Studies On Reproductive Relevant Biologic Properties Of Phytohemmaglutinin (PHA) And PHA Containing Gel From Kidney Bean

More and more studies have shown that plant lectins have effects on the reproductive process,including fertilization,embryo implantation,embryo development and pregnancy.We have been engaged in the research of kidney bean lectins(KBL)or phytohemmglutinin for over ten years.Studies have shown that KBL can safely and effectively prevent or block fertilization,inhibit embryo implantation and termination of pregnancy in different periods.Objective: In this study,the effects of Yunnan RKBL(red kidney bean lectin,or Phytohemmaglutinin,PHA)or RKBL containing gel on mouse vaginal structure after long period time administration have been observed;the effects of different concentrations of red kidney bean plant and its gel preparation on agglutination of sperm,especially on conditions of extremely low concentration have been recorded;the influence of RKBL on female mice and their offspring after twice repeatedly use RKBL for termination of pregnancy have been observed and recorded;also the influence of RKBL on the changes and distributions of RKBL receptor or binding sites in uterus after treatment with RKBL 12 h have been carried out.The aim of this study is to providing the experimental evidence and theoretical basis for understanding the mechanism of RKBL on its block fertilization and termination pregnancy,in order to develop a new microbicide which can be used for both contraceptive and prevention of sexually transmitted diseases including HIV,a new mathod for termination unwanted pregnancy.Methods: 1.Different concentrations of RKBL were injected into the mouse vagina through the vaginal opening contagiously for four months,mice were executed then the vagina and the surrounding tissues were took out and fixed in formaldehyde.Paraffin section and hematoxylin staining prepared,the morphological changes of vaginal epithelium and the underlying tissue were observed under a microscope.2,According to Sander-Cramer method,0.1ml semen was added into 0.5ml gels containing with different concentrations of RKBL.Recorded the lowest dose ofRKBL which causing death of sperms in 20 s and observe agglutination of sperms in different concentrations of the RKBL;At the bottom of the "V" tube,the volume of gel containing different concentrations of RKBL was added to observe the effect of killing or agglutining sperm.3.The effects of RKBL on termination of pregnant mice after 2 times repeated use RKBL for abortion then observe their third pregnancy and giving birth offsprings untill to 21d(weaning period),the babies development and related physiological and behavioral indexes were observed.4,Using fluorescein isothiocyanate(FITC)-RKBL lectin labeled as a probe to research the effects of uterine delivery(RKBL)on the changes and distribution of RKBL receptors or binding sites after 12 h treatment by RKBL.Results: 1.They were normal in all mice with long term(4 months)vaginal continuously using RKBL in vaginal,no any inflammation,disease,allergic reactions and other undesirable phenomena observed in vagina,vaginal mucosal epithelial cells were intact.2.When the concentration of RKBL in gel is higher than or equal to1mg/ml,it could make the sperm instant inactivation or death,that is the lowest dose of Sander-Cramer 20 s in death is 1mg/ml;When the concentration of RKBL was higher than 0.01mg/ml,different degrees of sperm agglutination were observed;3.Compared with the control group,the use of RKBL repeatedly treatment for abortion does not affect the third pregnancy in mice,however,the average litter size of mice number slightly decreased,the growth rate of babied slightly faster than the control group,there were no significant difference between them in the other indicators.4.RKBL receptors widely exists in mouse uterus,the uterine mucosa(i.e.endometrium)part of the fluorescence reaction is relatively weak,muscular layer of uterus and uterine serous parts were strong with fluorescence;after treated with RKBL,the distribution of receptor changes,uterine mucosa,myometrium and uterine serosa on the fluorescence intensity were different,the degree of enhancement of the fluorescence intensity of uterine wall muscle increased obviously.Conclusion: 1.Long term treatment with Red kidney bean lectin(RKBL)on the vagina will not cause adverse reactions on vaginal epithelial cells and under layers.2.The lowest concentration of RKBL gel for sperm death is 1mg/ml,and the low concentration of RBKL gel can make sperm aggregation in different degrees according to its concentration;in the "V" tube experiment,the concentration of RKBLgel can prevent sperm penetration;In mice,vaginal RKBL containing gel could stay in freely moving mouse at least 4h and play the effective role of contraception.3.Two times treatment for termination in the pregnancy mice were no obviously influence on further pregnancy and development of mouse babies,but the litter size decreased,no obvious effects on offspring growth,behavior and other physiological characteristics had been observed.4.RKBL receptor widely exists in mouse uterus,treatment with RKBL will induce the change in sites and distribution of RKBL receptors,uterine mucosa,myometrium and serosa were increased in different degree,which increase in uterine wall muscle is more obvious.