The Effects Of EEs On The Androgen Receptors In Mouse Genital System

Objective: There was great concern that the incidence of congenital disorders of male sexual differentiation was rapidly increasing. It suggests these could be made relationgship that the mother or the fetal exposed to the environment estrogen(EEs) in fetal period . However, all the mechanism how the EEs affect the genital system remains unclear. Recently, some studies indicated that the genitofemoral nerve (GFN) affected by the androgen secreted by testis, could has the effect of gubernaculum testis contraction and form exercise through the neurotransmitter calcitonin gene-related peptide(CGRP) released by GFN nerve fiber. The CGRP was believed directly effect gubernaculum testis as the CGRP receptor was also found in gubernaculum testis. Based on these studies, a new mechanism of"testis-GFN-gubernaculum testis"axis was found for the testicular descent and development. EEs was believed a course of factor which affects the genital system development. It is not now know whether EEs affect the genital system development through this axis above. This experiment was designed to apply the DES, a well-known EEs to the pregnant mice, and than study the expressions of androgen receptors (AR) in the male offspring testis, gubernaculum testis and also, the motor neurocyte of GFN in spinal cord. The aim of this study was mainly attempted to make sure whether EEs affect the"testis- GFN-gubernaculum testis"axis when the genital system was influenced by EEs?Materials and methods: 200 8-10 weeks old Kunming female mice were divided into eight groups randomly, that is, normal group (not given any drug), control group (given DMSO and 0.9%NS), and experimental groups 1-6: DES were be given 100.0, 50.0, 25.0, 10.0, 0.5 and 0.1μg.kg-1.d-1 respectively (DES dissolved in DMSO and NS). Female mice were placed in individual cages with male mice ,and the morning when a vaginal plug appeared the day was designated as gestation day 0 (GD0). Pregnant mice were injected subcutaneously from GD9 to GD17 with DES dissolved in DMSO and NS or with DMSO and NS (controls). The day the newborns birthed was designated as postnatal day 0(PD0), and male offspring mice were sacrificed on Days GD17, GD19, PD0, PD3 andPD7: transsection was makd from epigastric zone and the low parts of them were fixed in 10% formaldehyde solution. Samples were sliced serially in coronal and were stained with HE method, expression of AR in testis, gubernaculum testis and spinal cord were detected by immunohistochemical methods. The expression intensity was quantitated by image analysis in terms of their gray scales, and statistically analyzed thereafter. Histological changes were observed with light microscope.Results: 1. Severe atrophy of seminiferous epithelium and spermatozoa decrease of mice were found in the DES high dosags group (DES100.0 and DES50.0μg.kg-1.d-1 groups). 2. a, Gubernaculum testis of normal and control groups was well developed, with an inner mesenchymal core and muscular outerlayer clearly . In contrast, in DES-treated male fetus, gubernaculum testis seems to be poor developed and undifferentiated, there wasn't demarcation between the mesenchymal core and the muscular. As the dosage increase ,the disorder obviously. b, When GD17 ,the gubernaculums testis was big and round ,as the fetal grow it first become bigger and more round, but regressed rapidly after borned .When PD7, it only leave some fiber that appearance irregular. 3. The motor neurocyte of GFN in spinal cord of normal groups and control groups and low dosage groups have no differentia on the cell's morphous and the cell's quantity. In DES high dosags group (DES100.0 and DES50.0μg.kg-1.d-1 groups), it seemed to be poor developed, with fewer but bigger cells and appeared inordinate. b, when GD19, cell of motor neurocyte of GFN in spinal cord seem many more and morphous regular, as the fetal grows, cells decrease and became bigger. When PD7, cells were obviously decrease, and volume bigger. 4. During GD17 to PD7, expressions of AR continuesly increase from GD17 to PD7 in the testis. And in the gubernaculums testis, it increases from GD17 to PD0, but decrease from PD0 to PD7, so as the motoneurons of GFN. And in these three tissues all, AR expression was lower in the middle and high DES-dosage groups than in normal groups, control groups and low dosage groups. The higher the dose of DES, the lower the AR expression. At the same time, the testis, gubernaculums testis and spinal cord seems developed poor as the does of DES increased. 5. The expressions of AR were of positively relationship among the testis, the gubernaculums testis and the motoneurons of GFN.Conclusion: 1. DES could induce the testis and gubernaculums testis maldevelopment, paramorphia and structure disorder, and was correlation close with the dosage. High dosage DES could induce the motor neurocyte of GFN decrease and volume become bigger. It suggest that EEs certainly affect the testis, gubernaculums testis and the motor neurocyte of GFN development, EEs certainly has acverse effect to the male reproductive system, furthermore, it appear dosage-dependent. 2. AR express in the Leydig cell in testis, the mesenchymal cells in the certer of the gubernaculums testis, the motor neurocyte of GFN in spinal cord .When compare to the normal groups and the control groups, the AR expression were all inhibited in the DES groups, as the dosage increase, the inhibition incresse, was of dose-dependent effect. 3. As the fetal grows, the AR expression increase in the testis, but in the gubernaculums testis it was increase at first and decrease at last, in the motor neurocyte of GFN in spinal cord, it was fall-off as the gubernaculum testis. Take the morphology chang of the testis, gubernaculums testis and the spinal cord into consider, it suggest that the testis and the GFN and the gubernuculum testis-"testis-GFN-gubernaculum testis"asix manifest close correlation during the genital system development. Furthermore, the asix could maybe one of the important pathway that adjust descensus testis. 4. The AR expression in different developmental stage in the testis, gubernaculum testis and the motor neurocyte of GFN in spinal cord were changed under the affect of different dosage EDS, what's more, they have positive correlation. Combination with the morphology change that these 3 tissue did when under the effect of different dosage of DES, it was further to confirm that"testis-GFN-gubernaculum testis"axis exactly exist, and EEs do exert influence on this axis. It also indicate that EEs could certainly affect the descensus testiculorum mediated by the GFN by adjust the AR expression.