Effects And Mechanism Of Estrodiol And Progesteron On Mouse Uterus Macrophages

BACKThe uterus is the site of embryonic growth and development. Macrophages (M (?)) derived from blood monocytes, is resident immune cells of the uterus, participating in the local uterine immune response, and immune regulation of mammalian reproductive function, and is very important in pregnancy establishment and maintain. Domestic and foreign research, studing the density, spatial-temporal distribution patterns, migration, differentiation and activation of the uterine macrophages in person or mouse with normal menstrual cycle or estrous cycle, prompted that the function and distribution of macrophages may directly or indirectly regulated by the ovarian steroid sex hormones-estrogen and progesterone.Estrogen and progesterone plays important roles in pregnancy establishment and maintenance. Estrogen can thicken the endometrium, improve the sensitivty of endometrium for implantation,which is important for the start of embryo implantation. Progesterone is secreted by the luteal cells in the non-pregnant mouse.It can further thicken the endometrium in the proliferative phase, so to provide a suitable environment for the survival and implantation of the fertilized egg, reduce the excitability of the uterine muscle cell membrane, inhibit the rejection of the mother to the fetus, and lower uterine sensitivity to oxytocin. The ’window’ of opportunity for embryo implantation arises as the culmination of a spatially and temporally coordinated series of adaptations in the sequential changes in the structure,cellular composition and expression of genes in the endometrium to allow embryo implantation. At present, a large number of clinical studies suggest that excessive levels of estrogen and progesterone in IVF cycle can impair endometrial receptivity, and lead to low clinical pregnancy rate. The abundance and distribition of macrophages in uterine during peri-implantation is highly time and space specific.Abnormal phenotypes or altered abundance of uterine macrophages could contribute to impaired endometrium receptivity. At present, there are a lot of research on the effects of estrogen and progesterone on endometrial macrophages, but the results of this studies are still controversial.More over, there is no domestic research on comparing the effect of super-physiological levels of estrogen or progesterone on uterine macrophages number and distribition in uterine with the effect of physiological levels.In this study, ovariectomy mouse model were used. Inject the mouse with added different concentrations of estrogen or progesterone, study the effect of physiological levels and super-physiological levels of estrogen or progesterone on uterine macrophage migration, distribution and its mechanism was discussed. There are a critical means in the study of mechanisms of maternal-fetal immune tolerance, moreover to provide a new way for transplantation immunology research in some extent.OBJECTIVEThe aim is to establish the ovariectomy mouse model, and to study the changes of amount of macrophages in uterine and the expression of M-CSF with injection of progesteron or estrogen of different concentration and it’s mechanism.METHODSNormal mouse were under go with ovariectomy. The operated mouses were divided into one control groups and five experimental groups. The experimental groups were injected subcutaneously with does of100ng estrogen,1000ng estrogen,2mg progesterone,20mg progesterone,0.1ml sesame oil (negative control group) respectively.The mouses were killed6or24hours after injection,and the uterus and blood were conllected for examination.Using HE staining to observe the uterine morphology and structure.Detection the macrophages by the methods of immunohistochemistry in the uterus to study the change and distribution of and the expression of M-CSF..RESULTS1. The results of morphology, HE staining of mouse’uterus and concentrations of estrogen and progesteron showed that the ovariectomized mouse model was successful.2. The result of immunohischemistry showed that few macrophages were observed in uerine in blank control group and negative control group, and there is no significant different in the amount of macrophages between these two groups.Compared with negative control group, significant increased in the amount of macrophages was observed in mouses6h and24h after injected with100ng estrogen and1000ng estrogen(P=0.000,0.003). There is no significant increase in the amount of macrophages in other two groups(P=0.499,0.225). Analysis of the numbers of macrophages in each part of uterine of the mouse indicated that compared with negative control group, there is no significant difference in the numbers of macrophages in each part of uterine of the mouse6h and24h after injected with100ng and1000ng estrogen(in both100ng group and1000ng group, the number of macrophages significantly increased in endometrium, myometrium and perimetrium after6h injection and significant increase were observed only in myometrium and perimetrium after24h injection).3. The result of immunohischemistry showed that, compared with negative control group, significant increased in the amount of macrophages was observed in mouses24h after injected with2mg progesterone and mouses6h after injected with2mg progesterone. There is no significant icrease in the amount of macrophages in other groups.Analysis of the numbers of macrophages in each part of uterine of the mouse indicated that compared with negative group, there is no significant increase in the numbers of macrophages in each part of uterine of the mouse6h after injected with2mg progesterone, however significant increase was oberved in endometrium and myometrium24h after. In uterine of mouse injected with20mg progesterone, the numbers of macrophages significantly increased only in myometrium6h after injection.4. The result of immunohischemistry showed that M-CSF expression observed in endometrial glandular epithelium, luminal epithelium, vascular endothelium, myometrium, and perimetrium. Blank control group and negative control group, M-CSF express less, and no significant difference observed between the two groups. Compared with negative control group, the expression of M-CSF in uterine were significantly increased6h and24h after injiection of100ng or100ng estrogen(p=0.001,0.008). Analysis of the expression of M-CSF in each part of uterine of the mouse indicated that after injected with100ng estrogen or1000ng estrogen, M-CSF expression in endometrium, myometrium and perimetrium significantly increased6h and24h after injection. There is no significant difference in M-CSF expression in each part of uterine between100ng estrogen injection group and1000ng estrogen injection group at6h and24h after injection.5、The result of immunohischemistry showed that M-CSF expression observed in endometrial glandular epithelium, luminal epithelium, vascular endothelium, myometrium, and outer membrane. Blank control group and negative control group, M-CSF express less, and no significant difference observed between the two groups. Compared with negative control group, both of2mg and20mg progesterone can significantly improve the expression of M-CSF in uterine24h after injiection. Analysis of the numbers of macrophages in each part of uterine of the mouse indicated that after injected with2mg progesterone, only endometrium M-CSF expression significantly increased6h after injection, and M-CSF expression in endometrium, myometrium and perimetium significantly icreased24h after injection. There is no significant increased in M-CSF expression in each part of uterine untile24h after injected with20mg progesterone.4. The change tendency was consistent in the number of macrophages and M-CSF expression in the whole uterus. Howerver the number of macrophages and M-CSF expression levels in mouse uterus was not significantly correlated (estrogen group:r=0.498, P=0.152; progestrogen group:r=-0.051, P=0.889).CONCLUSIONS1、Both physiological dose and super physiological dose estrogen or progesterone can increase the number of macrophages in mouse uterine.2、Super physiological dose progesterone can advance the time macrophages migrated to uterine and change their distribution, which may be a mechanism super physiological level progesterone affect the endometrial maturation leading to impaired endometrial receptivity.3、Both physiological dose and super physiological dose estrogen or progesterone can improve the expression of M-CSF, and there is no significant difference observed in M-CSF expression between physiological dose and super physiological dose.4、The change tendency was consistent in the number of macrophages and M-CSF expression in the whole uterus. The number of F4/80macrophages and the expression of M-CSF in mouse uterus was not significantly correlated, which suggest that estrogen and progesterone regulated the number and distribution of uterine macrophages by regulating many factor expression in uterine.