The Expression And Function Of ATF4 On In Vitro-fertilized Embryos And Uterus Of Sheep During Early Pregnancy

The sheep industry is a pillar industry in Xinjiang province,and the reproductive capacity of sheep is a very important project in sheep breeding practice and the production efficiency of sheep breeding.Lambing percentage is one of the most important indexes to judge the reproductive traits in sheep.There is accumulating evidence that ATF4 may be directly involved in mammalian reproduction,especially in embryo implantation.This study was designed to explore the spatio-temproal expression pattern of ATF4 in ovine endometrium,to verify the funcation of ATF4 in cellular level,The role of ATF4 on the endometrial decidualization in sheep.The study provides reliable experimental evidence for the mechanism of ATF4 during embryo implantation.The main contents and conclusions of as follow:Experiment 1.Temporal and spatial expression of ATF4 during implantation and estrus cycle in sheepObjective:the purpose of this study was to investigate changes of ATF4 expression in the endometrium during implantation and to investigeate its role in embryo implantation.Methods:thirty multiparous?3-5 parity?were assigned to five groups?n=6/group?as follows:no-pregnant group?day 9,13,17,21 and 25 of the cycle?,phase of implantation groups?day 9,13,17,21 and 25 of pregnancy?.The mRNA and protein expressions of ATF4 in ovine endometrium during implantation and estrus cycle were compared using RT-PCR and Western blot.Immunohistochemistry was also performed to study the localization of ATF4 protein in ovine endometrium.Results:ATF4 mRNA and protein level went up with the advance of pregnancy and was higher than that of non-pregnant sheep?P<0.05?and gradually increased from Day 9 of pregnancy reaching a maximum level on Day 21 and then declining on Day 25.Localization of ATF4 in ovine uters was in luminal epitheliuml and glandular epithelium in non-pregnant sheep.But distinct immune-staining of ATF4 detected in strom in pregnancy ovine uterus except for luminal epithelium and glandular epithelium.Conclusion:the study showed that ATF4 may plays a certain role during early implantation and it may be involved in deciduation process of endometrial stromal cells in sheep.Experiment 2.Isolation,culture and identification of endometrial stromal cells in sheepObjective:to study the method of extracellular isolation culture of endometrial stromal cells in sheep,and lay the foundation for the functional verification of ATF4 gene in cell level.Methods:select on either side of the ewes uterine horn,aseptic processing operation,surgical separation of endometrial tissue in37?,5%CO₂ generation in primary culture.Endometrial stromal cells were isolated and purified by means of pancreatic enzyme differential digestion.Using wave protein is a specific protein expression of the characteristic of stromal cells,using immunocytochemistry method to the qualitative detection of stromal cells,determined by MTT method is used to draw a sheep endometrial stromal cells in vitro growth curve.Results:endometrial stromal cells of sheep endometrium with normal transmission and growth in vitro were successfully established by tissue block shearing method.Conclusion:after separation,culture and identification,the matrix cell in vitro model was successfully established,which laid the foundation for carrying out related functional verification.Experiment 3.The role of ATF4 on the endometrial decidualization on in sheep?1?Objective:to establish an ATF4 expression and interference system to lay a foundation for the study of ATF4 gene.Methods:to clone ATF4 cDNA full-length sequence and build pcDNA3.1?+?-atf4 eukaryotic expression vector;to design shRNA interference fragment of ATF4 gene,the shRNA expression box was constructed with pgenesil-1 vector,and the interference fragment was cloned into the pgenesil-1 vector,which was named pGenesil-ATshRNA196,pGenesil-ATshRNA 303,pGenesil-ATshRNA 633.The construction of bacterial liquid PCR detection,enzyme cutting,and sequencing of the company was successful.Results:the overexpression plasmid pcDNA3.1?+?-ATF4 and interference vectors of ATF4 gene were successfully constructed.Conclusion:the expression of the successful construction and the optimal interfering plasmid transfection cells were selected to provide an idea for further study of the function of ATF4 gene.?2?Objective:to explore the regulation effect of ATF4 expression and interference vector in cell proliferation,apoptosis and deciduation process.Methods:using the building a successful ATF4 two carrier induced decidual change?with progesterone,estradiol and ring adenosine phosphate inducing?together,decidual cell transfection 72 h,using real-time fluorescent quantitative PCR and protein imprinting technology evaluation after the corresponding plasmid transfection enhance or inhibit ATF4 expression effect,at the same time with Western blot detection prolactin protein changes.Results:the expression of mRNA andproteinindecidualcellswasup-regulatedbyATF4-pcDNA3.1,and pGenesil-ATshRNA303 was able to induce the reduction of mRNA and protein?P<0.05?.Conclusion:overexpression of ATF4 inhibits the process of decidualization of endometrial stromal cells in sheep,while interfering with ATF4 promotes the process of deciduation of the endometrium cells of pregnant sheep.Experiment 4.The molecular mechanism of the process of endometrial stromal cells deciduation in sheepObjective:to find the molecular mechanism of ATF4 gene in the process of deciduation of the endometrium stromal cells in pregnancy.Methods:the expression of ATF3,CHOP,death receptor DR4,DR5,caspase-8 and bcl-2 protein in ATF4-ATF3-CHOP-DR5 pathway were detected after Western blot was detected in sheep endometrial stromal cells induced decidulization.Results:both DR4 and DR5 protein were significantly increased in the induction of endometrial stromal cell decidualization 72 h?P<0.05?.The expression level of caspase-8 protein was increased,and the expression level of bcl-2 protein was significantly decreased?P<0.05?.Conclusion:ATF4-pcDNA3.1 cause apoptosis levels rise,while pGenesil-ATshRNA303 cause apoptosis levels drop,deduced decidual cells ATF4 protein may be through ATF4-ATF3-CHOP-DR5 signaling pathway and death receptor DR5/DR4 lead to procaspase-8 activated combination of caspase-8,the Bcl-2 protein expression cause decidual cell apoptosis through mitochondrial dependent pathways and blocking the endometrial decidualization process in pregnancy sheep.Experiment 5.Quantitative of ATF4 gene in vitro fertilization embryo and nuclear transplantation embryo at different developmental stagesObjective:to explore the expression change law of ATF4 in early in vitro development embryo and provide theoretical basis for further study on the role of ATF4 in embryonic development.Methods:to collect,ovary oocytes collection,in vitro matured respectively for in vitro fertilization?IVF?and nuclear transfer operation,collect 2-4 cell respectively,8-16cell,morula embryo and blastocyst stage embryos,with real-time PCR and indirect immunofluorescence,mRNA and protein localization quantitatively.Results:the quantitative results of RT-PCR showed that 2-4 cell,8-16 cell,morula embryo,with the development of the embryo,the expression of ATF4 gradually increased,and the expression of the blastocyst was reduced to the lowest level.In vitro fertilization embryo and nuclear transplantation embryo,2-4 cell,8-16 cell,the expression quantity of each period of morula was significant?P<0.05?.Conclusion:ATF4 plays an important role in early embryo development.The full text conclusion:this experimental study on the ATF4 in early pregnancy,the uterus expression of sheep,the results showed that ATF4 in pregnant ewes endometrial stromal cells can be clearly expressed,not pregnant ewes express is not obvious.ATF4 also verified in sheep endometrial stromal cells decidual can specifically expressed in the process of estrogen and progesterone positive regulatory role,the expression of ATF4 decidual cell apoptosis and decidual influence,ATF4-ATF3-CHOP-DR5 regulation of signaling pathways is a major pathway,but the caspase-8 protein increased expression and the Bcl-2 protein decreased expression through the regulation of apoptotic receptor DR5,through the mitochondrial depend on the way to induce the apoptosis of decidual stromal cells.The results also show that ATF4 has a certain effect on the development of early embryo.