| Heat stress is one of the most common stressors that severely affects the livestock productivity.The impacts of heat stress on animal reproductive functions have received wide attention in recent years.Studies have revealed that heat stress affects hormone secretion,germ cell proliferation and embryonic development processes,thereby reducing the reproductive performance of sows.Little is known about the effects of heat stress on the porcine endometrial epithelial during porcine pre-implantation,because most current studies have focused on ovarian function and embryonic development in pigs.Studies have demonstrated that heat stress cause hypertonic stress through altering cell integrity,leading to changes in the intracellular environment.The changes of the in uterine environment may influence the development and health of offspring during embryo implantation.Autophagy is considered an adaptive mechanism resistant to heat stress and hypertonic stress.Therefore,in this study,we investigated the effects of heat stress and hypertonic stress-induced autophagy in porcine endometrial epithelial cells on early embryo implantation and its regulatory mechanism through useing histomorphological assay,real-time fluorescence quantitative detection,western blot detection,signaling pathway assay and laser confocal microscopy observation,and thus provides theoretical support for the porcine rearing environment in early pregnancy to fully enhance the reproductive potential of sows.The main findings are as follows.1.Effect of heat stress on uterine and gene expression profiles during porcine pre-implantationThis experiment demonstrated the effect of heat stress on the endometrial tissue during porcine pre-implantation.Six primiparous sows were randomly divided into the control and heat stress groups.Heat treatment was started on day 11 of the critical period of embryo implantation,and the heat treatment lasted 4 days.Uterine tissues were collected on day 14 to evaluate cross sections for tissue damage and to detect the expression of tight junction protein and autophagy-related protein.(1)H&E staining results showed that the porcine endometrial epithelial thickness in the heat-stressed group(21.0613 ± 3.1871 mm)was extremely significantly lower than that of the control group(38.0225 ± 5.0119 mm)(P < 0.001).Transmission electron microscopy revealed that microvilli of porcine endometrial epithelial cells were unevenly distributed,adhered,inverted or absent,and formed holes by cell depression and cell membrane rupture under heat stress.(2)Western blot results showed that the expression of N-cadherin and E-cadherin in the endometrial tissue of the heat-stressed group was significantly lower than that of the control group(P < 0.05),and the expression of Rho A and ROCK1 was significantly lower than that of the control group(P < 0.05).(3)Western blot results showed that the expression of autophagy-related P62 protein was significantly reduced in the endometrial tissue of pigs under heat stress(P < 0.05);meanwhile,the phosphorylation levels of mTOR signaling pathway and its downstream effectors S6K1 and 4EBP1 were significantly reduced(P < 0.05).(4)RNA-Seq results showed that 338 genes were up-regulated and 378 genes were down-regulated in the heat-stressed group.The differentially up-regulated genes were mainly related to the molecular functions of important functions such as immune system processes,matrix metalloproteinase activity,and aminoacyl-t RNA ligase activity.The differentially down-regulated genes were associated with molecular functions that are important for calcium binding,extracellular domain,molecular sensor activity,signal sensor activity,and transmembrane signaling receptor activity.2.Effect of heat stress on porcine endometrial epithelial cellsTo investigate the regulatory mechanism of heat stress-induced endometrial damage in pigs,a heat stress model of porcine endometrial epithelial cells was constructed in this experiment.The porcine endometrial epithelial cells in logarithmic growth phase were transferred to a high temperature incubator(42 ℃)for different times(0 h,1 h,2 h and 4h)to induce heat stress response.(1)Significant changes in cell morphology and cell activity were observed in heat stress-treated porcine endometrial epithelial cells at 0 h,1 h,2 h and 4 h.At 0 h of heat stress,there was no significant difference in OD values between the two groups(P >0.05);at 1 h of heat treatment,the viability of porcine endometrial epithelial cells was significantly reduced(P < 0.001).(2)RT-q PCR was performed to detect the mRNA expression of heat shock proteins,and the results showed that the mRNA levels of HSP90,HSP27,HSPH1 and HSPA4 L were significantly increase in the heat stress group than in the control group,and their expression gradually increased with the extension of heat stress time in a time-dependent manner.The expression level of HSP70 mRNA increased and then decreased,and reached the peak at 1 h of heat treatment.(3)RT-q PCR was performed to detect the mRNA expression of embryo implantation-related genes,and the results showed that with the prolongation of heat treatment time,the MUC1 mRNA levels in the heat treatment group were lower than those in the control group;the expression of HBEGF,GNRHR,DKK1,OPN and FN1 first increased and then decreased.(4)The expression of BAX increased and then decreased after heat stress treatment of porcine endometrial epithelial cells,and the expression of BCL2 gene did not change at 1h and 2 h of heat treatment,and reached a peak at 4 h.(5)The cellular ROS and MMP levels were detected after heat stress treatment of porcine endometrial epithelial cells.The results showed that the ROS level in porcine endometrial epithelial cells first increased and then decreased,reaching a peak at 1 h,and both were higher than control group.The MMP showed a trend of first decreasing,then increasing and then decreasing,and reached the lowest value after 1 h of heat treatment.(6)The results showed that the expression of P62 in endometrial epithelial cells in the heat treatment group was significantly lower than that in the control group(P < 0.05);the expression of LC3II/LC3 I protein in the heat treatment group was significantly lower than that in the control group(P < 0.05);meanwhile,the expression of GRP78 protein in the heat stress group was significantly higher than that in the control group(P < 0.05).3.Effect of hyperosmotic stress the histomorphology of mouse endometrial tissue and the function of embryo implantationIn this experiment,a mouse embryo implantation hyperosmotic stress model was constructed using Na Cl.On the third day of pregnancy,Na Cl(0.5 M,1 M and 2 M)was injected intraperitoneally for 5 consecutive days to study the effects of hyperosmotic stress on the uterine morphology and embryo implantation function in mice.(1)After hyperosmotic stress,the changes in body weight of mice in the 2 M-treated group were extremely significantly higher than those in the control group(P < 0.001),while there was no significant difference between the 0.5 M and 1 M groups(P > 0.05).Compared with the control group,the embryo morphology changed significantly with the increase of Na Cl concentration,and the embryo implantation sites on both sides of the uterus were not evenly spaced;there was no significant difference in the wet weight of the uterus of mice in the 0.5 M and 1 M groups,while the wet weight of the 2 M group was significantly lower(P < 0.05).(2)H&E staining showed that the uterine cavity was loose in the 1 M group compared with the control group,while the uterine cavity was open and irregular in the 2M group,with incomplete development of the luminal epithelium and fewer glands.4.Effect of hyperosmotic stress on porcine endometrial epithelial cellsIn this experiment,a hyperosmotic stress model of porcine endometrial epithelial cells was constructed using Na Cl to study the effects of hyperosmotic stress on the proliferation,autophagy and integrity of porcine endometrial epithelial cells.The osmolarity was adjusted to 500 m Osmol/kg by adding Na Cl to the culture medium and the incubation time was 15 min,0.5 h,1 h,3 h and 6 h.(1)LC3II/LC3 I protein expression peaked at 1 h and 3 h of treatment for 15 min,0.5h,1 h,3 h and 6 h in hyperosmotic treated porcine endometrial epithelial cells.Meanwhile,CCK8 results showed that cell viability was significantly reduced at 3 h and 6h(P < 0.001)in hyperosmotic treated porcine endometrial epithelial cells.(2)Western blot results showed that the expression of β-catenin,N-cadhrein and E-cadherin was highly significantly reduced in the hyperosmotic treatment group compared with the control group(P < 0.01),while there was no significant difference in the expression of ZO-1(P > 0.05).The expression of ROCK1 and ROCK2 was significantly lower in the hyperosmotic stress group compared with the control group(P <0.01).Immunofluorescence results showed that the ZO-1 cell membrane staining was significantly disorganized and discontinuous in the hyperosmotic stress group,and there was also significant staining in the cytoplasm;the expression of E-cadherin was significantly reduced.Confocal results showed that the hyperosmotic treatment for 1 h showed rounding and cytoskeletal collapse of porcine endometrial epithelial cells as well as disrupted distribution pattern of F-ACTIN.(3)Western blot results showed that the expression of LC3II/LC3 I and GRP78 was significantly increased(P < 0.05),and the expression of P62,p-mTOR,p-S6K1 and p-4EBP1 was significantly decreased(P < 0.01)in the hyperosmotic stress group compared with the control group.(4)The RNA-Seq results showed that 148 up-regulated genes and 163down-regulated genes were screened in the hyperosmotic stress group.The biological processes of the differential genes were mainly involved in "stress response pathway","bio adhesion","cell component organization or synthesis","biological regulation","regulation of biological processes","reproductive processes","cellular processes","cell proliferation ","metabolism",etc.5.Functional study of differentially expressed gene S100A9(1)The expression of embryo implantation-related genes S100A8 and S100A12 was significantly down-regulated(P < 0.05)in porcine endometrial epithelial cells after S100A9 knockdown,which was not conductive to embryo implantation.(2)Significant decrease in G1 and G2 phase cells and significant increase in S phase cells after overexpression of S100A9 gene in porcine endometrial epithelial cells(P <0.05).(3)The percentage of total apoptotic cells was significantly decreased after overexpression of S100A9 gene in porcine endometrial epithelial cells,while the percentage of total apoptotic cells was significantly increased after S100A9 knockdown. |
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