Expression, Regulation And Function Of Polyamie-related Genes In Mouse Uterus During Peri-implantation Period

The polyamines,including putrescine,spermidine and spermine,exist widely in eucaryotic cells.Through binding different molecules,such as DNA and proteins,polyamines play key roles in cell proliferation and differentiation.Ornithine decarboxylase(ODC),which decarboxylates L-ornithine to form putrescine,is the rate-limiting enzyme of the polyamine biosynthsis.The rapid turnover of Odc protein is mediated by ornithine decarboxylase antizyme(Oaz).By a serial of biochemical reactions,polyamines are catalized by Odc as well as S-adenosylmethionine decarboxylase(Amd),spermidine synthase(Srm) and spermine synthase(Sms). Spermidine/spermine N1-acetyltransferase(Sat),peroxisomal N1-acetyl-spermine/spermidine oxidase(Paox) and spermine oxidase(Smox) are mainly in charge of polyamine metabolism.In this study,the expression of polyamine related genes in mouse uterus during peri-implantation period was examined by in situ hybridization,real-time RT-PCR and Western blot.By using mouse models of pseudopregnancy,delayed implantation and ovarian hormone treatments,the regulation on the expression of polyamine-related genes in uterus were also determined.The effects of Odc or polyamine levels on cell proliferation and the expressions of other polyamine-related genes were investigated in cultured uterine cells.The importance of Odc activity in embryo implanation was studied by treating pregnant mice withα-difluoromethylomithine(DFMO),a specific inhibitor for Odc.Our results showed that both uterine Odc mRNA and protein levels were strongly expressed at implantation sites on day 5 of pregnancy.The expression pattern of Oazl in uterus during days 1 to 5 of pregnancy was quite similar to Odc.The expression levels of Srm,Amd1,Sat and Smox were also up-regulated at implantation sites.Howev.er,there were no detectable signals of Sms and Paox mRNAs in uterus.In the uterus on day 5 of pseudopregnancy or under delayed implantation,the expression signals of Odc,Oazl,Srm,Amd1,Sat and Smox were hardly to be detected,but there were strong expression signals of these genes after the activation of delayed implantation.In the ovariectomized mice treated with estrogen and/or progesterone,Odc mRNA expression signals were strongly detected in uterus,but there were no detectable signals of Odc expression when co-treated with estrogen or progestone receptor antagonists.In estrogen-treated group,Oaz1 mRNA signals were evidently detected in uterus,but not in control or estrogen receptor antagonist-treated group.Besides,there were no expression signals of other polyamine-related genes in hormone-treated uterus.In cultured uterine cells,Oazl mRNA level was enhanced after the overexpression of Odc.Sat expression level was also up-regulated after administrated by exogenous spermidine or spermine. After administration of DFMO,the proliferation activity of uterine stromal cells was decreased, while the expression level of cell cycle regulator p21 was increased.The numbers of the mice with implantation sites were decreased significantly after injecting mice with DFMO during day 3 and/or 4 of pregnancy.The activation of delayed implantation was also inhibited by DFMO treatment.Nevertheless,trans-4-methylcyclohexylamine,a specific inhibitor for Srm,had no effect on embryo implantation.The expression level of Amdl at implantation sites was highly induced after the injections of DFMO,and Amdl mRNA level was also up-regulated by DFMO treatmen in cultured uterine cells.However,its expression level was decreased after the overexpression of Odc or putrecine treatment.These data demonstrated that the expression levels of Odc,Oaz1,Srm,Amd1,Sat and Smox were specifically up-regulated at implantation sites in a blastocyst-dependent manner,suggesting their important roles in embryo implantation.Furthermore,uterine expressions of Odc and Oaz1 were controled by steroid hormones.In cultured endometrial cells,expression levels of Oaz1,Sat and Amd1 were regulated by Odc or polyamines level.Finally,the embryo implantation was inhibited significantly by the injection of DFMO,indicating that Odc activity is essential for implantation.In conclusion,our results suggest that the coordinated expressions of polyamine-related genes in mouse uterus during early pregnancy may be important for implantation.