Dibutyl Phthalate Exposure Disrupts Progression Of Meiotic Prophase ? By Interfering Homologous Recombination In Fetal Mouse Oocyte

Objective: In mammals,the oocytes that are generated early in life represent the entirety of female reproductive potential over the life span.Unlike spermatocytes,oocytes enter meiosis in the embryonic stage,go through the leptotene,zytotene,pachytene stage,and arrest at the diplotene stage of the prophase of meiosis I.During this process,homologous recombination occurs,that provides genetic diversities of the species,and produces crossovers,ensuring the proper segregation of homologous chromosomes in the metaphase of meiosis I.Therefore,the completion of meiotic I prophase is essential for the early oogenesis.Many of the infertilities or compromised fertilities in humans stems from the meiosis abnormalities.Dibutyl phthalate(DBP)is a widely-used plastizer and an endocrine disrupting chemicals.With the estrogen-like activity,DBP shows a wide range of female reproductive toxicity that there is a close relationship between female fertility decline and DBP exposure.Although studies have shown that another phthalate compound,DEHP,impairs the progression of the prophase of the meiotic I,it is unclear whether DBP affect the early meiosis processes.Therefore,this study aims to investigate the effects of DBP exposure on the prophase of the meiotic I in oocytes and its mechanism.To explore the possible harm caused by DBP inhibition the progression of the prophase of the meiotic I,and provide experimental and scientific basis for prevention and control of plasticizer pollution and protection of female reproductive health.Methods: This study includes in vivo and in vitro experiments.In vivo experiment,CD1 pregnant mice at 14.5 days of gestation were randomly divided into control group 1 mg/kg-bw/d DBP group,10 mg/kg-bw/d DBP group and 1 mg/kg-bw/d DBP group,and orally administered for 3 days,and the fetal ovaries were collected for the following detection.The stage of meiosis was analyzed by chromosome spreading and immunofluorescence staining.In vitro experiment,the separated 14.5 days post coitus fetal ovaries of CD1 mice were randomly divided into DMSO group,10 ?mol/L DBP group and 100 ?mol/L DBP group and cultured for 3 days for examination.Analysis of the stage of meiosis and homologous recombination by chromosome spreading and immunofluorescence.Detection of expression levels of key factors of homologous recombination by western-blot,qRT-PCR and immunofluorescence.The ROS level of oocytes and the expression levels of antioxidant enzymes SOD1 and SOD2 were detected after DBP exposure.The apoptosis of ovarian cells was analyzed by TUNEL assay.The use of chromosome spreading and immunofluorescence to detect DBP acts on the receptor pathway in the prophase of meiosis I.Results:1.DBP exposure delays the progression of meiotic prophase I in oocytes.Both in vitro and in vivo experiments showed that DBP exposure significantly reduced the proportion of pachytene oocytes and increased the proportion of zygotene oocytes.2.DBP exposure disrupts homologous recombination.Chromosome spreading and immunofluorescence assay showed that the number of the foci of homologous recombination cross-site protein MLH1 increased after DBP exposure,indicating excessive DNA exchange between homologous chromosomes are induced.At the same time,the number of the foci of DNA double-stranded protein RAD51 and expression levels of ?H2AX,which is widely used as a DNA damage marker,also increased significantly,indicating that DNA double strand breaks were accumulated after DBP exposure.3.DBP exposure increases DNA damage by inducing oxidative stress in the fetal ovary.The ROS assay showed a significant increase of the ROS levels of oocytes in the ovary after exposure to DBP,and the western-blot assay also showed elevated levels of protein expression of the antioxidant superoxide dismutases SOD1 and SOD2.4.DBP exposure reduces the expression of key factors of HR in oocytes.The qRT-PCR and western-blot showed that DBP exposure decreased the expression levels of ATR,POL?,SMC3 and REC8,which were key factors of homologous recombination.At the same time,the immunofluorescence results of POL? and REC8 showed that the fluorescence signals in DBP group were also significantly weakened.These results indicated that DBP exposure down-regulated the expression of key factors that regulate DNA repair in the HR of oocytes,resulting in disrupted homologous recombination.5.DBP exposure induces apoptosis in fetal ovaries.The TUNEL assay showed that the number of apoptosis in the ovary increased after DBP exposure,and the BAX/BCL2 ratio and Caspase3 protein levels were significantly increased after DBP exposure.This result demonstrated that DBP exposure causes cell apoptosis in the ovary.6.DBP inhibits the progression of MI prophase via estrogen receptors(ERs).The estrogen receptor ?-specific antagonist MPP and the estrogen receptor ?-specific antagonist PTHPP were employed to investigate the involvement of ERs.Chromosome spreading showed that after the combined administration of DBP and MPP,or DBP and PTHPP,the delayed progression of meiosis I prophase oocytes was reversed,indicating that the effect of DBP was mediated through both ER? and ER?.Conclusion: This study demonstrates that DBP exposure induces increase oxidative stress in the oocytes and compromises DNA repair in the homologous recombination,resulting in accumulated DNA damage and the subsequent ovarian cell apoptosis.Furtheremore,DBP exposure disrupts meiotic prophase I through estrogen receptors,thereby affecting oogenesis.