Identification and characterization of human oviductal cell derived embryotrophic factor 3
Identification and characterization of human oviductal
cell derived embryotrophic factor 3
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 pre implantation 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 pre implantation 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 2~, cullin I 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 NaIK-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 ofhepsin 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 NaIK-ATPase-f31 subunit expression than medium alone culture indicated that ETF-3 produced by OE-E6/E7 increased the NaIK-ATPase-f31 expression of the treated embryos.
Monoclonal anti-ETF-3 antibody that abolished the embryotrophic activity ofETF-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
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) and mouse pre implantation embryo (Crry, CR3), respectively. The present data showed that the oviduct produced C3/C3b, which was converted to iC3b to stimulate embryo development. The mechanism of iC3b on preimplantation embryo development remained to be investigated.
School:The University of Hong Kong
School Location:China - Hong Kong SAR
Source Type:Master's Thesis
Keywords:oviduct epithelial cells rodents embryology gene expression
Date of Publication:01/01/2004