Objective To reveal the function of rabbit oviductin DPF 1 on overcoming the early embryo development block Method 1.9 kb DPF 1 cDNA derived from recombinant pBluescript IIKS was digested by HincII, then 1.8 kb D...Objective To reveal the function of rabbit oviductin DPF 1 on overcoming the early embryo development block Method 1.9 kb DPF 1 cDNA derived from recombinant pBluescript IIKS was digested by HincII, then 1.8 kb DPF 1 cDNA was obtained. Linear carrier pGEX 4T 3 was obtained by SmaI NotI digestion. The recombinant pGEX 4T 3/DPF 1 cDNA was constructed by ligating linear pGEX 4T 3 with 1.8 kb DPF 1 cDNA. Large quantity of fusion protein was expressed in E.coli DE3 induced by IPTG at 37℃. Cells were lysed by using lysozyme and the fusion proteins were purified. To induce anti DPF 1 antiserum, male BALB/c mice were immunized by using GST DPF 1 fusion protein. Conditioned cultured medium derived from rabbit oviduct mucosa epithelial cells was prepared and loss of function analysis of DPF 1 was done by adding anti DPF 1 antibodies in the conditioned medium co culture with mouse fertilized eggs. Result GST DPF 1 fusion protein was purified and antisera were prepared. After GST absorption, the antiserum shows high specificity to DPF 1. Anti DPF 1 antiserum absorbed with GST could totally inhibit the early embryos development of mouse cultured in the conditioned medium and there was a positive relation between the ratio of early embryos of mouse blocked at 2 cell stage and the dose of anti DPF 1 antiserum added to the conditioned medium. Conclusion 'Loss of function' analysis revealed that rabbit oviductin ' DPF 1'has the function of overcoming the early embryo development block.展开更多
DeveloPment Promoting Factor-1 (DPF-1) was derived from the rabbit oviductmucosa epithelial cells. fertilized eggs were treated with DPF-1 and it was adminis-tered to female mice in order to investigate its effect on...DeveloPment Promoting Factor-1 (DPF-1) was derived from the rabbit oviductmucosa epithelial cells. fertilized eggs were treated with DPF-1 and it was adminis-tered to female mice in order to investigate its effect on embryonic development and implantation. The results of experiment I indicated that of the 82 fertilized eggs cul-tured in the absence of DPF-1, only 9 were normally divided to the morula stage onday4. By contrast, in the medium containing DPF-1 109(90% ) of 121 embryos weredeveloped to the blastocyst stage. In eoperiment Ⅱ, the developed blastocysts weretransferred into the uterus of 3-day pseudopregnant DPF-1-treated or control recipi-ents. Three different combinations of intrauterine transfer were performed: the DPF-1 treated embryos transferred to the control reclpients(DPF- 1→C),the control embryosto the DPF- 1 treated recipients(C→DPF- 1), and the control embryos to the control re-cipients (C→C). The implantation rate was assessed on day 18 after transfer. Therewere significant differences in pregnancy rate per mouse between DPF-1→C and C→DPF- 1 or control groups(C→C). The embryo implantation rate was low in C→DPF-1 (26. 0%, 75/288) and C→ C (25 3%, 73/288) as compared with DPF-1→C(61. 8%, 178/288). These results suggest that the major site of the DPF-1 actionduring early pregnancy is the embryo itself. DPF-1 enables embryos to develop, buthas no obvious direct effect on uterine receptivity.展开更多
基金This work was supported by grant No.39730 46 0 of the National Nature Science F oundationsup-ported by grant No.G19990 5 5 90 2 of National"973" Projectsupported by National L aboratory ofContraceptives and Devices Research
文摘Objective To reveal the function of rabbit oviductin DPF 1 on overcoming the early embryo development block Method 1.9 kb DPF 1 cDNA derived from recombinant pBluescript IIKS was digested by HincII, then 1.8 kb DPF 1 cDNA was obtained. Linear carrier pGEX 4T 3 was obtained by SmaI NotI digestion. The recombinant pGEX 4T 3/DPF 1 cDNA was constructed by ligating linear pGEX 4T 3 with 1.8 kb DPF 1 cDNA. Large quantity of fusion protein was expressed in E.coli DE3 induced by IPTG at 37℃. Cells were lysed by using lysozyme and the fusion proteins were purified. To induce anti DPF 1 antiserum, male BALB/c mice were immunized by using GST DPF 1 fusion protein. Conditioned cultured medium derived from rabbit oviduct mucosa epithelial cells was prepared and loss of function analysis of DPF 1 was done by adding anti DPF 1 antibodies in the conditioned medium co culture with mouse fertilized eggs. Result GST DPF 1 fusion protein was purified and antisera were prepared. After GST absorption, the antiserum shows high specificity to DPF 1. Anti DPF 1 antiserum absorbed with GST could totally inhibit the early embryos development of mouse cultured in the conditioned medium and there was a positive relation between the ratio of early embryos of mouse blocked at 2 cell stage and the dose of anti DPF 1 antiserum added to the conditioned medium. Conclusion 'Loss of function' analysis revealed that rabbit oviductin ' DPF 1'has the function of overcoming the early embryo development block.
文摘DeveloPment Promoting Factor-1 (DPF-1) was derived from the rabbit oviductmucosa epithelial cells. fertilized eggs were treated with DPF-1 and it was adminis-tered to female mice in order to investigate its effect on embryonic development and implantation. The results of experiment I indicated that of the 82 fertilized eggs cul-tured in the absence of DPF-1, only 9 were normally divided to the morula stage onday4. By contrast, in the medium containing DPF-1 109(90% ) of 121 embryos weredeveloped to the blastocyst stage. In eoperiment Ⅱ, the developed blastocysts weretransferred into the uterus of 3-day pseudopregnant DPF-1-treated or control recipi-ents. Three different combinations of intrauterine transfer were performed: the DPF-1 treated embryos transferred to the control reclpients(DPF- 1→C),the control embryosto the DPF- 1 treated recipients(C→DPF- 1), and the control embryos to the control re-cipients (C→C). The implantation rate was assessed on day 18 after transfer. Therewere significant differences in pregnancy rate per mouse between DPF-1→C and C→DPF- 1 or control groups(C→C). The embryo implantation rate was low in C→DPF-1 (26. 0%, 75/288) and C→ C (25 3%, 73/288) as compared with DPF-1→C(61. 8%, 178/288). These results suggest that the major site of the DPF-1 actionduring early pregnancy is the embryo itself. DPF-1 enables embryos to develop, buthas no obvious direct effect on uterine receptivity.
