Autophagy In Abnormal Primordial Follicles Development Of Newborn Mice Induced By DEHP

Objective: DEHP is a kind of PVC plasticizers. It can be easily eluted from the plastic and directly affect human health. This is because it is not formed a covalent bond between the plastic substrate but connected by hydrogen bond and Fan Deli. Related articles have reported that DEHP is one kind of environmental estrogen. Once inside the body it can cause a wide range of effects on human health.Effect of DEHP on the reproductive health of women has been received widespread concern. Existing studies had shown that ovaries were the main target organ for female with toxicity. Based on our previous study, DEHP could induce abnormal development in primordial follicles of newborn mice ovaries, while the specific mechanism was not clear. Autophagy is a common life phenomenon in eukaryotic cells. It is a metabolic process involving transport the distortional, damage and aging proteins in cells and organelles into the soluble enzyme then digestion and degradation eventually, which plays an important role in maintaining cell survival, updating, recycling materials and stabling internal environment. Therefore, this paper aims to study whether DEHP participate in the process of abnormal development in primordial follicles of newborn mice ovaries.Methods: Adult female Kun Ming mice of six to eight weeks at cleaning stage were randomly divided into the control groups and the DEHP groups(30 in each group). The newborn mice were injected intraperitoneally based on the concentration of drugs and its weight. The concentration were 0.62 mg /kg/d,2.5mg /kg/d,5 mg /kg/d,10 mg /kg/d respectively. The control group injected with the same volume of corn oil. We started injection from newborn rats born on day 1(1 day post-parturition, 1 dpp), once a day and continuous injection of 5 days, collecting ovarian tissue of newborn rats on the 6th day morning(6 dpp). We had done the following researches.(1) Effection of DEHP on the development in primordial follicles of newborn mice ovaries was studied by HE staining and TEM. Expression of the follicular marker molecule(GDF9) was detected by RT-PCR.(2) We had studied effect of DEHP on the formation of autophagy in primordial follicles of newborn mice ovaries. The level of autophagosome iconic protein(LC3) was determined using western blot and autophagic morphology was observed by TEM.(3) ULK1,Beclin 1 and Atg5 are improntant molecules for the formation of autophagy in primordial follicles of newborn mice ovaries. So we used RT-PCR and western blot to detect their mRNA levels and protein levels.(4) The levels of AMPK,p-AMPK,mTOR,p-mTOR,p-P70s6k( T389) and p-4EBP1 were detected by western blot.(5) We studied the effects of DEHP on autophagic degradation. Expressions of LAMP2,P62 and Cathepsin B were detected by RT-PCR and western blot.(6) We studied the effects of DEHP combined with autophagy inhibitor(3-MA) on the development in primordial follicles of newborn mice ovaries. HE staining was used to observe the follicular development situation in the control group, DEHP, DEHP and 3- MA mixed group and 3-MA groups.Results: HE staining results showed primordial follicle number of DEHP group decreased significantly, and the higher concentration of DEHP, the more obvious decrease in the number of primordial follicles. TEM results showed that, compared with controls, DEHP groups appeared the oocytes decreased. Transparent and chorionic were not obvious. The number of follicles decreased. Oocytes appeared developmental retardation. Dealing with the newborn mice with 0.62 mg/kg DEHP, autophagosomes could be found in the newborn mice ovarian tissues. Western blot results showed that, compared with controls, DEHP groups revealed the expression of LC3-Ⅱ, Atg5, Beclin 1, p-p70s6k(T389), p-AMPK, p-mTOR, Cathepsin L, Cathepsin B, P62 and LAMP2 increased significantly, the expression of p-ULK1, p-4EBP1 decreased significantly. The Real- time PCR results were consistent with the results of protein expression. After blending with 3-MA, the results showed that the expression of LC3-Ⅱ, Atg5, P62 decreased. HE staining results showed that the number of primordial follicles increased.Conclusion: After intraperitoneal injection with DEHP, autophagosome of newborn mouse ovaries increased. Autophagy formation process enhanced. Lysosome degradation of autophagy weakended. Autophagy pathway was damaged. These results suggested that DEHP might affect the primordial follicles of newborn mice ovaries through autophagy.