The Study On The Relationships Between Lysophosphatidicacid Receptor 3, Cyclooxygenase-2, Pinopodes And Endometrial Receptivity In Mouse

Embryo implantation is one of the vital factors for successful pregnancy and the establishment of endometrial receptiblity is the determinative factor for a well embryo implantation. As a result, the reseach on endometrial embryo receptiblity is very important. The endometrial receptiblity is a special condition of endometrium, during this period a series of changes in morphosis and secreted protein within the endometrium occurre under the control of ovarial steriod homone. As a consequence the allowance is available of apposition, adhesion, invasion and embryo implantation by the alteration in endometrial mesenchymal. This special period aslo be called"the window of implantation". The implantation procedure can accomplish in this short peroid by the reaction between endometrium and embryo. On the contrary, the implantation is impossible before or behind this peroid. It can be drawed from researchs on endometrial receptiblity that many factors play important roles in the implantation procedure, which include steroid hormone, growth factor, cell factor signal, adhesion molecule etc. those factors expression and action in different time and under the control of different space enviromental form the molecular basis of embryo implantation. The expression level of any factors is abnormal will contribute to change the enviroment of endometrium and the endometrial receptivity and lead to infertility. The knowledge about the mechanism of implantation has not completely understood. It becomes the great handicape to improve the assisted reproductive technology level. To explore the pathway about the signal that participate the moderation procedure of the endometrial receptiblity will benefit us to get information about the failure of embryo implantation and increase the pregnancy rate accordingly.Lysophosphatidic acid (LPA)-a new growth factor, has been founded by Mills in abdominal fluids resulted from ovrian cancer, is a kind of derivation of cell membran lipid functioning as an intercell phospholipid signal. LPA, secreted by actived blood platelet, cancer cell, fibroblast and adipocyte or produced by some inflammatory cell and endothelial cell, exists in blood serum, ocular humor and blood plasma. LPA mainly exhibit multiple biological functions by connecting to the special G-protein couple receptor in the target cell that invovlved in many cellular processes including cellular survice, proliferation, differentiation, cytoskeletal reorganization, interaction with cell and tumour formation and cancer cell invasion and so on in many physiological and pathological process.There are at least four receptors for LPA, LPAR1-4. LPAR1 and LPAR2 are expressed in almost all tissues,but LPAR3 shows a limited expression pattern in the ovary, heart, testes and prostate. The lately research showed, LPAR1 and LPAR2 are participate in the development of neurology and the formation of craniofacial. They are closely correlated with neurologic disease. LPAR3 in particular is important in female reproductive system: The female mouse whose PLAR3 gene have been knocked out appeared a delayed embryo implantation, abnormal space distribution, decreased expression of COX-2 gene. COX-2 is a rate-limiting enzyme of prostaglandin synthesis. The fact that giving exogenous PGE2 and cPGI can compensate the delayed embryo implantation in LPAR3 gene absent showing the closed relationship between LPAR3, COX-2 and embryo implantation. Nowadays, the majority of researchs on LPA and its receptor were focus on the function in tumor invasion and migration. The latest study indicated that high level expression of PLA and its receptor LPAR2, LPAR3 can promote cancer cellular proliferation, migration, adhension and induce the activation of matrix metalloproteinase (MMP) and the secretion of angiogenesis factor in objects with ovarian cancer. The process of implantation was similar, in some sense, to the process of tumor cell invasion and migration. In the generation domain, there is lack of information about the function of LPAR3 on establishing endometrial receptiblity and embryo implantation until now. Pinopodes is a smooth bigger size prominence formed by endometrial epithelium. The occurrence of completely developed pinopodes during implantation peroid is regarded as the morphological characteristic of endometrial receptiblity which existed only for 24 hours and match the endometrial receptivity period. In rodent, pinopodes accordance to the window of implantation. In huaman, mature pinopodes are seen only during a brief window following the midcycle luteinizing hormone (LH) surge between LH+6 and LH+8. It is the period that blastocyst begin to attach to endometrium. As an ultramicrostructure marker of implantation window openning, about pinopodes there have many correlated studies in its function on reproduction. But no literature can be found at home and abroad about the relationship with LPAR3.Controlled ovarian hyperstimulation (COH) can get many ovum by using ovulation stimulants, is an important element in determination of whether the IVF-ET can success or not. Long protocol controlled ovarian hyperstimulation is the most commonly used protocol by Gonadotrophin releasing hormone agonist. Researchs demonstrated that this plan can improve the quality of ovum and got many ovum but compared with the high retrieval rate, the clinical pregnancy rate still in a low level.This may be because that the GnRHa long time superovulation program put a negative effect in the balance of internal hormone which affected the morphous and the secrete function of endometrium and resulted in asynchronization between the developtment of the endometrium and embryo. Decrease the endometrial receptiblity. Now the mechanism about the relationship between COH and decreasing in endometrial receptiblity is still unclear. So to evaluate the mechanism of decreasing in endometrial receptiblity and find an effective method to solve the problem plays a significent role in increasing the pregnancy rate by IVF-ET. Now it is absent in the document about the temporal and spatial interrelation with GnRHa long protocol controlled ovarian hyperstimulation on the expression of LPAR3 and pinopodes in mouse uterine endometrium during periimplantation periode. In view of that, to study the expression of LPAR3 and COX-2 in uterine endometrium and the development of pinopodes during the periimplantation of mouse and the relationship of coexpression will provide clue of theory for the building endometrial receptivity and reveal the maybe molecule mechanism of endometrial receptivity.The specimens of humen are limited, kunming mice and human are all mammals, the procedure of embryo implantation is similar, so we select kunming mouse as our study objectives.The partΙ: The expression of LPAR3 and COX-2 in uterine endometrium during the period of periimplantation of mouse embryos and the development of pinopodesObject: To evaluate the dynamic change of lysophosphatidic acid receptor 3 mRNA, protein and distribution. The expression and location of COX-2 in mouse uterine endometrium during the period of periimplantation; To evaluate the pinopodes development in window of implantation.Method: The expression of LPAR3 mRNA and protein in uterine endometrium tissues during periimplantation of pregnant and pseudopregnant mice were detected by using semiquantitative of PT-PCR, Western blotting and immunohistochemistry methods. The COX-2 expression and location was detected by immunohistochemistry SP methods. Using scanning electronmicroscope to observe the development of pinopodes during periimplantation peroid.Result: LPAR3 mRNA and protein were positive in endometrium of unpregnancy, pregnancy and pseudopregnancy mouse. LPAR3 protein mainly distributed in luminal and glandular epithelium and part of stromal cells of mouse uterus. Around the embryo implantation procedure in mouse, the level of LPAR3 mRNA and protein began to increase on 3 days post coitus (3d.p.c), peak around 4d.p.c and returned suddenly to basal level on 5d.p.c and 6d.p.c. The temporal profile of LPAR3 mRNA in the uterus of pseudopregnancy mouse was similar to those observed in normal pregnant mouse. There was no significant different between the two groups ( P > 0.05). The tendency of LPAR3 protein in pregnant and pseudopregnancy was regularity to LPAR3 mRNA. The COX-2 mainly existed in luminal, glandular epithelium vascular endothelial cell and part of stromal cell, didn't express in normal and before 3d.p.c, but began to express on 4d.p.c and extend to stromal cell along with the progression of pregnancy. The expression of COX-2 increased and deepened positive staining in stromal cells. The mode expression of COX-2 in pregnancy mouse was similar to that of pseudopregnancy. The mode of pinopodes: developing pinopodes appeared on 3d.p.c; mature pinopodes on 4d.p.c and degenerated pinopodes on 5d.p.c.which was coincidence with the expression peak of LPAR3 in uterine endometrial. The pattern was similar in pregnancy and pseudopregnancy.Conclusion: LPAR3, COX-2 and pinopodes may participate in the building of endometrial receptiblity. The concordance of LPAR3 and pinopodes was in the temporal and spatial and could be as a marker in judgement of endometrial receptibity. There was no relationship between the expression of LPAR3, the development of pinopodes and whether or not the presentation of embryo in uterus.That showed they were mainly regulated by matermal signal. COX-2 possiblely participated in the procedure of embryo implantation.The partΠ: The regulatory effect of estradiol progesterone on the expression of LPAR3 in uterine endometrium tissue and Ishikawa cell linesObject: To investigate the regulatory effect of estradiol, progesteronge and progensterone antagonist RU486 on the expression of LPAR3 gene and protein in uterine endometrium and Ishikawa cell lines.Method: Ovariectomized kunming mice were randomly divided into four groups: estradiol group, progesterone group, combined with estradiol and progesterone group, progesterone and RU486 group.The expression of LPAR3 mRNA and protein in uterine endometrium were detected by using semiquantitative of PT-PCR, Western blotting methods. The highly differentiated endometril adenocarcinoma cells(Ishikawa cell line) were cultured in vitro with 17-beta estradiol (E2)(10-9M, 10-8M, 10-7M), progesterone (10-7M, 10-6M, 10-5M), E2 combined with progesterone (10-8M+10-6M), for 48 hours respectively and the expression of LPAR3 protein was detected by immunocytochemistrical methods.Result: There was a positive LPAR3 mRNA expression in ovariectomized Kunming mouse endometrium tissue; Progesterone alone could improve the expression of LPAR3 mRNA in mouse endometrium markedly and showed a time dependent; Estradiol alone could not put significantly effect on LPAR3 mRNA expression; Estradiol combined with progesterone could counteracted the up-regulation effect of progesterone; RU486 could decrease the up-regulation effect of progesterone. The tendency expression of LPAR3 protein has a positive correlation with the mRNA. There was no significant effect on the expression of LPAR3 protein in Ishikawa cell lines by variety concentration of estradiol, progesterone, estradiol combined with progesterone.Conclusion: In vivo animal experiment, progesterone alone could increase the expression of LPAR3, but estradiol alone had no effect on it. Estradiol combined with progesterone could counteracted the up-regulation effect of progesterone; RU486 could decrease the up-regulation effect of progesterone. There was no significant effect on the expression of LPAR3 protein in Ishikawa cell lines by estradiol and progesterone.The partШ: The effect of long protocol controlled ovarian hyperstimulation by GnRHa on the expression of LPAR3 and the development of pinopodes in mouse uterine endometrium during periimplantation periodObject: To investigate the influence of controlled ovarian hyperstimulation by GnRHa/HMG/HCG long protocol treatment to the expression of LPAR3 mRNA and protein and the development of pinopodes; To evaluate the maybe molecular mechanism of adverse effect on endometrial receptivity by COH which may provide theory for selecting an ideal programe of ovarian hyperstimulation in clinical. Method: Healthy femal unpregnant Kunming mice were randomly divided into COH group and natural conception group(NC).The expession of LPAR3 mRNA and protein in uterine endometrium tissue was detected by PT-PCR and Western blotting during the periimplantation period. The development of pinopodes on 3d.p.c~6d.p.c by using scanning electron microscope. The estrogen and progesterone level in mouse blood serum was detected by Abbott Laboratories A4sym automatic immune radiomete.Result: The peak expression of LPAR3 mRNA and protein on 3d.p.c in COH group, which was similar to natural conception group on 4d.p.c. There was no significant difference in other days. In the COH group, on 3d.p.c, the development of pinopodes was out-of-step, which characteristic of variation in size and in alignment. Some were mature pinopodes, most were developing ones, which had an uneven surface and microvilli. The number of pinopodes in COH group was less than in natural conception group and the style of expression was focal. On 4d.p.c, the development of pinopodes were derangement and the size was vary and were out of sync. On 5d.p.c, there were few degenerated pinopodes and most endometrium was covered with microvilli cells. In natural conception group: on 4d.p.c, there were a lots of coincidence pinopodes, the development was synchron, homogeneous distribution, equal size, smooth surface and looked like mushroom which progectured the surface of cells. On 5d.p.c , the surface of pinopodes were introcession and were degenerated pinopodes. The concentration of estrogen and progesterone in blood serum ascensus obviously in both groups with the progress of pregnancy, but it was higher in COH group than in natural conception group at each time. There was significantly different between two groups.Conclusion: COH impacted the balance of endogenous hormone in mouse which resulting in an abnormal higher level of estrogen and progesteron. COH altered the endometrial receptibility through changing the relative factors for implantation and the change of ultrastructural in the temporal and spatial.