The Expression Of Placenta-specific 8 (PLAC8) In Human Early Embryonic Development And In-vitro Implantation

BackgroundPlacenta-specific 8 (PLAC8) was shown to be located and enriched in the placenta of mice in recent years. It was reported in early embryonic development of zebrafish and placenta development of mice. A number of research results show that PLAC8 may be a key molecule in the process of bovine embryonic implantation, and it can be used as one of the biomarkers to predict pregnancy outcome of bovine. However, little is known about PLAC8 expression in human oocytes and embryos.ObjectiveTo explore PLAC8 expression at molecular and cellular level in the process of early embryonic development and in-vitro implantation of human, to provide theoretical support for further function study.MethodsWe utilized human oocytes and embryos that could not be used in the infertility treatment and had been abandoned and donated for research by patients who had undergone IVF-ET therapy. We first extracted RNA from single oocyte,2-,4-,8-cell embryo, morula and blastocyst, total thirty-three samples, including three oocytes.six every stage embryos (three normal fertilized, three abnormal fertilized), then applied the quantitative polymerase chain reaction experiments for quantitative analysis, to detect PLAC8 mRNA expression; Using embryo immunofluorescence staining technique, a total of fifty-three human oocytes/embryos, including twelve oocytes, four 2-cell embryos, four 4-cell embryos, eight 8-cell embryos, seven morulae and eighteen blastocysts, were stained to detect PLAC8 protein expression and location; Embryo-endometrial stromal cell co-culture models were subsequently established; To investigate the expression of PLAC8 on implantation,25 hatched blastocysts were co-cultured with artificial decidual endometrial stromal cells and the implanted models were stained to detect whether PLAC8 protein is expressed and its expression pattern.Results1. There was no PLAC8 mRNA expression in all mature oocytes,2-,4-,8-cell embryos, abnormal fertilized morulae. In normal fertilized morulae, PLAC8 mRNA detection rate was 33%, in blastocyst,100%. There was no statistical differencein PLAC8 mRNA relativeexpression between normal and abnormal fertilized blastocysts.In the process of human early embryo development, proteasome subunit beta 6 (PSMB6) was more stablethan ACTB (beta actin).2. The immunofluorescence assay confirmed the presence of PLAC8 protein in the cytoplasm of all human oocytes and embryos preceding implantation.A distinct, stronger immunoreactivity was exhibited in the cell cortex, which diminished toward the cell center and disappeared completely in the nuclear region.3. Successfully developed human endometrial stromal cells-human blastocysts co-culture model. The satisfied decidualization was induced with 1μM medroxyprogesterone 17-acetate and 0.5 mM 8-Br-cAMP in 96 h.4. PLAC8 protein was transported into the nucleolus following blastocyst implantation and invasion into endometrial stromal cells.Conclusions1. In humans, PLAC8 mRNA is detectable in morulae and blastocysts, and PLAC8 protein is located in the cytoplasm of oocytes and preimplantation embryos.2. Human endometrial stromal cells-human blastocysts co-culture model was a powerful tool to study the process of human embryonic in-vitro implantation.3. After the blastocysts implanting and invading endometrial stromal cells, PLAC8 protein was located in the nucleolus, relating to the state of embryonic implantation.