1. Association Studies Between Complex Phenotypes In Response To Male TU Contraception And Polymorphism Of Aromatase Gene 2. Study Model Of Uterus Bleeding-like Changes In Mice Using RU486

From a public health perspective,the need for contraception has been greater. For man,although there existed few male-specific methods(withdrawal,condoms, and vasectomy),the shortcomings of the existing methods not being negligible,and so some new male contraceptive methods were in researching such as hormonal contraceptive,contraceptive vaccine and so on.Recent studying have demonstrated that the hormonal contraception is the new choice for male contraceptive methods,which is the most prospective in clinical application.Investigators have sought to develop a male hormonal contraceptive based on the observation that spermatogenesis depends on stimulation by gonadotropins,follicle-stimulating hormone(FSH) and luteinising hormone(LH). Most clinical trials demonstrated that exogenous high-dosage testosterone combined with or without progestin is a safe,effective and fully reversible method of male contraception.Compared with withdrawal and condoms,the participants' satisfaction with testosterone contraception was good.In those completed clinical trails,however,a consistent finding in these studies is that azoospermia is achieved in only 40- 70%of Caucasian men and 97%of Chinese, and the remainder show exhibited various degrees of oligozoospermia.This diverse polymorphism of response to TU were observed inter- and intra-races.The explanation for the nonuniform induction of azoospermia by testosterone remains unknown.Therefore,understanding the basis of the heterogeneity in response would not only provide important insights into the physiology of the male reproductive system but could,as a second step of development,also lead to a male contraceptive tailored to individual requirementsIn this study,based on extensive reviewing research of male hormone contraception,it is indicated that estrogen is important for spermatogenesis and is a major regulator of the gonadal-pituitary feedback for the gonadotropin axis in male hormone contraception with exogenous high-dosage testosterone.In other words, androgen contraceptives may inhibit spermatogenesis by negative feeback effect on hypothalamus-pituitary-testis axis of estrogen transformed from testosterone.Aromatase is the key enzyme responsible for the formation of estrogens from androgens.The aromatase gene encoding aromatase P450(estrogen synthetase) is expressed in several extragonadal sites and regulated in a tissue-specific fashion. Activation of each promoters give rise splicing of these untranslated alternative exons to form the mature transcript occurs at a common 3'-splice junction that is upstream of the translational start site.This means that although transcripts in different tissues have different 5'-termini,the coding region and thus the protein expressed in these various tissue sites are always the same.However,the promoter regions upstream of each of the several untranslated first exons have different cohorts of response elements,and so regulation of aromatase expression in each tissue that synthesizes estrogens is different.In human males,a number of tissues have the capacity to express aromatase and hence synthesize estrogens including testes and numerous sites in the brain such as several areas of the hypothalamus,limbic system,and cerebral cortex.So,we set out association studies between complex phenotype in response to male TU contraception and polymorphism of aromatase gene aiming at providing clues to explain the diversity of contraceptive action among the subjects using present TU contraceptive regimen.Methods:In this research,to identify and characterize of cyp19 gene polymorphism was performed by denaturing high-performance liquid chromatograph and sequencing in the population of which used TU contraception from China Hebei. The sequence of special promotor PI.f with 5' flanking region and all the cyp19 coding exons with some up and downstream sequences respectively were analysed. Then,we study the association within those found variation allele and phenotype in the population from hormone contraception by using ANOVA and some bioinformatic tools such as haplotyping etc.Finally,to determine whether we found the sequences strong related with nonuniform induction of azoospermia by testosterone in PI.f 5'flanking region can binding a certain transfactor,the electrophoretic mobility shift assay(EMSA) was used in analysis of DNA-protein interactions.Results:In our study,fourteen variable sites were identified within a span of 7kb and deep divergence in the distribution of some SNPs was noted.We have performed an association study using these 14 biallelic polymorphism of cyp19 and determined there are two loci are associated with variation in height in 142 adult males from Han Chinese(p＜0.05).In association analysis within pheonotype and polymorphism of cyp19 gene used with haplotyping and haplotype blocking,we identified 15 haplotypes and 4 common haplotyps represent 81.8%of the study population with 8 variate allelels.Moreover,a haplotype block with two loci was found strong association with diverse induction of azzospermia by testosterone,which located that PI.f 5'flanking region.EMSA conformed that this special region in PI.f 5' flanking region can binding a certain protein.Conclusion:These observations indicate that in men,genetic variation in cyp19 are involved in determining normal adult height,and contribute to the diverse induction of azoospermia by testosterone.In PI.f promoter region,there exits a transcript regulation region which have two found viriate site.Moreover,the analyses of haplotype and haplotype block defined by the grouping and interaction of several variants rather than any individual SNP correlated with complex phonotypes is more effective. Humans and old-world primates differ from most other animals in that their endometrium undergoes bleeding,shedding,remodelling and subsequent regeneration if pregnancy is not established at the opportune time.Human endometrium is thus a dynamic tissue that,to prepare for implantation,undergoes well-defined cycles of proliferation,differentiation,and shedding(menstruation) in response to the prevailing endocrine and paracrine environment.The endometrium is consequently a site of recurrent physiological injury and repair。Research into menstrual mechanisms is hindered by the lack of availability of rodent models to assess the role of various factors in regulating menstrual function.Up to now,there are two reports about mouse menstrual-like model by physical progesterone withdrawl.In some clinical researching,RU 486,given to normally cycling women at midluteal and lateluteal phase,provokes uterine bleeding,shedding and menstruating in advanced.So,we set out study on model of uterus bleeding-like changes in mice by using mifepristone.Methods:Ovarectomized NIH female mice were administered with mifepristone following oil-induced decidualization.Morphology,hormone levels were evaluated at an interval of 8 hours in 48 hours after treatment and bleedings were monitored by using vaginal smears examination.Expression ofVEGF mRNA and protein were evaluated by in situ hybridization and immunohistochemistry during menstrual-like progressing in mice model.Results:Decidual endometrium was breakdown drastically by 16 h,and shed by 24h accompanying the bleeding.The endometrium regenerated from 24 h and restored completely by 48 h.Progesterone levels in serum remained invariable,estradiol levels in serum increased during the process.VEGF mRNA and protein were localized in both the stroma and glandular epithelium.Expression is increased in the decidual endometrium zone during early bleeding and in remnant stroma zone and regenerated epitheliums fllowing that.The high levels of VEGF were correlated with the increase of serum estradiol levels in control uterus com.Conclusions:Menstrual-like changes in artificial decidualization mice were provoked by using mifepristone which mimic withdrawal of progesterone on pharmacologicl level.The expression of the vascular endothelial growth factor suggested that VEGF plays an important role in bleeding and regeneration in the endometrium in mice menstrual-like model using mifepristione.