Expression Of Cyclin-E In Mouse Endometrium And Its Effect During Blastocyst Implantation

BackgroundBlastocyst implantation is a complex and significant vital process that the embryo invasion into the endometrium by interaction between embryo and endometrium. It is one of the earliest events in reproduction of humans and mammals that determines whether pregnancy will develop successfully. Successful implantation depends on invasive blastocyst, acceptive endometrium and their synchronization. Initiation of embryo implantation occurs at the blastocyst stage and there is a short period defined'window'of uterine receptivity for implantation, the so-called"implantation window". The regulation of early embryo development and the detailed molecular mechanism of implantation by which embryos invade into the uterus remains poorly understood, due to the large number of genes and the complexity of the systems involved. Numbers of researches showed that there are striking similarities between normal embryonic development and neoplasm metastasis, many of protooncogene and tumor suppressor gene which producing a marked effect in tumor plays an important part in blastocyst nidation. So protooncogene and tumor suppressor gene is becoming hot spot in blastocyst implantation .The Cyclins, which plays an important part in cell multiplication as a protooncogene, has been highly thought for its roles in blastocyst implantation.Cyclin-E is a neucleoprotein which controls the G1 cell cycle. The Cyclin-E gene is located on human chromosome 19q12～13, including 4 exons and 3 introns, encodes a protein with 395aa. The molecular weight of Cyclin-E protein is about 5×10kD, contains a highly stenoplastic area with 87aa called"cyclin box". This"cyclin box"remained in every species can conjunction with CDK (cyclin-dependent Kinases).The express of Cyclin-E gene steps up at the intermediate G1 stage, and reaches a peak at G1/S juncture of the advanced G1 stage. Then it is decreased with the cell enter in S stage and drop to nought at G2/M stage. A research of Wei Yu et al indicated that the Cyclin-E gene expressed negatively in normo-endometrium. It has been discovered that Cyclin-E shows a hypso-expression at the embryo implantation situs of pregnancy mouse by Reese J et al with the technique of gene-array. This study manifests that Cyclin-E maybe one of the regulating factor of embryo implantation. So it is a significant study to investigate the expression rule of Cyclin-E in the endometrium of early pregnant mice.ObjectiveTo investigate the expression rules of Cyclin-E in the mice endometrium during the early pregnancy and its roles in mice blastocyst implantation.Methods1. Mouse endometrium in pregnant mice on days 1,2, 3, 4, 5, 6, 7 of pregnancy were used to study. There are 7 study groups, signed D1~7, each group has 10 mice. RT-PCR (Reverse transcript-polymerase chain reaction) was applied to detect Cyclin-E mRNA expression in endometrium from pregnant mice on days 1~7.2. Collect uterus of pregnant mice on days 1-7 to study. There are 7 study groups, each group has 4 mice. Immunohisto-chemistry techniques was applied to detect Cyclin-E protein expression in endometrium from pregnant mice on days 1~7.3. The difference of implanted blastocyst number was registered on the pregnant d8 by injected Anti-cyclin-E antibody in horn of uterus at 20:00 on day 3 of pregnancy.Result1. RT-PCR: Cyclin-E mRNA were detected in the mice endometrium throughout the days 1~7 of pregnancy. The expression of Cyclin-E gene in pregnant mouse were steady increasing from day 2 of pregnancy and reaching the maximal level on day 5 of pregnancy, then it were decreased from day 6 of pregnancy and remained more express than day 1 of pregnancy. The maxlmum of express was on day 5 of pregnancy, comparing with other groups (P<0.05). 2. Immunohistochemical analysis showed the same result as RT-PCR. Cyclin-E protein extensive express in endometrium of mouse , especially in the epithelial cell, while stromal cell also express considerable Cyclin-E protein, the positive chromatosis present at cellular nucleus. On day 1 of pregnancy there were small amounts of Cyclin-E protein expressed at glandular epithelium, on day 2 of pregnancy the major express located at glandular epithelium and lumina epithelium. Then, from day 3 to day 5 of pregnancy the positive chromatosis appeared extensively at glandular epithelium and lumina epithelium and stromal cells, increasing with the days up. On day 6 of pregnancy the positive express at stromal cells decreased, but it was still intensive expressed at glandular epithelium and lumina epithelium. On day 7 of pregnancy there were only slight positive chromatosis at lumina epithelium.3. The Anti-cyclin-E antibody were injected into uterine horn of pregnant mice on days 3 of pregnancy and the number of implantation sites was recorded on day 8, the results showed that the number of Anti-cyclin-E antibody injection group significantly decreased, comparing with saline injection (p<0.05).ConclusionCyclin-E were persistently expressed in mouse endometrium during the early pregnancy and showed a maxlmum express on nidation window phase (the day 5 of pregnancy). It might be concerned with proliferation and differentiation of endometrium tissues, and facilitated the decidualization of endometrium. Interrupted by Anti-cyclin-E antibody, the number of implantation sites was significantly decreased, manifesting that Cyclin-E might participate in the regulation process of mouse blastocyst implantation.