http://informahealthcare.com/sts ISSN: 1025-3890 (print), 1607-8888 (electronic) Stress, 2014; 17(6): 494–503 ! 2014 Informa UK Ltd. DOI: 10.3109/10253890.2014.966263
ORIGINAL RESEARCH REPORT
Restraint stress alters immune parameters and induces oxidative stress in the mouse uterus during embryo implantation Guanhui Liu*, Yulan Dong*, Zixu Wang, Jing Cao, and Yaoxing Chen
Stress Downloaded from informahealthcare.com by Monash University on 12/03/14 For personal use only.
Laboratory of Veterinary Anatomy, College of Animal Medicine, China Agricultural University, Beijing, China
Abstract
Keywords
The influence of stress on embryo implantation is not well understood. Prior studies have focused on later gestational stages and the long-term impact of stress on immune function. The objective of this study is to investigate the effects of restraint stress on the immune parameters and the oxidative states of the uterus during implantation. In this study, pregnant CD1 mice were subjected to restraint stress (4 h/d) on embryonic day 1 (E1) and sacrificed on E3, E5, and E7. Maternal plasma corticosterone (CORT) secretion and implantation sites in the uterus were examined. The uterine (excluding embryos) homogenate and uterine lymphocytes were collected to examine oxidative stress states and associated immune parameters. The results demonstrated that restraint stress increased maternal plasma CORT secretion and reduced the number of implantation sites by 15.3% on E5 and by 26.1% on E7. Moreover, restraint stress decreased the density of uterine natural killer (uNK) cells in the endometrium by 22.1–47.9% and increased the density of mast cells in the myometrium by 55.6–76.9%. Restraint stress remarkably decreased the CD3+CD4+ T/CD3+CD8+ T cell ratio (by 26.2–28.9%) and attenuated uterine lymphocyte proliferation and secretion of cytokines. In addition, restraint stress threatened the intracellular equilibrium between oxidants and antioxidants, resulting in decreased glutathione peroxidase (GSH-PX) (32.2% and 45.7%), superoxide dismutase (SOD) (15.5% and 26.1%), and total antioxidant capacity (T-AOC) (18.4% and 18.2%) activities and increased malondialdehyde (MDA) (34.4% and 43.0%) contents on E5 and E7. In conclusion, these findings demonstrate that restraint stress causes abnormal implantation and negatively impacts immune parameters in association with oxidative stress in mice.
Immune parameters, implantation sites, oxidative stress, pregnant mice, restraint stress, uterus
Introduction Maternal stress is common during both pregnancy and the postnatal period. During pregnancy, maternal stress (i.e. pregnancy-specific anxiety, depressive symptoms, and stressful life events) is associated with lower birth weight and preterm delivery. In 2009–2010, nearly 75% of mothers in the USA reported experiencing at least one stressful event prior to childbirth (Centers for Disease Control and Prevention, Pregnancy Risk Assessment Monitoring System). In South Asia, over 25% of mothers suffered from depressive disorders during their third trimester of pregnancy (Rahman et al., 2003). In China, approximately 54% and 37.1% of mothers experienced antenatal anxiety and depression, respectively (Lee et al., 2007). Since 1986, restraint stress has been generally used as a tool for the evaluation of physiology, pharmacology,
*These authors contributed equally to this work. Correspondence: Yaoxing Chen, Laboratory of Veterinary Anatomy, College of Animal Medicine, China Agricultural University, Haidian, Beijing 100193, PR China. Tel: +86 10 62733778. Fax: +86 10 62733199. E-mail: [email protected]
History Received 25 March 2014 Revised 19 August 2014 Accepted 27 August 2014 Published online 10 November 2014
immunology, and behavioral neurobiology (Wiebold et al., 1986). The response of animals to this stressor typically involves the triggering of two endocrine systems, including (1) the hypothalamic–pituitary–adrenal (HPA) axis and glucocorticoid production and (2) the sympathetic nervous system and the release of catecholamines (epinephrine and norepinephrine). It has been reported that pregnant rats subjected to restraint stress for 45 min (three times a day) from E15 to E21 showed subsequent decreases in body weight gain and food intake (Mairesse et al., 2007). Maternal psychosocial stress causes pregnancy complications, including implantation failure, recurrent spontaneous abortions, spontaneous preterm birth, and developmental abnormalities of the fetus in rodents (Zhang et al., 2011). Uterine microenvironment, including the inflammatory response and oxidative stress states, is also important for normal pregnancy progression and for the development of offspring. Immune challenge during late pregnancy has been shown to induce preterm birth and affect cognitive and affective functioning in offspring (Paris et al., 2011). Psychological stress applied on E19 has been shown to cause anxiety in rat dams in association with decreased glutathione (GSH) levels with a concomitant increase in MDA levels (Toumi et al., 2013). However, few studies have
Stress Downloaded from informahealthcare.com by Monash University on 12/03/14 For personal use only.
DOI: 10.3109/10253890.2014.966263
focused on changes in immune function and oxidative stress states in animals under restraint stress during implantation. In the process of implantation, the uterine environment undergoes serial remodeling, which involves leukocyte infiltration, trophoblast invasion, and specific cytokine and chemokine profiles at the maternal–fetal interface. Oxidative stress decreases the numbers of implantation sites and causes embryonic resorption in mice (Shahin et al., 2013). Psychological stress increases the ‘abortive’ cytokine TNFa and decreases the ‘anti-abortive’ cytokine TGF-P2, which may intricately interfere with implantation (Craig, 2001). Based on the previous studies, we hypothesize that oxidative stress and/or the inflammatory response act as crucial links between restraint stress and early pregnancy outcome. Therefore, this study used an animal model of restraint stress during pre- and post-implantation. It examined whether restraint stress disturbed the balance of the uterine microenvironment and caused alterations in the immune parameters and oxidative stress states, blastocyst implantation, and embryonic development in mice.
Materials and methods Ethical approval The present study conformed to the Guide for the Care and Use of Laboratory Animals published by the Animal Welfare Committee of the Agricultural Research Organization, China Agricultural University (Beijing, China). Animals A total of 210 female CD1 mice (8 weeks of age; Vital River Laboratory Animal Technology Co. Ltd., Beijing, China) were housed under conventional conditions (at a temperature of 21 ± 1 C, a relative humidity of 50 ± 10%) with a regular 14-h light:10-h dark cycle (with lights on at 7:00 A.M.). The mice were allowed ad libitum access to water and food. The animals were maintained in a small, quiet room for 1 week before the studies were initiated to minimize stress. Estrous mice were then placed with a sexually experienced male at night. The next morning, vaginal smears were obtained from the females with vaginal sperm plugs, and sperms were microscopically evaluated. Females regarded as pregnant were separated from the males and were considered to be at E1. Restraint stress was initiated on E1 until the mice were sacrificed on E3, E5, or E7. Specifically, pregnant female mice were individually placed into transparent and ventilated plastic centrifuge tubes (so that they were able to breathe freely but were unable to escape) to limit their movements for 4 h (from 8:00 A.M. to 12:00 P.M.) on E1. Because of the limitation of the restraint tubes, the stressed mice temporarily had no access to water or food. The control pregnant females were left untreated in their home cages, from which water and food were also temporarily removed for 4 h, to create experimental conditions similar to those of the stressed mice and to allow for increased accuracy in the assessment of the experimental factors (restraint stress and gestational stage). After 4 h of restraint stress, the animals were again provided access to water and food. According to the circadian rhythms of mice (nocturnal animals) and a previous study
Restraint stress and embryo implantation
495
(Pallare´s et al., 2013), the control mice should not have been affected by the lack of water and food. Plasma and tissue preparations A number of 90 pregnant females were anesthetized by 2% pentobarbital (4 mL/kg body weight) on E3, E5, and E7 immediately following the restraint stress. Plasma samples were collected for the detection of corticosterone (CORT). Thirty uterine specimens were fixed in 4% paraformaldehyde and embedded in paraffin for histological analysis. Some of the uteri were rapidly dissected with excluding the embryos and stored at 80 C for cytokine analysis (30 mice) and antioxidant enzyme analysis (30 mice). The total number of implantation sites on the walls of the uteri was identified following the intravenous injection of 0.5 mL 0.5% trypan blue dye on E5, and the implantation sites were counted directly on E7. Measurement of plasma CORT concentration The total plasma CORT concentration was measured using a competitive enzyme-linked immunosorbent assay (CEA540Ge, Uscn Life Science Inc., Wuhan, China). All the tests were performed according to the manufacturer’s instructions. The limit of detection for the assay ranged from 6.17 to 500 ng/mL. Eight plasma samples were included in each group. Each sample was tested in triplicate. Histological analyses of uNK cells and mast cells Uteri with implantation sites (2–3 mm in length) were routinely processed into paraffin sections. Five-micron-thick sections were mounted onto 10% polylysine-coated slides. The uNK cells and mast cells were stained with periodic acidSchiff (PAS) and toluidine blue, respectively. Positively stained cells were observed under a microscope. The number of uNK cells per mm2 in the endometrium was calculated, while the number of mast cells per mm2 in the myometrium was counted in five random fields from six cross-sections of the four uterine specimens from each group on E3, E5, and E7. Measurements of cytokines and oxidative stressrelated enzymes Immediately after the mice were sacrificed, the uteri were rapidly dissected, and the embryos were removed, frozen on dry ice, and stored at 80 C. The uteri with removed embryos were weighted and placed in a 0.9% saline solution (1:9) to prepare a uterine homogenate. Finally, supernatants were extracted by centrifugation (1000 g, 20 min) to determine cytokine concentrations, oxidative molecule production, and the presence of anti-oxidative enzymes. IL-2 and IL-4 cytokines To quantitatively measure the concentrations of uterine IL-2 and IL-4, a routine sandwich enzyme-linked immunosorbent assay was performed (IL-2: EK0398; IL-4: EK0405, Boster Biological Technology, Ltd., Wuhan, China). All tests were conducted according to the manufacturer’s instructions. The samples and reagents were equilibrated to room temperature
496
G. Liu et al.
before testing. The limit of detection for the IL-2 assay ranged from 15.6 to 1000 pg/mL and that for the IL-4 assay ranged from 7.8 to 500 pg/mL. The data describing uterine IL-2 and IL-4 levels were expressed as pg/mg of protein. Approximately five whole uterine samples were required for each group. Each sample was tested in triplicate.
Stress, 2014; 17(6): 494–503
compensated for by the use of cells FITC-only, and PE-only antibodies. analyzed with a FACSCalibur Biosciences, San Diego, CA) using (BD Biosciences, San Diego, CA).
stained with APC-only, The lymphocytes were flow cytometer (BD the CellQuest program
Lymphocyte proliferative activity assay Oxidative stress-related enzymes GSH-PX and SOD are well-known scavenger enzymes that protect cells from oxidative stress, while MDA is responsible for inducing oxidative stress. T-AOC, SOD, GSH-PX, and MDA assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). T-AOC, SOD, and GSH-PX were expressed as U/mg of protein, and MDA was expressed as nmol/mg of protein.
Stress Downloaded from informahealthcare.com by Monash University on 12/03/14 For personal use only.
Uterine lymphocytes isolation Uterine lymphocytes were isolated as previously described (Blois et al., 2004) with some modifications. In brief, a total of 120 pregnant mice were sacrificed, the uterus was rapidly dissected from each, and the embryos/fetuses were removed. After removing the fatty and connective tissues, the obtained uterus was washed in 0.01 mol/L phosphate-buffered saline (PBS), sliced longitudinally into small pieces, and incubated with 0.1% collagenase in a 15-mL centrifuge tube at 37 C for 60 min. The digestive tissues were passed through a nylon tissue sieve (200 nylon mesh per 2.5 cm) and washed twice with RPMI-1640 medium (Gibco-BRL, Grand Island, NY). After the centrifugation step, the supernatant was discarded, and the precipitate was suspended in 3 mL RPMI-1640 complete medium and combined with 3 mL lymphocyte separation medium (LTS1077, Tianjin Haoyang Biological Technology Co., Ltd., Tianjin, China) for centrifugation (800 g for 10 min). The isolated lymphocytes were washed twice with 1.5 mL RPMI-1640 complete medium. The cell viability (498%) was detected using 0.5% trypan blue dye, and the cell suspension was cultured in 1 mL RPMI-1640 at a density of 1 106 cells/mL. T lymphocyte subsets and lymphocyte proliferative activities were analyzed in the uterine lymphocytes. Approximately 10 whole uterine samples were required for each group for the lymphocyte isolation. Flow cytometry analysis Flow cytometry was used to determine the phenotypes of the uterine lymphocytes. As previously described (Schuerwegh et al., 2001), isolated uterine lymphocyte cells were stained for 30 min in the dark at room temperature with 1 mL of specific monoclonal antibodies directed against T-cell surface markers, including APC-conjugated anti-CD3 (553066, BD Biosciences, San Diego, CA), FITC-conjugated anti-CD4 (553046, BD Biosciences, San Diego, CA), and PEconjugated anti-CD8 (553032, BD Biosciences, San Diego, CA). Next, the lymphocytes were washed with 0.2 mL PBS and centrifuged at 500 g for 5 min (three times). Finally, the cells were re-suspended in 0.2 mL PBS and subjected to flow cytometric analysis. Fluorochrome-equivalent IgG isotype controls were used to detect non-specific binding. The spectral overlap of three types of fluorochromes was
The 200-mL lymphocyte suspensions were seeded into each well of a 96-well flat-bottom microwell plate (Costar 3599, Corning Inc., Corning, NY) in the presence of 15 mg/mL LPS (L2630, Sigma, St. Louis, MO) or ConA (C2272, Sigma, St. Louis, MO). After incubating the lymphocytes at 37 C in a humidified, 5% CO2 atmosphere for 68 h, the proliferation index was assayed using 5 mg/mL methyl thiazolyl tetrazolium (MTT, Sigma, St. Louis, MO) (Ali et al., 2013). Control cells were incubated with RPMI-1640 alone. Specifically, the plate was incubated for 4 h after the addition of 10 mL MTT per well, and then, 90 mL of 10 mg/mL sodium dodecyl sulfate (SDS, Sigma, St. Louis, MO) was pipetted into each well. After incubation at 37 C for 4 h, the microplate was read using a Bio-Rad microplate reader (Model 550, Bio-Rad, Berkeley, CA) at a wavelength of 570 nm. The proliferative activity of the lymphocytes was expressed as the stimulation index (SI) as follows (Li et al., 2013): SI ¼ OD570 ðstimulated cellsÞ=OD570 ðunstimulated cellsÞ Statistical analysis The data were analyzed with SPSS 18.0 (SPSS Inc., Chicago, IL) as described in a previous study (Ortolani et al., 2014). To analyze the effects of restraint stress and different gestational stages on the experimental data, a two-way analysis of variance (ANOVA) was performed followed by Duncan’s post hoc test. The statistical difference between the control and stressed mice at the same gestational stage was evaluated by independent samples t test. The data were expressed as the mean ± SD. *p50.05 and #p50.01 were used to denote the significance compared with the corresponding control groups.
Results Plasma CORT secretion Plasma CORT secretion of the pregnant mice was influenced by restraint stress (Figure 1; the two-way ANOVA followed by Duncan’s post hoc test: F(1, 16) ¼ 45.678, p50.001) and gestational stage (F(2, 16) ¼ 13.238, p50.001). Furthermore, plasma CORT secretion in the control mice showed an inverted V profile with a peak at E5 that leveled off at E7. Although the stressed mice displayed the same pattern, plasma CORT secretion increased compared with the control mice at the same stage (independent t-test: t ¼ 10.49, p50.001 for control versus stressed mice on E3; t ¼ 5.31, p ¼ 0.003 for control versus stressed mice on E5; t ¼ 3.02, p ¼ 0.019 for control versus stressed mice on E7). Number of implantation sites As shown in Figure 2, the uterus was smooth in appearance on E3 because blastocyst implantation occurred on E5 after mating. To observe the implantation sites in the uterus, the
DOI: 10.3109/10253890.2014.966263
mice were injected with 0.5% trypan blue through the caudal vein on E5. The implantation sites were stained blue and arranged as a string of beads in the uterus. Restraint stress negatively influenced the number of implantation sites (Figure 2; the two-way ANOVA followed by Duncan’s post hoc test: F(1, 47) ¼ 5.145, p ¼ 0.028). Because the uterus became receptive to the blastocyst at a fixed time (E5), the
Restraint stress and embryo implantation
497
gestational stage had no effect on the number of implantation sites (F(1, 47) ¼ 1.250, p ¼ 0.269). The implantation sites in the uterus on E7 appeared as round or oval enlargements, and the number of implantation sites significantly decreased in the stressed mice compared with the control mice (Figure 2; control ¼ 13.5 ± 1.84; stressed ¼ 10.0 ± 4.51; independent t-test: t ¼ 3.30, p ¼ 0.003). The number of implantation sites decreased on E5 with no statistically significant differences observed between the control and stressed mice.
Stress Downloaded from informahealthcare.com by Monash University on 12/03/14 For personal use only.
Densities of uNK cells and mast cells
Figure 1. Effects of restraint stress on plasma CORT secretion on E3, E5, and E7 pregnant mice. The data (ng/mL) were expressed as the mean ± SD. Uppercase letters represented the differences among E3, E5, and E7 in the control group (p50.05), and lowercase letters represented the differences among E3, E5, and E7 in the stressed group (p50.05). *p50.05 and #p50.01 were used to denote significance compared with the corresponding control groups. Figure 2. Effects of restraint stress on the number of implantation sites on E3, E5, and E7 pregnant mice. There were no implantation sites on E3, while on E5, implantation sites were detected as round or oval embryos that were stained blue by 0.5% trypan blue. As expected, the implantation sites were larger and arranged as a string of beads in the uterus. Scale bars ¼ 1 cm. The short arrows show the implantation sites on E5 and E7. Values were expressed as the mean ± SD. # p50.01 versus the E7 control group.
Normally, uNK cells are the predominant uterine lymphocytes in mice during the early gestational stage. In this study, the uNK cells appeared oval in shape and rose-red by PAS staining. As shown in Figure 3, these cells were scattered throughout the endometrium and were non-granulated (approximately 5 mm in diameter) on E3 and E5. On E7, the majority of uNK cells was large (approximately 13 mm in diameter), heavily granulated, and distributed within the decidua basalis. The density of uNK cells in the endometrium showed an inverted V profile, with a peak on E5 that waned to basic levels on E7 in the control mice. Although the stressed mice exhibited a similar trend, the density of endometrial uNK cells decreased relative to that of the control mice (Figure 3a; the two-way ANOVA followed by Duncan’s post hoc test: F(1, 79) ¼ 200.231, p50.001 for restraint stress effects; F(2, 79) ¼ 166.787, p50.001 for gestational stage effects). The density of uNK cells in the endometrium of
Stress Downloaded from informahealthcare.com by Monash University on 12/03/14 For personal use only.
498
G. Liu et al.
Stress, 2014; 17(6): 494–503
Figure 3. Photomicrographs show the effects of restraint stress on the distribution and density of uNK cells and mast cells in the uteri of pregnant mice on E3, E5, and E7. The morphology of uNK cells in the endometrium and mast cells in the myometrium was examined by staining with PAS and toluidine blue, respectively. Long arrows indicate positive-staining uNK cells in the endometrium, and short arrows indicate positive-staining mast cells in the myometrium. Scale bars ¼ 50 mm. The density of uNK cells (a) in the endometrium and mast cells (b) in the myometrium are shown. Data (cells/mm2) were expressed as the mean ± SD. The meaning of the letters in the bar chart is the same as Figure 1. #p50.01 were used to denote significance compared with the corresponding control groups. uNK cells, uterine natural killer cells; MCs, mast cells; End, endometrium; UL, uterine lumen; DB, decidua basalis; RUL, residual uterine lumen; LSM, outer longitudinal smooth muscle of uterine wall; CSM, inner circular smooth muscle of uterine wall; Ug, uterine gland; VLa, vascular layer.
stressed mice decreased compared with the corresponding control mice (Figure 3a; independent t-test: t ¼ 4.72, p50.001 on E3; t ¼ 12.10, p50.001 on E5; and t ¼ 5.79, p50.001 on E7). Mast cells showed some correlation with pregnancy failure in the mice. In this study, toluidine blue staining resulted in red mast cells that were present in large numbers in the myometrium but that were scarce in the endometrium. As summarized in Figure 3(b), we observed a dramatic increase in the number of mast cells per mm2 in the myometrium of the pregnant mice exposed to restraint stress compared with the control mice at the corresponding gestational stage. The density of mast cells in the myometrium was influenced by restraint stress (Figure 3b; the two-way ANOVA followed by Duncan’s post hoc test: F(1, 59) ¼ 73.569, p50.001) and gestational stage (F(2, 59) ¼ 117.317, p50.001). In contrast to the inverted V profile of uNK, the density of mast cells in the myometrium displayed a typical V profile with a drop on E5.
In comparison with the respective control mice, the density of mast cells in the myometrium of stressed mice increased at the three gestational stages (Figure 3b; independent t-test: t ¼ 5.97, p50.001 on E3; t ¼ 4.14, p ¼ 0.002 on E5; and t ¼ 8.50, p50.001 on E7). T lymphocyte subsets The results from the flow cytometric analysis are shown in Figure 4. Restraint stress (Figure 4; the two-way ANOVA followed by Duncan’s post hoc test: F(1, 15) ¼ 33.082, p50.001) and gestational stage (F(2, 15) ¼ 24.235, p50.001) both affected the CD3+CD4+ T/CD3+CD8+ T cell ratio. The stressed mice showed lower percentages of CD3+CD4+ T/CD3+CD8+ T cells at the three gestational stages compared with the corresponding control mice (Figure 4; independent t-test: t ¼ 3.09, p ¼ 0.027 on E3; t ¼ 4.67, p ¼ 0.005 on E5; and t ¼ 7.01, p ¼ 0.001 on E7).
Stress Downloaded from informahealthcare.com by Monash University on 12/03/14 For personal use only.
DOI: 10.3109/10253890.2014.966263
Restraint stress and embryo implantation
499
Figure 4. Effects of restraint stress on the T lymphocyte subsets in the uterus on E3, E5, and E7. The bar chart shows the CD3+CD4+ T/CD3+CD8+ T cell ratio between the stressed and control groups. Values were expressed as the mean ± SD. The meaning of the letters in the bar chart is the same as Figure 1. *p50.05 and #p50.01 were used to denote the significance compared with the corresponding control groups.
Lymphocyte proliferation and cytokine secretion The proliferative activities of uterine T and B lymphocytes were significantly lower in the stressed group than in the control group (Figure 5; the two-way ANOVA followed by Duncan’s post hoc test: F(1, 33) ¼ 19.829, p50.001 and F(1, 15) ¼ 79.871, p50.001). Gestational stage had no influence on the proliferative activities of uterine T and B lymphocytes (the two-way ANOVA followed by Duncan’s post hoc test: F(2, 33) ¼ 1.408, p ¼ 0.259 and F(2, 15) ¼ 2.892, p ¼ 0.087). These findings indicated that exogenous restraint stress predominately affected the proliferative activities of uterine lymphocytes. Comparative analysis showed that the proliferative activities of uterine T lymphocytes from the stressed mice were consistently decreased at the three gestational stages (Figure 5a; independent t-test: t ¼ 5.28, p ¼ 0.002 on E3; t ¼ 2.76, p ¼ 0.022 on E5; and t ¼ 2.60, p ¼ 0.018 on E7) compared with the corresponding control mice. Similarly, the stressed mice showed decreased proliferative uterine B lymphocyte activities on E5 and E7 in comparison with the respective control mice (Figure 5b; independent t-test: t ¼ 7.35, p ¼ 0.017 on E5 and t ¼ 5.03, p ¼ 0.002 on E7).
The contents of uterine IL-2 and IL-4 were influenced by restraint stress (Figure 5; the two-way ANOVA followed by Duncan’s post hoc test: F(1, 50) ¼ 9.12, p50.001 for IL-2 and F(1, 39) ¼ 184.869, p50.001 for IL-4) and gestational stage (F(2, 50) ¼ 2421.83, p50.001 for IL-2 and F(2, 39) ¼ 16647.451, p50.001 for IL-4). The results showed that stress led to the simultaneous enhancement of the proinflammatory and anti-inflammatory cytokine responses, including IL-2 and IL-4 secretion, on E3 and E5, respectively. There were increases in IL-2 (Figure 5c; independent t-test: t ¼ 10.37, p50.001 on E3 and t ¼ 9.65, p50.001 on E5) and IL-4 (Figure 5d; independent t-test: t ¼ 17.20, p50.001 on E3 and t ¼ 8.61, p50.001 on E5) in the stressed mice compared with the control mice. However, the levels of IL-2 and IL-4 significantly decreased on E7 (independent t-test: t ¼ 82.40, p50.001 for IL-2 and t ¼ 47.05, p50.001 for IL-4) compared with the control mice. As displayed in Figure 5(e), restraint stress (the two-way ANOVA followed by Duncan’s post hoc test: F(1, 50) ¼ 75.314, p50.001) and gestational stage (F(2, 50) ¼ 151.900, p50.001) significantly influenced the IL-2/IL-4 ratio in the uterus. Following treatment with
Stress Downloaded from informahealthcare.com by Monash University on 12/03/14 For personal use only.
500
G. Liu et al.
Stress, 2014; 17(6): 494–503
Figure 5. Effects of restraint stress on proliferation and cytokine secretion by uterine lymphocytes on E3, E5, and E7. The results show the proliferation ability of uterine lymphocytes induced by ConA (a) and LPS (b) in the control and the stressed group on E3, E5, and E7. A comparative statistical analysis shows the changes in IL-2 (c), IL-4 (d), and the IL-2/IL-4 ratio (e) in the uterus between the control and stressed groups on E3, E5, and E7. Values were expressed as the mean ± SD. The meaning of the letters in the bar chart is the same as Figure 1. *p50.05 and #p50.01 were used to denote the significance compared with the corresponding control groups.
Figure 6. Effects of restraint stress on the oxidative stress states in the uterus on E3, E5, and E7. The bar chart shows the contents of GSH-PX (a), SOD (b), T-AOC (c), and MDA (d) in the uterus of the control and stressed groups on E3, E5, and E7. Values were expressed as the mean ± SD. The meaning of the letters in the bar chart is the same as Figure 1. *p50.05 and #p50.01 were used to denote the significance compared with the corresponding control groups.
restraint stress, the IL-2/IL-4 ratio was increased at the three gestational stages (Figure 5e; independent t-test: t ¼ 5.76, p50.001 on E3; t ¼ 4.92, p ¼ 0.001 on E5; and t ¼ 6.10, p50.001 on E7) compared with the control mice.
Oxidative stress Statistical analysis showed that the activity levels of GSH-PX and SOD were influenced by restraint stress (Figure 6; the
Stress Downloaded from informahealthcare.com by Monash University on 12/03/14 For personal use only.
DOI: 10.3109/10253890.2014.966263
two-way ANOVA followed by Duncan’s post hoc test: F(1, 24) ¼ 26.375, p50.001 for GSH-PX and F(1, 33) ¼ 5.873, p ¼ 0.021 for SOD) and gestational stage (F(2, 24) ¼ 12.865, p50.001 for GSH-PX and F(2, 33) ¼ 29.022, p50.001 for SOD). As shown in Figure 6(a) and (b), the uterine activities of GSH-PX and SOD in the stressed mice showed statistically significant decreases on E5 (Figure 6a; independent t-test: t ¼ 4.56, p ¼ 0.011 for GSH-PX and t ¼ 2.53, p ¼ 0.045 for SOD) and E7 (Figure 6a; independent t-test: t ¼ 9.35, p50.001 for GSH-PX and t ¼ 2.82, p ¼ 0.014 for SOD) compared with the corresponding control mice. Additionally, restraint stress (F(1, 30) ¼ 8.850, p ¼ 0.006 for T-AOC and F(1, 29) ¼ 5.646, p ¼ 0.024 for MDA) and gestational stage (F(2, 30) ¼ 182.458, p50.001 for T-AOC and F(2, 29) ¼ 7.746, p ¼ 0.002 for MDA) significantly affected the contents of T-AOC and MDA. T-AOC contents significantly decreased in the stressed group on E5 and E7 (Figure 6c; independent t-test: t ¼ 5.09, p50.001 on E5 and t ¼ 9.61, p ¼ 0.001 on E7). MDA contents as an indicator of lipid peroxidation significantly increased on E5 and E7 (Figure 6d; independent t-test: t ¼ 2.49, p ¼ 0.042 on E5 and t ¼ 2.86, p ¼ 0.019 on E7). However, no significant differences were found in the activities of GSH-PX and SOD or in T-AOC and MDA contents on E3 between the control and stressed mice.
Discussion Restraint stress increased plasma CORT secretion The use of an animal model of restraint stress is a recognized technique for assessing the effects of psychological stress, which induces the activity of the HPA axis (Zafir & Banu, 2009). Therefore, cortisol (in humans) or CORT (in mice) is generally used as a stress marker because it is related to energetic, immunological, and psychological challenges (Nepomnaschy et al., 2006; Padgett & Glaser, 2003). Our findings demonstrated that the plasma CORT secretion of the stressed mice significantly increased from E3 to E7, indicating that the animal model was valid. Restraint stress interfered with the number of implantation sites Investigators have traditionally focused on the offspring of maternal mice exposed to persistent stress. The offspring of mice that experienced maternal restraint stress at gestational day (GD) 1–4, GD5–8, GD9–12, and GD13–16 have been shown to suffer from various malformations, such as cleft palate, bipartite ossification, and growth retardation (Lee et al., 2008). Moreover, the offspring from maternal restraint stress have also been reported to demonstrate reduced mobility and delayed task learning (Fuentes et al., 2007). However, few studies have focused on the effects of restraint stress on blastocyst implantation and uterine implantation. Pare´ & Glavin (1986) have found that pregnant mice that experience restraint stress for 6 d show fewer implantation sites than control mice and that the implantation sites of stressed mice are small and appear to undergo resorption. In the present study, the number of implantation sites significantly decreased on E7. Although restraint stress
Restraint stress and embryo implantation
501
led to a slight decrease in the number of implantation sites on E5, the uterine microenvironment may have undergone some internal changes. To further investigate the molecular mechanisms underlying the establishment of the implantation platform, this research mainly focused on the uterine immune microenvironment and oxidative states. Restraint stress altered local uterine immune activity The immune system is crucial for female receptivity to embryonic implantation (Robertson, 2010). uNK cells, which are large granular leukocytes, are considered to be instrumental to uterine vasculature remodeling and the maintenance of early pregnancy (Agarwal et al., 2012). In contrast to uNK cells, mast cells secrete histamine and disrupt the process of trophoblast invasion and growth. Although Tayade et al. (2007) documented that uNK cells peak in number on GD10 and subsequently decrease from GD12, our results showed that the density of these cells in the endometrium first increased and then declined from E3 to E7. This finding was attributed to the statistical approach used, which mainly analyzed the density in the uterine endometrium. Although the density of uNK cells in the endometrium was decreased on E7, the cell diameter was increased. The number of uNK cells has been shown to be lower in socially stressed rats than in control rats (Stefanski et al., 2005); however, uterine mast cells are activated and degranulated following the exposure of pregnant mice to sonic stress in association with increased abortion rates (Arck et al., 2001). Similarly, our results showed that the density of uNK cells in the endometrium significantly decreased while the mast cell density in the myometrium dramatically increased in the stressed groups from E3 to E7, indicating that cell number and cell function may be linked. Our findings suggest that restraint stress disturbed the uterine immune microenvironment by altering the uNK and mast cell densities in the uterus. In addition to uNK cells and mast cells, T and B lymphocytes are the most important uterine immunocytes in pregnant mice. The numbers, proportions, and functions of lymphocytes represent the immune microenvironment of the uterus. Previous studies have demonstrated that chronic stress dysregulated the immune response by weakening the responses of lymphocytes to mitogens (Silberman et al., 2002), decreasing the T helper/T suppressor cell ratio (Segerstrom & Miller, 2004), and disturbing the balance of Th1/Th2 cells (Dhabhar, 2009). The down-regulation of the CD4+ T/CD8+ T cell ratio is accompanied by abnormal behavior (Stefanski et al., 2005), body weight loss (Kick et al., 2012), and the abnormal distribution of blastocysts (Wongweragiat et al., 1999). In the present study, restraint stress decreased the proliferative activities of uterine lymphocytes, and the CD3+CD4+ T/CD3+CD8+ T cell ratio. Moreover, following treatment with 6-hydroxydopamine, the ratio of IL-2/IL-4 in the uterus has been shown to significantly increase and embryo implantation has been shown to decrease from E3 to E7 (Dong et al., 2007). Restraint stress administered from GD12 to parturition induces a basal proinflammatory state in the hippocampus in pregnant C57BL/6 mice (Diz-Chaves et al., 2013). In this study, the ratio of IL-2/ IL-4 was dramatically up-regulated from E3 to E7. This
502
G. Liu et al.
finding revealed that there was a distinct Th1-bias in association with restraint stress, leading to a imbalance of the uterine microenvironment. In addition, blastocysts typically implant into the uterus on E5. The numbers and functions of the immune cells in the uterus on E5 in the stressed mice showed distinct changes compared with the control mice. Our results suggest that restraint stress disturbed the balance of the uterine immune microenvironment during the implantation period, which altered the numbers of uNK and mast cells, decreased the CD3+CD4+ T/CD3+CD8+ T cell ratio and the proliferative activities of T and B lymphocytes, and increased the IL-2/IL-4 ratio. Therefore, we hypothesize that restraint stress decreased the number of implantation sites due to alterations in the uterine immune microenvironment.
Stress Downloaded from informahealthcare.com by Monash University on 12/03/14 For personal use only.
Restraint stress aggravated the oxidative stress states of the uterus Psychological stress not only stimulates the HPA axis but also causes oxidative damage by disturbing the balance between the oxidant and the antioxidant state. The exogenous administration of CORT may have caused an effect similar to that of restraint stress, decreasing the activities of SOD and GSH-PX and increasing MDA levels in the brain, liver, and heart (Zafir & Banu, 2009). Few studies have investigated the direct correlations between the induction of oxidative stress under psychological stress and pregnancy outcomes. One study has shown that two consecutive days of heat stress elevate reactive oxygen species levels and impair the development of pre-implantation embryos (Koyama et al., 2012). In addition, psychological stress attenuates enzymatic antioxidant activities and increases MDA levels (El-Far et al., 2007). In this study, we found that restraint stress threatened the intracellular equilibrium between oxidants and antioxidants, leading to a state of oxidative stress characterized by decreased GSHPX, SOD, and T-AOC activities and increased MDA levels at implantation (E5) and post-implantation (E7). These findings raise questions regarding the complex mechanism by which psychological stress induces adverse pregnancy outcomes and the correlation between oxidative stress and the immune response in association with restraint stress. Psychological stress is involved in the production of oxidative metabolites, which in turn exacerbate chronic inflammatory disorders (Kang & McCarthy, 1994). The stress-induced inflammatory response releases cytokines and stimulates macrophages to produce free-radical nitric oxide in association with oxidative stress (Sen & Chakraborty, 2011). Additionally, psychological stress induces the secretion of hormones, such as cortisol and catecholamine, which alter serum glucose concentrations, culminating in an excess of detrimental free radicals (Mitra, 2008). The active glucocorticoid receptor binds with nuclear factors, such as AP-1, NF-kB, the cAMP response element-binding protein, and the signaling and transcription factors STAT3 and STAT5 to initiate complex signaling pathways, leading to T cell apoptosis (Jamieson & Yamamoto, 2000) and the suppression of IL-2 production (Mittelstadt & Ashwell, 2001). Based on the previous studies and our current findings, we hypothesize that oxidative stress and/or the inflammatory response act as crucial links between restraint stress and early pregnancy
Stress, 2014; 17(6): 494–503
outcomes. However, the manner by which restraint stress is internally recognized, consequently affecting the uterine immune microenvironment and the oxidative state requires further research.
Conclusion In summary, we conclude that restraint stress acts as a psychological stressor responsible for inducing oxidative stress and that it negatively impacts immune parameters. The dysregulation of uterine immune activity and the exacerbation of oxidative stress lead to a reduction in the number of implantation sites. However, further research will be necessary to explore the manner by which stressful signals are internalized, thereby disturbing antioxidant activities and altering immune parameters. In addition, further research is required to elucidate whether certain substances or specific signaling molecules participate in crosstalk, linking oxidative stress mechanisms with the immune system.
Declaration of interest The authors disclose that there is no conflict of financial interest that influenced this work. This work was supported by the National High-tech Research and Development Projects (863) (No. 2013AA102306) and the National Natural Science Foundation of China (Nos. 31272483, 31272516, and 31372332).
References Agarwal A, Aponte-Mellado A, Premkumar BJ, Shaman A, Gupta S. (2012). The effects of oxidative stress on female reproduction: a review. Reprod Biol Endocrinol 10:49. Ali A, Khajuria A, Sidiq T, Kumar A, Thakur NL, Naik D, Vishwakarma RA. (2013). Modulation of LPS induced inflammatory response by Lawsonyl monocyclic terpene from the marine derived Streptomyces sp. Immunol Lett 150:79–86. Arck PC, Handjiski B, Hagen E, Joachim R, Klapp BF, Paus R. (2001). Indications for a ‘brain-hair follicle axis (BHA)’: inhibition of keratinocyte proliferation and up-regulation of keratinocyte apoptosis in telogen hair follicles by stress and substance P. FASEB J 15: 2536–8. Blois SM, Joachim R, Kandil J, Margni R, Tometten M, Klapp BF, Arck PC. (2004). Depletion of CD8+cells abolishes the pregnancy protective effect of progesterone substitution with dydrogesterone in mice by altering the Th1/Th2 cytokine profile. J Immunol 172:5893–9. Craig M. (2001). Stress and recurrent miscarriage. Stress 4:205–13. Dhabhar FS. (2009). Enhancing versus suppressive effects of stress on immune function: implications for immunoprotection and immunopathology. Neuroimmunomodulation. 16:300–17. Diz-Chaves Y, Astiz M, Bellini MJ, Garcia-Segura LM. (2013). Prenatal stress increases the expression of proinflammatory cytokines and exacerbates the inflammatory response to LPS in the hippocampal formation of adult male mice. Brain Behav Immun 28:196–206. Dong Y, Chen Y, Wang Z, Naito J, Chen JL. (2007). Role of sympathetic nerves on early embryonic development and immune modulation of uterus in pregnant mice. Auton Neurosci 131:87–93. El-Far M, El-Sayed IH, El-Motwally Ael-G, Hashem IA, Bakry N. (2007). Tumor necrosis factor-alpha and oxidant status are essential participating factors in unexplained recurrent spontaneous abortions. Clin Chem Lab Med 45:879–83. Fuentes S, Colomina MT, Vicens P, Domingo JL. (2007). Influence of maternal restraint stress on the long-lasting effects induced by prenatal exposure to perfluorooctane sulfonate (PFOS) in mice. Toxicol Lett 171:162–70. Jamieson CA, Yamamoto KR. (2000). Crosstalk pathway for inhibition of glucocorticoid-induced apoptosis by T cell receptor signaling. Proc Natl Acad Sci USA 97:7319–24.
Stress Downloaded from informahealthcare.com by Monash University on 12/03/14 For personal use only.
DOI: 10.3109/10253890.2014.966263
Kang DH, McCarthy DO. (1994). The effect of psychological stress on neutrophil superoxide release. Res Nurs Health 17:363–70. Kick AR, Tompkins MB, Flowers WL, Whisnant CS, Almond GW. (2012). Effects of stress associated with weaning on the adaptive immune system in pigs. J Anim Sci 90:649–56. Koyama H, Ikeda S, Sugimoto M, Kume S. (2012). Effects of folic acid on the development and oxidative stress of mouse embryos exposed to heat stress. Reprod Domest Anim 47:921–27. Lee AM, Lam SK, Sze Mun Lau SM, Chong CS, Chui HW, Fong DY. (2007). Prevalence, course, and risk factors for antenatal anxiety and depression. Obstet Gynecol 110:1102–12. Lee YE, Byun SK, Shin S, Jang JY, Choi BI, Park D, Jeon JH, et al. (2008). Effect of maternal restraint stress on fetal development of ICR mice. Exp Anim 57:19–25. Li J, Wang Z, Cao J, Dong Y, Chen Y. (2013). Melatonin receptor subtypes Mel1a and Mel1c but not Mel1b are associated with monochromatic light-induced B-lymphocyte proliferation in broilers. Domest Anim Endocrinol 45:206–15. Mairesse J, Lesage J, Breton C, Bre´ant B, Hahn T, Darnaude´ry M, Dickson SL, et al. (2007). Maternal stress alters endocrine function of the feto-placental unit in rats. Am J Physiol Endocrinol Metab 292: 1526–33. Mitra A. (2008). Diabetes and stress: a review. Ethno-Med 2:131–5. Mittelstadt PR, Ashwell JD. (2001). Inhibition of AP-1 by the glucocorticoid-inducible protein GILZ. J Biol Chem 276:29603–10. Nepomnaschy PA, Welch KB, McConnell DS, Low BS, Strassmann BI, England BG. (2006). Cortisol levels and very early pregnancy loss in humans. Proc Natl Acad Sci USA 103:3938–42. Ortolani D, Garcia MC, Melo-Thomas L, Spadari-Bratfisch RC. (2014). Stress-induced endocrine response and anxiety: the effects of comfort food in rats. Stress 17:211–18. Padgett DA, Glaser R. (2003). How stress influences the immune response. Trends Immunol 24:444–8. Pallare´s ME, Adrover E, Baier CJ, Bourguignon NS, Monteleone MC, Brocco MA, Gonza´lez-Calvar SI, Antonelli MC. (2013). Prenatal maternal restraint stress exposure alters the reproductive hormone profile and testis development of the rat male offspring. Stress 16: 429–40. Pare´ WP, Glavin GB. (1986). Restraint stress in biomedical research: a review. Neurosci Biobehav Rev 10:339–70. Paris JJ, Brunton PJ, Russell JA, Frye CA. (2011). Immune stress in late pregnant rats decreases length of gestation and fecundity, and alters later cognitive and affective behaviour of surviving pre-adolescent offspring. Stress 14:652–64.
Restraint stress and embryo implantation
503
Rahman A, Iqbal Z, Harrington R. (2003). Life events, social support and depression in childbirth: perspectives from a rural community in the developing world. Psychol Med 33:1161–7. Robertson SA. (2010). Immune regulation of conception and embryo implantation-all about quality control? J Reprod Immunol 85:51–7. Segerstrom SC, Miller GE. (2004). Psychological stress and the human immune system: a meta-analytic study of 30 years of inquiry. Psychol Bull 130:601–30. Sen S, Chakraborty R. (2011). The role of antioxidants in human health. In: Andreescu S, Hepel M, editors. Oxidative stress: diagnostics, prevention, and therapy. ACS symposium series. Washington, DC: American Chemical Society, p. 1–37 (Chapter 1). Schuerwegh AJ, De Clerck LS, Bridts CH, Stevens WJ. (2001). Wasp venom immunotherapy induces a shift from IL-4-producing towards interferon-gamma-producing CD4+ and CD8+ T lymphocytes. Clin Exp Allergy 31:740–6. Shahin S, Singh VP, Shukla RK, Dhawan A, Gangwar RK, Singh SP, Chaturvedi CM. (2013). 2.45 GHz microwave irradiation-induced oxidative stress affects implantation or pregnancy in mice, Mus musculus. Appl Biochem Biotechnol 169:1727–51. Silberman DM, Wald M, Genaro AM. (2002). Effects of chronic mild stress on lymphocyte proliferative response. Participation of serum thyroid hormones and corticosterone. Int Immunopharmacol 2: 487–97. Stefanski V, Raabe C, Schulte M. (2005). Pregnancy and social stress in female rats: influences on blood leukocytes and corticosterone concentrations. J Neuroimmunol 162:81–8. Tayade C, Hilchie D, He H, Fang Y, Moons L, Carmeliet P, Foster RA, Croy BA. (2007). Genetic deletion of placenta growth factor in mice alters uterine NK cells. J Immunol 178:4267–75. Toumi ML, Merzoug S, Baudin B, Tahraoui A. (2013). Quercetin alleviates predator stress-induced anxiety-like and brain oxidative signs in pregnant rats and immune count disturbance in their offspring. Pharmacol Biochem Behav 107:1–10. Wiebold JL, Stanfield PH, Becker WC, Hillers JK. (1986). The effect of restraint stress in early pregnancy in mice. J Reprod Fert 78: 185–92. Wongweragiat S, Searle RF, Bulmer JN. (1999). Decidual T lymphocyte activation in hydatidiform mole. J Clin Pathol 52:888–94. Zafir A, Banu N. (2009). Modulation of in vivo oxidative status by exogenous corticosterone and restraint stress in rats. Stress 12:167–77. Zhang SY, Wang ZJ, Li JJ, Wei DL, Sui SH, Zhang ZH, Zhou P, Tan JH. (2011). Maternal restraint stress diminishes the developmental potential of oocytes. Biol Reprod 84:672–81.
ORIGINAL RESEARCH REPORT
Restraint stress alters immune parameters and induces oxidative stress in the mouse uterus during embryo implantation Guanhui Liu*, Yulan Dong*, Zixu Wang, Jing Cao, and Yaoxing Chen
Stress Downloaded from informahealthcare.com by Monash University on 12/03/14 For personal use only.
Laboratory of Veterinary Anatomy, College of Animal Medicine, China Agricultural University, Beijing, China
Abstract
Keywords
The influence of stress on embryo implantation is not well understood. Prior studies have focused on later gestational stages and the long-term impact of stress on immune function. The objective of this study is to investigate the effects of restraint stress on the immune parameters and the oxidative states of the uterus during implantation. In this study, pregnant CD1 mice were subjected to restraint stress (4 h/d) on embryonic day 1 (E1) and sacrificed on E3, E5, and E7. Maternal plasma corticosterone (CORT) secretion and implantation sites in the uterus were examined. The uterine (excluding embryos) homogenate and uterine lymphocytes were collected to examine oxidative stress states and associated immune parameters. The results demonstrated that restraint stress increased maternal plasma CORT secretion and reduced the number of implantation sites by 15.3% on E5 and by 26.1% on E7. Moreover, restraint stress decreased the density of uterine natural killer (uNK) cells in the endometrium by 22.1–47.9% and increased the density of mast cells in the myometrium by 55.6–76.9%. Restraint stress remarkably decreased the CD3+CD4+ T/CD3+CD8+ T cell ratio (by 26.2–28.9%) and attenuated uterine lymphocyte proliferation and secretion of cytokines. In addition, restraint stress threatened the intracellular equilibrium between oxidants and antioxidants, resulting in decreased glutathione peroxidase (GSH-PX) (32.2% and 45.7%), superoxide dismutase (SOD) (15.5% and 26.1%), and total antioxidant capacity (T-AOC) (18.4% and 18.2%) activities and increased malondialdehyde (MDA) (34.4% and 43.0%) contents on E5 and E7. In conclusion, these findings demonstrate that restraint stress causes abnormal implantation and negatively impacts immune parameters in association with oxidative stress in mice.
Immune parameters, implantation sites, oxidative stress, pregnant mice, restraint stress, uterus
Introduction Maternal stress is common during both pregnancy and the postnatal period. During pregnancy, maternal stress (i.e. pregnancy-specific anxiety, depressive symptoms, and stressful life events) is associated with lower birth weight and preterm delivery. In 2009–2010, nearly 75% of mothers in the USA reported experiencing at least one stressful event prior to childbirth (Centers for Disease Control and Prevention, Pregnancy Risk Assessment Monitoring System). In South Asia, over 25% of mothers suffered from depressive disorders during their third trimester of pregnancy (Rahman et al., 2003). In China, approximately 54% and 37.1% of mothers experienced antenatal anxiety and depression, respectively (Lee et al., 2007). Since 1986, restraint stress has been generally used as a tool for the evaluation of physiology, pharmacology,
*These authors contributed equally to this work. Correspondence: Yaoxing Chen, Laboratory of Veterinary Anatomy, College of Animal Medicine, China Agricultural University, Haidian, Beijing 100193, PR China. Tel: +86 10 62733778. Fax: +86 10 62733199. E-mail: [email protected]
History Received 25 March 2014 Revised 19 August 2014 Accepted 27 August 2014 Published online 10 November 2014
immunology, and behavioral neurobiology (Wiebold et al., 1986). The response of animals to this stressor typically involves the triggering of two endocrine systems, including (1) the hypothalamic–pituitary–adrenal (HPA) axis and glucocorticoid production and (2) the sympathetic nervous system and the release of catecholamines (epinephrine and norepinephrine). It has been reported that pregnant rats subjected to restraint stress for 45 min (three times a day) from E15 to E21 showed subsequent decreases in body weight gain and food intake (Mairesse et al., 2007). Maternal psychosocial stress causes pregnancy complications, including implantation failure, recurrent spontaneous abortions, spontaneous preterm birth, and developmental abnormalities of the fetus in rodents (Zhang et al., 2011). Uterine microenvironment, including the inflammatory response and oxidative stress states, is also important for normal pregnancy progression and for the development of offspring. Immune challenge during late pregnancy has been shown to induce preterm birth and affect cognitive and affective functioning in offspring (Paris et al., 2011). Psychological stress applied on E19 has been shown to cause anxiety in rat dams in association with decreased glutathione (GSH) levels with a concomitant increase in MDA levels (Toumi et al., 2013). However, few studies have
Stress Downloaded from informahealthcare.com by Monash University on 12/03/14 For personal use only.
DOI: 10.3109/10253890.2014.966263
focused on changes in immune function and oxidative stress states in animals under restraint stress during implantation. In the process of implantation, the uterine environment undergoes serial remodeling, which involves leukocyte infiltration, trophoblast invasion, and specific cytokine and chemokine profiles at the maternal–fetal interface. Oxidative stress decreases the numbers of implantation sites and causes embryonic resorption in mice (Shahin et al., 2013). Psychological stress increases the ‘abortive’ cytokine TNFa and decreases the ‘anti-abortive’ cytokine TGF-P2, which may intricately interfere with implantation (Craig, 2001). Based on the previous studies, we hypothesize that oxidative stress and/or the inflammatory response act as crucial links between restraint stress and early pregnancy outcome. Therefore, this study used an animal model of restraint stress during pre- and post-implantation. It examined whether restraint stress disturbed the balance of the uterine microenvironment and caused alterations in the immune parameters and oxidative stress states, blastocyst implantation, and embryonic development in mice.
Materials and methods Ethical approval The present study conformed to the Guide for the Care and Use of Laboratory Animals published by the Animal Welfare Committee of the Agricultural Research Organization, China Agricultural University (Beijing, China). Animals A total of 210 female CD1 mice (8 weeks of age; Vital River Laboratory Animal Technology Co. Ltd., Beijing, China) were housed under conventional conditions (at a temperature of 21 ± 1 C, a relative humidity of 50 ± 10%) with a regular 14-h light:10-h dark cycle (with lights on at 7:00 A.M.). The mice were allowed ad libitum access to water and food. The animals were maintained in a small, quiet room for 1 week before the studies were initiated to minimize stress. Estrous mice were then placed with a sexually experienced male at night. The next morning, vaginal smears were obtained from the females with vaginal sperm plugs, and sperms were microscopically evaluated. Females regarded as pregnant were separated from the males and were considered to be at E1. Restraint stress was initiated on E1 until the mice were sacrificed on E3, E5, or E7. Specifically, pregnant female mice were individually placed into transparent and ventilated plastic centrifuge tubes (so that they were able to breathe freely but were unable to escape) to limit their movements for 4 h (from 8:00 A.M. to 12:00 P.M.) on E1. Because of the limitation of the restraint tubes, the stressed mice temporarily had no access to water or food. The control pregnant females were left untreated in their home cages, from which water and food were also temporarily removed for 4 h, to create experimental conditions similar to those of the stressed mice and to allow for increased accuracy in the assessment of the experimental factors (restraint stress and gestational stage). After 4 h of restraint stress, the animals were again provided access to water and food. According to the circadian rhythms of mice (nocturnal animals) and a previous study
Restraint stress and embryo implantation
495
(Pallare´s et al., 2013), the control mice should not have been affected by the lack of water and food. Plasma and tissue preparations A number of 90 pregnant females were anesthetized by 2% pentobarbital (4 mL/kg body weight) on E3, E5, and E7 immediately following the restraint stress. Plasma samples were collected for the detection of corticosterone (CORT). Thirty uterine specimens were fixed in 4% paraformaldehyde and embedded in paraffin for histological analysis. Some of the uteri were rapidly dissected with excluding the embryos and stored at 80 C for cytokine analysis (30 mice) and antioxidant enzyme analysis (30 mice). The total number of implantation sites on the walls of the uteri was identified following the intravenous injection of 0.5 mL 0.5% trypan blue dye on E5, and the implantation sites were counted directly on E7. Measurement of plasma CORT concentration The total plasma CORT concentration was measured using a competitive enzyme-linked immunosorbent assay (CEA540Ge, Uscn Life Science Inc., Wuhan, China). All the tests were performed according to the manufacturer’s instructions. The limit of detection for the assay ranged from 6.17 to 500 ng/mL. Eight plasma samples were included in each group. Each sample was tested in triplicate. Histological analyses of uNK cells and mast cells Uteri with implantation sites (2–3 mm in length) were routinely processed into paraffin sections. Five-micron-thick sections were mounted onto 10% polylysine-coated slides. The uNK cells and mast cells were stained with periodic acidSchiff (PAS) and toluidine blue, respectively. Positively stained cells were observed under a microscope. The number of uNK cells per mm2 in the endometrium was calculated, while the number of mast cells per mm2 in the myometrium was counted in five random fields from six cross-sections of the four uterine specimens from each group on E3, E5, and E7. Measurements of cytokines and oxidative stressrelated enzymes Immediately after the mice were sacrificed, the uteri were rapidly dissected, and the embryos were removed, frozen on dry ice, and stored at 80 C. The uteri with removed embryos were weighted and placed in a 0.9% saline solution (1:9) to prepare a uterine homogenate. Finally, supernatants were extracted by centrifugation (1000 g, 20 min) to determine cytokine concentrations, oxidative molecule production, and the presence of anti-oxidative enzymes. IL-2 and IL-4 cytokines To quantitatively measure the concentrations of uterine IL-2 and IL-4, a routine sandwich enzyme-linked immunosorbent assay was performed (IL-2: EK0398; IL-4: EK0405, Boster Biological Technology, Ltd., Wuhan, China). All tests were conducted according to the manufacturer’s instructions. The samples and reagents were equilibrated to room temperature
496
G. Liu et al.
before testing. The limit of detection for the IL-2 assay ranged from 15.6 to 1000 pg/mL and that for the IL-4 assay ranged from 7.8 to 500 pg/mL. The data describing uterine IL-2 and IL-4 levels were expressed as pg/mg of protein. Approximately five whole uterine samples were required for each group. Each sample was tested in triplicate.
Stress, 2014; 17(6): 494–503
compensated for by the use of cells FITC-only, and PE-only antibodies. analyzed with a FACSCalibur Biosciences, San Diego, CA) using (BD Biosciences, San Diego, CA).
stained with APC-only, The lymphocytes were flow cytometer (BD the CellQuest program
Lymphocyte proliferative activity assay Oxidative stress-related enzymes GSH-PX and SOD are well-known scavenger enzymes that protect cells from oxidative stress, while MDA is responsible for inducing oxidative stress. T-AOC, SOD, GSH-PX, and MDA assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). T-AOC, SOD, and GSH-PX were expressed as U/mg of protein, and MDA was expressed as nmol/mg of protein.
Stress Downloaded from informahealthcare.com by Monash University on 12/03/14 For personal use only.
Uterine lymphocytes isolation Uterine lymphocytes were isolated as previously described (Blois et al., 2004) with some modifications. In brief, a total of 120 pregnant mice were sacrificed, the uterus was rapidly dissected from each, and the embryos/fetuses were removed. After removing the fatty and connective tissues, the obtained uterus was washed in 0.01 mol/L phosphate-buffered saline (PBS), sliced longitudinally into small pieces, and incubated with 0.1% collagenase in a 15-mL centrifuge tube at 37 C for 60 min. The digestive tissues were passed through a nylon tissue sieve (200 nylon mesh per 2.5 cm) and washed twice with RPMI-1640 medium (Gibco-BRL, Grand Island, NY). After the centrifugation step, the supernatant was discarded, and the precipitate was suspended in 3 mL RPMI-1640 complete medium and combined with 3 mL lymphocyte separation medium (LTS1077, Tianjin Haoyang Biological Technology Co., Ltd., Tianjin, China) for centrifugation (800 g for 10 min). The isolated lymphocytes were washed twice with 1.5 mL RPMI-1640 complete medium. The cell viability (498%) was detected using 0.5% trypan blue dye, and the cell suspension was cultured in 1 mL RPMI-1640 at a density of 1 106 cells/mL. T lymphocyte subsets and lymphocyte proliferative activities were analyzed in the uterine lymphocytes. Approximately 10 whole uterine samples were required for each group for the lymphocyte isolation. Flow cytometry analysis Flow cytometry was used to determine the phenotypes of the uterine lymphocytes. As previously described (Schuerwegh et al., 2001), isolated uterine lymphocyte cells were stained for 30 min in the dark at room temperature with 1 mL of specific monoclonal antibodies directed against T-cell surface markers, including APC-conjugated anti-CD3 (553066, BD Biosciences, San Diego, CA), FITC-conjugated anti-CD4 (553046, BD Biosciences, San Diego, CA), and PEconjugated anti-CD8 (553032, BD Biosciences, San Diego, CA). Next, the lymphocytes were washed with 0.2 mL PBS and centrifuged at 500 g for 5 min (three times). Finally, the cells were re-suspended in 0.2 mL PBS and subjected to flow cytometric analysis. Fluorochrome-equivalent IgG isotype controls were used to detect non-specific binding. The spectral overlap of three types of fluorochromes was
The 200-mL lymphocyte suspensions were seeded into each well of a 96-well flat-bottom microwell plate (Costar 3599, Corning Inc., Corning, NY) in the presence of 15 mg/mL LPS (L2630, Sigma, St. Louis, MO) or ConA (C2272, Sigma, St. Louis, MO). After incubating the lymphocytes at 37 C in a humidified, 5% CO2 atmosphere for 68 h, the proliferation index was assayed using 5 mg/mL methyl thiazolyl tetrazolium (MTT, Sigma, St. Louis, MO) (Ali et al., 2013). Control cells were incubated with RPMI-1640 alone. Specifically, the plate was incubated for 4 h after the addition of 10 mL MTT per well, and then, 90 mL of 10 mg/mL sodium dodecyl sulfate (SDS, Sigma, St. Louis, MO) was pipetted into each well. After incubation at 37 C for 4 h, the microplate was read using a Bio-Rad microplate reader (Model 550, Bio-Rad, Berkeley, CA) at a wavelength of 570 nm. The proliferative activity of the lymphocytes was expressed as the stimulation index (SI) as follows (Li et al., 2013): SI ¼ OD570 ðstimulated cellsÞ=OD570 ðunstimulated cellsÞ Statistical analysis The data were analyzed with SPSS 18.0 (SPSS Inc., Chicago, IL) as described in a previous study (Ortolani et al., 2014). To analyze the effects of restraint stress and different gestational stages on the experimental data, a two-way analysis of variance (ANOVA) was performed followed by Duncan’s post hoc test. The statistical difference between the control and stressed mice at the same gestational stage was evaluated by independent samples t test. The data were expressed as the mean ± SD. *p50.05 and #p50.01 were used to denote the significance compared with the corresponding control groups.
Results Plasma CORT secretion Plasma CORT secretion of the pregnant mice was influenced by restraint stress (Figure 1; the two-way ANOVA followed by Duncan’s post hoc test: F(1, 16) ¼ 45.678, p50.001) and gestational stage (F(2, 16) ¼ 13.238, p50.001). Furthermore, plasma CORT secretion in the control mice showed an inverted V profile with a peak at E5 that leveled off at E7. Although the stressed mice displayed the same pattern, plasma CORT secretion increased compared with the control mice at the same stage (independent t-test: t ¼ 10.49, p50.001 for control versus stressed mice on E3; t ¼ 5.31, p ¼ 0.003 for control versus stressed mice on E5; t ¼ 3.02, p ¼ 0.019 for control versus stressed mice on E7). Number of implantation sites As shown in Figure 2, the uterus was smooth in appearance on E3 because blastocyst implantation occurred on E5 after mating. To observe the implantation sites in the uterus, the
DOI: 10.3109/10253890.2014.966263
mice were injected with 0.5% trypan blue through the caudal vein on E5. The implantation sites were stained blue and arranged as a string of beads in the uterus. Restraint stress negatively influenced the number of implantation sites (Figure 2; the two-way ANOVA followed by Duncan’s post hoc test: F(1, 47) ¼ 5.145, p ¼ 0.028). Because the uterus became receptive to the blastocyst at a fixed time (E5), the
Restraint stress and embryo implantation
497
gestational stage had no effect on the number of implantation sites (F(1, 47) ¼ 1.250, p ¼ 0.269). The implantation sites in the uterus on E7 appeared as round or oval enlargements, and the number of implantation sites significantly decreased in the stressed mice compared with the control mice (Figure 2; control ¼ 13.5 ± 1.84; stressed ¼ 10.0 ± 4.51; independent t-test: t ¼ 3.30, p ¼ 0.003). The number of implantation sites decreased on E5 with no statistically significant differences observed between the control and stressed mice.
Stress Downloaded from informahealthcare.com by Monash University on 12/03/14 For personal use only.
Densities of uNK cells and mast cells
Figure 1. Effects of restraint stress on plasma CORT secretion on E3, E5, and E7 pregnant mice. The data (ng/mL) were expressed as the mean ± SD. Uppercase letters represented the differences among E3, E5, and E7 in the control group (p50.05), and lowercase letters represented the differences among E3, E5, and E7 in the stressed group (p50.05). *p50.05 and #p50.01 were used to denote significance compared with the corresponding control groups. Figure 2. Effects of restraint stress on the number of implantation sites on E3, E5, and E7 pregnant mice. There were no implantation sites on E3, while on E5, implantation sites were detected as round or oval embryos that were stained blue by 0.5% trypan blue. As expected, the implantation sites were larger and arranged as a string of beads in the uterus. Scale bars ¼ 1 cm. The short arrows show the implantation sites on E5 and E7. Values were expressed as the mean ± SD. # p50.01 versus the E7 control group.
Normally, uNK cells are the predominant uterine lymphocytes in mice during the early gestational stage. In this study, the uNK cells appeared oval in shape and rose-red by PAS staining. As shown in Figure 3, these cells were scattered throughout the endometrium and were non-granulated (approximately 5 mm in diameter) on E3 and E5. On E7, the majority of uNK cells was large (approximately 13 mm in diameter), heavily granulated, and distributed within the decidua basalis. The density of uNK cells in the endometrium showed an inverted V profile, with a peak on E5 that waned to basic levels on E7 in the control mice. Although the stressed mice exhibited a similar trend, the density of endometrial uNK cells decreased relative to that of the control mice (Figure 3a; the two-way ANOVA followed by Duncan’s post hoc test: F(1, 79) ¼ 200.231, p50.001 for restraint stress effects; F(2, 79) ¼ 166.787, p50.001 for gestational stage effects). The density of uNK cells in the endometrium of
Stress Downloaded from informahealthcare.com by Monash University on 12/03/14 For personal use only.
498
G. Liu et al.
Stress, 2014; 17(6): 494–503
Figure 3. Photomicrographs show the effects of restraint stress on the distribution and density of uNK cells and mast cells in the uteri of pregnant mice on E3, E5, and E7. The morphology of uNK cells in the endometrium and mast cells in the myometrium was examined by staining with PAS and toluidine blue, respectively. Long arrows indicate positive-staining uNK cells in the endometrium, and short arrows indicate positive-staining mast cells in the myometrium. Scale bars ¼ 50 mm. The density of uNK cells (a) in the endometrium and mast cells (b) in the myometrium are shown. Data (cells/mm2) were expressed as the mean ± SD. The meaning of the letters in the bar chart is the same as Figure 1. #p50.01 were used to denote significance compared with the corresponding control groups. uNK cells, uterine natural killer cells; MCs, mast cells; End, endometrium; UL, uterine lumen; DB, decidua basalis; RUL, residual uterine lumen; LSM, outer longitudinal smooth muscle of uterine wall; CSM, inner circular smooth muscle of uterine wall; Ug, uterine gland; VLa, vascular layer.
stressed mice decreased compared with the corresponding control mice (Figure 3a; independent t-test: t ¼ 4.72, p50.001 on E3; t ¼ 12.10, p50.001 on E5; and t ¼ 5.79, p50.001 on E7). Mast cells showed some correlation with pregnancy failure in the mice. In this study, toluidine blue staining resulted in red mast cells that were present in large numbers in the myometrium but that were scarce in the endometrium. As summarized in Figure 3(b), we observed a dramatic increase in the number of mast cells per mm2 in the myometrium of the pregnant mice exposed to restraint stress compared with the control mice at the corresponding gestational stage. The density of mast cells in the myometrium was influenced by restraint stress (Figure 3b; the two-way ANOVA followed by Duncan’s post hoc test: F(1, 59) ¼ 73.569, p50.001) and gestational stage (F(2, 59) ¼ 117.317, p50.001). In contrast to the inverted V profile of uNK, the density of mast cells in the myometrium displayed a typical V profile with a drop on E5.
In comparison with the respective control mice, the density of mast cells in the myometrium of stressed mice increased at the three gestational stages (Figure 3b; independent t-test: t ¼ 5.97, p50.001 on E3; t ¼ 4.14, p ¼ 0.002 on E5; and t ¼ 8.50, p50.001 on E7). T lymphocyte subsets The results from the flow cytometric analysis are shown in Figure 4. Restraint stress (Figure 4; the two-way ANOVA followed by Duncan’s post hoc test: F(1, 15) ¼ 33.082, p50.001) and gestational stage (F(2, 15) ¼ 24.235, p50.001) both affected the CD3+CD4+ T/CD3+CD8+ T cell ratio. The stressed mice showed lower percentages of CD3+CD4+ T/CD3+CD8+ T cells at the three gestational stages compared with the corresponding control mice (Figure 4; independent t-test: t ¼ 3.09, p ¼ 0.027 on E3; t ¼ 4.67, p ¼ 0.005 on E5; and t ¼ 7.01, p ¼ 0.001 on E7).
Stress Downloaded from informahealthcare.com by Monash University on 12/03/14 For personal use only.
DOI: 10.3109/10253890.2014.966263
Restraint stress and embryo implantation
499
Figure 4. Effects of restraint stress on the T lymphocyte subsets in the uterus on E3, E5, and E7. The bar chart shows the CD3+CD4+ T/CD3+CD8+ T cell ratio between the stressed and control groups. Values were expressed as the mean ± SD. The meaning of the letters in the bar chart is the same as Figure 1. *p50.05 and #p50.01 were used to denote the significance compared with the corresponding control groups.
Lymphocyte proliferation and cytokine secretion The proliferative activities of uterine T and B lymphocytes were significantly lower in the stressed group than in the control group (Figure 5; the two-way ANOVA followed by Duncan’s post hoc test: F(1, 33) ¼ 19.829, p50.001 and F(1, 15) ¼ 79.871, p50.001). Gestational stage had no influence on the proliferative activities of uterine T and B lymphocytes (the two-way ANOVA followed by Duncan’s post hoc test: F(2, 33) ¼ 1.408, p ¼ 0.259 and F(2, 15) ¼ 2.892, p ¼ 0.087). These findings indicated that exogenous restraint stress predominately affected the proliferative activities of uterine lymphocytes. Comparative analysis showed that the proliferative activities of uterine T lymphocytes from the stressed mice were consistently decreased at the three gestational stages (Figure 5a; independent t-test: t ¼ 5.28, p ¼ 0.002 on E3; t ¼ 2.76, p ¼ 0.022 on E5; and t ¼ 2.60, p ¼ 0.018 on E7) compared with the corresponding control mice. Similarly, the stressed mice showed decreased proliferative uterine B lymphocyte activities on E5 and E7 in comparison with the respective control mice (Figure 5b; independent t-test: t ¼ 7.35, p ¼ 0.017 on E5 and t ¼ 5.03, p ¼ 0.002 on E7).
The contents of uterine IL-2 and IL-4 were influenced by restraint stress (Figure 5; the two-way ANOVA followed by Duncan’s post hoc test: F(1, 50) ¼ 9.12, p50.001 for IL-2 and F(1, 39) ¼ 184.869, p50.001 for IL-4) and gestational stage (F(2, 50) ¼ 2421.83, p50.001 for IL-2 and F(2, 39) ¼ 16647.451, p50.001 for IL-4). The results showed that stress led to the simultaneous enhancement of the proinflammatory and anti-inflammatory cytokine responses, including IL-2 and IL-4 secretion, on E3 and E5, respectively. There were increases in IL-2 (Figure 5c; independent t-test: t ¼ 10.37, p50.001 on E3 and t ¼ 9.65, p50.001 on E5) and IL-4 (Figure 5d; independent t-test: t ¼ 17.20, p50.001 on E3 and t ¼ 8.61, p50.001 on E5) in the stressed mice compared with the control mice. However, the levels of IL-2 and IL-4 significantly decreased on E7 (independent t-test: t ¼ 82.40, p50.001 for IL-2 and t ¼ 47.05, p50.001 for IL-4) compared with the control mice. As displayed in Figure 5(e), restraint stress (the two-way ANOVA followed by Duncan’s post hoc test: F(1, 50) ¼ 75.314, p50.001) and gestational stage (F(2, 50) ¼ 151.900, p50.001) significantly influenced the IL-2/IL-4 ratio in the uterus. Following treatment with
Stress Downloaded from informahealthcare.com by Monash University on 12/03/14 For personal use only.
500
G. Liu et al.
Stress, 2014; 17(6): 494–503
Figure 5. Effects of restraint stress on proliferation and cytokine secretion by uterine lymphocytes on E3, E5, and E7. The results show the proliferation ability of uterine lymphocytes induced by ConA (a) and LPS (b) in the control and the stressed group on E3, E5, and E7. A comparative statistical analysis shows the changes in IL-2 (c), IL-4 (d), and the IL-2/IL-4 ratio (e) in the uterus between the control and stressed groups on E3, E5, and E7. Values were expressed as the mean ± SD. The meaning of the letters in the bar chart is the same as Figure 1. *p50.05 and #p50.01 were used to denote the significance compared with the corresponding control groups.
Figure 6. Effects of restraint stress on the oxidative stress states in the uterus on E3, E5, and E7. The bar chart shows the contents of GSH-PX (a), SOD (b), T-AOC (c), and MDA (d) in the uterus of the control and stressed groups on E3, E5, and E7. Values were expressed as the mean ± SD. The meaning of the letters in the bar chart is the same as Figure 1. *p50.05 and #p50.01 were used to denote the significance compared with the corresponding control groups.
restraint stress, the IL-2/IL-4 ratio was increased at the three gestational stages (Figure 5e; independent t-test: t ¼ 5.76, p50.001 on E3; t ¼ 4.92, p ¼ 0.001 on E5; and t ¼ 6.10, p50.001 on E7) compared with the control mice.
Oxidative stress Statistical analysis showed that the activity levels of GSH-PX and SOD were influenced by restraint stress (Figure 6; the
Stress Downloaded from informahealthcare.com by Monash University on 12/03/14 For personal use only.
DOI: 10.3109/10253890.2014.966263
two-way ANOVA followed by Duncan’s post hoc test: F(1, 24) ¼ 26.375, p50.001 for GSH-PX and F(1, 33) ¼ 5.873, p ¼ 0.021 for SOD) and gestational stage (F(2, 24) ¼ 12.865, p50.001 for GSH-PX and F(2, 33) ¼ 29.022, p50.001 for SOD). As shown in Figure 6(a) and (b), the uterine activities of GSH-PX and SOD in the stressed mice showed statistically significant decreases on E5 (Figure 6a; independent t-test: t ¼ 4.56, p ¼ 0.011 for GSH-PX and t ¼ 2.53, p ¼ 0.045 for SOD) and E7 (Figure 6a; independent t-test: t ¼ 9.35, p50.001 for GSH-PX and t ¼ 2.82, p ¼ 0.014 for SOD) compared with the corresponding control mice. Additionally, restraint stress (F(1, 30) ¼ 8.850, p ¼ 0.006 for T-AOC and F(1, 29) ¼ 5.646, p ¼ 0.024 for MDA) and gestational stage (F(2, 30) ¼ 182.458, p50.001 for T-AOC and F(2, 29) ¼ 7.746, p ¼ 0.002 for MDA) significantly affected the contents of T-AOC and MDA. T-AOC contents significantly decreased in the stressed group on E5 and E7 (Figure 6c; independent t-test: t ¼ 5.09, p50.001 on E5 and t ¼ 9.61, p ¼ 0.001 on E7). MDA contents as an indicator of lipid peroxidation significantly increased on E5 and E7 (Figure 6d; independent t-test: t ¼ 2.49, p ¼ 0.042 on E5 and t ¼ 2.86, p ¼ 0.019 on E7). However, no significant differences were found in the activities of GSH-PX and SOD or in T-AOC and MDA contents on E3 between the control and stressed mice.
Discussion Restraint stress increased plasma CORT secretion The use of an animal model of restraint stress is a recognized technique for assessing the effects of psychological stress, which induces the activity of the HPA axis (Zafir & Banu, 2009). Therefore, cortisol (in humans) or CORT (in mice) is generally used as a stress marker because it is related to energetic, immunological, and psychological challenges (Nepomnaschy et al., 2006; Padgett & Glaser, 2003). Our findings demonstrated that the plasma CORT secretion of the stressed mice significantly increased from E3 to E7, indicating that the animal model was valid. Restraint stress interfered with the number of implantation sites Investigators have traditionally focused on the offspring of maternal mice exposed to persistent stress. The offspring of mice that experienced maternal restraint stress at gestational day (GD) 1–4, GD5–8, GD9–12, and GD13–16 have been shown to suffer from various malformations, such as cleft palate, bipartite ossification, and growth retardation (Lee et al., 2008). Moreover, the offspring from maternal restraint stress have also been reported to demonstrate reduced mobility and delayed task learning (Fuentes et al., 2007). However, few studies have focused on the effects of restraint stress on blastocyst implantation and uterine implantation. Pare´ & Glavin (1986) have found that pregnant mice that experience restraint stress for 6 d show fewer implantation sites than control mice and that the implantation sites of stressed mice are small and appear to undergo resorption. In the present study, the number of implantation sites significantly decreased on E7. Although restraint stress
Restraint stress and embryo implantation
501
led to a slight decrease in the number of implantation sites on E5, the uterine microenvironment may have undergone some internal changes. To further investigate the molecular mechanisms underlying the establishment of the implantation platform, this research mainly focused on the uterine immune microenvironment and oxidative states. Restraint stress altered local uterine immune activity The immune system is crucial for female receptivity to embryonic implantation (Robertson, 2010). uNK cells, which are large granular leukocytes, are considered to be instrumental to uterine vasculature remodeling and the maintenance of early pregnancy (Agarwal et al., 2012). In contrast to uNK cells, mast cells secrete histamine and disrupt the process of trophoblast invasion and growth. Although Tayade et al. (2007) documented that uNK cells peak in number on GD10 and subsequently decrease from GD12, our results showed that the density of these cells in the endometrium first increased and then declined from E3 to E7. This finding was attributed to the statistical approach used, which mainly analyzed the density in the uterine endometrium. Although the density of uNK cells in the endometrium was decreased on E7, the cell diameter was increased. The number of uNK cells has been shown to be lower in socially stressed rats than in control rats (Stefanski et al., 2005); however, uterine mast cells are activated and degranulated following the exposure of pregnant mice to sonic stress in association with increased abortion rates (Arck et al., 2001). Similarly, our results showed that the density of uNK cells in the endometrium significantly decreased while the mast cell density in the myometrium dramatically increased in the stressed groups from E3 to E7, indicating that cell number and cell function may be linked. Our findings suggest that restraint stress disturbed the uterine immune microenvironment by altering the uNK and mast cell densities in the uterus. In addition to uNK cells and mast cells, T and B lymphocytes are the most important uterine immunocytes in pregnant mice. The numbers, proportions, and functions of lymphocytes represent the immune microenvironment of the uterus. Previous studies have demonstrated that chronic stress dysregulated the immune response by weakening the responses of lymphocytes to mitogens (Silberman et al., 2002), decreasing the T helper/T suppressor cell ratio (Segerstrom & Miller, 2004), and disturbing the balance of Th1/Th2 cells (Dhabhar, 2009). The down-regulation of the CD4+ T/CD8+ T cell ratio is accompanied by abnormal behavior (Stefanski et al., 2005), body weight loss (Kick et al., 2012), and the abnormal distribution of blastocysts (Wongweragiat et al., 1999). In the present study, restraint stress decreased the proliferative activities of uterine lymphocytes, and the CD3+CD4+ T/CD3+CD8+ T cell ratio. Moreover, following treatment with 6-hydroxydopamine, the ratio of IL-2/IL-4 in the uterus has been shown to significantly increase and embryo implantation has been shown to decrease from E3 to E7 (Dong et al., 2007). Restraint stress administered from GD12 to parturition induces a basal proinflammatory state in the hippocampus in pregnant C57BL/6 mice (Diz-Chaves et al., 2013). In this study, the ratio of IL-2/ IL-4 was dramatically up-regulated from E3 to E7. This
502
G. Liu et al.
finding revealed that there was a distinct Th1-bias in association with restraint stress, leading to a imbalance of the uterine microenvironment. In addition, blastocysts typically implant into the uterus on E5. The numbers and functions of the immune cells in the uterus on E5 in the stressed mice showed distinct changes compared with the control mice. Our results suggest that restraint stress disturbed the balance of the uterine immune microenvironment during the implantation period, which altered the numbers of uNK and mast cells, decreased the CD3+CD4+ T/CD3+CD8+ T cell ratio and the proliferative activities of T and B lymphocytes, and increased the IL-2/IL-4 ratio. Therefore, we hypothesize that restraint stress decreased the number of implantation sites due to alterations in the uterine immune microenvironment.
Stress Downloaded from informahealthcare.com by Monash University on 12/03/14 For personal use only.
Restraint stress aggravated the oxidative stress states of the uterus Psychological stress not only stimulates the HPA axis but also causes oxidative damage by disturbing the balance between the oxidant and the antioxidant state. The exogenous administration of CORT may have caused an effect similar to that of restraint stress, decreasing the activities of SOD and GSH-PX and increasing MDA levels in the brain, liver, and heart (Zafir & Banu, 2009). Few studies have investigated the direct correlations between the induction of oxidative stress under psychological stress and pregnancy outcomes. One study has shown that two consecutive days of heat stress elevate reactive oxygen species levels and impair the development of pre-implantation embryos (Koyama et al., 2012). In addition, psychological stress attenuates enzymatic antioxidant activities and increases MDA levels (El-Far et al., 2007). In this study, we found that restraint stress threatened the intracellular equilibrium between oxidants and antioxidants, leading to a state of oxidative stress characterized by decreased GSHPX, SOD, and T-AOC activities and increased MDA levels at implantation (E5) and post-implantation (E7). These findings raise questions regarding the complex mechanism by which psychological stress induces adverse pregnancy outcomes and the correlation between oxidative stress and the immune response in association with restraint stress. Psychological stress is involved in the production of oxidative metabolites, which in turn exacerbate chronic inflammatory disorders (Kang & McCarthy, 1994). The stress-induced inflammatory response releases cytokines and stimulates macrophages to produce free-radical nitric oxide in association with oxidative stress (Sen & Chakraborty, 2011). Additionally, psychological stress induces the secretion of hormones, such as cortisol and catecholamine, which alter serum glucose concentrations, culminating in an excess of detrimental free radicals (Mitra, 2008). The active glucocorticoid receptor binds with nuclear factors, such as AP-1, NF-kB, the cAMP response element-binding protein, and the signaling and transcription factors STAT3 and STAT5 to initiate complex signaling pathways, leading to T cell apoptosis (Jamieson & Yamamoto, 2000) and the suppression of IL-2 production (Mittelstadt & Ashwell, 2001). Based on the previous studies and our current findings, we hypothesize that oxidative stress and/or the inflammatory response act as crucial links between restraint stress and early pregnancy
Stress, 2014; 17(6): 494–503
outcomes. However, the manner by which restraint stress is internally recognized, consequently affecting the uterine immune microenvironment and the oxidative state requires further research.
Conclusion In summary, we conclude that restraint stress acts as a psychological stressor responsible for inducing oxidative stress and that it negatively impacts immune parameters. The dysregulation of uterine immune activity and the exacerbation of oxidative stress lead to a reduction in the number of implantation sites. However, further research will be necessary to explore the manner by which stressful signals are internalized, thereby disturbing antioxidant activities and altering immune parameters. In addition, further research is required to elucidate whether certain substances or specific signaling molecules participate in crosstalk, linking oxidative stress mechanisms with the immune system.
Declaration of interest The authors disclose that there is no conflict of financial interest that influenced this work. This work was supported by the National High-tech Research and Development Projects (863) (No. 2013AA102306) and the National Natural Science Foundation of China (Nos. 31272483, 31272516, and 31372332).
References Agarwal A, Aponte-Mellado A, Premkumar BJ, Shaman A, Gupta S. (2012). The effects of oxidative stress on female reproduction: a review. Reprod Biol Endocrinol 10:49. Ali A, Khajuria A, Sidiq T, Kumar A, Thakur NL, Naik D, Vishwakarma RA. (2013). Modulation of LPS induced inflammatory response by Lawsonyl monocyclic terpene from the marine derived Streptomyces sp. Immunol Lett 150:79–86. Arck PC, Handjiski B, Hagen E, Joachim R, Klapp BF, Paus R. (2001). Indications for a ‘brain-hair follicle axis (BHA)’: inhibition of keratinocyte proliferation and up-regulation of keratinocyte apoptosis in telogen hair follicles by stress and substance P. FASEB J 15: 2536–8. Blois SM, Joachim R, Kandil J, Margni R, Tometten M, Klapp BF, Arck PC. (2004). Depletion of CD8+cells abolishes the pregnancy protective effect of progesterone substitution with dydrogesterone in mice by altering the Th1/Th2 cytokine profile. J Immunol 172:5893–9. Craig M. (2001). Stress and recurrent miscarriage. Stress 4:205–13. Dhabhar FS. (2009). Enhancing versus suppressive effects of stress on immune function: implications for immunoprotection and immunopathology. Neuroimmunomodulation. 16:300–17. Diz-Chaves Y, Astiz M, Bellini MJ, Garcia-Segura LM. (2013). Prenatal stress increases the expression of proinflammatory cytokines and exacerbates the inflammatory response to LPS in the hippocampal formation of adult male mice. Brain Behav Immun 28:196–206. Dong Y, Chen Y, Wang Z, Naito J, Chen JL. (2007). Role of sympathetic nerves on early embryonic development and immune modulation of uterus in pregnant mice. Auton Neurosci 131:87–93. El-Far M, El-Sayed IH, El-Motwally Ael-G, Hashem IA, Bakry N. (2007). Tumor necrosis factor-alpha and oxidant status are essential participating factors in unexplained recurrent spontaneous abortions. Clin Chem Lab Med 45:879–83. Fuentes S, Colomina MT, Vicens P, Domingo JL. (2007). Influence of maternal restraint stress on the long-lasting effects induced by prenatal exposure to perfluorooctane sulfonate (PFOS) in mice. Toxicol Lett 171:162–70. Jamieson CA, Yamamoto KR. (2000). Crosstalk pathway for inhibition of glucocorticoid-induced apoptosis by T cell receptor signaling. Proc Natl Acad Sci USA 97:7319–24.
Stress Downloaded from informahealthcare.com by Monash University on 12/03/14 For personal use only.
DOI: 10.3109/10253890.2014.966263
Kang DH, McCarthy DO. (1994). The effect of psychological stress on neutrophil superoxide release. Res Nurs Health 17:363–70. Kick AR, Tompkins MB, Flowers WL, Whisnant CS, Almond GW. (2012). Effects of stress associated with weaning on the adaptive immune system in pigs. J Anim Sci 90:649–56. Koyama H, Ikeda S, Sugimoto M, Kume S. (2012). Effects of folic acid on the development and oxidative stress of mouse embryos exposed to heat stress. Reprod Domest Anim 47:921–27. Lee AM, Lam SK, Sze Mun Lau SM, Chong CS, Chui HW, Fong DY. (2007). Prevalence, course, and risk factors for antenatal anxiety and depression. Obstet Gynecol 110:1102–12. Lee YE, Byun SK, Shin S, Jang JY, Choi BI, Park D, Jeon JH, et al. (2008). Effect of maternal restraint stress on fetal development of ICR mice. Exp Anim 57:19–25. Li J, Wang Z, Cao J, Dong Y, Chen Y. (2013). Melatonin receptor subtypes Mel1a and Mel1c but not Mel1b are associated with monochromatic light-induced B-lymphocyte proliferation in broilers. Domest Anim Endocrinol 45:206–15. Mairesse J, Lesage J, Breton C, Bre´ant B, Hahn T, Darnaude´ry M, Dickson SL, et al. (2007). Maternal stress alters endocrine function of the feto-placental unit in rats. Am J Physiol Endocrinol Metab 292: 1526–33. Mitra A. (2008). Diabetes and stress: a review. Ethno-Med 2:131–5. Mittelstadt PR, Ashwell JD. (2001). Inhibition of AP-1 by the glucocorticoid-inducible protein GILZ. J Biol Chem 276:29603–10. Nepomnaschy PA, Welch KB, McConnell DS, Low BS, Strassmann BI, England BG. (2006). Cortisol levels and very early pregnancy loss in humans. Proc Natl Acad Sci USA 103:3938–42. Ortolani D, Garcia MC, Melo-Thomas L, Spadari-Bratfisch RC. (2014). Stress-induced endocrine response and anxiety: the effects of comfort food in rats. Stress 17:211–18. Padgett DA, Glaser R. (2003). How stress influences the immune response. Trends Immunol 24:444–8. Pallare´s ME, Adrover E, Baier CJ, Bourguignon NS, Monteleone MC, Brocco MA, Gonza´lez-Calvar SI, Antonelli MC. (2013). Prenatal maternal restraint stress exposure alters the reproductive hormone profile and testis development of the rat male offspring. Stress 16: 429–40. Pare´ WP, Glavin GB. (1986). Restraint stress in biomedical research: a review. Neurosci Biobehav Rev 10:339–70. Paris JJ, Brunton PJ, Russell JA, Frye CA. (2011). Immune stress in late pregnant rats decreases length of gestation and fecundity, and alters later cognitive and affective behaviour of surviving pre-adolescent offspring. Stress 14:652–64.
Restraint stress and embryo implantation
503
Rahman A, Iqbal Z, Harrington R. (2003). Life events, social support and depression in childbirth: perspectives from a rural community in the developing world. Psychol Med 33:1161–7. Robertson SA. (2010). Immune regulation of conception and embryo implantation-all about quality control? J Reprod Immunol 85:51–7. Segerstrom SC, Miller GE. (2004). Psychological stress and the human immune system: a meta-analytic study of 30 years of inquiry. Psychol Bull 130:601–30. Sen S, Chakraborty R. (2011). The role of antioxidants in human health. In: Andreescu S, Hepel M, editors. Oxidative stress: diagnostics, prevention, and therapy. ACS symposium series. Washington, DC: American Chemical Society, p. 1–37 (Chapter 1). Schuerwegh AJ, De Clerck LS, Bridts CH, Stevens WJ. (2001). Wasp venom immunotherapy induces a shift from IL-4-producing towards interferon-gamma-producing CD4+ and CD8+ T lymphocytes. Clin Exp Allergy 31:740–6. Shahin S, Singh VP, Shukla RK, Dhawan A, Gangwar RK, Singh SP, Chaturvedi CM. (2013). 2.45 GHz microwave irradiation-induced oxidative stress affects implantation or pregnancy in mice, Mus musculus. Appl Biochem Biotechnol 169:1727–51. Silberman DM, Wald M, Genaro AM. (2002). Effects of chronic mild stress on lymphocyte proliferative response. Participation of serum thyroid hormones and corticosterone. Int Immunopharmacol 2: 487–97. Stefanski V, Raabe C, Schulte M. (2005). Pregnancy and social stress in female rats: influences on blood leukocytes and corticosterone concentrations. J Neuroimmunol 162:81–8. Tayade C, Hilchie D, He H, Fang Y, Moons L, Carmeliet P, Foster RA, Croy BA. (2007). Genetic deletion of placenta growth factor in mice alters uterine NK cells. J Immunol 178:4267–75. Toumi ML, Merzoug S, Baudin B, Tahraoui A. (2013). Quercetin alleviates predator stress-induced anxiety-like and brain oxidative signs in pregnant rats and immune count disturbance in their offspring. Pharmacol Biochem Behav 107:1–10. Wiebold JL, Stanfield PH, Becker WC, Hillers JK. (1986). The effect of restraint stress in early pregnancy in mice. J Reprod Fert 78: 185–92. Wongweragiat S, Searle RF, Bulmer JN. (1999). Decidual T lymphocyte activation in hydatidiform mole. J Clin Pathol 52:888–94. Zafir A, Banu N. (2009). Modulation of in vivo oxidative status by exogenous corticosterone and restraint stress in rats. Stress 12:167–77. Zhang SY, Wang ZJ, Li JJ, Wei DL, Sui SH, Zhang ZH, Zhou P, Tan JH. (2011). Maternal restraint stress diminishes the developmental potential of oocytes. Biol Reprod 84:672–81.
