The Role Of M~6A Modification-mediated Rap1/PI3K/AKT Signaling Pathway In TDCIPP-induced GC-2 Spd Cell Apoptosis

TDCIPP（Tri（1,3-dichloro-2-propyl）phosphate）,a novel environmental endocrine disruptor and one of the widely used organophosphate flame retardants,has been reported to exhibit certain toxic effects on several systems such as the nervous,liver,kidney,and reproductive systems.Population-based epidemiological studies have shown that TDCIPP adversely affects male semen quality,and zebrafish experimental models have preliminarily shown that TDCIPP causes reproductive toxicity.However,the mechanisms underlying TDCIPP-induced testicular injury in mammals remain largely unknown.To address this gap,this study aimed to establish a TDCIPP-induced testicular injury model in male mice and investigate the specific mechanisms underlying the testicular damage caused by TDCIPP.Objective:To study the damage of testicle induced by TDCIPP and the mechanism of spermatocyte apoptosis in male mice.Methods:1.Abnormal m⁶A modification mediated by TDCIPP induces testicular injury in male miceTwenty-four 4-week-old SPF C57BL/6 male mice were randomly divided into control group,5 mg/（kg·d）,25 mg/（kg·d）,and 125 mg/（kg·d）TDCIPP-treated groups according to body weight and orally gavaged with TDCIPP for 28 days.The daily changes in body weight were recorded,and after 28 days,the testes and epididymides were dissected and weighed.The paraffin sections of testicular tissues were stained with hematoxylin and eosin（HE）to observe morphological changes,and sperm were stained with HE to observe sperm morphology and calculate sperm count and abnormal rate.The m⁶A qualitative detection kit was used to detect the m⁶A methylation level in testicular tissue,and RT-qPCR and Western blot experiments were performed to detect the expression levels of m⁶A methyltransferase and demethylase in the testes.Me RIP-seq sequencing was used to observe the enrichment of differentially expressed genes with m⁶A modification.TUNEL and Hoechst33342 staining were used to study the apoptotic level of testicular tissue,and Western blot experiments were performed to detect the expression levels of apoptosis-related proteins.2.Apoptosis of mouse spermatocyte induced by abnormal m⁶A modification via Rap1/PI3K/AKT signaling pathwayFirstly,the protein expression levels of Rap1,PI3K/AKT signaling pathway in mouse testis were detected by Western blot.Subsequently,mouse spermatocytes（GC-2spd）were used to explore the mechanism at the cellular level.CCK8 assay was used to detect the viability of GC-2 spd cells after TDCIPP exposure,and to determine the concentration of subsequent exposure.After GC-2 spd cells were exposed to TDCIPP,the expression levels of m⁶A methyltransferase and demethyltransferase were detected by RT-qPCR and Western blot.The apoptosis level of GC-2 spd cells was detected by flow cytometry and Western blot.The expression levels of Rap1,PI3K/AKT signaling pathway proteins in the TDCIPP-exposed GC-2 spd cells were detected by Western blot.To validate the regulatory role of Rap1 on the PI3K/AKT signaling pathway,ESI09 was used to inhibit the expression of Rap1 protein in GC-2 spd cells.The protein expression levels and apoptosis levels of Rap1 and the PI3K/AKT signaling pathway in GC-2 spd cells was measured.Results:1.Abnormal m⁶A modification mediated by TDCIPP induces testicular injury in male mice（1）Since day 21 of exposure,the weight gain rate of 125 mg/（kg·d）group was slower than that of control group（P<0.05）.After exposure,compared with control group,testis and epididymal organ coefficients of mice in exposed group had no significant change.The morphological structure of testicular tissue was significantly damaged in 25 and 125 mg/（kg·d）TDCIPP exposed groups.Sperm count decreased and sperm malformation rate increased in 25 and 125 mg/（kg·d）TDCIPP exposed groups（P<0.01）.（2）The m⁶A modification level in 125 mg/（kg·d）TDCIPP exposed group was significantly higher than that in control group（P<0.01）.The results of RT-qPCR and Western blot indicated that the expression level of Mettl14 was significantly higher than that of control group（P<0.01）.Me RIP-seq sequencing of KEGG signaling pathway enrichment analysis results showed that m⁶A modified differential genes were enriched into apoptosis,Rap1,PI3K/AKT signaling pathways.（3）TUNEL and Hoechst33342 staining results showed that the apoptosis level of testicular tissue in TDCIPP exposed group increased compared with the control group（P<0.05）.Bax/Bcl-2 ratio,Cleaved Caspase-3 and Caspase-9 expression levels were significantly increased in the 25 and 125 mg/（kg·d）TDCIPP exposed groups（P<0.01）,Cleaved PARP expression increased at 125 mg/（kg·d）TDCIPP exposure group（P<0.001）.2.Apoptosis of mouse spermatocyte induced by abnormal m⁶A modification via Rap1/PI3K/AKT signaling pathway（1）Western blot indicated that Rap1 protein expression in testicular tissues of mice exposed to TDCIPP at 5,25 and 125 mg/（kg·d）was increased compared with the control group（P<0.001）.The expression levels of PI3K and p-AKT decreased（P<0.01）while the expression level of AKT decreased in 5 and 125 mg/（kg·d）TDCIPP exposed groups（P<0.001）.（2）CCK8 assay suggested that the median lethal dose(IC₅₀)of TDCIPP against GC-2spd cells was 74μM,and subsequent TDCIPP concentrations were 0,15,30,and 60μM.（3）Flow cytometry revealed that the apoptotic rate of GC-2 spd cells in the TDCIPP-exposure group increased significantly（P<0.01）.Furthermore,Western blot analysis showed increased protein expression of Bax/Bcl-2 ratio,Cleaved PARP,Caspase-9,and Cleaved Caspase-3 in the TDCIPP-exposure group（P<0.001）.（4）Compared with the control group,the protein expression of Rap1 in GC-2spd cells was upregulated in the TDCIPP-exposure group（P<0.001）.Moreover,the expression levels of PI3K protein were decreased in GC-2 spd cells exposed to 30 and60μM of TDCIPP（P<0.01）,while the expression levels of AKT and p-AKT proteins were also decreased（P<0.01）.（5）After applying the Rap1 protein inhibitor ESI09,compared with the TDCIPP-exposure group,the inhibitor group（TDCIPP+ESI09）showed a decrease in the expression of Rap1 protein（P<0.001）and an increase in the expression levels of PI3K,AKT,and p-AKT proteins（P<0.05）.Additionally,flow cytometry indicated that the apoptotic rate of GC-2 spd cells in the inhibitor group was reduced（P<0.01）,and Western blot analysis showed a decrease in the protein expression levels of Bax/Bcl-2ratio,Cleaved PARP,Caspase-9,and Cleaved Caspase-3（P<0.01）.Conclusions:1.TDCIPP exposure induced testicular damage in male mice,with abnormal elevation of m⁶A modification levels and enrichment of differentially expressed genes mainly involved in apoptosis,Rap1,and PI3K/AKT signaling pathways.2.TDCIPP-mediated abnormal m⁶A modification induced overexpression of Rap1 protein,which in turn blocked the PI3K/AKT signaling pathway,ultimately leading to apoptosis of mouse spermatocyte cells.