Enhancement of Embryo Implantation Ability by LPA supplementation through RGS2 Signaling Pathway

• Main Authors: I-Ching Chen, 陳逸菁
• Other Authors: 高淑慧
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/80499748635195393370

碩士 === 臺北醫學大學 === 醫學檢驗生物技術學研究所 === 95 === To improve the implantation rate is an important issue in the assisted reproductive techonology. The successful implantation depends upon the implantation-competent blastocyst and the receptive uterus. This process is initiated via blastocyst-endometrium interaction and delicately regulated by multiple factors. Lysophosphatidic acid (LPA) is a lipid-based signaling molecule and essential to mammalian oocyte maturation and preimplantation development. LPA3 had been demonstrated that it participates in implantation process through receptor-mediated signaling in uterus but the detail mechanisms are unclear. In this study, we try to explore the role of LPA in this process. As the result, LPA supplementation enhanced the attachment rate of blastocysts on the matrigel, especially following 100 mM LPA treatment (62.4% ± 1.6% vs. 9.0% ± 9.0, p<0.05). In vivo mouse mode showed the LPA treated group had significantly higher implantation rate than the control group (87.5% ± 7.2% vs. 66.7% ± 4.2%, p<0.05). Moreover, the implantation rate was inhibited by Ki16425 (LPA inhibitor) (25.0% ± 14.4% vs. 70.8% ± 4.2%, p<0.05). To gain insights into the molecular mechanisms, we identified the candidate genes involved in this process including 35 up-regulated genes (Normalized Expression Ratio, NER＞2) and 22 down-regulated genes (NER＜0.5) by oligonucleotide microarray. The top five up-regulated gene was Obox1 (47 folded increase), Omt2b (37), Oog1 (28), Btg4 (25), Trim61 (22), and RGS2 (21). Content of the regulator of G-protein signaling protein 2 (RGS2) mRNA was confirmed with 27.0 ± 5.5 folded increase by real-time quantitative PCR analysis. By use of pharmalogical LPA inhibitor, RGS2 mRNA was reduced to 4.5 ± 0.8-folded (p=0.06). The augmented RGS2 expression was suppressed by LPA inhibitor and significantly reduced the attachment rate compared with LPA alone group (77.5% ± 4.2% vs. 21.7% ± 4.5%, p<0.01). Through RGS2 signaling pathway, LPA enhance blastocyst implantation ability .In addition, stress fiber organized via RGS2 activation and required for embryo implantation process. All the treated embryos were stain with FITC-labeled phalloidin to observe the distribution of actin filament. We found the well-organized stress fiber distributed in the LPA-treated embryos, and localized at the embryo migration facade (38.9% vs. 20.8%). However, in the inhibitor groups the granules-like actin filament only generally distributed in blastocyst cytoplasm. These results indicate that through RGS2 signaling pathway, LPA may enhance blastocyst implantation ability by regulation of actin filament organization.