Expression And Function Of P38 Mitogen-Activated Protein Kinase In Early Embryo And Peri-Implantation Endometrium In Mouse

Objective:Implantation is a key event of the human and mammals reproductive process,it is a complicated process involving the blastocyst adheres to and invades the maternal endometrium gradually at the specific step of implantation window.In the complicated process,a number of factors mediate a series of molecular biology events which include steroid hormones(estrogen,progesterone),growth factors,cytokines,adhesion molecules,proteinums,peptides and enzymes.At the subtle regulation of these factors,some intracellular signal conducting pathways were activated,and regulate the expression of special genes,to coordinate the development of embryo trophblast and the maternal endometrium, accordingly to make the well-developed blastocyst invade the endometrium successfully.Recently,although much progress on the mechanism of blastocyst implantation have been made,many key questions are still to be further explored.For example,what intracellular signal conductiong pathways were activated? The signals activating these conductiong pathways come from maternal or embryonic body,or from both ? How is a relationship between various intracellular signal conductiong pathways during implantation? and etc.MAPK pathways mediate some cellular processes,including cell proliferation,growth,differentiation,apoptosis and death.The MAPK superfamily of proteins consists of four separate signaling cascades:the c-Jun N-terminal kinase or stress-activated protein kinases (JNK/SAPKs),the extracellular signal-regulated kinases(ERKs),the ERK5 or big MAP kinase 1,and the p38 MAPK group of protein kinases,all of which are highly conserved throughout eukaryotic systems.These proteins and enzymes make a signaling cascades which is interlace each other, and play an important role in the intracellular signal transmission.In recent years,studies oversea showed that MAPK pathway play important roles in many process such as embroygenesis,uterus decidualization,blastocyst adhesion,trophoblastic cell proliferation and invades the maternal endometrium.The p38 MAPK signaling cascade regulates a variety of cellular activities in different cells and tissue types.It is influenced by a variety of stimulus including growth factors,cytokines,and pathogens. So it is considered as the center of the intracellular signal transmission.Lately,there was study showing that p38MAPK is activated in mammalian perimplantation development,and its activation is required by uterus decidualization,This idea is not yet reported in domestic literature. Studies both about the expression of p38MAPK in peri-implantation endometrium and injection inhibitor SB203580(specific inhibitors of p38 MAPKα/β)in vivo to interfere with implantation are not reported at home and abroad.To further explore the effect of p38 MAPK on mouse embryo development and implantation,the methods including immunohistochemistry,embryo culture in vitro and intrauterine injection in vivo were used in our experiments in order to further understand the mechanism of implantation, enrich the theory of implantation in reproductive biology,facilitate the successful rate of assisted reproductive technology such as IVF-ET and offer reliable experimental evidence and animal models for developing new types of contraceptives.Methods:1.Pregnant and pseudopregnant uterus of 1～8 days in mice were obtained.The specimens were sliced into section(of 5um) Immunohistochemistry method and image analysis were employed to explore the expression and change rules of p38MAPK in peri-implantation mouse endometrium,uterus of nonpregnancy as control.2.Early embryos were obtained at the following stages:two-cell, four-cell,eight-cell,morulae and blastocyst.The expression and changes of p38MAPK in early embryos were examined by immunohistochemistry and immunofluorescence.3.Interfering experiment by p38MAPK inhibitorsIn vivo:Mice were obtained on day 4 of pregnant,intrauterine in jection of p38 MAPK inhibitors were used and morphological and histological methods were employed to observe the embryo number of implantation on day 8 and the variances in the embryo and the endometrium.In vitro:Murine embryos were flushed from oviducts of timed pregnant mice at the two-cell stage(48 h post-hCG),pooled,washed,and divided into five groups.They were cultured with different culture treatments:①1:1DMEM/F12 alone,②1:1DMEM/F12 + DMSO(inactive analogue),③1:1DMEM/F12 + 0.2μmol/L SB203580,④1:1DMEM/F12 + 2.0μmol/L SB203580, and⑤1:1DMEM/F12 + 20μmol/L SB203580.Embryos were assessed for morphology and progression through cleavage divisions at 24 h postdrug treatment(72 h post-hCG),at which point half of the embryos in groups 3,4,and 5 were removed from culture,washed,and placed in fresh medium culture drops for the remainder of the experiment.All embryos were then assessed at 48 h postdrug treatment(96 h post-hCG)to observe the developmental information(from cleavage to blastocyst).Cell viability and cell numbers were determined at this time by assaying for uptake of a vital dye(trypan blue).The expression of p38MAPK in blastocysts of each treated group were examined by immunofluorescence.Results:1.The p38MAPK-positive staining was found in the cytoplasm of the lumina epithelium,glandular epithelium and decidual cell.In the pregnant group,p38MAPK-positive expression in lumina epithelium was weak at 3 days and peak at 4～5 days in the endometrium of pregnant mouse, and then declined:in glandular epithelium,p38MAPK-positive expression is observed from 4^thday and then keep at this level,p38MAPK-positive expression in decidual cell is increased gradually from the 5^thday of gestation.In the pseudopregnant group,weak staining of p38MAPK was only observed on the 4^thday of gestation,and negative staining were observed on the other days.p38MAPK was expressed weakly in the endometrium of nonpregnant mouse.2.The p38MAPK-positive staining was found in the pre-implanted embryos and gradually increased with development;3.Interfering experiments:In vivo:The average of embryo-implantation(2.6250±0.5175)in p38MAPK inhibitor(10μM)treated uterus were significantly lower than that of control uterus given saline solution(6.2500±0.8864),P＜0.01.The microscopic observation demonstrated that the embryo degenerated,and even necrosed in some parts.In the decidual cells there was obvious adipose degeneration,while many blood cells invaded the embryos and the decidua.In vitro:Two-cell stage embryos cultured in the presence of SB203580(specific inhibitors of p38 MAPKα/β),progressed to the eight-cell stage with the same frequency as controls;however,treated embryos halted their development at the 8- to 16-cell stage.In inhibitor-treated groups,the embryos were composed of a mixture of compacting and noncompacting cells, and the embryo development were delayed for one to two cell cycle than that of controls.Treated embryos remained viable as the developmental blockade was rescued by removing embryos from the drug treatment and placing them in drug-free medium until they progressed to the blastocyst stage.Conclusions:1.p38MAPK activity may be required to support embryo development through the murine preimplantation interval.2.p38MAPK,in addition to correlating to blastocyst implantation,may participate indirectly in the process of endometrial decidualization.3.The expression of p38MAPK in mouse endometrium is regulated not only by maternal factors(steroid hormones),but also by the implanted embryos.4.p38MAPK inhibitors can inhibit the blastocyst implantation and the development of embryos and decidual cells in vivo.