| Objective①To investigate the expression of macrophage migration inhibitory factor (MIF) in the peri-implantation mouse endometrium;②To investigate the effects of ISO-1,a selective macrophage migration inhibitory factor (MIF) tautomerase activity inhibitor,on the mice embryos implantation and growth;③To investigate the change of the morphology and the expression of MIF in mice endometrium after treating with ISO-1 before implantation. Methods We took mature and nulliparous Kunming female mice as subject, and three Kunming adult female mice were caged with a male overnight. The following morning, females were checked for the presence of a vaginal sperm plug. The day of vaginal plug was regarded as day 1 of pregnancy. Firstly,120 mice of days 1-8 of pregnacy were put to death through cervical vertebrae luxation, then the uteri were disemboweled and the unpregnant mice uteri was(estrous stage)taken as controls. The uteri were fixed in 4% paraformaldehyde. Immunohistochemical SP staining and in situ hybridization were used to measure the expression of MIF protein and mRNA respectively. Pictures were taken by microscopic imaging system and then analysed by computer. Another 120 pregnant mice were randomly divided into four groups (low-dose group, middle-dose group, high-dose group and control of 1% DMSO). Low- dose group, middle-dose group, high-dose group were given ISO-1 (2, 6, 18 mg/kg) by i.p. injection on the day 3 of pregnancy and the control group was treated with the equal volume of 1% DMSO. Half mice were killed by the same method above on the day 4 of pregnancy, then the uteri were taken and fixed in 4% paraformaldehyde. HE staining was used to observe the changes of structure of mice endometrium on the day 4 of pregnancy . In mice endometrium on day 4 of pregnancy, the expression of MIF protein and mRNA were studied by immunohistochemical SP staining and in situ hybridization. On the day 8 of pregnancy, the other half was killed and the number of the embryos and the uterine organ coefficient were calculated. Results①The results of immunohistochemisty and in situ hybridization showed that the expression of MIF protein was parallel to that of mRNA. MIF was detectable in each group mice endometrium and more marked in the glandular epithelium and luminal epithelium and cell aggregates scattered throughout the stroma. The expression of MIF was predominant in the control group. Compared with the control, during the pregnancy of days 1-3, the expression of MIF was stronger and then decreased obviously on days 4-5 in luminal epithelium and glandular epithelium of uterus. On days 6-8 the expression of MIF gradually increased again. With the decrease of endometrial glands and the closing of uterine lumen, MIF was mostly expressed in the decidua basalis and the secondary decidual zone(SDZ) besides glandular epithelium and luminal epithelium.But the expression of MIF was negative in the primary decidual zone (PDZ). Furthermore, MIF was expressed strongly in the developing embryos after implantation. Image analysis showed that the mean optical density (MOD) of MIF in each group of days 1-3 and 6-8 of pregnancy had no significant difference, compared with the control(P>0.05). And there were no significant differences among the six groups(P>0.05). Compared with the control and days 1-3 and 6-8 of pregnancy, the MOD of MIF in each group of days 4-5 of pregnancy had significant difference(P<0.05). But there were no significant differences between days 4 and 5 of pregnancy(P>0.05).②In the control group treated with 1% DMSO, the number of the embryos was 10.27±1.71 and the uterine organ coefficient was (1.037±0.201)%.In the low-dose group, the number of the embryos was 10.33±1.80 and the uterine organ coefficient was (1.040±0.235)%. There were no significant differences in the number of the embryos and the uterine organ coefficient between the low-dose group and the control(P>0.05). In the middle-dose group, the number of the embryos was 10.93±2.09 and the uterine organ coefficient was (1.119±0.244)%. Compared with the control, there were also no significant differences in the number of the embryos and the uterine organ coefficient(P>0.05). In high-dose group, the number of the embryos was 12.40±2.16 and the uterine organ coefficient was (1.094±0.190)%. Compared with the control, the number of the embryos were distinctly increased(P<0.05),but there were no significant differences in the uterine organ coefficient(P>0.05).③Effect of ISO-1 on morphological changes of mice endometrium and the expression of MIF: HE staining showed that the structure of mice endometrium in each group had no distinct difference. The results of immunohistochemisty and in situ hybridization showed that MIF protein and mRNA were both detectable in the glandular epithelial cell and luminal epithelial cell in each group mice endometrium. Compared with the control , the mean optical density (MOD) of MIF protein and mRNA in the low-dose group,middle-dose group and high-dose group had no significant differences respectively (P>0.05). Conclusion①MIF is expressed continuously in mouse endometrium on day 1～8 of pregnancy, which suggests that MIF be involved in the process of implantation and embryo development of mouse after implantation.②ISO-1 may promote mice blastocysts implantation with a dose-dependent manner.③ISO-1 may improve mice blastocysts implantation by antagonizing the biological function of MIF. |
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