Gestational Cadmium Exposure Impairs Fetal Growth And Offspring Reproductive Development And Its Placental Parkin-regulated Mechanism

Part 1:Gestational exposure to Cd induces fetal growth restriction(FGR)and continuously impairs the reproductive development in offspring.Objective:The present study was aimed to explore whether gestational exposure to Cd induces FGR and continuously impairs the reproductive development in offspring,and its placental parkin-regulated mechanism.Methods:In the current study,we aimed to investigate the role of Parkin mitochondrial translocation in Cd impaired fetal growth restriction(FGR)and the reproductive development of offspring via population case-control study,animal and cell experiments.Based on the population case-control study,we explored the association of Parkin mitochondrial translocation,MCL-1 reduction,placental apoptosis,mitophagy,placental progesterone(P4)synthesis inhibition and all-cause FGR.The animal study contains 7 experiments.In experiment 1,CD-1 pregnant mice were exposed to low-dose Cd(50 mg/L)and high-dose Cd(150 mg/L)through drinking water from GD8 to GD18.Maternal sera,placentae and fetal testes were collected on GD18,and the fetal crown-rump length and weight were recorded to investigate the effect of maternal Cd exposure on fetal growth and testis development.In experiment2,CD-1 pregnant mice were exposed to low-dose Cd(50 mg/L)and high-dose Cd(150mg/L)through drinking water from GD8 to GD18,and delivered naturally on GD18.The rate of testicular descent and anal genital distance were recorded on PND22.The offspring male mice were performed euthanasia on PND35/70.The mice weight,testicular weight and prostate weight were recorded.The mice serum and testes were collected.The testicular primary leydig cells were separated.The sperm count was recorded on PND70.The purpose of experiment 2 was to investigate whether maternal Cd exposure continuously impaired testicular development,testosterone synthesis and spermatogenesis.In experiment 3,to verify the speculation that maternal exposure to environmental Cd promotes placental Parkin mitochondrial translocation,placental apoptosis and P4 synthesis,CD-1 pregnant mice were performed intraperitoneal injection with Cd Cl₂(4.5 mg/kg)on GD8.In experiment 4,to understand whether Parkin mitochondrial translocation contributes to environmental Cd-triggered placental apoptosis and P4 synthesis inhibition,CD-1 pregnant mice were performed intraperitoneal injection with Cd Cl₂(4.5 mg/kg)on GD8 with or without M-1 treatment(25 mg/kg/day,i.p.)from GD7-GD11.In experiment 5,to examine whether mitochondrial targeted antioxidant MT represses environmental Cd-triggered mitochondrial Parkin transposition and apoptosis in placental trophoblasts,CD-1pregnant mice were performed intraperitoneal injection with Cd Cl₂(4.5 mg/kg)on GD8with or without MT treatment(5 mg/kg/day,i.p.)from GD7-GD11.In experiment 6,CD-1 pregnant mice were exposed to low-dose Cd(50 mg/L)and high-dose Cd(150mg/L)through drinking water from GD8 to GD18 with or without NAC(500 mg/kg/day,i.g.)treatment from GD7-GD17.Maternal sera,placentae and fetal testes were collected on GD18,and the fetal crown-rump length and weight were recorded to investigate the effect of NAC on Cd-impaired fetal growth and testis development.In experiment 7,CD-1 pregnant mice were exposed to low-dose Cd(50 mg/L)and high-dose Cd(150mg/L)through drinking water from GD8 to GD18 with or without NAC(500mg/kg/day,i.g.)treatment from GD7-GD17,and delivered naturally on GD18.The rate of testicular descent and anal genital distance were recorded on PND22.The offspring male mice were performed euthanasia on PND35/70.The mice weight,testicular weight and prostate weight were recorded.The mice serum and testes were collected.The testicular primary leydig cells were separated.The sperm count was recorded on PND70.The purpose of experiment 2 was to investigate whether maternal NAC supplement mitigated Cd-induced the sustained impairment of testicular development,testosterone synthesis and spermatogenesis.The cell study contains 5 experiments.In experiment 1,human JEG-3 cells were administrated with Cd Cl2(20?M)for 0-24 h to examine whether environmental Cd exposure promoted Parkin mitochondrial translocation,apoptosis and P4 synthesis in human placental trophoblasts.In experiment 2,to understand whether Parkin mitochondrial translocation promoted environmental Cd-induced apoptosis and P4 synthesis inhibition,and its mechanism,short interfering RNAs targeting homo sapiens Parkin was administrated before Cd(20?M)treatment for 6 h.In experiment 3,To further verify that the Parkin-modulated ubiquitin modification aggravated the degradation of MCL-1,MG132(5?M)was used to pretreat the cells for 1 h.In experiment 4,in order to evaluate the role of mitochondrial ROS in environmental stress-promoted mitochondrial Parkin transposition,Mito-TEMPO(10?M),a specific inhibitor for mitochondrial ROS,was supplied in the cells for 1 h before Cd(20?M)stimulated for 6 h.In experiment 5,in order to investigate the mechanism of parkin-dependent mitophagy activation in environmental Cd-stimulated placental trophoblasts,the cells were pretreated with PERK si RNA before Cd stimulation.Results:The results showed that gestational exposure to Cd significantly lowered the fetal weight and crown-rump length,suggesting that Cd impaired fetal growth.The results also showed maternal cadmium exposure significantly delayed the time of testicular descent and decreased the distance of anal genital distance in offspring male mice.Further results showed that the absolute weight and relative weight of reproductive organs(testes,prostate and epididymis)in Cd group were significantly lower than in Ctrl group.As compared to the Ctrl,the sperm count in Cd group was also significantly reduced on PND70 in offspring male mice.In addition,maternal Cd exposure significantly reduced testosterone levels of testes and sera in offspring male mice.Correspondingly,gestational exposure to Cd also reduced the levels of testosterone synthase(St AR,TSPO and CYP11A1)in testes and testicular primary leydig cells.Here,our human case-control study firstly showed that there was a positive association of Parkin mitochondrial translocation,MCL-1 reduction,placental apoptosis,mitophagy,placental P4 synthesis inhibition and all-cause FGR.Subsequently,Cd was administered to establish in vitro and in vivo models of placental apoptosis or FGR.Our model demonstrated that Parkin mitochondrial translocation was observed in Cd-administrated placental trophoblasts.Meaningfully,Parkin si RNA(si R)dramatically mitigated Cd-triggered apoptosis in placental trophoblasts.Mdivi-1(M-1),an inhibitor for Parkin mitochondrial translocation,mitigated Cd-induced apoptosis in placental trophoblasts,which further ameliorated the effect of attenuated placental size in Cd-exposed mice.Furthermore,the interaction of MCL-1 with Parkin or Ub in Cd-stimulated cells was stronger than that in controls.MG132,an inhibitor for proteasome,abolished MCL-1 degradation in Cd-stimulated cells.Importantly,Parkin si R and M-1 memorably abolished the ubiquitin-dependent degradation of MCL-1 in placental trophoblasts.Interestingly,mito-TEMPO and melatonin,two mitochondria-targeted antioxidants,obviously rescued Cd-caused mitochondrial membrane potential(MMP)decrease,Parkin mitochondrial translocation,MCL-1 degradation,and apoptosis in placental trophoblasts.Based on our case-control study,we also investigated the association of placental mitophagy with reduced progesterone(P4)level and all-cause FGR.We firstly found environmental Cd exposure lowered the P4content in maternal sera,placentae and amniotic fluids of mice.The level of three mitochondrial P4 synthases,including St AR,CYP11A1 and 3?-HSD,was also reduced in Cd-treated placentae.Furthermore,Cd triggered mitophagy,as determined by the degradation of two mitochondrial proteins HSP60 and COX IV,and the accumulation of co-localizations of TOM20 with LC3B or Parkin in placental trophoblasts.Correspondingly,Cd elevated mitochondrial Parkin level in placental trophoblasts.Mdivi-1,a mitophagy inhibitor,obviously attenuated Cd-induced reduction of placental P4 and FGR in mice.Moreover,mdivi-1 and Parkin si RNA(si R)markedly reversed Cd-caused P4 synthesis inhibition in human placental trophoblasts.Interestedly,the PERK/ATF4 signaling was activated in Cd stimulated-placental trophoblasts.PERK si R inhibited mitochondrial proteins degradation in Cd-stimulated placental trophoblasts.In particularly,mitophagy activation and P4 synthesis suppression occurred in small-for-gestational-age placentae based on our case-control study.In addition,our results on animal experiment 6 showed that gestational supplement with NAC reversed Cd-lowered the fetal weight and crown-rump length,suggesting that NAC abolished Cd-induced FGR.The results on animal experiment 7 showed maternal NAC supplement significantly alleviated Cd-induced delay of testicular descent and decreased the distance of anal genital distance in offspring male mice.Further results showed that the absolute weight and relative weight of reproductive organs(testes,prostate and epididymis)in NAC+Cd group were significantly higher than that in Cd group.As compared to Cd group,the sperm count in NAC+Cd group was also significantly elevated on PND70 in offspring male mice.In addition,maternal NAC supplement significantly mitigated Cd-reduced testosterone levels of testes and sera in offspring male mice.Correspondingly,gestational supplement with NAC abolished Cd-reduced the levels of testosterone synthases(St AR,TSPO and CYP11A1)in testes and testicular primary leydig cells.Conclusion:Taken together,the above results allowed us to draw three conclusions.Firstly,gestational exposure to Cd induces FGR and reproductive development impairment in male offspring.Secondly,environmental Cd exposure induced placental apoptosis via ROS-mediated Parkin-modulated ubiquitin-mediated degradation of MCL-1.Next,environmental Cd exposure induced progesterone synthesis inhibiting via activating PERK-regulated mitophagy in placental trophoblasts.Finally,gestational supplement with NAC alleviated Cd-induced FGR and reproductive development impairment in male offspring.