| BACKGROUND AND OBJECTIVES:Embryo implantation is an successive complex physiological process,which the embryo adhere to and implant into uterine.Embryo implantation not only is regulated by many factors from uterus and embryo,but also by a lot of biological moleculars,such as osteopontin, integrinανβ3 and so on.Osteopontin(OPN) is a secreted glycosylated phosphoprotein,which contains an Arg-Gly-Asp(RGD)sequence.It belongs to extracellular matrix protein(ECM).The original observation shows that OPN is expressed by bone tissue,and the subsequent research found that OPN is expressed in uterine endometrium of secretion phase, the invade trophoblastic cell,the glandular epithelium of deciduas and placenta.OPN is both cytokine and adhesion molecule.Although OPN binds to various integrin receptors,theανβ3 integrin has been recognized as a primary receptor for OPN.OPN binds primarily to integrin receptors via its RGD sequence,and mediates cell adhesion and migration.Theανβ3 integrin and osteopontin play an important role in embryo implantation.So it is necessary to establish an animal model and elucidate their mechanism in embryo implantation.In this project,(1) Mice early pregnancy model was established using PMSG and HCG.(2) OPN and integrinβ3 expression were detected in the peri-implantation mouse endometrium,and the expression profile of OPN and integrinβ3 were determined.(3) Inhibitory effect on mice blastocyst implantation was observed after intra-uterus injection of antibodies against osteopontin and integrinβ3. METHODS:1.Establishment of mice early pregnancy model20 mice at proestrus stage were equally divided into two groups:the induced ovulation group(PMSG+HCG) were injected with PMSG and HCG,and the control group was injected with saline solution.Mice were sacrificed at 8 days after pregnancy(n=10 per group;day 1 is served as the day of vaginal plug),and anatomized.The implantation number of mice blastocyst was counted.The pregnancy rates were got.According, estradiol and progesterone concentrations were detected in serum and endometrium tissue homogenate of mice.The morphology of endometrium and ovary was observed under microscopy.2.OPN and integrinβ3 expression were detected in the peri-implantation mouse endometrium.Of 44 mice,24 female mice at proestrus,20 male mice at sexual maturity.Female mice were injected with PMSG and HCG.Four female mice were sacrificed at estrus(Day0).20 female mice assigned to pregnant status were mated to intact fertile 20 males of the same strain. Uteri were then collected on Day 4,5,6,7,and 8 of pregnancy(n=4 per group;Day 1 defined as the day of vaginal plug).The implantation number of mice blastocyst was counted.Endmetrium tissues were stored at liquid nitrogen or 4%paraformaldehyde.OPN and integrinβ3 expression were detected using immunohistochemistry and western-blotting.OPN and integrinβ3 gene expression was analyzed by RT-PCR.3.Effect of intra-uterus injection of antibody against osteopontin and antibody against integrinβ3.20 mice were respectively injected with antibodies against osteopontin or integrinβ3.The control group was injected saline solution. No treatment served as the blank control group.Mice were sacrificed at Day 8 of pregnancy,and anatomized.The implantation numbers of mice blastocysts were counted.The endometrium morphology was observed under microscopy.RESULTS:1.Establishment of mice early pregnancy modelThe results showed that the pregnancy rate in PMSG+HCG group was 90%,and implantation number of mice blastocysts was 9.5±3.6.In the control group,the pregnancy rate was 60%,and implantation number of mice blastocyst was 3.8±3.9.Both pregnancy rate and the numbers of implantation sites were higher in PMSG+HCG group than the control group.In PMSG+HCG group,estrodiol and progesterone concentrations were also higher than the control group.2.OPN and integrinβ3 expression were detected in the peri-implantation mouse endometriumThe result of our study showed that the expression of OPN and integrin in mouse endometrium luminal epithelium on Day0 of pregnancy was very weak,OPN and integrinβ3 protein expression gradually increased along pregnancy process.OPN and integrinβ3 gene expression also increased along pregnancy process,the expression of OPN and integrinβ3 on Day5 was increased to the extreme,and but on Day6 was decreased,and on Day7or Day8 was decreased dramatically.3.To study the effect s of osteopontin and integrinβ3 antibodies on mice blastocyst implantation.The results showed that the pregnancy rate was 60%in osteopontin antiserum group,implantation number of mice blastocyst was 1.6±1.19. The pregnancy rate was 70%in integrinβ3-antiserum group,implantation number of mice blastocyst was 1.7±1.28.All of pregnancy rate and implantation number of mice blastocyst were significantly low in the osteopontin-antiserum and integrinβ3-antiserum group compared with N.S group(90%,10.0±1.63) and the blank control group(100%, 10.5±1.58).These suggest that osteopontin-antibody and integrinβ3-antibody block mice blastocyst attachment and implantation.CONCLUSIONS:1.Early pregnancy model of mice was established using PMSG and HCG injection.The mice pregnancy model using PMSG and HCG model is better than nature pregnancy one.Both pregnancy rate and the numbers of implantation sites were higher in PMSG+HCG group than the control.This model provides a novel technology for investigating mechanism of embryo implantation and uterine endometrial receptivity.2.Both gene and protein expression profile of osteopontin and integrinβ3 show that osteopontin and integrinβ3 increase along pregnancy process,peaked on Day 5.The expression of osteopontin and integrinβ3 in mouse endometrium frames the "implantation window", which is a candidate biochemical marker of uterine receptivity.This indicates that osteopontin and integrinβ3 involve in embryo implantation. This provides a novel clue for elucidating mechanism of embryo implantation.3.Osteopontin-antibody and integrinβ3-antibody could block mice blastocyst attachment and implantation,and induced the morphological changes of uterine endometrial.This indicates that osteopontin and integrinβ3 are associated with uterine endometrial receptivity at embryo implantation.Osteopontin and integfinβ3 may participate in mice blastocyst anchor into uterine.
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