Study Of Expression Of HLA-G MRNA Isoforms In Human Preimplantation Embryos And The Effect Of LIF On HLA-G Expression And Related Signaling Pathway

Objective: 1. To investigate the expression of HLA-G isoforms in human preimplantation embryos. 2. To investigate the effect of LIF of HLA-G expression on protein level and the signaling pathways involved in LIF modulation of HLA-G expression.Materials and Methods: The frist part of the study: Human embryo mRNA was isolated using Dynabeads mRNA DIRECTTM Kit. Nested RT-PCR was performed using SuperScriptTM One-Step RT-PCR Platinum Taq Kit and Platinum PCR SuperMix High Fidelity Kit. Immunofiuorescence staining was used to detect HLA-G protein in human embryos.The second part of the study: JEG-3 trophoblast cells (an HLA-G expressing choriocarcinoma cell line) were grown in DMEM/F12 mediumand stimulated by Recombinant LIE Then they were labeled by immunofluoresence with anti-HLA-G monoclonal antibody MEM-G/9 and analysed by flow cytometry. JEG-3 cells were stimulated by Recombinant LIF and treated with or without AG490 or PD98059. The HLA-G protein level was analysed by flow cytometry.Results: The first part of study: 1. HLA-G mRNA isoforms in human cleavage stage embryos: A total of twenty individual cleavage stage human embryos were examined for HLA-G mRNA isoform expression by nested RT-PCR (five 2-cell, five 4-cell, five 6-cell and five 8-cell). Only 7/20 embryos expressed HLA-G 2. HLA-G mRNA isoforms in human morulas: Five individual day 4 human embryos were examined. All of them expressed HLA-G. 3. HLA-G mRNA isoforms in human blastocysts: Twenty five day 6 expanded blastocysts were studied. All of them were found to express HLA-G mRNA. G1 was detected in 20/25 (80%), G2 in 4/35 (16%), G3 in 25/25 (100%), G4 in 24/25 (96%), G5 in 5/25 (20%), G6 in 8/25 (32%). 4. HLA-G protein in human pre-implantation embryos: Twenty 2-cell to 8-cell human embryos were stained with MEM-G/9, a monoclonal antibody specific for HLA-G1 and G5. HLA-G labeling was found in 15/20 2-cell to 8-cell human embryos. HLA-G positive labeling was observed in all 3 morulas and 5 blastocysts examined.The second part of the study: The HLA-G protein level in JEG3 cells was evaluated by flow cytometry. It was found that recombinant LIF upegulated the HLA-G protein level in manner of both dose response and time course. This upregulation was blocked by PD98059, a specific inhibitor of MARK/ERK signaling pathway, but not AG490, a specific inhibitor of JAK/STAT signaling pathway.