| Objective: The placenta is a temporary organ for communication between the pregnant body and the fetus.Placental hypogenesis or dysfunction leads to severe pregnancy-related diseases,resulting in significant economic losses to the dairy industry.Lacking of placental types differs from humans and rodents and functional cow placental organoid models,the mechanisms of dairy cows placental formation,development,and functional regulation,as well as their molecular associations with pregnancy complications are far from elucidated.There is also a lack of scientific and effective technical strategies for the prediction and intervention of related pregnancy diseases.The purpose of this experiment is to construct the trophoblast organoids of dairy cow placenta cultured in vitro and provide a good organoid model for the in vitro study of pregnancy diseases related to dairy cow placenta development.Methods: Firstly,H ＆ E staining technique was used to observe the histological characteristics of bovine placental trophoblast in early pregnancy;Immunohistochemical staining was used to compare the localization and expression of Ki67 in placental trophoblast cells of pregnancy at 2,3 and 4 months;The primary trophoblast cells were cultured and identified in TOM medium.Then,primary cells and BTCs were put into matrigel to generate organoids;Immunofluorescence technique was used to detect the secretion of placental prolactin（CSH1）and syncytial markers CD46 and CD71;RNA sequencing was used to screen organogenesis regulation genes.The trophoblast organoids constructed in vitro were co-cultured with endometrial epithelial cells（EECs）.The expression changes of uterine receptivity related factors in EECs before and after co culture were detected and analyzed by Western blot.Results:（1）The results of H ＆ E staining showed that the trophoblast of bovine placenta in early pregnancy contained monocytes and binucleate cells,and the two cells grew alternately without stratification boundary.（2）Immunohistochemical results showed that the proportion of proliferating cells in the trophoblast of the placenta in the third trimester was the highest compared with other placenta in the early trimester,so the placenta in third trimester was taken for follow-up test.（3）The primary trophoblast cells cultured in TOM medium grew as cell colonies,similar to trophoblast progenitor cells.（4）Immunofluorescence staining showed that the cultured trophoblastic organs had the function of CSH1 secretion and expressed syncytial marker factor.（5）RNA sequencing analysis showed that organogenesis regulation gene cluster was associated with placental development regulation at third months of gestation.（6）Western blot results showed that the protein expressions of receptivity regulators Integrin αV β3,Wnt7 a,HOXA10,IFNAR-1 and IFNAR-2 in EECs were significantly increased after co-culture with trophoblastic organs（p<0.05）.Conclusion:（1）The proportion of proliferating cells in bovine placenta is high in third months of gestation,which is suitable for the culture of primary trophoblast and the construction of trophoblast organoids in vitro.（2）Bovine placental trophoblast organoids have successfully generated with primary placental trophoblast cells and BTCs in Matrigel and TOM medium,those have the functions of secreting binucleate secreted proteins and regulating uterine receptivity.Our research provides an in vitro trophoblastic organs model for the further study of bovine placental development and pregnancy in the future. |
