Effect Of β₂-AR-ERK Pathway On Restraint Stress Induced Endometrial Regeneration Of Mice During Embryo Implantation

The rate of early embryo loss reached approximately40%in the sows, which was related to the intensive rearing pattern and the gestational cages (restricted motion). However, it was not fully clear that the mechanism of restraint stress from the gestational cages on the early embryo loss. Our previous studies demonstrated that sympathetic nerve participated in the process of embryo implantation in pregnant mice. Therefore, according to perspective of sympathetic-aderenal system, this study focused on the mechanism of restraint stress on the implantation of embryo during the early pregnant mice. In this study, the pregnant CD1mice (the appearance of vaginal plug was considered to be at embryonic day1) were divided into the control and restraint stressed group. The latter was subjected to restraint stress (4h/day) on embryonic day1(E1) and till being sacrificed on E3, E5and E7. The plasma and uterine tissue were prepared to be studied. Results as followed:1. The effect of restraint stress on the number of implantation sites and the index of sexual organs during embryo implantationE1pregnant mice was subjected to3d,5d and7d restraint stress (4h/d), and the concentration of plasma corticosterone (CORT), noradrenaline (NE) and blood glucose in maternal mice (E3-E7) were significantly increased by69.00%-88.27%(P=0.000-0.019),11.03%-39.49%(P=0.002-0.049) and7.93%-26.97%(P=0.000-0.032) respectively, compared with the corresponding control mice. Therefore, those results demonstrated that the animal model of restraint stress was valid. Restraint stress decreased the number of embryo implantation sites by15.29%(E5, P=0.346) and26.07%(E7, P=0.003), and decreased the index of sexual organs (uterus and ovary) by16.15%(E5, P=0.004) and19.97%(E7, P=0.019), compared with the corresponding control mice.2. The effect of restraint stress on the endometrial regeneration of pregnant mice during embryo implantationThe endometrial receptivity was related to the endometrial development. In this study, restraint stress decreased the ratio of endometrial area (endometrial area/uterine area) by8.94%-18.80%(E3-E7, P=0.000-0.021) and decreased the ratio of uterine gland area by30.63%(E3, P=0.000) and44.52%(E5, P=0.000). Restraint stress decreased the expression of MMP-9(regulating the endometrial regeneration and the development of blood vessel) by24.67%-63.45%(E3-E7, P=0.000-0.028). The mean optical density (MOD) of VEGF-positive cells was significantly lower14.55%-45.87%(E3-E7, P=0.000-0.018) and CD34-positive cells was lower37.16%(E5, P=0.003) and70.54%(E7, P=0.000) than the corresponding control mice. The proliferation by PCNA immunochemistry and apoptosis by TUNEL of endometrial cells were evaluated. It showed that MODPCNA/MODTUNEL was decreased by32.37%-39.75%(E3-E7, P=0.002-0.011) in the stressed mice. Caspase-3as an apoptosis molecule, was significantly higher30.10%-44.69%(E3-E7, P=0.001-0.019) than the corresponding control mice. Thus, it demonstrated that restraint stress induced the apoptosis of endometrial cells, inhibited the development of endomentrium and blood vessel then resulted in the decrease of endometrial receptivity, which ultimately decreased the number of implantation sites. 3. The effect of restraint stress on the uterine immune activity of pregnant mice during embryo implantationThe density of uNK cells in the endometrium of stressed mice decreased by22.14%～47.90%(E3-E7, P=0.000) but the density of mast cell (MC) in the myometrium of stressed mice increased by55.65%-76.91%(E3-E7, P=0.000～0.002), compared with the corresponding control mice. Restraint stress decreased the proliferation of T lymphocytes by5.61%-7.30%(E3-E7, P=0.002～0.022), and decreased the proliferation of B lymphocytes by17.58%(E5, P=0.002),8.28%(E7, P=0.017). It found that restraint stress decreased CD3+CD4+T/CD3+CD8+by26.16%-28.91%(E3-E7, P=0.001～0.027) and decreased IL-2/IL-4by7.91%-16.21%(E3-E7, P=0.000). The results indicated that restraint stress disturbed the uterine immune response and led to a Th1/Th2imbalance, which displayed that decreasing the density of uNK, increasing the density of MC, weakening the responses of lymphocytes to mitogens and decreasing CD3CD4+/CD3+CD8+4. The effect of restraint stress on the uterine antioxidant activity of pregnant mice during embryo implantationThe activities of uterine GSH-PX were decreased by32.16%(E5, P=0.011) and45.71%(E7, P=0.000), and T-SOD were decreased by15.52%(E5, P=0.045) and26.13%(E7, P=0.014) in the stressed mice when compared with the corresponding control mice. Thus, the total antioxidant capacity (T-AOC) decresed by18.45%(E5, P=0.000) and18.19%(E7, P=0.001), but malonydialdehyde (MDA) increased by34.37%(E5, P=0.042) and42.96%(E7, P=0.019) in stressed mice. In vitro, endometrial stromal cells (ESCs) being isolated from maternal mice was treated with different concentration of exogenous H2O2at0,100,200,400,800,1000μM. The result found that the proliferative activity of ESCs were reduced by22.74%-61.01%(100-1000μM H2O2, P=0.000) in a dose-dependent manner. Therefore, it indicated that restraint stress induced an imbalance in the uterine oxidative microenvironment and subsequently inhibited the the proliferation of ESCs during the implantation and post-implantation periods of pregnancy.5. The intracellular signal mechanism of restraint stress on the endometrial regeneration of pregnancy mice during embryo implantationPrevious researches indicated the ERK and NF-κB were involved in the signal pathway of apoptosis induced by oxidative stress. In present study, the expression of uterine pERK1/2detected by Western Blot (WB) was significantly increased by38.85%-52.30%(E3-E7, P=0.025-0.049), and the expression of uterine pNF-KBp65was significantly increased by54.38%-74.47%(E3-E7, P=0.001～0.019) in stressed mice, compared with the corresponding control mice. Restraint stress induced the release of NE, which binded β-AR to show its biologic influence. There were three subtypes of P-AR mRNA in the uterus of E3-E7pregnancy mice. Statistically, restraint stress upregulated the expression of uterine β2-AR mRNA, but it had no effect on the expression of β1-AR mRNA and β3-AR mRNA when compared the corresspoding control mice. In addition, it found that the expression of β2-AR was significantly increased by52.30%(E5, P=0.003) and28.77%(E7, P=0.042), compared with the corresponding control mice. It appeared by immunohistochemical staining that β2-AR-positive cells were located in the myometrium, glandular epithelial cells, luminal epithelial cells and decidual cells. The expression of β2-AR in the ESCs separated from uterus of E5pregnant mice was found. Those results indicated that β2-AR played a major role in the restraint stress model of pregnant mice.In vitro, ESCs were treated with exogenous β-AR agonist isoproterenol (ISO). It found that the proliferative activity of ESCs was decreased by38.60%(P=0.000), and increased the expression of Caspase-3by98.98%(P=0.026). In addition, effect of adenylate cyclase agonist-forskolin (FSK) on the proliferation and apoptosis of ESCs was similar to the effect of ISO, which suggested that β-AR induced the activation of intracellular cAMP, then inhibited the proliferation and promoted the apoptosis of ESCs. However, the exogenous β2-AR blocker (Butoxamine) and PKA inhibitor H-89, can significantly reverse the the effect of ISO on the proliferation and apoptosis of ESCs. Adding exogenous MEK inhibitor PD98059and NF-κB inhibitor PDTC, which induced the proliferative activity of ESCs increased by36.31%(P=0.001) and40.80%(P=0.000), respectively, but the expression of Caspase-3was significantly decreased by52.15%(P=0.017) and74.79%(P=0.002), respectively. It found that the trend of the expression of pERK1/2was consistent with that of Caspase-3in different drug group, which indicated that ERK1/2was involved in the apoptosis of ESCs. Therefore, it suggested that restraint stress induced the activation of β2-AR (with the classical cAMP-PKA pathway), and thus induced phosphorylation of ERK1/2and NF-κB, increased the expression of Caspase-3, ultimately inhibited the proliferation and promoted the apoptosis of ESCs.In conclusion, restraint stress actived SAS of pregnant mice, which induced the secretion of stress hormons (CORT and NE). The signal of restraint was mediated by β2-AR, activating intracellular cAMP and downstream PKA signaling, directly or indirectly induced phosphorylation of ERK1/2by oxidative stress. On the one hand, pERK1/2directly induced NF-κB into the nucleus, initiated the transcription of target genes, and induced the production of inflammatory cytokine (IL-2). This process caused a shift of Thl/Th2balance toward Thl-inflammatory status, which led to uterine immune imbalance during embryo implantation. On the other hand, pERK1/2upregulated the expression of Caspase-3, inducing the apoptosis of endometrial cells. It blocked the adaptive regeneration of the endometrium and resulted in the decrease of endometrial receptivity, which was not conducive to embryo implantation and development.