Effects Of Hypoxia On The Circadian Clock And Endoplasmic Reticulum Stress Signaling Pathways In Goat Trophoblast Cells

Trophoblast cells play a crucial role in embryo implantation and pregnancy establishment.Accumulating evidence have shown that physiological or pathological hypoxia affects various physiological functions of trophoblast cells.However,the regulatory mechanism of hypoxia on the physiological functions of trophoblast cells remains largely elusive.In this study,we hypothesized that hypoxia regulates physiological function in goat trophoblastic cells?GTC?by affecting the circadian clock and endoplasmic reticulum stress signaling pathways.CoCl₂ and dimethyloxalylglycine?DMOG?were selected to establish hypoxia models of GTC.Real time PCR?qPCR?,Real time luminometer system?Kronos Dio AB-2550?,and Western blotting methods were utilized to detect the effects of hypoxia on the circadian clock,endoplasmic reticulum?ER?stress and key genes in steroid hormones synthesis of GTC.The results are as follows:1.Cell viability at different time points?12 h,24 h,and 36 h?of GTC treated with different concentrations?0?M,100?M,200?M,and 400?M?of CoCl₂ was detected by the CCK-8 kit.200?M of CoCl₂ is the largest concentration of GTC without significant cytotoxicity.According to reports in the literature and verified that 500?M DMOG is an appropriate concentration for inducing GTC hypoxia.After synchronizing GTC with 100 nM dexamethasone,GTC were treated with or without 200?M CoCl₂ or 500?M DMOG.The expression of target genes of HIF1?protein?Glut1 and Vegf??,was measured by qPCR at different time points?12 h,16 h,20 h,24 h,28 h,and 32 h?.The results showed that the expression of Glut1 in GTC treated with CoCl₂was significantly higher than that in the control group?P<0.001?,but there was no significant difference in the expression of Vegf?compared with the control group?P>0.05?.The expression of Glut1 and Vegf?in GTC treated with DMOG increased significantly?P<0.001?compared with the DMSO group.The above results indicated that the hypoxia model of GTC has been successfully established.2.Lentivirus system was used to transfect the recombinant lentiviral vector pLV6-Bmal1-Luc into GTC cells.The cells were then treated with 100 nM DXM for 2 h and divided into 2 groups.One group was treated with 500?M DMOG and the other group was treated with the same volume of DMSO.Luciferase?Luc?activity was chrononically measured using the Kronos Dio AB-2550 system.The results showed that there were robust Bmal1-Luc circadian oscillations in GTC in the presence of DMOG or not.Interestingly,DMOG treatment decreased the amplitude and lengthened the period of Bmal1-Luc circadian oscillations in comparison with DMSO group?P<0.001?.The above results indicated that hypoxia has a significant effect on the circadian clock system of GTC.3.After synchronization with 100 nM dexamethasone,GTC was treated with 200?M CoCl₂ or 500?M DMOG.qPCR was untilized to detect the core clock genes transcriptions at different time points?12 h,16 h,20 h,24 h,28 h and 32 h?.Cosinor analysis was used to detect the rhythmicity of clock genes expression profiles,and Two-way ANOVA was adopoted to analysis the treatment factor on clock gens expression levels.The results showed that:?1?Under normal condition,the core circadian clock genes?Bmal1,Cry2,Dbp,and Per1?behaved a clear rhythmic expression pattern in GTC?P<0.05?.?2?After treatment with 200?M CoCl₂,the expression of Bmal1 and Cry2 was significantly decreased?P<0.05?,the expression of Per1 was significantly increased?P<0.001?,and the expression of Dbp was not alerted?P>0.05?;The rhythm expression of Dbp and Cry2 disappeared?P>0.05?,Bmal1 and Per1 still maintained the rhythmic expression pattern?P<0.05?.?3?After treatment with 500?M DMOG,the expression of Bmal1was significantly decreased?P<0.001?,the expression of Per1 was significantly increased?P<0.001?,but the expression of Dbp and Cry2 was not changed significantly?P>0.05?;Bmal1,Dbp,Per1,and Cry2 still exhibited rhythmic expression patterns in GTC with DMOG treatment?P<0.001?.Threse results further indicated that hypoxia has a significant effect on the circadian clock system of GTC.The mechanism of the hypoxia induced by CoCl₂ and DMOG may be different.Thus the changes caused by CoCl₂ and DMOG are somewhat different.4.Western blotting was used to detect the expression of endoplasmic reticulum stress signaling pathway-related proteins at 12 h and 32 h after GTC treatment with 200?M CoCl₂.The results showed that GTC hypoxia induced by CoCl₂ significantly alerted the signal pathway of endoplasmic reticulum stress with the increase of GRP78 and ATF6 protein and the decrease of phospho IRE1a protein?P<0.05?.5.q PCR was used to further detect the expression of key genes of steroid hormones synthesis at different time points after treatment with 200?M CoCl₂ and 500?M DMOG.The results showed that the expression of Star is significantly decreased?P<0.001?after CoCl₂-induced GTC hypoxia.Consistently,the expression of Hsd3?is significantly decreased?P<0.001?but Star is not alerted?P>0.05?after DMOG treatment,suggesting that hypoxia stimulation may have an important effect on the synthesis of steroid hormones in GTC.In summary,CoCl₂ and DMOG can induce hypoxia in GTC.Hypoxia can cause changes in the expression and rhythm of core circadian clock genes,and changes in expression of Proteins related to Endoplasmic Reticulum Stress Signaling Pathway and genes related to Steroid Hormone Synthesis in GTC.