| Background: Blastocyst implantation is one of the earliest events in reproduction of humans and mammals that determines whether pregnancy will develop successfully or not. Successful implantation depends on invasive embryo, acceptive endometrium and their synchronization. There is an extremely short period in uterine receptivity for blastocyst implantation, which is called"implantation window". Implantation in all mammals involves shedding of zona pellucida, followed by orientation, attachment and adhesion, and invasion of the blastocyst to the endometrium. Blastocyst implantation is involved in the regulation of large number of genes and the enrollment of complicated regulatory systems. The mammalian uterus changes dramatically in response to the blastocyst implantation during the early pregnancy, and apoptosis plays an important role in this process[1][2].Animal studies showed that apoptosis of luminal epithelial cells facing the trophoblasts could exclude the barrier of blastocyst permeation and the regression of the decidual cells allowed a restricted and coordinated invasion of trophoblast cells into the maternal compartment.[3] Apoptosis is in connection with retaining mater-fetal immune tolerance, too. Endometrial apoptosis, triggered by blastocyst through paracrine secretion or justacrine, starts with apposition of the blastocyst and adhesion of the trophoblast in the receptive uterine epithelium and is more dramatic in the uterine endometrial epithelia surrounding the blastocyst[4]. P16INK4A gene is considered that it could prompt aging through its inducing apoptosis. Recently, P16INK4A gene was detected during"implantation window"in mouse endometrium. However, there is no report about the expression rule of P16INK4A gene in mouse endometrium, which is a meaningful subject.Objective: To investigate the expression of P16INK4A gene in mouse endometrium during early pregnancy and to analyze its role in blastocyst implantation.Methods:1. Mouse endometria of un-pregnancy and early pregnancy were gathered in the study (d0, d2-d7). There were 7 groups, and each group contained 20 mice. P16INK4AmRNA expression was detected in mouse endometrium by Real-time fluorescent quantitative PCR (FQ-PCR).2. Mouse endometria of un-pregnancy and early pregnancy were gathered in the study. There were 7 groups, and each group contained 20 mice. P16INK4A protein expression was detected in mouse endometrium by immunohistochemistry.3. Mouse endometria during the early pregnancy were gathered in the study. There were 6 groups (d2-d7), and each group contained 20 mice. P16INK4A protein expression was detected in mouse endometrium by Western Blot.4. To analyze the influence of P16INK4A on embryonic implantation, the uterine of the pregnant mice was injected with P16INK4A antibody and the number of implanted blastocysts was recorded.Results:1. FQ-PCR: P16INK4AmRNA was expressed in mouse endometrium during early pregnancy. The expression of P16INK4AmRNA in pregnant groups was higher than that in un-pregnant group (P<0.05) and appeared an increased trend as time passing by, reaching the maximum at pregnancy d5.2. Immunohistochemistry: P16INK4A protein was located in kytoplasm and apical surface of luminal epithelium and gland epithelium, and in stromal cell during pregnancy d3-d6. Immunohistochemical analysis showed the same result as FQ-PCR.3. Western blot: The expression of P16INK4A protein in pregnant groups appeared an increased trend as time passing by, reaching the maximum at pregnancy d5.4. The number of implanted blastocysts in the left horn (P16INK4A antibody) was less than in the right horn (normal saline) in the experiment group (P<0.05)Conclusion: P16INK4AmRNA and protein in mouse endometrium during early pregnancy were higher than of un-pregnancy, and they were extremely expressed during d3-d6. The apoptosis of luminal epithelial cells and decidual cells induced by P16INK4A might participate in the process which endometrium changes to the receptive one to accommodate blastocyst implantation.
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