interestingly, in contrast to pregnant animals, lactating rats tended to be less anxious on the elevated plus-maze. A reduced anxiety in lactating rats has been shown by studying the duration of freezing in response to an auditory stimulus (Hard & Hansen, 1984). These behavioural alterations, however, might be related to the complex patterns of maternal behaviour which include an increased aggressive behaviour towards conspecifics (Erskine, Barfield & Goldman, 1978). Generally, in both pregnant and lactating rats, the results indicate an independent regulation of HPA axis activity and anxiety-related behaviour thus confirming recent results (Pich, Heinrichs, Rivier, Miczek, Fisher & Koob, 1993; Walker et al. 1995).

In conclusion, we have demonstrated reduced responsiveness of the HPA axis to various stressors in pregnancy. This involves reduced corticotroph responsiveness to CRH with reduced CRH receptor density at the adenohypophysis. This adaptation may protect the fetuses from lifelong adverse effects of exposure to excessive glucocorticoids.

Acknowledgements

We thank Gabriele Kohl, Patrick Lörscher, Renate Simchen and Phillip Bull for excellent technical assistance including quantitative analysis of plasma hormones. This study was supported by Deutsche Forschungsgemeinschaft, DAAD/ARC and Swiss National Science Foundation. I. D. N. is a recipient of the Heisenberg stipend of the DFG. H. J. is a BBSRC Research Student.

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