Spatiotemporal Expression And Function Of Bone Morphogenetic Protein 7 In Decidual Cells In Mice

Objectives: Embryo implantation is an important and complicated physiological process in viviparous mammalian and human reproduction, which is also a precisely regulated and quickly accomplished physical phenomenon synchronized with development of maternal uterus and embryo at a particular time named "implantation window". Pregnancy needs extensive reconstruction of uterus as well as growth and differentiation of endometrial cell, which is decidualization. In addition, the decidualization process involves the interaction and regulation of adhesion molecules, proteolytic enzymes, growth factors, cytokines, vasoactive factors and multiple genes. Comparative research shows BMPs were expressed during embryo development in mice. To further study the expression pattern and roles of BMP-7 in uterus, we use various approaches including primary cell culture, quantitative PCR, immunohistochemistry, immunocytochemistry, western blotting. We detect the levels of BMP-7 mRNA and protein in the pregnant mouse uterine endometria in order to explore the role of BMP-7 in decidualization process.Methods: 1. RT-PCR and immunohistochemistry were applied to analyze BMP-7 mRNA and protein in mouse uterine endometrium from day 5 to 8 of pregnancy. 2. Mouse uterine stromal cells were isolated from uterus of pregnant day 4 and cultured in DMEM containing 10% fetal bovine serum. Decidualization of stromal cells was induced using progesterone, estradiol, and cAMP followed by transfected with 1.0 ng/ml, 10 ng/ml, 100 ng/ml recombinant BMP-7 adenovirus, respectively. After 72 h of culture, the expression of prolactin, a typical marker of decidual cells, was detected by RT-PCR and Western blotting. 3. Ovariectomized mice were randomly divided into 3 groups. Each group were subcutaneously injected with sesame oil (0.1ml/mouse), progesterone (2mg/mouse), RU486 (2mg/mouse) and progesterone (2mg/mouse), respectively. All mice were killed at 0h, 6h, 12h, 24h after injection to collect the utera samples in order to achieve uterine RNA and total protein from which BMP-7 levels was later examined by quantitative PCR and Western blotting.Results: 1. The expression level of BMP-7 mRNA was higher in the implantation sites than that in inter-implantation sites during day 5 to 8 of pregnancy (P<0.01), and the level increased gradually as days passed by in the implantation sites (P<0.05). BMP-7 protein was expressed in the decidualizing stromal cells tightly surrounding the blastocyst at day 5 and in the primary decidual zone at day 6. Progessively, the positive detection appeared mainly in the secondary decidual zone especially in the stromal cells of mesometrial pole at day 7 and day 8. 2. During decidualization of stromal cells, the expression of prolactin protein in recombinant BMP-7 adenovirus group were significantly increased (P<0.01), especially in 10 ng/ml group (P<0.01). 3. Progesterone was demonstrated with ovariectomized mouse models to up-regulate the expression of BMP-7 mRNA (P<0.01). RU486, an specific antagonist of progesterone receptor, is capable of abrogating progesterone effect on BMP-7.Conclusions: BMP-7 gene was demonstrated to be involed in inducing the decidualization of uterine stromal cells in early pregnant mice and this effect was regulated by progesterone.