The Expression Rule And Significance Of Calreticulin Of Early Pregnancy

Objective:To investigate the molecular mechanism of calreticulin(CRT) during the implantation process, RT-PCR,indirect immunofluorescence histochemistry, western blotting and hybridization in situ techniques have been applied to detect CRT mRNA and protein expression, distribution and dynamic change of CRT in endometria from non-pregnant mice and pregnant mice on day1 to day7, respectively.Methods:1. Found the animal model of NIH pregnant mice. Endometrium from day 1-7 in early pregnancy mice were used to following study. pregnant mice (d1, d2,d3, d4, d5, d6,d7) were randomly divided into 7groups and non-pregnant (d0) mice were control group.Both non-pregnant and pregnant mice endometria was collected, and then immediately stored -80℃for further test.2. The expression of CRT mRNA was deteced by RT-PCRT and hybridization in situ in non-pregnant and the endometria of early pregnant mice. 3. The expression of CRT protein was detected by western blotting and indirect immunofluorence histochemistry in non-pregnant and the endometria of early pregnant mice.4. CRT antisense oligonucleotide was injected into uterus horns of pregnant mice on day 3 and observed the number of blastocyst implantation at pregnant day 8.Result:1. The result our study showed that The expression of CRT mRNA in pregnant mice endometria is higher than that of non-pregnant(P<0.05). The results showed a gradual raise trend with pregnant days passed from day1 to day5 and reached the maximum level on pregnant day4,5.2. The result of hybridization in situ is identical with result of RT-PCR and positive expression of CRT was located in the cytoplasm of epithelia and stromal cells.3. By indirect immunofluorence histochemistry, the positive expression of CRT protein has been observed in endometria of pregnant and non-pregnant mice and mainly located in the cytoplasm of epithelia and stromal cells. The CRT expression of endometria in pregnant is higher than that of non-pregnant and reached the maximum level on pregnant day5. On pregnant day6 to day7, the expression level of CRT porotein is down-regulated, but there are no statistically significant difference compared with d4 and d5(P>0.05).4. The result of western blot is consistent with result of the immuno- fluorence histochemistry.5. The numbers of blastocyst in horn of uterus which was injected CRT antisense oligonucleotide is fewer than that of saline injected .Conclusion1. CRT mRNA and protein has observed in the endometria of non-pregnant and pregnant mice,and is higher in pregnant mice than that of non-pregnant mice.The result indicated that CRT might involved in the whole process of blastocyst implantation and play an important role.2. According to the high expression of CRT in implantation window(day 5) and its distribution rule in the endometria of early pregnant mice,we suggested that CRT might participated in the adhesive process of blastocyst implantation.It influences the action between trophoblast and endometrium to make the best reception of endometrium via promoting the adhesion of cell-extracellular matrix .3..CRT antisense oligonucleotide was used to hold back it`s transcribe and to furthermore block up its physiological function.This result of this study show that number of embryo implantation where injected antisense oligonucleotide is fewer than that of injection saline. It suggested that CRT might play an important roles.