IK Cytokjne Expression And Function In Bovine Preimplantation Embryos And Bovine Fetal Fibroblast Cells

IK cytokine is a down-regulator of class Ⅱ major histocompatibility complex (MHCII). The protein encoded by this gene contains multiple repeats of arginine (R), glutamic acid (E) and aspartic acid (D), and thus also is named protein RED. MHCII antigens express only on the surface of antigen-presenting cells (APC), such as macrophage cells, B lymphocytes, activated T lymphocytes, dendritic cells and so on. As for situations of non-APCs, IK cytokine suppresses MHCⅡ expression and inhibits MHCⅡ induction by interferon y (IFN y), which is important for maintain of the stability of the immune system.In this study, the transcript of IK cytokine in bovine preimplantation embryos was found to be differentially expressed according to different development stages by mRNA differential display reverse transcription polymerase chain reaction (mRNA DDRT-PCR). The results of quantitative real-time PCR (qPCR) showed that the mRNA expression of IK cytokine decreased during maturation from germinal vesicle (GV) oocyte to second meiotic metaphase (Mil) oocyte, and kept down through in vitro fertilization (IVF) and cleavage to2-cell embryo. When developed to8-cell embryo stage, the IK cytokine transcript increased to reach a peak among the whole process of preimplantation development. During progression after8-cell embryo to blastocyst, the ik transcript dropped. Then IK cytokine was defined as a differential expression gene in bovine preimplantation embryo. Embryos of different development stages including IVF zygote,2-cell embryo,8-cell embryo, morula and blastocyst were subjected to immunofluorescence staining using IK cytokine-specific antigen. The results demonstrated that no IK cytokine protein expressed in embryos till8-cell stage, and then there was IK cytokine protein in nuclear in morula and blastocyst, with no differential expression in inner cell mass (ICM) and trophoblast.Zygotes immediately after IVF were microinjected to down-regulate ik mRNA with small interference RNA (siRNA), negative control zygotes were injected with in vitro culture (IVC) medium of the same volume as that of siRNA. The results showed that the interference rate of siRNA detected by qPCR was79%(P<0.01). The cleavage rate and8-cell embryo rate of zygotes injected with siRNA decreased in comparison with that of negative control embryos (P<0.05). However, the blastocyst rate was not influenced(P>0.05). qPCR detection of siRNA-microinjection embryos showed an ascent of IK cytokine transcript in8-cell embryos to catch up with that in negative control8-cell embryos after the intense fall in2-cell embryos. The IK cytokine transcript in siRNA-microinjection blastocysts was0.74times as that in negative control blastocysts. Immunofluorescence staining demonstrated that there is no shift in IK cytokine protein expression caused by ik-target siRNA. These results suggested that during the zygote genome activation (ZGA) occurred in8-cell embryo, ik gene got activated and the new transcribed ik mRNA replenished the transcript stored in GV oocyte, and thus reduced the interference induced by siRNA. However, as for the occasion of blastocyst, when ik transcription attenuated, siRNA effect arose.Although there was no IK cytokine protein expressed in embryos of stages before8-cell stage, down-regulation of ik mRNA in this episode negatively affected cleavage and8-cell embryo rate, suggesting that there could be minute quantity of IK protein which played an important role in cleavage and8-cell embryo development. That ik mRNA reached a peak and the protein began to express numberously in8-cell embryo indicated that IK cytokine made difference in8-cell embryo. It was the substantial increasing ik transcript that covered the siRNA effect, and thus made it difficult to estimate the effect of ik to8-cell rate. In blastocyst, though the mRNA expression of ik decreased as a result of siRNA, the protein expression of ik got no influence, so that the siRNA injection did not affect blastocyst rate.The qPCR detection of ik down-stream gene, bos taurus class Ⅱ major histocompatibility complex (DRB3) and transactivator of class Ⅱ major histocompatibility complex(ciita), showed the mRNA expression of DRB3and ciita in8-cell embryo was2.4and3.7times of that in MⅡ oocyte, and in blastocyst, it was0.39and0.44times. However, the results of immunofluorescence staining of IVF2-cell embryo,4-cell embryo,8-cell embryo, morula and blastocyst and siRNA-injection8-cell embryo, morula and blastocyst demonstrated there was no CIITA protein expression in embryo of any preimplantation development stage, suggesting that interference of ik mRNA would not alter MHCⅡ expression.In summary, ik expression in bovine preimplantation embryo was under a strict control, and was not easy to be altered by exogenous siRNA, which played an important role in maintaining low expression of MHCII molecules on the surface of blastocyst.qPCR was used to detect ik expression in bovine fetal fibroblast cells (BFFC) and revealed high abundance of ik transcript. Meanwhile, researches on Hela cell indicated a new function of IK cytokine to activate spindle assembly checkpoint (SAC). In order to determine whether ik functioned in association with SAC in BFFC, mRNA abundance of SAC main member, including mad1, mad2and cdc20were detected in ik over-and down-regulated BFFC. The results showed no changes in expression level of the three SAC members in ik down-regulated BFFC while ik over expression elevated the mRNA level of mad2and cdc20. This inferred that ik affected SAC function sufficiently but not essentially, that is, down-regulation of ik would not inhibit SAC functions, but ik over-expression would promote SAC function.This research assessed IK cytoking functions in bovine preimplantation embryo and bovine fetal fibroblast cell and this will provide basis for cytokine researches.