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This dissertation, Identification and Characterization of Human Oviductal Cell Derived Embryotrophic Factor 3 by Yin-lau, Lee, 李燕柳, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Identification and characterization of human oviductal cell derived embryotrophic factor 3 Submitted by LEE YIN LAU For the degree of Doctor of Philosophy at The University of Hong Kong in June, 2004 The objectives of this study are to investigate the effect of a human oviduct derived embryotrophic factor, embryotrophic factor-3 (ETF-3) on the gene expression of mouse preimplantation embryo and to determine the identity of ETF-3. Human oviductal epithelial cells (OE) were immortalized (OE-E6/E7) and characterized. OE-E6/E7 retains a number of characteristics of OE. It possessed human oviductal specific glycoprotein, estrogen receptors, cytokeratin and strong telomerase activities. The development of preimplantation mouse embryo was significantly better after cocultured with OE-E6/E7 and cultured in medium supplemented with OE-E6/E7 derived ETF-3 when compared to medium alone culture. This cell line was used for subsequent studies. The mRNA expression patterns of the ETF-3 treated embryos were studied at the blastocyst stage by mRNA differential display (DDRT-PCR). Twelve of the differentially expressed genes that had high homology with cDNA sequences in the GenBank were selected for further characterization. The differential expressions of ezrin, heat shock 70kD protein 5, cytochrome c oxidase subunit VIIa-L precursor, proteinase activated receptor 2, eukaryotic translation initiation factor 2β, cullin 1 and proliferating cell nuclear antigen were confirmed by RT-PCR. The results demonstrated that OE-E6/E7 produced ETF-3 that influenced gene expression of mouse blastocyst. It is hypothesized that the higher hatching and blastulation rate after ETF-3 treatment may be due to the alteration of gene expression related to these processes related genes. Hepsin and Na/K-ATPase expression had been implicated in these processes respectively. TaqMan real-time quantitative PCR (qPCR) was used to quantify the mRNA copy number of these two genes in mouse embryos with or without ETF-3 treatment. The expression of hepsin in mouse blastocysts was very low but detectable and unaffected by ETF-3 treatment. ETF-3 treated and in vivo developed embryos had significantly higher Na/K-ATPase-β1 subunit expression than medium alone culture indicated that ETF-3 produced by OE-E6/E7 increased the Na/K-ATPase-β1 expression of the treated embryos. Monoclonal anti-ETF-3 antibody that abolished the embryotrophic activity of ETF-3 recognized a 115-kDa protein in the ETF-3 preparation. The protein was identified by mass spectrometry analysis to be complement C3. Immuno-cross-reactivities between ETF-3 and C3 proteins using anti-C3 and anti-ETF-3 antibodies confirmed the identities of ETF-3. Derivatives of C3, C3b and iC3b but not C3, were embryotrophic. iC3b was most efficient in enhancing the development of blastocysts with larger size and higher hatching rate, consistent with the previous reported embryotrophic activity of ETF-3. Embryos treated with iC3b contained iC3b immunoreactivity. The oviductal epithelium produced C3 as C3 immunoreactivity and mRNA were detected in epithelium of human fallopian tube and OE-E6/E7. Cyclical changes of C3 expression were also found in the mouse oviduct with the highest expression at II the estrus stage. Molecules involving in the conversion of C3b to iC3b and for binding of iC3b were present in the human oviduct (factor I)

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