The Role Of Akt/mTOR Signaling In Rat Reproductive Toxicity Induced By Nonylphenol

Part I Nonylphenol induces damage in Sertoli cells involving Akt/mTOR signaling pathwayObjective:Nonylpheol(NP)is one of endocrine disrupting chemicals(EDCs)which is widespread and induces reproductive toxicology.Sertoli cells(SCs)are the only somatic cells in the seminiferous tubules,and they play pivotal roles in spermatogenesis,which promote spermatogonial proliferation and differentiation,germ cell development.The aim of this research was to explore the detrimental effect of NP to SCs.Methods:SCs were isolated,purified,incubated by immunofluorescence with FSHR antibody to detect the purity by laser confocal microscopy.They were pretreated with the PI3K inhibitor wortmannin for 1h,then treated with NP(0,10,20,and 30 ?M)for 12h.Quantitative RT-PCR was used to measure targeted gene expressions.The protein expressions were detected by western blotting and immunofluorescence.Result:More than 95%of the cells were SCs.The protein levels of PI3K/Akt/mTOR pathway were downregulated at middle-and high-dose groups,which also disturbed the mRNA levels in the pathway and increased the p-AMPK protein.Furthermore,the protein levels of pro-caspase-3 and LC3B-? were decreased,those of cleaved-caspase-3 and LC3B-? were increased,and the ratio of LC3B-?/LC3B-? was upregulated.NP also inhibited protein levels of ABP,FSHR,N-cadherin,transferrin,vimentin;disturbed the gene levels of all,but vimentin.Pretreatment with wortmannin,alleviated an NP-induced reduction in protein levels of PI3K and PTEN.Conclusions:Excess NP exposure induces apoptosis and autophagy,damages the structure and function of SCs involving the PI3K/Akt/mTOR pathway.Part ? Reproductive toxicities and mechanisms of NP in prepuberty male ratsObjective:The period from prepuberty to sexual maturity stage is vital for the development and maturation of reproductive system.The purpose of this part was to explore the effect of NP to prepubertal SD male rats on the structure and function of testes,the quality of sperm in epididymis.Methods:Forty-two 3-week-old rats were randomly divided into six groups,which were treated with NP(0,NAC,25,50,100,100+NAC mg/kg/2d for 30 consecutive days)by intraperitoneal injection,7 per group.NAC(1 g/1)was provided in the animals5 drinking water.Animals were sacrificed by cervical dislocation 48h after the last treatment.The plasma levels of FSH,LH and T were measured by ELISA kits;SOD,MDA and GSH-PX were determined by colorimetry.HE staining was carried out to observe histological changes in testes,and TUNEL assay was used to assess the apoptosis.The expressions of targeted gene and protein were detected by RT-PCR and western blotting.Result:NP induced a decrease in levels of T(15.58%,17.23%,13.38%in 25,50,100 mg/kg groups,respectively).The plasma level of SOD was downregulated in the high-dose group(22.49%),and MDA was upregulated(46.01%).Moreover,NP disturbed mRNA and/or protein levels of PI3K,PTEN,PDK1,p-Akt,p-mTOR,p70S6K,caspase-3,LC3B.It triggered morphological abnormality in epididymal sperm(2.00-,3.02-fold in 50,100 mg/kg group,respectively).Furthermoer,the number of apoptotic cells increased in 50 and 100 mg/kg groups(2.03-,2.87-fold,respectively).Pretreatment with NAC,attenuated NP-induced MDA production;ameliorated the toxic effect in epididymal sperm and the apoptosis in the testes.Conclusions:NP exposure disturbs endocrine system,triggers oxidative stress,induces apoptosis and autophagy in testis,reduces semen quality involving the PI3K/Akt/mTOR pathway.Part ? Reproductive toxicities and mechanisms induced by NP prenatal exposure in male offspringObjective:Pregnancy is high sensitive,vulnerable and susceptible,which play a dominant role on the development of reproductive system in male offspring.The goal of this part was to explore the effect of prenatal NP exposure on the structure and function of testes,the quality of sperm in epididymis in male offspring rats(21,35,and 60 days old).Methods:Forty female Sprague-Dawley rats and twenty male were cohabited,then randomly divided into four groups,and gavaged with NP(0,10,50,and 200 mg/kg/day)from gestation days 14 to 20.Six male offspring per group were acquired at random from different dams on postnatal day(PND)21,35,and 60,respectively.They were sacrificed by cervical dislocation.ELISA technique was used to detect the plasma FSH,LH and T.HE staining was processed to evaluate the morphometric and pathological feature.Apoptosis was evaluated using the TUNEL assay.Effects on ultrastructure were investigated by performing transmission electron microscopy(TEM).RT-PCR and western blotting was carried out to measure the expressions of targeted gene and protein.Epididymal sperm were evaluated by computer-assisted semen analysis(CASA).Result:NP prenatal expose decreased body weight in the low-and high-dose groups at PND 60,testis weight at all three time points in the low-dose groups,and testis coefficient only in the low-dose group at PND 21.It also disturbed plasmatic levels of FSH,LH,and testosterone;triggered apoptosis and autophagy;affected mRNA and/or protein levels of ABP,FSHR,INHB,N-cadherin,transferrin,and vimentin involving the Akt/mTOR signaling pathway at different time points.Moreover,NP induced sperm malformation in the epididymis,reduced the density and quality of sperm.Of note,apoptosis was up-regulated at PND 35,down-regulated at PND 60 in NP-treated groups.Conclusions:Prenatal NP exposure disturbs endocrine system,triggers apoptosis and autophagy in testis,damage the structure and function of testes,reduces semen quality involving the PI3K/Akt/mTOR pathway.NP results in reproductive lesions in utero,which was much serious at puberty.