Research On The Mechanism Of Immature Testicular Developmental Injury Induced By DEHP Exposure During Pregnancy

Objective: To clarify the developmental damage and underlying mechanism on offspring testis induced by DEHP exposure during pregnancy.To investigate the effects of DEHP exposure during pregnancy on testicular developmental toxicity and its potential mechanism combined with single-cell sequencing technology.To elucidate the role of ROS-mediated PI3K/Akt/m TOR signaling pathway in gonocyte developmental injury in vitro experiments.To provide a scientific basis for the prevention and treatment of DEHP-induced reproductive toxicity.Methods: Part Ⅰ: SD pregnant mice were given 750 mg/kg.bw DEHP by gavage every day(from E13 to birth)to construct an animal model of DEHP exposure during pregnancy.(1)The body weights of newborn male mice of PND 1,PND 3,PND 6,and PND 9 offspring,testicular masses were observed.The hematoxylin-eosin staining was used to detect testicular histopathology.Western blot(WB)was used to detect the important proteins of intrinsic apoptosis pathway(Bax,Bcl-2,cytochrome C,Cleaved-Caspase3)and the proteins about spermatogenic markers(PLZF,c-Kit).The object of this part is to clarify the etiological correlation between DEHP exposure during pregnancy and testicular developmental damage in offspring neonatal male mice.(2)We used WB to detect expression levels of antioxidant enzymes(Nrf2,HO-1)and important proteins of PI3K/Akt/m TOR signaling pathway(PI3K,p-Akt,Akt,p-m TOR,m TOR)in testis tissue of PND 1,PND3,PND 6 and PND 9offspring male mice.The correlation between the inhibition of PI3K/Akt/m TOR signaling pathway mediated by oxidative stress and the developmental damage of immature testes of offspring male mice was determined.Part Ⅱ: C57 pregnant mice were given 750 mg/kg b.w.DEHP by gavage every day(from E13 to birth),and an animal model of DEHP exposure during pregnancy was constructed.Single-cell sequencing technology comprehensively and deeply analyzed the damage of DEHP during pregnancy on the immature testes of offspring neonatal male mice.(1)The testicular tissue of the PND 5 newborn male rat was dissociated into a single-cell suspensions,and the c DNA library was constructed after passing the quality control.(2)Dimensionality reduction,clustering,and visualization of cells were performed under unsupervised clustering to obtain 23 cell clusters,which were defined based on marker genes.(3)Focusing on germ cell subgroups,definitions,transcriptome difference analysis,and pseudo-time analysis of germ cell subgroups were conducted to clarify the toxic effect of DEHP on germ cells.(4)Transcriptome difference analysis,cell cycle analysis and pseudo-time analysis were performed on Sertoli cell subsets to clarify the toxic effect of DEHP on Sertoli cells.(5)Transcriptome difference analysis of stromal cell subsets was performed to clarify the toxic effect of DEHP on Leydig cells.(6)Transcriptome difference analysis and pseudo-time analysis of PTMs were performed to clarify the toxic effect of DEHP on PTMs.(7)The intercellular communication relationship in the testis of the offspring neonatal male mice was explored to clarify the damage of DEHP on the intercellular relationship in the testis.Part Ⅲ: The mouse teratoma cell line P19(an in vitro model of gonocytes)was used for in vitro culture.(1)Concentration gradient MEHP was used to intervene P19 cells,and CCK8 was used to detect the cell survival rate to clarify the toxic damage effect of MEHP on P19 cells and determine the appropriate intervention concentration of MEHP in vitro.(2)The important proteins regarding intrinsic apoptosis pathway(Bax,Bcl-2,cytochrome C,Cleaved-Caspase3)were detected via WB assays.WB and immunofluorescence experiments were used to detect the protein expression level and cell localization of the stemness markers(Oct4,Sox2,Nanog)of P19 cells to clarify the effect of MEHP on apoptosis and stemness of P19 cells.(3)The model of P19 cell differentiation into germ cells was constructed via RA.Flow cytometry was used to detect the level of apoptosis and cell cycle to detect cell viability and proliferation at this RA concentration.WB and immunofluorescence were used to detect the protein expression and cell localization of germ cell stemness markers(Oct4,Sox2,Nanog)and differentiation markers(Stra8,c-Kit),which confirmed that the RA-induced P19 cell differentiation model was successful.(4)MEHP and RA were used to intervene P19 cells at the same time,and the protein expression and cell localization of germ cell differentiation markers(Stra8,c-Kit)were detected by WB and immunofluorescence to clarify the toxic effect of MEHP on P19 cell differentiation.(5)The level of reactive oxygen species(ROS)in P19 cells was detected.Oxidative enzymatic indicators(Nrf2,HO-1),and important proteins in the PI3K/Akt/m TOR signaling pathway(PI3K,p-Akt,Akt,p-m TOR,m TOR)were detected to make it clear that MEHP can lead to ROS burst,oxidative stress damage,PI3K/Akt/m TOR signaling pathway inhibition.(6)The ROS scavenger N-acetyl-L-cysteine ??(N-Acetyl-L-cysteine,NAC)were carried out to rescue the toxic effect of MEHP 400μM and RA+MEHP 400 μM groups.It was confirmed that NAC could partially improve the increase of apoptosis,stemness inhibition and differentiation disorder of P19 cells caused by MEHP.The ROS levels,oxidative stress damage indicators,and PI3K/Akt/m TOR signaling pathway in P19 cells were also detected to further clarify the role of ROS-mediated inhibition of PI3K/Akt/m TOR signaling pathway in MEHP-induced P19 cell damage.Results: Part Ⅰ: Exposure to DEHP during pregnancy can significantly reduce the body weight and testicular mass of the offspring newborn male mice.The HE staining revealed that the migration and differentiation of gonocytes were injured,and multinucleated gonocytes appeared;The expression levels of important proteins in intrinsic apoptosis pathway such as Bax/Bcl-2,cytochrome C and Cleaved-caspase3 were significantly increased.The protein expression levels of Nrf2 and HO-1,were significantly increased.PI3 K,p-Akt/Akt,and p-m TOR/m TOR were significantly decreased.Part Ⅱ: After DEHP exposure,the testicular cell composition of offspring neonatal male mice was significantly different,and the cells were identified as germ cells,Sertoli cells,Leydig cells,peritubular myeloid cells(PTMs),endothelial cells,stromal cells,macrophages and innate lymph based on marker genes.GO and KEGG enrichment analysis of marker genes were performed.Subgroup analysis of germ cells was performed and defined as spermatogonial stem cell group SSC-1,spermatogonial progenitor cell group SSC-2,differentiated spermatogonia group Differentiated-SG.Pseudo-time analysis was performed,it revealed that germ cell development began at SSC-1,ended up with Differentiated-SG.SSC-2 is an intermediate transition state cell.The top 50 differential genes along the pseudo-time line were divided into three clusters according to their expression trends.After DEHP exposure,the number of germ cells was reduced,the distribution and the developmental trajectory changed obviously.Germ cells were mainly concentrated in SSC-1 or state1.The differential expression genes of germ cell subsets were mainly enriched in gene sets related to negative regulation of cell differentiation,stem cell stemness maintenance,oxidative stress and p53-mediated intrinsic apoptosis pathway.Subgroup analysis of Sertoli cell population showed that DEHP lead to the decrease of Sertoli cells,abnormal distribution,abnormal developmental trajectory,and cell cycle arrest of Sertoli cells.The differential expression genes between the two groups were mainly enriched in functional gene sets such as cytoskeleton,cell adhesion,cell cycle,oxidative stress,and p53-mediated intrinsic apoptosis pathway.The differential expression genes were mainly enriched in gene sets such as mitochondria,mitochondrial inner membrane,endoplasmic reticulum,cholesterol metabolism,and oxidative stress.Subgroup analysis of PTMs showed that after DEHP exposure,the cell composition and developmental trajectories of PTMs were significantly different.The differential expression genes were mostly related to hypoxia,oxidative stress,P53-mediated intrinsic apoptosis pathway,male gonadal development,and cell junctions.Via cell phone DB,it was found that the number and types of intercellular receptor-ligand pairs were changed after DEHP exposure.The expression levels of GDNF and WNT signaling pathway receptors Gfra1 and Ret in germ cells in DEHP group were up-regulated.The expression levels of TGF-β,Hedgehog signaling pathway ligands BMP4,BMP2 and Dhh in somatic cells in DEHP group were down-regulated.Part Ⅲ: MEHP exposure decreased the relative viability of P19 cells.After MEHP treatment,Bax,Bcl-2,cytochrome C and Cleaved-Caspase3 in P19 cells were significantly increased.The stemness markers Oct4,Sox2,and Nanog were significantly decreased.4μM RA intervention in P19 Cells for 24 h had no significant effect on the apoptosis level and cell cycle of P19 cells.The stemness markers Oct4,Sox2,and Nanog significantly decreased,and their localization transferred from the nucleus to the cytoplasm.The germ cell differentiation markers Stra8 and c-Kit increased significantly,but the localization did not change significantly.After MEHP and RA treatment,the differentiation markers(Stra8,c-Kit)decreased significantly,and the localization did not change significantly.After MEHP treatment,ROS in P19 burst,and the oxidative enzymatic indexes(Nrf2,HO-1)increased significantly.PI3 K,p-AKt/Akt and p-m TOR/m TOR obviously decreased.NAC can clear ROS level in P19 cells and avoided oxidative stress injury.It can relieve the inhibition of PI3K/Akt/m TOR signaling pathway caused by MEHP,and rescue apoptosis caused by activation of intrinsic apoptotic pathway,and restore the stemness and differentiation potential of P19 cells.Conclusion: Exposure to DEHP during pregnancy can damage the development of immature testes of offspring neonatal male mice,mainly injure the development of gonocytes.MEHP can cause P19 cell apoptosis,inhibiting stemness,and blocking differentiation of P19 cells.ROS-mediated inhibition of PI3K/Akt/m TOR signaling pathway is involved in this injury process.