The Effects Of Fertilization Of Mice Sperm In Vitro With Anti-uPA Antibody Secreted By Positive Hybridoma Cells

PartⅠThe preparation of anti-uPA antibody secreted by positive hybridoma cellsObjective: To prepare supernatant containing anti-urokinase-type plasminogen activator antibody with high titer, secreted by positive hybridoma cells.Methods: BALB/c mice of 6 to 8 weeks were immunized by monoclonal antibody technology. After determination the titer of antibody by indirect ELISA with tail vein blood, spleen cells suspension of the mice was fused with myeloma cells named SP2/0 . Then the hybridoma cells were screened by the HAT medium. Once the 2/3 areas of culture hole were occupied by them, the supernatant was immediately tested.Results: After routine immunization, the titer of antibody in serum was 1:12800. Hybridoma cell clones were observed growing bigger and bigger under microscope at the seventh day and the tenth day after cell fusion. In the supernatant, the titer of antibody was 1:6400, and its class was IgG. by indirect ELISA . Among of 196 holes, there were 140 positive holes, so the fusion rate was 71.4 percent. The numbers of chromosome were 84～104, equaled to the sum of spleen and myeloma.Conclusion: By routine immunization process, we prepared IgG–type antibody of urokinase with high titer and hybridoma cells secreting antibody endlessly.PartⅡThe effect of prepared anti-uPA antibody supernatant on sperm function of miceExperiment 1 The effects of prepared anti-uPA antibody supernatant on mice sperm viability and motility rate. Objectives: To observe the changes of mice sperm viability and motility at different time points after adding different dilutions hybridoma cell supernatant containing anti-uPA antibody into sperm.Methods: The swum-up sperm of mice was divided into four groups:The group A and C were positive controls, and the group B and D were negative controls. In the group A and B, cell supernatant with dilution of 1 / 5 was added, and in C and D groups cells supernatant with dilution of 1 / 10. Then the changes of sperm viability and motility at different time points ( 10min,20min,30min,40min,50min,60min) were observed. The motility was counted under a light microscope, and the viabilty was determined by eosin Y staining.Results: Compared with group B, the viability and motility of group A were lower at 30min,40min,50min,60min points after anti-uPA antibody added in sperm suspension. Especially the viability at 50min was obviously lower than that of B. (P <0.01). Compared with group D, the viability and motility of group C were with the same as group A. Compared with group A and B, the viability and motility were declined from 30min to 60min, especially at 60min with a significant difference (P <0.01). Although viabity and motility in group C were declined, it was not obviously as that of group A, but the difference has no statistical significance. .Conclusions:The viability and motility are obviously inhibited by the cell supernatant containing anti-uPA antibody,and this inhibition is sustainable. As difference of the titer of dilution , the effects on sperm viability and motility are different . The results show that anti-uPA antibody could obviously inhibit the fuction of uPA in vitro. Experiment 2 The effects of prepared anti-uPA antibody supernatant on mice sperm fertilization in ivtroObjectives: To observe the changes of sperm fertilization rate with anti-uPA antibody secreted by hybridoma cells.Methods: The strum up sperm was divided into three groups: the blank group,the positive control group and the negative control group. The eggs after promoting ovulation were divided into three groups with the same as sperm . Then sperm with eggs were fused in order to be fertilized, ultimately there were eight groups. The groups of antibody include four groups.Group A was blank of sperm and eggs .Group C was blank of eggs and positive sperm. Group E was the positive control of eggs and sperm . Group G was negative eggs and positive control of sperm. The others are opposited to the groups of antibody. After 24 hours , the changes of their fertilization rate were obversed .Results: Compared with group A and B, there is a significant difference in their fertilization rate (P <0.05). The fertilization of A group was 39.1%, lower than that of group B. Compared with group C and D, the fertilization of group C was 35.6% . There are a significant difference from group D (P <0.05). Compared with group E and F, there was a significant difference between group E (19.6%)and group F (P <0.05). Compared with group G and H, the difference was obvious (P <0.05), and that of group C only was 36.2%.Conclusions: Supernatant containing anti-uPA antibody could effectively inhibit the fuction of uPA , and lower the fertilization rate of mice. The mechanisms maybe: the one hand , the supernatant could lower the the activity and motility of sperm, and reduce the number of sperm aroud eggs.On the other hand , it could inhibit the function of uPA to guide sperm chemotaxis and inhibits precontact sperm-egg communication and lower the capability of sperm fertilization in vitro. So it maybe a new contraceptive drug in the future.