The Expression Of AMH And Its Recepter Gene In Rat With The Development Of Polycystic Ovarian Syndrome(PCOS)

Polycystic ovary syndrome(PCOS)is a most complicated and common endocr-inopathy of women in reproductive age.,Which is caused by polygenic inheritance and environmental factors.The pathogenesis and etiology of this syndrome remain unclear.In recent years,the research point for the pathogenesis of PCOS gradually turned to the level of molecular biology,found that many cytokines,such as the anti-Mullerian hormone,insulin-like growth factor(IGF),tumor necrosis factor(THF),adiponectin.leptin,and visfatin,ect.are involved in the pathogenesis of PCOS,especially AMH.in addition to sexual differentiation,also participating in the egg development,maturation,ovulation,fertilization ect..It was reported that serum AMH can not only predict ovarian reserve capacity,but also an independent predictor of in vitro fertilization and embryotransfer(IVF-ET),AMH is also important ovarian marker of ovarian granulosa cell tumor,taken as an useful tool for the diagnosis of polycystic ovary syndrome.In order to understand the pathogenesis of PCOS,we built polycystic ovary syndrome rat model to study the effects of dehydroeplandrosterone(DHEA)on reproductive organs development,hormones level in serum and AMH mRNA expression with the development of rat polycystic ovarian syndrome(PCOS).1.Identification and bioinformatics analysis of AMHR2 gene splices variantA novel splice variant was identified in the ovary of SD rat through RT-PCR,cloning and sequencing,named AMHR2-splice variant(AMHR2-SV1).Compard with AMHR2 reference sequence,it was deleted 137 bp(exon 10).Using NCBI ORF Finder and ExPASy-ProtParam tool,we found that.lacking exon 10 led to earlier termination codon.AMHR2-SV1 protein sequence deleted 128 aa and encoded 429 aa and the predicted molecular mass was 45.9907 kDa.Analysis of conserved domains suggested that AMHR2-SV1 lost part of the Serine/Threonine Kinase domain compared to AMHR2,owing to the absence of a new stop codon.RT-PCR was used to explore the expression of the potentiol splice variant in various tissues of rat.The tissue expression analysis indicated that both AMHR2 and AMHR2-SV1 transcripts could be detected in most rat tissues,including heart,liver,spleen,lung,kidney,adrenal,uterus and ovary,but the AMHR2 was the primary transcript;RT-PCR results showed that AMHR2 presented the highest expression in ovary and the lowest in liver,spleen and lung;while the AMHR2-SV1 was highly expressed in ovary and presented lower expression level in spleen.The mRNA for the spliced variants was relatively less abundant than AMHR2 mRNA in all tissues.The results show that AMHR2 gene was regulated by alternative splicing mechanism.The Serine/Threonine Kinase domain is the critical structures of AMHR2 and the AMHR2 is specific to AMH target tissues.AMHR2-SV1(without complete domain)may serve as a negative factor in AMH signaling.2.Building of Rat Polycystic Ovarian Syndrome(PCOS)model and related indexs detection.To investigate the relationships between organs and levels of hormones with the development of Rats Polycystic Ovarian Syndrome(PCOS)using DHEA.The organ coefficients and levels of serum hormone were compared at different ages of SD rats.Eighty immature female SD rats(21 d)were randomly divided into 2 groups:control group with no DHEA(n=40)and treatment group with DHEA(6 mg/100 g body weight,n=40).Rats were injected daily with 0.2 mL seame oil for up to 20 days.Ten rats of each groups were killed at 5,10.15 and 20 days.The blood samples were obtained,and organ indexes were calculated by weights of body,ovary,uterus,livers,kidney,and spleen.The result indicated that the body weights in treated groups were significantly higher than that in control group on both 15 and 20 days-old(P<0.01);Normalized by body weight,the uterus weights were significantly different((P<0.01);the ovarian weights in treated groups were higher than that in control groups on 5 and 10 days old(P<0.01).Serum AMH and T were significantly higher in treated groups than those in control groups(P<0.01 or P<0.05),whereas,the serum P level on]5 and 20 days old,LH and E2 levels on 20 days old were higher in treated group when compared with the control groups(P<0.01 or P<0.05).The levels of AMH mRNA were obvious different between the two groups,while the 5th and 10th days old were significantly higher(P<0.05).The dynamic development of organs coefficients suggested that the sexual organs were given priority to development during early stage of pre-puberty in treated group.Compared with the serum sex hormone,abnormal AMH production could be cause of pathogenesis and development marker of ovarian failure in PCOS rats.The expression of AMH mRNA in ovary and serum AMH level were not exactly the same.The results may supply the reference data for further research on PCOS in female rats.3.The expression analysis of AMH and its receptor mRNA during the development of PCOS rat.The expression levels of AMH,AMHR2 and AMHR2-SV1 during the development of rat ovarian PCOS were determined and their relationships were investigated by using real-time PCR in this experiment.The results obtained were as follows:1.With the same changing tendency between treatment and control group,the expression increasing of AMH mRNA is transient on the 10th day,and then decreased.Compared with the control group,the expression levels of AMH mRNA of treatment group were significantly higher on 5th and 10th days(P<0.01).2.AMHR2-complate and AMHR2-SV1 mRNA in the treatment group had the same changing tendencies.Compared with the control group,the expression levels were lower as compared with the control group except for the 20th day.Expression of AMHR2-SV1 mRNA were significant different between groups(P>0.05 or P>0.01),decreased until the 15th day(P<0.05).then increased on 20th day(P>0.05).The results suggested that exogenous DHEA may promote the expression of AMH mRNA and serum AMH at a time,but a double effect on AMHR2,especially AMHR2-SV1,may have some associations with rat PCOS.