Effects of angiogenin on the pre-implantation development of mouse embryos and establishment of an in vitro implantation model

• Main Authors: Tzu-Yen Fu, 傅子彥
• Other Authors: Pin-Chi Tang
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/56062218550924643632

碩士 === 中興大學 === 動物科學系所 === 95 === A group of homologous ribonucleases (RNases) belonged to the RNase A superfamily has been isolated and characterized from various species. During the past decades, a lot of efforts have been devoted to the study of RNases, including DNA cloning, analyses of their catalytic activities and structures. However, little is known about the biological functions of the ubiquitous RNases in vivo, especially RNase 5, the angiogenin. Recently, it was demonstrated that the gene expression could be blocked after injection of double-stranded RNA (dsRNA) into organisms and the phenomenon is called RNA interference (RNAi). The RNAi is mediated by 19-23 nucleotide dsRNAs homologous in sequence to the target genes to silence cognate genes post-transcriptionally, which involves mRNA degradation. Consequently, it shows the gene knockdown or knockdown-like effects. Hence, the aims of this study were to investigate the effects of angiogenin on the embryonic implantation by knocking down the expression of angiogenin (Ang) in mouse blastocysts, in addition to establishment of an in vitro culture system for early implantation study. In Experiment 1, the specific primers for Ang-1, Ang-2 and Ang-4 were designed to analyze the expression of pattern in mouse blastocysts by reverse transcription polymerase chain reaction (RT-PCR). The results showed that only Ang-1 was expressed at the blastocyst stage in mice. In Experiment 2, the different regions in the DNA sequences of Ang-1 and Ang-2 were selected and subcloned into RNAi expression vector, in which short hairpin RNAs were derived by U6 promoter. After transfected into B16-F10 mouse cell line, the expression of Ang-1 and Ang-2 were analyzed by RT-PCR. The RT-PCR results demonstrated that the expression of angiogenin was decreased to at least 40% by various RNAi expression vectors. In Experiment 3, the U6-sh-1 (RNAi expression cassette for knocking down Ang-1) or U6-shR-1 (RNAi expression cassette for knocking down RNase 1) fragments were injected into the pronuclei of mouse zygotes. The cleavage rates in the U6-sh-1-injected and U6-shR-1 injected groups were significantly lower than those in the uninjected control group after 72 h culture in vitro (77 % and 81% v.s. 95%, P < 0.05). No significant differences of morula/blastocyst formation were found between U6-sh-1-injected and U6-shR-1-injected groups (77% v.s. 81%, P > 0.05), but the development of morula/blastocyst in the U6-sh-1-injected or U6-shR-1 group was significantly decreased, compared to the control group (49% and 48% v.s. 90%, P < 0.05). The defective effects of U6-sh-1 and U6-shR-1 on the development of preimplantation embryos were demonstrated. Immunofluorescent staining showed that U6-sh-1 injection reduced Ang-1 protein levels in the blastocysts compared to those in the control group. In Experiment 4, the endometrial epithelial cells and stromal cells were isolated from mouse uteruses. For construction of the 3-dimensional culture system, epithelial cells were seeded on an artificial basal membrane (ECMatrix™) with underlying stromal cells embedded in the type I collagen matrix. The whole system was settled in a Millicell® (Millipore) hanging in a 24-well culture plate. The morphology of epithelial cells on the matrix became cuboidal after culture. Additionally, the columnar appearance with a basal nucleus was observed on the paraffin wax sections of epithelial cells. The mouse blastocysts were recovered and cultured in this model system. Normal hatching and attachment of the blastocyst were observed. The endometrial cells grown in the established in vitro culture system appeared similar morphology as those in vivo, suggesting this model system might facilitate further understanding of the cellular and molecular mechanisms involving in the implantation of mammalian embryos. In conclusion, the DNA-based shRNA constructs produced in this study could effectively decrease the expression of Ang-1. Further studies would be required to elicit the optimal culture conditions for the in vitro implantation model before applied to the study of the effect of angiogenin on the implantation.