Localization And Function Analysis Of Rabbit Oviductin, Cloning And In Vitro Expression Of Human Oviductin Gene

The Development Promoting Factor-1 (DPF-1, a rabbit oviductin) gene was early cloned from cDNA library derived from rabbit oviduct mucosal epithelial cells. There are much homology between DPF-1 and other species oviductins, especially in their N-terminal peptide (1 - 400 aa., containing a chitinase domain), whose homologies are up to 80%. The pi of DPF-1 is ranging from 7.2 to 8.1. In previous experiments, the GST-DPF-1, GST-NTP-DPF-1 (14-380 aa. no enclosing signal peptide) and GST-CTP-DPF-1 (381-454 aa.) were prepared, and antisera generated against these peptides were produced. The results obtained from in vitro experiments proved that antibodies against the C-terminal peptide of the rabbit oviductin could inhibit mouse early embryo development. Based on these previous experiments, we decided to analyze the distribution of DPF-1 in oviduct, and its effect on promoting early embryo development; to express DPF-1 in vitro and obseve its distribution in oocyte; to clone human oviductin gene and express it in vitro.Oviductin was expressed to a great extent during pre-ovulation stage under the control of ovarian hormones. Immunohistochemistry analysis of DPF-1 in estrus rabbit oviduct revealed that DPF-1 appeared strictly in the ampullary epithelium and the isthmic epithelium, and much more in the isthmic epithelium, not in the infundibulum. Western blotting analysis indicated antibodies generated against GST-CTP-DPF-1 reacted to a protein of-50 K.D protein derived from mouse oviduct secretion, and immunohistochemistry results indicated the -50 KD protein was expressed in the infundibulum epithelial cells and isthmic epithelial cells.The percentage of development from early embryo to blastocyst could be increased significantly by adding GST-CTP-DPF-1 in the culture medium and decreased by adding antiserum against GST-CTP-DPF-1. The results obtained from "neutralization experiment" also ascertained the promoting effect of GST-CTP-DPF-1 on early embryo development by counteracting the inhibition effect of the antiserum. Indirect immunofluorescense analysis of mouse embryo cultured in rabbit oviduct epithelium conditioned medium indicated the rabbit oviductin was presented on the outerface of cytoplasmic membrane of early embryo, which raised a suggestion that ovidcutin may function through direct binding onto the cell membrane of embryo.In order to clarify the localization of DPF-1 in oocyte/early embryo, an eukaryotic expression plasmid pEGFP-Nl/DPF-1 was constructed by fusing DPF-1 gene to the 5' terminus of eGFP gene. After transfecting the recombinant plasmid into HeLa cells, we got some cell strains expressing and secreting eGFP-DPF-1 stably. The apparent molecular weight of the fusion protein was up to 120 KD indicating the fusion protein had been submitted to a post-tranlation modification. The dynamic distribution of DPF-1 in rabbit oocyte-cumulus cells complexes (COC), the cumulus cells-deprived oocytes or oviductal oocytes co-cultured with the cell strain or cultured in the conditioned medium derived from the cell strain was studied. The results revealed that DPF-1 but no GFP alone associated with zona pellucidae (ZP) of oocytes, especially in the inner layer of ZP, and was evenly distributed in dot-like shape on the outer surface of the cytoplasmic membrane. In contrast, the cumuluscells around the oocyte did not interfere the association of DPF-1 with oocyte, and no trace of DPF-1 was found in the cumulus cells. It seems that it is the first time to prove the oviductin expressed and secreted by eukaryotic cells in vitro could bind to oocytes, and to provide the facility in observing the dynamic binding process directly. Undoubtedly, these results also furnished some interesting informations for the further studying on the oviductin function and its mechanism.In order to reveal the universal charateristics of oviductins through comparative analysis, two complete human oviductin cDNAs were screened from human oviductepithelial cells cDNA library by using 32P-labelled DPF-1 cDNA as probe...