The Influence Of Anti-human Urokinase-type Plasminogen Activator Antibody On The Reproduction Of The Female Mice

PARTⅠThe Method of Inducing Female Mice to Produce Anti-human uPA Antibody and the Potency AnalysisObjectiveTo set up an effective and stable method of inducing female mice to produce the anti-human uPA antibody.MethodsDivid the female mice into five group randomdly, including Blank control group (granting no treatment), Adjuvant control (granting normal saline 100μl and equal volume of adjuvant), Low dose of immunized group(granting the uPA antigen of human 20μg/100μl and the equal volume of adjuvant), Middle dose of immunized group (granting the uPA antigen of human 40μg/100μl and the equal volume of adjuvant) and High dose of immunized group(granting the uPA antigen of human 80μg/100μl and the equal volume of adjuvant). The mice were immunized every two weeks.After the first week of the three times of the immunity of the female mice, the titres of antibody of the serum were determined by enzyme-labeled immunosorbent assay (ELISA). ResultsThe value of the optical density (OD) of anti-human uPA antibody of the immunized groups were significantly higher than that of the three control groups. By computing the valence of antibody of the serum the mice by the method of relative value, we found that the valence of antibody of the serum of the immunized groups were all higher than the three groups (Negative control group, Blank control group and the Adjuvant control). The difference was statistically significant(P<0.05).The valence of antibody of the serum of the immunized groups reached 1:10240 or above. ConclusionUsing human uPA as the antigen to immunize the female mice, we found that the empirical method was not only a method of inducing the mice to produce the stable anti-human uPA antibody, but also a method of inducing the mice to achieve a higher level of antibody.PARTⅡThe Influence of Anti-human Urokinase-type Plasminogen Activator Antibody on the Reproduction of the Female MiceExperiment One The Influence of Inducing Female Mice to Produce Anti-human uPA Antibody on the pregnancy in vivoObjectiveTo observe the influence on the pregnant situation of the female mice, which were immunized by using urokinase-type plasminogen activator of human.MethodsThe immunized mice were coupled with the adult male mice on the fourth time of immune. The pregnant mice were dissected on the seventh day and the pregnancies rate and the average of implanted embryos were recorded. ResultsComparing with the groups of blank control and adjuvant control, the pregnancies rate and the average number of implanted embryos of the three immunized groups were decreased. The difference was statistically significant(P<0.05). But the difference was not statistically significant between the groups of blank control and adjuvant control(P>0.05).ConclutionThe marked decline of the fertility of the female mice, which were immunized by human uPA, suggested uPA could be an effective target for immunocontraception.Experiment Two The Influence of Inducing Female Mice to Produce Anti-human uPA Antibody on the Fertilizability in Vivo FertilizationObjectiveBy observing the fertilization of the ovums in vitro, we would observe the influence on the reproduction of the female mouse immunized by uPA of human.MethodsEggs were collected from the immunized female mice synchronized with 10 units of pregnant mare serum gonadotropin (PMSG) and 10 units of human chorionic gonadotropin (hCG) 48 hours and 16 hours before collection, respectively, and fertilized with the sperm of the healthy male mice in vitro. The number of ovulation was recorded and the fertilization was scored. The number of corepus luteum of ovaries serial section was recorded under a light microscope.Results1.Average of ovulationComparing with the groups of blank control and adjuvant control, the average of ovulation of the three groups of immunized by uPA were decreased. The difference was statistically significant(P < 0.05). But the difference was not statistically significant between the groups of the blank control and the adjuvant contro(lP>0.05).2.The pregnant rate in vitroComparing with the group of blank control, the pregnant rate of the groups of adjuvant control and the three groups of immunized by uPA in vitro were decreased. But the difference was not statistically significant(P>0.05).3.The information of merogenesispThe rate of the grade of A and B embryos of the high dose and middle dose groups of immunized by uPA were lower than the group of blank control and adjuvant control. The difference was statistically significant(P<0.05). Comparing with the groups of blank control and adjuvant control, the rate of the grade of A and B embryos of the group of low dose was decreased. But the difference was not statistically significant(P>0.05).4.Average of corepus luteumThe average of corepus luteum of the three groups of immunized by uPA were decreased by comparing with the group of blank control and adjuvant control. The difference was statistically significant(P<0.05).ConclutionUsing human uPA as the antigen to immunize the female mice, then the average of ovulation, the pregnant rate in vitro,the average of corepus luteum and the rate of the grade of A and B embryos were decreased when there were stable anti-human uPA antibody in the female mice. It revealed that the empirical method could influence the ovulation, the fertilizability and the growth of the ovum.Experiment Three The Influence of Using the Serum Contained Anti-human uPA Antibody on the Fertilizability IVFObjectiveBy using the serum which was collected from the five groups of mice to interfere in the ovums of the mice and observe the state of the fertilization in vitro. We would observe the influence on the fertilizability and the state of survivorship of the ovums.MethodsBy using the method of PMSG/hCG, we could achieve the mouse cumulus cell-oocyte complex. Put the health mouse cumulus cell-oocyte complex in the culture fluid which contained the serum of the every group of mice. Then, the oocytes were fertilized with the sperm of the health male mice in vitro. We recorded the pregnant rate and the death rate of the oocytes.Results1.The Pregnant RateThe pregnant rate of the ovotids, which were cultured with the serum of the immunized groups of the female mice, were lower than that of groups of blank control and adjuvant control. The difference was statistically significant(P<0.05). But the difference was not statistically significant between the group of blank control and adjuvant control(P>0.05).2.The death rate of oocytesThe oocytes cultured with the serum of the uPA immunized groups of the mice had the lower pregnant rate and the higher death rate, especially the group of high dose of immunized by uPA. The difference was statistically significant between the group of high dose of immunized compared with other groups(P <0.01). There were increase in the groups of low dose and the middle dose of immunized by uPA in the death rate, but the difference was not statistically significan(tP >0.05). The difference was not statistically significant between the group of blank control and adjuvant control(P>0.05).ConclutionBy using the serum which was collected from the five groups of mice to interfere in the oocytes of the mice and observe the state of the fertilization in vitro. There were increase of the death rate of oocytes and decrease of the pregnant rate. It revealed that the serum which contain anti-human uPA antibody could decrease the fertilizability and the state of survivorship of the ovums.