| Background: With the development of modern industry, the adverse health effects of environmental endocrine disruptors on human and animals have arisen great concern worldwide. Pre-study demonstrated that some widely distributed exogenous agents (esp. synthetic chemicals) exist widely in the environment and can concentrate in the human body through multiple routes of exposure. It has been hypothesized that they may interfere with the production, release, transport, metabolism, binding, biologic action and elimination of natural hormones. By presenting the effects of mimicking or inhibiting the action of natural ligands, they can induce a series of adverse effects such as declined of male reproductive function and increased in incidence of urogenital anomalies and cancers. These compounds are called environmental endocrine disruptors (EEDs). Recently DEHP has been listed as one of the most important EEDs, while it is widely used as a prominent plasticizer in plastic and chemical industry.Numerous reports of epidemiology reveled that there was a declining trend of semen quality in global extent. Experimental studies have suggested that this phenomenon was induced by anti-androgenic effect of EEDs. Epididymis is an important place for sperm maturing and acquiring energy, and epididymal epithelial cell acts an important role in this process. We presume that the lesions of epididymal epithelial cell are crucial to epididymal dysfunction and should be correlated to the declining semen quality induced by EEDs. Thus, through studies on both pathogenesis of pathologic and functional lesions of epididymis in mice induced by DEHP and protectional effect of Zinc, it becoming necessary to reducing environmental pollutions, preventing or alleviating the incidence of male reproductive dysgenesis, and improving the health of human as well.Objective: To study the reproductive toxicity and the pathogenesis of endocrine disrupting induced by DEHP, find the antagon which can against its reproductive toxicity.Methods: The whole experiment concludes two parts: experiment in vitro and in vivo. The animal we selected was healthy male KM mice. (1) Experiment in vivo: animal were divided into two stages, the stage in mature and the stage in immature, in each stage mice were randomly divided into four groups: normal control group, corn oil group, DEHP group and DEHP+ Zinc gluconate group. Intragastric administration was used to establish animal models, after been administered medicine 10 days mice were killed. The following results in each group were recorded: epididymal weight, mean epididymal tubule diameter and height of epididymal epithelial cells. AR and ER expression level were measured by immunohistochemistry. (2)Experiment in vitro: the stages were identical with the experiment in vivo,mice in each stage were executed to obtain epididymis, from them epididymal epithelial cells were isolated by 0.1 % collagenase (type I) and 0.125 % Pancreatin to be cultured in DMEM medium. After cultured for 48 hours cells were randomly divided into four groups: normal control group, testosterone group, DEHP group and DEHP+ Zinc gluconate group. After cultured 10 days the following results in each group were recorded: The viability of epididymal epithelial cells were measured using MTT assay, proliferation of epididymal epithelial cells was measured using inverted microscope, content of Sialic acid(SA) was detected and the activity ofα-1,4-glucosidase and lactic acid dehydrogenase(LDH) were investigated from the supernatant of medium.Results:Experiment in vivo:In PND20 stage:(1)Male mice epididymal weight showed in DEHP group reduced significantly compared to normal control group, corn oil group and DEHP+ Zinc gluconate group(p<0.01);(2)Mean epididymal tubule diameter and height of epididymal epithelial cells showed in DEHP group diminished dramatically compared to normal control group, corn oil group and DEHP+ Zinc gluconate group ( p<0.01 ) ;(3)Using light microscope, vacuolar degeneration of epididymal epithelial cells and cells shedding from tubal wall were found in DEHP group, cilia of cells arranged disorderly, interstitial tissue hyperplasia and reduction of sperm counts were also seen in these groups,it showed no dramatic variation of pathologic in epididymis in normal control group, corn oil group and DEHP+ Zinc gluconate group;(4)Using transmission electron microscope, mitochondrion swelling, rough endoplasmic reticulum distension, lysosome increasing, smooth muscle cells of epididymal tube fibrosis were found in DEHP group, mitochondrion swelling was seen in DEHP+ Zinc gluconate group, it showed no significant variation of epididymis in normal control group and in corn oil group;(5)The AR expressions in epididymis were reduced in DEHP group compared to normal control group, corn oil group and DEHP+ Zinc gluconate group, the ER expressions showed inverted trends ( p<0.01 ) ;(6)It showed no significant variation of experimental results between each two groups of normal control group, corn oil group and DEHP+ Zinc gluconate group(p>0.05).In PND50 stage:(1)Male mice epididymal weight showed no significant variation between each two groups(p>0.05);(2)Mean epididymal tubule diameter and height of epididymal epithelial cells showed no significant variation between each two groups(p>0.05);(3)It showed no significant variation of epididymal pathologic histology in each group;(4)It showed no significant ultrastructure change of epididymis in each group; (5)The AR expressions in epididymis were reduced in DEHP group compared to normal control group, corn oil group and DEHP+ Zinc gluconate group, the ER expressions showed inverted trends(p<0.01);(6)It showed no significant variation of experimental results between each two groups of normal control group, corn oil group and DEHP+ Zinc gluconate group(p>0.05).Experiment in vitro:In PND20 stage:(1)The viability of epididymal epithelial cells showed in DEHP group decreased significantly compared to normal control group, testosterone group and DEHP+ Zinc gluconate group(p<0.01);(2)Activities of theα-1,4-glucosidase and LDH showed in DEHP group decreased significantly compared to normal control group, testosterone group and DEHP+ zinc gluconate group(p<0.01);(3)Contents of SA were detected increased in DEHP group compared to normal control group, testosterone group and DEHP+ Zinc gluconate group(p<0.01);(4)It showed no significant variation of experimental results between each two groups of normal control group, testosterone group and DEHP+ Zinc gluconate group(p>0.05).In PND50 stage:(1)The viability of epididymal epithelial cells showed in DEHP group decreased significantly compared to normal control group and testosterone group(p<0.01);(2)Activities of theα-1,4-glucosidase and LDH showed no significant variation between each two groups(p>0.05);(3) Contents of SA showed no significant variation between each two groups(p>0.05);(4)It showed no significant variation of experimental results between each two groups of normal control group, testosterone group and DEHP+ Zinc gluconate group(p>0.05).Conclusions:(1)DEHP is a definite environmental endocrine disruptor, and possibly contribute to development and function of epididymis;(2)DEHP can induce lesions of epididymal structure and function;(3)Epididymal reproductive toxicity induced by DEHP more occurred in adolescence;(4)Zinc has antagonism to toxicity of DEHP. |
PDF Full Text Request