Mouse PSCs Secrete Activin A To Improve Their EPI Competency In The Host Blastocyst

Mouse pluripotent stem cells(PSCs)have important research and application value,because PSCs not only can provide perfect research models for developmental biology,but also be used for transgenic animal production.At present,the embryo injection is the main way of transgenic mice production through PSCs.According to the stage of host embryos,the embryo injection can be divided into the blastocyst injection,4-cell or 8-cell stage embryo injection,2-cell stage embryo injection and 1-cell stage embryo injection.The 4-cell or 8-cell stage embryo injection and blastocyst injection are the most commonly used ways to generate transgenic mice.Compared with the blastocyst injection,PSCs have a higher contribution in the chimeras,and F0 mice which were completely derived from exogenous PSCs(called PS-mice)can be obtained by the 4-cell or 8-cell stage embryo injection.However,the mechanism of 4 or 8-cell stage embryo injection is not clear and remains to be studied.Studies have shown that PSCs injected into early embryos affect the cell fate of recipient embryos,and secreted proteins are important components in the cell-cell regulation.Therefore,this study explored the effects of PSCs secretory proteins on the development of mouse early embryos.In order to reduce the influence of the compaction of 8-cell stage embryos,this study mainly focused on the 4-cell stage embryo.First,through 4-cell embryo injection,we confirmed that exogenous PSCs injected into host embryos could hinder the development of Epiblast(EPI),while exogenous PSCs instead of host embryos developed into EPI and further developed into the fetus.Second,the PSCs-conditional medium was used to treat 4-cell stage embryos.It is found that the conditional medium of PSCs can effectively obstruct the EPI development,indicating that PSCs can regulate the early embryo development fate by secreting certain proteins.To further clarify effective components that impede the development of EPI cells in PSCs-conditional medium,we used mass spectrometry to detect components of PSCs-conditional medium,and obtained candidate proteins by analysis.Through screening,we found that Activin A is an important effective component of PSCs secretory proteins.To explore effects of Activin A on early embryo development,4-cell embryos were cultured in the medium containing Activin A.When these embryos developed into blastocysts,the marker genes and proteins of EPI,TE and PE were detected by immunofluorescence staining and transcriptome sequencing.These results showed that Activin A could impede the EPI development and promote the development of TE.In addition,we analyzed the expression pattern of main receptors(such as ALK4)of Activin A in mouse pre-implantation embryos.The data showed that these receptors were expressed in embryos at various stages,but the expression of receptors in blastocysts was lower than that in other stage embryos.Then we detected the influence of Activin A on the contribution of ESCs to chimeras.The results showed that compared with the control group,ESCs injected into Activin A-treated embryos were not only easier to contribute into EPI cells of E4.5 embryos,but also had a higher contribution to E10.5 fetuses.After reconstructed embryos produced by 4-cell stage embryo injection were treated by Activin A,we found that the ESCs could more easily contribute to EPI cells of E4.5 embryos.These results suggest that Activin A can enhance the contribution of exogenous ESCs to EPI cells and even replace the receptor embryo to develope into the fetus by hindering the development of the host embryo EPI.In summary,this study explored the effect of PSCs secretory proteins on the development fate of early embryos.Taking Activin A as an example,we explored the mechanism that PSCs have higher contributions to chimeras produced by 4-cell stage embryo injection than blastocyst injection.Compared with blastocysts,the earlier stage embryos(such as 4-cell stage embryos)have a more sensitive response to the PSCs secretion due to the higher expression of receptors,which can more effectively change the early embryo development fate and improve the competency of ESCs contributions to chimeras.This study not only provides a theoretical basis for further optimizing the chimera production system,but also expands the understanding of the role of Activin A(a member of PSCs secreted proteins)in the development of mouse pre-implantation embryos.