Study On Expression Differences Of IFN-Ï„ On Early Implantation Between In Vivo And In Vitro Ovine Embryos

In this study,it was aimed to investigate the expression differences of IFN-Ï„on early implantation between in vivo and in vitro ovine embryos. The IFN-Ï„on early implantation sites of both in vivo and IVF (in vitro fertilization) ovine embryos were detected by in situ hybridization, semi-quantitative RT-PCR, immunohistochemical staining and Western Blotting Hybridization. There are total five experiments in it.Experiment 1: The H-E staining was initiated for checking up the sheep embryonic status implanted on their uterus of both in vivo and IVF ones on Day10, Day12, Day14and Day16 after their estrus.Experiment 2: The in situ hybridization analysis was initiated to detect mRNA expression of IFN-Ï„on the early implantation sites of both in vivo and IVF ovine embryos. The freezing cross-sections of these implantation tissues were hybridized with the radioactive-labeled antisense or sense ovine IFN-Ï„cRNA probes, and IFN-Ï„mRNA was detectable primarily on conceptus trophectoderm of the implantation tissues of in vivo embryos on Day16.Experiment 3: The semi-quantitative RT-PCR was initiated to detect IFN-Ï„mRNA Expression on the early implantation sites of both in vivo and IVF ovine embryos. The expression level of IFN-Ï„mRNA on implantation sites of ovine in vivo embryos on Day16, was significantly higher than that of IVF ovine embryos on Day16(P<0.05).Experiment 4: The immunohistochemical staining was initiated to detect IFN-Ï„protein expressional distributions on the early implantation sites of both in vivo and IVF ovine embryos. The IFN-Ï„signal was detected with rabbit anti-ovine IFN-Ï„polyclonal antibody, and was distributed primarily in their endometrial LE and conceptus trophectoderm.Experiment 5: The Western Blotting Hybridization was initiated to detect IFN-Ï„expression differences on the early implantation sites of both in vivo and IVF ovine embryos. An appropriate protein of 22 kD was detected by this method with specific antiserum. The expression level of IFN-Ï„on implantation sites of ovine IVF embryos on Day14, was significantly higher than that of the in vivo one on Day10(P<0.05), while among the other three in vivo groups, there were no significant differences (P>0.05). However, the expression levels of IFN-Ï„on implantation sites of all the other ones, were significantly lower than that of ovine IVF embryos on Day16 (P<0.05). In addition, that protein level on implantation sites of ovine IVF embryos on Day10, was significantly higher than those on the other IVF ones. (P<0.05)Collectively, it can be concluded as following: 1) The embryo was not found in all IVF groups, but it was found in in vivo ones on both Day12,14 and 16 and IVF ones on Day12 and 16. 2) IFN-Ï„mRNA was detectable primarily in conceptus trophectoderm on early implantation sites of in vivo ovine embryos on Day16. 3) The expression level of IFN-Ï„mRNA on implantation sites of ovine in vivo embryos on Day16, which was significantly higher than that of the in IVF ovine embryos. 4) Immunoreactive IFN-Ï„protein was predominantly localized in endometrial LE and conceptus trophectoderm detected with a rabbit polyclonal antibody. 5) The expression level of IFN-Ï„on early implantation sites of IVF groups was significantly higher than that of the in vivo on Day10.