| Placenta is an important factor affecting embryo development and growth during goat pregnancy.It will be caused miscarriage,fetal dysplasia,developmental delay and many other problems by abnormal placental development in the early pregnancy.However,the mechanism of regulating placental development in early pregnancy is very complex.Ten-eleven translocation?TET?family proteins play a key role in regulating the development of embryo and placenta,and the development of gametes and early embryos will be regulated by the balance of cell death in oviduct and endometrial.Whether TETs are involved in the vital activity of cells in oviduct or endometrium during early embryonic development is unclear.Research has shown that exosomes exists in all kinds of body fluids,containing a variety of proteins,lipids,nucleic acids and other biological ingredients,which can be used as vector transferring information between cells and tissues,and then regulating the function of target cells or tissues.At present,there are few studies on the separation,identification and function of placental exosomes in goats.In conclusion,we choose goats and take research as follows.Firstly,we examined the expression patterns of TETs and genes related programming cell death and inflammation in the oviduct and uterine horn of goat between 19d?C19?and 19d?P19?,and analysed the correlation between TETs and programmed cell death genes.Secondly,the exosomes of uterine fluid in group C19 and group P19 were extracted and identified,and the effect of goat uterine fluid on the proliferation of placental trophoblast cells was studied.Finally,we extracted,identified goat plasma exosomes and sequenced mi RNA from non-pregnant?NP?and pregnant 30?P30?groups.Then we predicted target genes of miRNAs and analyzed their function,which screen out miRNAs from goat plasma exosome that regulating placental trophoblastic cells.The research results are as follows:1.There is correlation between TETs and programmed cell death relative genes of oviduct and uterine horn in early pregnancy goats.After gene expression and correlation analysis between C19 group and P19 group,we found that TET2?P < 0.01?,NF-kB?P < 0.01?,Caspase1?P < 0.01?,GSDMD?P < 0.05?which expressing in goats' oviduct were significantly increased in P19 group.The correlation analysis showed that TET2 and TET3?P < 0.01?,NF-kB?P < 0.01?,Caspase9?P < 0.05?were significantly positive correlation in the oviduct.Goat uterine horn glands in P19 group increased and varied in morphology.OPN is the key genes for embryo implantation and its expression and BAX/BCL2 expression were increased significantly?P < 0.05?.The correlation analysis showed that OPN was positively correlated with cell death related genes Caspase1?P < 0.01?,NLRP3?P < 0.05?and GSDMD which is the key gene of cell death,?P < 0.05?.However,GSDMD was positively correlated with Bax?P < 0.05?,Caspase1?P < 0.01?and NLRP3?P < 0.05?in oviduct and uterine horn.2.The effects of goat uterine fluid exosomes on proliferation of placental trophoblast cells.Through the detection of exosome proteins in uterine fluid and uterine fluid,it was found that the total protein content of exosomes in P19 group was higher than that in C19 group.According to a study that effect of the uterine fluid and its exosome on placental trophoblast cells proliferation,we found that both C19 group and P19 group goats' uterine fluid could significantly improve the expression of PCNA,which is the marker gene of placental trophoblast cells proliferation?P < 0.05?,Exosomes of uterine fluid were absorbed by placental trophoblast cells,and PCNA expression was improved only in P19 group?P < 0.01?.3.Identification of plasma exosome miRNAs in early pregnancy goats and its effect on the proliferation of placental trophoblastic cells.?1?Compared with NP group,goat plasma exosomes concentrations in P30 group was higher,which were determined by concentration test.However,the proliferation of placental trophoblast cells in P30 group were inhibited?P < 0.05?.?2?Six small RNA libraries of goat plasma exosomes in NP group and P30 group were constructed,and 217 differential miRNAs were detected by exosome miRNA sequencing,which 116 miRNAs were up-regulated and 101 miRNAs were down-regulated.Results showed that mdo-mir-31-5p,ssa-mir-137-3p,novelmiR1355,novelmiR734,and novelmiR736 were only specifically expressed in the P30 group,demonstrating a certain role in early pregnancy and possibly serving as a marker for the diagnosis of early pregnancy.?3?We predicted target genes of miRNA and analyzed their function that the target genes were significantly enriched into Notch signaling pathway,Wnt signaling pathway and Hippo signaling pathway.All in all,we got the following conclusions from the whole study:?1?TETs involved in regulating apoptosis and inflammatory-related genes expression in oviduct during early pregnancy,and there was a synergistic effect on the development of the oviduct and uterus between cell apoptosis and proptosis.In addition,the key genes for embryo implantation were significantly correlated with pyroptosis related genes which suggesting that proptosis has a certain influence on the attachment of early embryos.?2?We isolated and identified exosomes of uterine fluid and plasma on early pregnancy goats and figured out the effect of goat exosomes of uterine fluid and plasma on the proliferation of placental trophoblast cells.?3?We found out differential miRNAs in the plasma exosomes of goats.The target genes projections and function were analyzed,and identified some potential miRNAs which regulat early pregnancy in goat plasma exosomes... |
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