Effects And Regulatory Mechanisms Of Gja1 During Uterine Decidualization In Mice

Decidualization,a process during which uterine stromal cells undergo extensive proliferation and differentiation into decidual cells,is essential for continued embryonic development and successful pregnancy.Inadequate decidualization causes embryo miscarriage and early pregnancy loss irrespective of whether development of the blastocyst is normal.It has been previously reported that Acvr1 played an important role in modulating uterine decidualization.Conditional ablation of Acvr1 in the uterus exhibited decidualization failure in response to an artificial stimulus.Although microarray analysis of oil-induced deciduoma in Acvr1-deficient mice has demonstrated that decidualization-related gene Hand2 was down-regulated,the underlying molecular mechanism of Acvr1 remains poorly understood.Gap junctions,which are membranous channels for the exchange of small molecules directly between/among adjacent cells or between cells and their extracellular environment,are critical for decidualization,because its blockade could suppress the proliferation and differentiation of uterine stromal cells.Cx43 was the principal and most well-studied component of the gap junctions.Loss of Gja1 that codes Cx43 in mouse uterus led to severe fertility defects due to the impaired stromal responsiveness to implanting embryo and deciduogenic stimulus accompanied with defective angiogenesis.Similar results have been obtained in human endometrial stromal cells where attenuation of Gja1 expression disrupted the gap junctional communication between neighboring cells and impeded the differentiation of stromal cells.However,there is still very limited information available regarding the regulatory mechanism of Gja1 in the process of decidualization.This study was designed to investigate the expression,function and regulation of Gja1 during peri-implantation period in mouse uterus.In situ hybridization was performed to examine the distribution of Gja1 mRNA in the uterus of early pregnancy,delayed and activated implantation and artificial decidualization.The results found that no obvious Gja1 mRNA signal in the uteri from days 1 to 4 of pregnancy.On day 5 of pregnancy,Gja1 mRNA was highly expressed in the subluminal stroma surrounding the implanting blastocyst,but weakly detected at the inter-implantation sites.From day 6 to 8 of pregnancy,a high level of Gja1 mRNA signal was observed in the decidua.Under in vivo artificial decidualization,Gja1 expression was significantly elevated.Gja1 could induce the proliferation of uterine stromal cells and enhance the expression of Ccnd3 and Cdk4.Inhibition of Gja1 with specific siRNA could reduce the expression of Prl8a2 and Prl3c1 which were two well-known differentiation markers for decidualization,whereas overexpression of Gja1 could enhance their expression.Further analysis revealed that Gja1 might act downstream of Acvr1 and c AMP to regulate the differentiation of uterine stromal cells.Administration of cAMP analogue 8-Br-cAMP to Acvr1 siRNA-transfected stromal cells resulted in an obvious increase of Gja1 expression,whereas PKA inhibitor H89 impeded the induction of Gja1 elicited by Acvr1 overexpression,indicating that cAMP-PKA signal mediates the regulation of Acvr1 on Gja1 expression.In uterine stromal cells,knockdown of Gja1 blocked the cAMP induction of Hand2.Moreover,siRNA-mediated down-regulation of Hand2 impaired the stimulatory effects of Gja1 overexpression on the expression of Prl8a2 and Prl3c1,while constitutive expression of Hand2 reversed the inhibitory effects of Gja1 siRNA on stromal differentiation.Meanwhile,Gja1 might play a vital role in the crosstalk between Acvr1 and Hand2.Collectively,Gja1 may act downstream of cAMP-PKA signal to mediate the effects of Acvr1 on the differentiation of uterine stromal cells through targeting Hand2.