| Studies performed over the past several years have shown that excepting paracrine and endocrine, interferon-tau (IFN-Ï„) also conduct its function via an autocrine manner. ISG15(interferon-stimulated gene15) could be strikingly up-regulated by I type IFN in mostly tissues. But whether ISG15was also involved in the autocrine function of IFN-Ï„? In this study, in vitro produced bovine embryos were used to investigate the role of ISG15in development of bovine embryo. Firstly, the expression pattern of ISG15mRNA during early developmental stages was exposed by RT-PCR. Then lentivirus vector with ISG15RNA interferance (ISG15i) was injected into bovine2-cell embryos for down-regulating the expression of ISG15mRNA. Finally, rbIFN-Ï„ was added into embryo culture medium. The result showed that the level of ISG15mRNA expression was increased from day0to day2(P<0.01), and disappeared in day4. However, the expression of ISG15mRNA was detectable in day6and day8, and the expression level was higher in day6when compared with that in day8(P<0.05). After ISG15i, the hatch out rate of blastocyst and total cell number per diameter of blastocyst were significantly reduced (P<0.05). Connexin43was also down-regulated after1SG15i(P<0.05). Moreover, the expression level of IFNT mRNA was reduced in ISG151group in day8blastocysts (P<0.05). Ets2is the key factor in regulating IFNTexpression. We found that ISG15i also decreased the mRNA levels of Ets2in day8embryos when compared with the control (P<0.05). We preciously found that supplementation of rbIFN-Ï„ has promoting effect on bovine embryo development. In this study, ISG15mRNA was significantly increased in day6and day8bovine embryos after rbIFN-Ï„ treatment (P<0.05), while, endogenous IFNT mRNA was decreased. These results showed that ISG15played a role in bovine embryo development and regulated the expression of IFNT. It is tempting to speculate that there exists a negative feedback loop in IFN-Ï„ regulation, which could insure the suitable levels of IFN-Ï„ expression during early embryo developmental stages.As informed in the front, ISG15mRNA was expressed in day0zygotes. Whether ISG15was also expressed during bovine oocytes maturation? We found that there was no expression of ISG15mRNA during GV to MI stage in bovine oocytes. However, the level of ISG15mRNA was strikingly increased in MII stage (P<0.01), which could be inhibited by addition of actinomycin D (specific RNA polymerase II inhibitor) during MI to MII stages (P<0.05). After ISG15mRNA overexpression, there is no effect on oocyte maturation and cleavage (P>0.05), but rates of blastocyst were significantly promoted (P<0.05). In conclusion, ISG15was expressed during MI to MII stage during bovine oocyte maturation, and had promoting effect on bovine embryo development as a maternal gene.In order to investigate the genes expression patterns of ISG15,IFNT, Cdx2andOct4in bovine embryos\at late developmental stages, alginate hydrogel was used to build two types of3-D (three-dimensional) culture systems for day8or hatched-out bovine embryos in vitro culture. The first is alginate hydrogel embeded embryo culture system (AECS), and another is alginate hydrogel overlay embryo culture system (AOCS). The results showed that embryos cultured in AECS were elongated in morphology to a certain degree and cell number also sharply increased. Moreover, differentiation occurred in bovine embryos during in vitro culture. Dinuclear cells were visible in day31embryos. Moreover, the expression of IFNT and ISG15were still detectable in day26and day32IVF bovine embryos. And the expression ratio of Cdx2and Oct4mRNA was increased from day6to day40(P<0.05).In conclusion, these two alginate hydrogel embryo culture systems could support elongation, differentiation and development of in vitor produced post-hatching bovine embryos. IFNT was continuously expressed in bovine embyros unless implantation was happen. |
