Study On The Function And Mechanism Of IFRG15 Protein In Mouse Embryonic Development Before Implantation

In mammals, the genome of germ cells is transcriptionally silencing during meiosis. In mice, a wide range of embryonic genome activation starts at the late stage of 2-cell embryo; while in human it occurs at the 4-8-cell stage. Before the zygotic genome activation (ZGA), embryonic development is under the regulation of maternal factors (including mRNA and protein), which are accumulated and stored in cytoplasm of mature oocytes. After fertilization or activation of nuclear transfer embryos, these maternal factors start to regulate early embryonic development, and simultaneously initiate embryonic genome transcription, leading to express new genes and synthesize new factors, and inducing a cascade reaction to synthesize more cytokines, which maintain the early embryonic development. Even after the activation of the embryonic genome, the persistence of maternal factors can precisely regulate the early embryonic development by acting with other maternal factors or interacting with the new genes expressed in zygote. So it will be helpful to better understand the preimplantation events during embryonic development and regulatory mechanisms, as well as providing a theoretical basis to improve the success rate of in vitro culture of embryos by the study on maternal factors.Interferon alpha responsive gene 15 (IFRG15) was identified from the proteomic map of mouse Mil oocytes in our lab, which was abundantly expressed in the oocytes. As a relatively new protein, the physiological functions of EFRG15 in the oocytes and early embryos remain unknown. It has been reported that Ifrgl5 mRNA was expressed in rabbit oocytes and embryos, suggesting that it has a certain effect on the development of preimplantation embryos. In order to better understand the function and mechanism of IFRG15 during mouse preimplantation embryonic development, we performed the present study focusing on the following three aspects.Firstly, based on the bioinformatics analysis of IFRG15 using NCBI database and real-time PCR detection, we found that IFRG15 was encoded by Torlaip2 gene that produced a total of nine transcripts, only four among which encode 131 amino acids of the IFRG15 protein. IfrglS mRNA was highly expressed in the reproductive system, and constitutively expressed in GV oocytes, MII oocytes and preimplantation embryos. Thus we speculated that IFRG15 might play an important role in regulating preimplantation embryonic development as a maternal protein.Secondly, to study the important role of IFRG15 in preimplantation embryonic development, we used the microinjection of siRNA against EFRG15 to block its function. Our results indicated that IFRG15 knockdown in oocytes led to retarded growth from the one cell stage of the embryos and reduced number of two cells-containing oocytes. Such inhibition of development showed a dose-dependent manner. In contrast, IFRG15 knockdown at the two-cell stage did not prevent oocytes from developing into the three-cell stage. Thus we concluded that the blockage of zygote division induced by Ifrgl 5 silence was due to the disrupted cleavage process in the preimplantation embryonic development, rather than the interference in the first division. At the same time, we transfected EFRG15 siRNA into the mouse fibroblast cells and found that the cell cycle arrest did not occur in somatic cells. Therefore, EFRG15 protein plays an important physiological role only in mouse preimplantation embryonic development.Thirdly, we used the living cell imaging system to determine the exact time-point of the developmental retardant induced by EFRG15 knockdown. Our results showed that the pronucleus could normally move closely to each other, but the infusion was abnormal when EFRG15 was knocked down. The DNA replication was detected by using an EdU incorporation Kit. We found that EdU was correctly incorporated into DNA in the S phase in both groups, suggesting that the DNA replication was blocked in the late S period or G2 period by IFGR15 knockdown. We also detected the expression levels of yH2AX, a marker gene involved in the DNA double-strand break and found that yH2AX had a higher expression level in Ifrg15 siRNA-injected oocytes. Furthermore, ATM, a biomarker of G2 checkpoint, was also expressed at a higher level. In contrast, the expression levels of MuERV-L (murine endogenous retrovirus-like), EIF-la (eukaryotic translation initiation factor A), HSP70.1 (heat shock protein 70.1) and U2AFBP were significantly inhibited when IFGR15 was knocked down. Thereofore, the silence of Ifrg15 induced the DNA damage, activated the G2 checkpoint, and subsequently inhibited the activation of the zygotic transcription and arrested the cell cycle.Finally, we tried to find the potential mechanism through which IFRG15 affected mouse preimplantation of the embryonic development by using the high-throughput RNA-Seq sequencing approach. Our results indicated that 1445 genes were differently expressed when Ifrg15 was knocked down,1086 of which were up-regulated and 359 were down-regulated. Many genes were involved in the regulation of cell cycle and reproduction process, suggesting that they might be the mediators of IFRG15 in regulating preimplantation embryonic development.In summary, this is the first time to show that IFRG15 plays an important role in regulating preimplantation embryonic development. We also explored the detailed mechanism through which IFRG15 exerts its functions. Further, by using transcriptional sequencing in zygote with Ifrg15 knockdown, we identified some candidate genes interacting with IFRG15. Our findings are helpful to better understand the molecular regulation of preimplantation embryonic development and zygotic gene activation.