Roles Of Pregnancy-associated Plasma Protein A And Protein O-fucosyltransferases In Embryo Implantation

I. ObjectiveEmbryo implantation and development is a complex biological process for theestablishment of the successful pregnancy. The zygote develops into morula (2-cellstage,4-cell stage and8-cell stage), then the early blastocyst, which transfers from theoviduct to the uterine cavity. In the uterine cavity, embryo implantation typicallyincludes blastocyst apposition, adhesion to the uterine epithelium and penetration intothe stroma of the endometrium. The formation of matured embryo and embryoimplantation are pivotal to establish and maintain the successful pregnancy in mammals.Embryo expresses and secretes a variety of molecules which are involved in the processof embryo implantation and development.Pregnancy-associated plasma protein A (PAPPA) was mainly secreted by placenta,and it could be detected in serum after the fifth week of pregnancy. It is significantlyincreased during the first trimester of pregnancy, and continuously elevates until the endof pregnancy. It showed that low maternal serum level of PAPPA is associated withabnormal embryo development, such as threatened abortion, in the first trimester ofpregnancy, which indicates that PAPPA has an important role in embryo implantationand development.Glycosyltransferases and its product play critical roles in embryo development,inflammatory reaction and tumorigenesis. Fucosyltransferases catalyze the synthesis offucosylated protein through N-linkage or O-linkage. Fucosyltransferases (FUTs) catalyze the synthesis of N-linked fucosylated protein, such as FUT4, FUT7, whileprotein O-fucosyltransferases (poFUTs) catalyze the synthesis of O-linked fucosylatedprotein, such as poFUT1and poFUT2. It reported that FUT4and FUT7were requiredin embryo development. However, the exact roles of poFUT1and poFUT2duringembryo migration and invasion have not been well characterized.Hormonal and cytokines are essential factors in embryo implantation anddevelopment. Among these factors, progesterone and leukemia inhibitory factor (LIF)play key roles in embryo implantation and development. Progesterone promotes embryodevelopment by increasing cell growth, and LIF regulates the uterine receptivity forembryo implantation. Progesterone and LIF regulate many down-stream geneexpressions through the receptors mediated pathway in embryo implantation anddevelopment.Here, we found the roles and regulation of PAPPA and poFUT1in embryoimplantation and development. We identified that PAPPA or poFUT1were up-regulatedby progesterone or LIF, and played important role in embryo implantation anddevelopment, which provided experimental evidence for clinical diagnosis and therapy.II. Methods1. The serum level of PAPPA, progesterone and LIF were measured by ELISAassay.2. The gene expression of PAPPA and poFUT1were analyzed by RT-PCR orReal-time PCR.3. The expressions of PAPPAand poFUT1protein were detected by Western Blot.4. The expression and localization of PAPPA, poFUT1and poFUT2were checkedby immunohistochemistry and immuofluorenscence.5. The recombinant vector or siRNAs were transfected into cells.6. The cell migration and invasion were detected by transwell assay.7. MMP-2activity was analyzed by gelatin zymography assay.8. The cell proliferation was detected by CCK-8and cell counting assay.9. The adhesion rate was measured by in vitro adhesion model. 10. The expressions of PI3K/Akt signal molecules were measured by WesternBlot.III. Results1. Pregnancy-associated plasma protein A up-regulated by progesterone promotesadhesion and proliferation of trophoblastic cells(1) Serum progesterone and PAPPAlevel correlated in pregnant women.(2) The PAPPA expression was high in villi tissue while not detectable in deciduastissue in both gene and protein level.(3) Progesterone up-regulated the expression of PAPPA in trophoblastic (JAR)cells.(4) Down-regulated PAPPA decreased the adhesion and proliferation oftrophoblastic cells.(5) Progesterone promoted trophoblastic cell adhesion and proliferation viaup-regulated PAPPAexpression.2. LIF promotes trophoblast cell migration and invasion through upregulation ofpoFUT1expression at fetal-maternal interface(1) PoFUT1was highly expressed in normal placental trophoblast cells from thefirst trimester, but lowly expressed in threatened abortion trophoblast cells.(2) The expression of poFUT2was no significantly changed in normal placentaltrophoblast cells and threatened abortion trophoblast cells.(3) Serum level of LIF in threatened abortion patients was significantly lower thanthat in normal pregnant women.(4) PoFUT1was expressed in trophoblast cell line.(5) Silencing of poFUT1suppressed trophoblast outgrowth in extravillous explantcultures and inhibited the invasion and migration of JAR cells.(6) Over-expression of poFUT1promoted migration and invasion of JAR cells.(7) LIF up-regulated poFUT1expression in JAR cells and promoted trophoblastoutgrowth in extravillous explant cultures.(8) LIF promoted invasion and migration of JAR cells through poFUT1. (9) Up-regulation of poFUT1by LIF promoted cell invasion and migration throughPI3K/AKT signal pathway.IV. Conclusions1. Pregnancy-associated plasma protein A up-regulated by progesterone promotesadhesion and proliferation of trophoblastic cells(1) The serum PAPPA level correlated with the serum progesterone level inpregnant women.(2) PAPPAwas expressed only in villi tissue.(3) Progesterone up-regulated the expression of PAPPAin JAR cells.(4) PAPPApromoted trophoblastic cell adhesion in vitro adhesion model.2. LIF promotes trophoblast cell migration and invasion through upregulation ofpoFUT1expression at fetal-maternal interface(1) The expression of poFUT1and LIF decreased in threatened abortion patients.(2) LIF promoted the expression of poFUT1in JAR cells.(3) LIF and poFUT1promoted trophoblast outgrowth in extravillous explantcultures and the invasion and migration of JAR cells.(4) Up-regulation of poFUT1by LIF promoted cell invasion and migrationthrough PI3K/Akt signal pathway.