Study On The Inheritance Mechanism Of Nedd4 - Mediated Fertilization Injury Of Ethylene

Objective:Vinclozolin (Vnz) is one of environmental hormone androgen deprivation distracters which widely exist in the environment. Vnz is usually absorbed by human body through occupational exposure, food surface residual and contaminated water. Vnz is widely used for preventing and controlling disease of grapes, fruit and vegetables, such as grey mould, brown spot and sclerotium. The xinjiang region is the largest grape production areas in China. Vnz is used by high frequency in the xinjiang grape planting. Vnz residual is generally found in wine and grape surface, which results in the potential risks for food safety. Unlike traditional antiandrogen drug flutamide, Vnz exposure can cause male reproductive system of F1-F4 male offspring appear the epigenetic across generations inherit the phenotype during the key period of embryo sex differentiation. Therefore, the long-term potentially harms of Vnz to life and reproduce of human, livestock and wild animals is remarkable. At present, the mechanism that male reproductive system of F1-F4 male offspring appear the epigenetic across generations inherit the phenotype caused by Vnz is unclear. Now several hypothesis theory were put forward, one of the hypothesis theory is Nedd4 mediated IGF-1R antiapoptotic mechanisms. Nedd4 belong to ubiquitin ligase E3 family. Nedd4 involved in multiple cellular processes and regulation of signaling pathways.IGF-1-R, androgen receptors AR, PTEN are substrates of Nedd4 ubiquitin. Nedd4 regulate the growth and cell apoptosis through IGF-1-R. Nedd4’s ubiquitin regulation to AR affects the growth and proliferation of prostate cancer cells. Vnz reacts by interfering with the combination of androgen and AR. Based on the above theoretical basis, some scholars put forward that Nedd4 mediated IGF-1-R antiapoptotic mechanisms may be originated from the molecular mechanisms of epigenetic across generations inherit phenotype caused by Vnz exposure. this mechanisms has not been confirmed by experiment up to now. Possible mechanism of Nedd4 mediated IGF-1R antiapoptotic and germ cell impairment by Vnz in vitro experiments were discussed in this paper. Through the modern variety of biological experiment technology, the possible mechanism of reproductive damage by embryonic exposure Vnz was researched in order to understand the reproductive toxicity of Vnz and provide theoretical reference for Vnz science protectionMethods:including the in vivo test of animals and the cell culture in vitro experiment.一、The in vivo test of animals Set up experimental animal model, select SD pregnant rats, intraperitoneal injection of E8-E14 Vnz (100 mg/kg/day), set up corresponding comparison at the same time, sampling the F1 progeny in P20 (20 days after birth) and P60 (60 days after birth), sampling mothers after the breast-feeding.(1) General measurement:pregnant rat body weight growth during the molding time; liver, kidney, testis, ovary weight and viscera than of organization P20, P60 F1 progeny;(2) Sperm count and morphological changes observation of P60 male offspring;(3) Serum testosterone (T) content of P60 male offspring and Serum estradiol (E2) and progesterone (P) content of P60 female offspring by using chemiluminescence method; testing P60 offspring serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) content by using Enzyme-linked immunoassay (ELISA);(4) Morphological detection:liver, kidney, testis, ovary tissue morphology observation of P20 and P60 offspring through conventional HE staining pathological; Observation of P20, P60 testis and ovary tissue apoptosis using In situ end labeling technique (TUNEL);(5) Testing Bcl-2, caspases3, caspases9 protein expression level of the testes and ovarian tissue of P20, P60 offspring using immunohistochemical method;(6) Testing Nedd4, Grb10, IGF-1R, Bcl-2, caspases3, caspases9 gene expression level of testes and ovarian tissue of P20, P60 offspring using Real-Time PCR;二、The cell culture in vitro experiment(1) Set up a rat ovarian granulosa cells in vitro culture model, analyze the influence of Vnz on rat granulosa cell growth, proliferation, differentiation and the effect of steroid hormone secretion; By observing Vnz cultivating granular cell IGF-1R concentration change and Estradiol secretion influence mechanism, study the possible mechanisms that the effects of Vnz on steroid hormones of granulosa cells;(2)Set up a rat leydig cells in vitro culture model, study the influence of Vnz on growth, proliferation and secretion of testosterone of rat mesenchymal cells. By analyze the change of the concentration of IGF-1 of Vnz cultivating mesenchymal cells cAMP, study the possible mechanisms that the effects of Vnz on testosterone secretion of mesenchymal cells.Results:一、Animal in vivo experiment results1. There was no significant difference of weight gain between Vnz exposed group and control group of pregnant rats (P> 0.05); There was no significant difference of born rat proportion of male and female, birth weight and birth survival rate between Vnz exposed group pregnant rats and control group pregnant rats (P> 0.05); There was no significant difference of body weight, liver weight, kidney weight and testicles weight between Vnz exposed group P20、P60 offspring male rat and control group offspring male rat; There was no significant difference of liver weight, kidney weight between P20 offspring male rat and P20 offspring female rat (P>0.05); The liver weight, kidney weight of P60 offspring male rat is higher than that of P60 offspring female rat;2、The number of sperm of Vnz exposed group P60 offspring male rat reduce (P< 0.05).3、Serum estradiol content of Vnz P60 exposed group offspring female rat was obviously lower than that of the control group (P< 0.05); Progesterone levels was also lower than the control group, but there was no significant difference (P> 0.05); Serum testosterone content of Vnz exposed group P60 offspring male rat was obviously lower (P< 0.05);4、Liver steatosis, liver cable arrangement is not neat, testicular tissue fine small tube cavity largen, sperm cells reduce and edema was found in P20、P60 offspring caused by Vnz. Uneven distribution of the ovarian granular cell layer was found in P20 offspring, Oocyte boundary is not clear, thickening of basement membrane, transparent with shrinkage of oocytes, radial boundary is not clear, follicle collapse, follicular atresia was found in P60 offspring, and these were also caused by Vnz.5、Obvious apoptosis of testicles and ovaries were found in P20, P60 offspring of Vnz exposed group (P< 0.05);6. The immunohistochemical results:csapases3, caspases9 increased was found in The testes and ovarian tissue of Vnz exposed group P20, P60 offspring (P< 0.05);7. In experimental P20, P60, male offspring and female offspring P60 Nedd4 content are lower than the corresponding control group (P< 0.05),Grb10 gene in each group were higher (P> 0.05). IGF-1-R in each group were increased, but only the experimental group P20 and P60 male offspring increased significantly (P <0.05). The Bcl-2 level were significantly lower (P< 0.05) except experimental P60 female offspring. In experimental group, P60 caspases3 expression of male offspring is significantly higher (P< 0.05). In xperimental P60, caspases9 expression in testis and ovary tissue were increased (P< 0.05).二、In vitro cell culture experiments result1. Vnz effect granule cells experiment results:The serum estradiol content decreased obviously was found in high Vnz dose group (28 um) cultured granulosa cell (P< 0.05). Serum estradiol content is obviously lower was found in FSH+28, FSH+14, FSH+7 group compared with FSH group(p< 0.05); there was no significant difference between groups (p> 0.05) FSH+28. FSH+14、FSH+7; Serum estradiol content of E+28、E+14’E+7 group is slightly higher than that of group used exemestane individually, but there was no significant difference between E+28、E+14、E+7 groups (P> 0.05); 3β-HSD and P secretion level in Vnz cultivating granular has no significant difference compared with control group. IGF-1R level is obviously lower in Vnz culture granular than that of control group (P<0.05)2.The testosterone secretion content decreased obviously was found in high Vnz dose(28uM)cultured role stroma cells(P< 0.05); cAMP concentration and IGF-1R level is obviously lower was found in high Vnz dose (28uM) cultured role stroma cells(P< 0.05).Conclusions:1. Vnz has no influence on body weight gain and fetiferous number of pregnant rats, Vnz has no influence on born proportion of male and female, birth weight and birth survival rate of F1 offspring. Vnz has no effect on the general growth of F1 offspring. Vnz can cause serum testosterone and estradiol levels of F1 male offspring and female offspring lower. But Vnz has no influence on progesterone, FSH, LH secretion of F1 offspring2. Vnz can lower the number of sperm of F1 adult male offspring;3. Vnz can damage liver, testes and ovaries tissue of F1 offspring;4. Vnz can induce reproductive cells of Fl offspring toapoptosis, Csapases3, caspases9 may be mediated the apoptosis process;5. Vnz has relatively weak inhibitory effect to the granular cell secretion of E2; Vnz don’t affect the normal granular cell proliferation and differentiation; Vnz has inhibiting effect on ovarian granulosa cell estradiol secretion through the cAMP-PKA pathway; Vnz reduces the expression of IGF 1 r granulosa cells;6. Vnz inhibits the testosterone secretion of mesenchymal cells. The possible mechanism is that Vnz inhibit the IGF-1r level of ley dig cells, thereby inhibiting the cAMP signal pathways and then inhibit the synthesis of testosterone. The detailed mechanism needs further study.In the male offspring that exposed Vnz during the crucial embryo sex differentiation stage, Nedd4/Grb10 complex’s degradation on IGF ubiquitin lower the expression of reproductive cells IGF-1R, reduces the promoting apoptosis inhibition of IGF-1R mediated PI3K/Akt and MAPK signal transduction pathway acting on caspases3, aggravates the apoptosis. It is speculated that the ubiquitin chemical degradation pathway of Nedd4/Grb10 complex acting on IGF is the epigenetic across generations inherit the phenotype pathogenesis of the F1-F4 male offspring exposure Vnz in the embryonic period.Vnz inhibit the cAMP and PAK signal transduction pathway of synthesis of steroid by influencing the IGF-IR expression of germ cells, the in the signaling pathways, affect the secretion of the reproductive cells further.