Physiological Significance Of Pcgf2 During Decidualization In Mice

Pcgf2(Polycomb group ring finger 2)belongs to Polycomb gene family,which has the functions of regulating various signaling pathways,cell cycle,target gene expression,and protein ubiquitination modification.At present,there is no report on the function of Pcgf2 in the reproductive system.In this study,uterine-specific Pcgf2 knockout mice was used as a model to research the physiological function of Pcgf2 in the period of peri-implantation during pregnancy.The main research results include the following aspects.First,mice with uterine loss of Pcgf2 have significant deficiency of fecundity.We used the technique of in situ hybridization and Western blotting to find that Pcgf2 was expressed during the process of mouse uterine receptivity establishment and decidualization.Subsequently,we crossed Pgr(Progesterone receptor)-Cre mice with Pcgf2-floxp mice to obtain uterine-specific Pcgf2 knockout mice.Female knockout mice mating with wild-type male mice resulted in a significant reduction in the number of offspring mice.Second,Pcgf2 depletion leads to decidual defect and abortion in the mid-gestation.Knockout mice showed normal embryo implantation on day 5 of pregnancy,and there was no significant difference in the number of implantation sites with control mice.We found that the expression patterns of marker genes for uterine receptivity establishment on day 4 of pregnancy and marker genes for embryo attachment on day 5 of pregnancy in knockout mice were similar with those in control mice.The results indicated that uterine loss of Pcgf2 underwent normal embryo implantation.However,during the process of uterine decidualization,from day 7 of pregnancy,the decidual volume and weight in knockout mice were significantly lower than that in control mice.Moreover,abortion occured in the middle of gestation in knockout mice.Third,uterine Pcgf2 deficiency exerts ectopic expression of ERa(Estrogen receptor a)in decidua,and the reproductive defects can be rescued by ERa antagonist,ICI 182 780.To determine the effect of Pcgf2 deletion on decidual development in mice,we examined the expression of several uterine decidual marker genes,such as Bmp2,Hoxa10,Cx43.These genes displayed no significant abnormalities in the knockout mice.Thus,we examined the expression of genes that should be down-regulated during decidualization,such as Esr1.The results revealed that there was no difference of Esrl mRNA level between knockout mice and control mice,but the protein level of ERa was significantly increased in knockout mice.In addition,supplementation with proper doses of ICI 182 780 from day 6 to day 8 of pregnancy could rescue the decidual defects in knockout mice.Fourth,Pcgf2 interacts with ERa directly and regulates ligand-dependent ERa ubiquitination and degradation.To investigate the mechanism by which Pcgf2 regulates ERa protein stabilization,mouse uterine stromal cells were isolated and then treated with estradiol.We found that the degradation rate of ERa protein was significantly reduced in knockout mice.Subsequently,we blocked the intracellular protein degradation in mouse uterine stromal cells with proteasome inhibitor MG 132 treatment and then examined the ubiquitination level of ERa.The results revealed that ERa protein ubiquitination level was significantly decreased in the Pcgf2 deficient uterine stromal cells.Moreover,co-immunoprecipitation results showed that Pcgf2 regulated ERa ubiquitination by interacting with ER? directly.Fifth,ERa inhibits angiogenesis and secondary decidual cell differentiation during decidualization.In order to clarify how the high ERa protein level inhibits the process of uterine decidualization in vivo,we investigated the mesometrial and anti-mesometrial decidual zone separately.In the mesometrial side,the vascular branches were reduced,accompanied with the decreased expression of angiogenic marker genes such as Vegfa,Angpt2,Fgf2 and Fgf10,which could be rescued by ICI 182 780 supplementation.In the anti-mesometrial side,the proportion of polyploidiztion decidual cells significantly reduced,and the expression of cell differentiation-related marker genes such as Prl8a2,Prl3c1 were significantly decreased,which could also be rescued by ICI 182 780 supplementation.In summary,this study shows that Pcgf2 interacts with ERa directly and regulates ligand-dependent ERa ubiquitination and degradation.The excessive level of ERa protein in the knockout mice further hindered the vascularization in the mesometrial side and secondary decidual cell differentiation in the anti-mesometrial side during the process of mouse uterine decidualization,which led to miscarriage in mid-gestation and infertility.The uterine-specific Pcgf2 knockout mouse provides a good animal model for exploring the clinical recurrent abortion.Meanwhile,the abnormal expression of Pcgf2 and ERa also provides a new clinical diagnosis method for recurrent miscarriage caused by uterine decidualization defects,and supplys a solid theoretical basis for the therapy of related pregnancy diseases.