Expression And Effects Of L-Selectin And Its Ligand During Embryonic Implantation In Mice

ObjectivesEmbryonic implantation is the key step of human and mammal pregnancy,and is controlled by the concerted reactions between invasive blastocyst and receptive endometrium. Embryonic implantation includes the following processes:the development from ovigerm into blastula in the"window phase", the removement of zona pellucida,the interaction between trophoblastic cells and decidual cells (which induce the recognition,apposition,and invasion of blastocyst to the endometrium).The"window phase"is regulated by a lot of regulatory factors, but the mechanism is still unclear. Recently, the bind between adhesion molecules and corresponding glucoprotein ligands is considered as one of the most important steps during embryonic implantation.To our knowledge,there are no exact reports about the expression and effect of L-seletin (one adhesion molecules),SLeX (ligand of L-selectin in mouse endometrium) and FucT-VII (key synthetase of SLeX) on the embryonic implantation.It may be helpful for uncovering the molecular mechanism on the concerted reaction between blastocyst and endometrium by study the expression of three molecules above and their effect on embryonic implantation.MethodsPart one1. Real-time fluorescent quantitative PCR (FQ-PCR) was applied to detect L-selectin mRNA expression in mouse ovigerms on day 1 to day 4 of pregnancy.2. Immunofluorescence (IF) was used for location and semiquantitative analysis of L-selectin protein in mouse ovigerms on day 1 to day 4 of pregnancy.3. To analyze the influence of L-selectin on embryonic implantation, uterine of pregnant mice was injected with L-selectin monoclonal antibody and the number of implanted sites was recorded.Part two1. RT-PCR was used to semiquantitatively analyze expression of FucT-VII mRNA in mouse endometria on day 1 to day 5 of pregnancy.2. Immunohistochemistry was adopted to locate SLeX protein expression in mouse endometria on day 1 to day 5 of pregnancy.3. Western Blot was used to detect expression of SLeX protein in mouse endometria on day 1 to day 5 of pregnancy.4. To analyze the influence of SLeX on embryonic implantation,the uterine of the pregnant mice was injected with SLeX monoclonal antibody and the number of implanted embryonics was recorded.ResultsPart one1. FQ-PCR:L-selectin mRNA is expressed in different stages of ovigerms, and gradually increased with the ovigerms differentiation and reached the maximum on the blastocyst stage (P<0.05).2. IF:Expression of L-selectin in uni-cellular- and four-cell-stage is few and most located in cytoplasm, but during morula stage it was expressed in the perimeter region of ovigerms and the strongest expression could observed in trophoblastic cells of blastocyst stage (P<0.05).3. L-selectin antibody interference in vivo:the number of implanted embryonics in the experiment group (injected with L-selectin monoclonal antibody) is fewer than that of control group (injected with saline solution).The number of the implanted embryonics showed a decreasing trend as the antibody concentration increasing (P<0.05).Part two1. RT-PCR:FucT-VII mRNA expression was detected in mouse endometria from day 1 to day 5 of pregnancy. It showed an in increasing trend, arrived at the maximum on day 4 and then decreased.2. Immunohistochemistry:SLeX expressed in endometrial cavity and glandular cells from day 1 to day 5 of pregnancy.3. Western Blot:SLeX expressed in mouse endometria from day 1 to day 5 of pregnancy.Its expression showed an increased trend,arrived the highest level on day 4 and then decreased (P<0.05).4. SLeX antibody interfere in vivo: the number of implanted embryonics in the experiment group (injected with SLeX monoclonal antibody) is fewer than that of control group (injected with saline solution). The number of the implanted embryonics showed a decreasing trend as the antibody concentration increasing (P<0.05).ConclusionExpression of L-selectin in ovigerms,SleX and FucT-VII in mouse endometrial,indicate that L-selectin might involved in the differentiation and exocytosis of ovigerms.The exocytosis could bind the ligands or receptors in mouse endometria, and then regulate receptive capability for embryonic implantation.The results showed that the expression of SleX and FucT-VII reached the highest level in mouse endometrium on day 4 of pregnancy and L-selectin had the strongest expression on blastocyst stage. When L-selectin was blocked by its antibody,we found the number of the implanted embryonics showed a decreasing trend as the antibody concentration increasing. We also found that the embryonic implantation could be blocked by SleX antibody.We speculate that L-selectin and its ligand SLeX might play a part in the signal transduction between blastocysts and endometrium via ligand-receptor interaction.This might be one important regulation pathway for the embryonic implantation including the position,adhesion and invasion of blastocysts and endometrium.