The Role And Machanism Of BCL2L15 In Regulating Goat Endometrial Receptivity

The loss of embryos in early pregnancy is one of the main problems that plague the efficiency of cattle and goat breeding in our country.In ruminants,the receptivity of the endometrium is essential for the establishment of pregnancy.Although a variety of molecules have been found to be involved in the regulation of endometrial receptivity,the regulation mechanism of goat endometrial receptivity is still unclear.This study explored the functional role and signal transduction process of BCL2L15 in regulating goat endometrial receptivity,and provided a theoretical basis for the treatment of early pregnancy embryo loss in ruminants and the improvement of livestock reproductive rate.(1)In this study,the goat endometrial epithelial cells(EECs)were treated by progesterone(P4),estradiol(E2)and interferon tau(IFNT)to simulate the hormone environment in the endometrial receptive phase in vivo.And then the expression of BCL2L15 was detected by q PCR and Western blot.The results showed that compared with the group which was treated without hormones and IFNT,the BCL2L15 gene and protein expression in the EECs treated with P4,E2 and IFNT were significantly increased.This result indicates that BCL2L15 is related to the regulation of goat endometrial receptivity.(2)In this study,the sh BCL2L15 lentiviral interference vector was successfully constructed.After the packaged lentivirus was infected with EECs,the BCL2L15 knockdown cell line was obtained whose interference efficiency was detected by fluorescence microscope,q PCR and Western blot.The endometrial receptivity of BCL2L15 knocked down cell line was detected in which the proliferation rate was detected by CCK8 and Ed U,the pregnancy elongation factor(ISG15,CXCL10 and RSAD2),key marker molecules of endometrial receptivity(HOXA10,HOXA11 and LIF),hormone and interferon homologous receptors(ESR1,PGR,IFNAR1 and IFNAR2)and cell adhesionrelated genes(SPP1,ITGB1,ITGB3 and ITGB5)were detected by q PCR,Western blot and immunofluorescence,the adhesion was tested by cell co-culture,the number of microvilli on the surface of EECs was observed by scanning electron microscope.The results showed that,compared with the control group,the proliferation rate of the BCL2L15 knockdown cell line was significantly increased,the pregnancy elongation factor,the key marker molecule of endometrial receptivity,the hormone and interferon homologous receptor and the cell adhesion-related genes were significantly decreased,the adhesion of EECs was decreased and the number of microvilli on the cell surface was increased significantly.Those results indicate that knocking down BCL2L15 will damage the endometrial epithelial cell receptivity,so BCL2L15 is necessary for regulating goat endometrial receptivity.(3)In this study,the expression of p-STAT1 and p-STAT3 was detected by Western blot,which showed that the expression of p-STAT1 and p-STAT3 were increased significantly in the BCL2L15 knockdown cell line.This result indicated that knocking down BCL2L15 gene will activate STAT1 and STAT3 pathways.After that,the EECs were pretreated with STAT1 inhibitor Fludarabine and STAT3 inhibitor Stattic,respectively,and the receptivity was evaluated by the detection method described in(2).The results showed that,compared with the control group,in the BCL2L15 knockdown cell line group pretreated with Fludarabine,the cell proliferation rate was significantly reduced,the pregnancy elongation factor,the key marker molecules of endometrial receptivity,hormones and interferon homologous receptors and the genes related to cell adhesion were increased significantly,the adhesion of EECs was increased,and the number of microvilli on the cell surface was decreased significantly,while the Stattic pretreatment group did not change significantly compared with the control group.This result shows that the STAT1 inhibitor Fludarabine but the STAT3 inhibitor Stattic can restore the endometrial receptivity damage in the BCL2L15 knockdown cell line.Therefore,BCL2L15 regulates goat endometrial receptivity through the STAT1 signaling pathway.The above results indicate that BCL2L15 is involved in the regulation of goat endometrial receptivity and plays a role through the STAT1 signaling pathway.Clarifying the function of the BCL2L15-STAT1 signaling pathway in endometrial receptivity can help increase the pregnancy rate and reproduction rate of livestock,and provide new ideas for early diagnosis and treatment of pregnancy failure and embryo loss.