Mechanism Of Tri-ortho-cresyl Phosphate (TOCP) Induced-rat Spermatogonial Stem Cells Toxicity

Objectives: Tri-o-cresyl phosphate has been widely used in industry andreported to induce reproductive toxicity in human, but the precise mechanism is yet tobe elucidated. The present study focuses on the toxicity and impact of TOCP on thespermatogonial stem cells in rats, and explores the mechanism of TOCP inducingreproductive toxicity.Methods:1. MTT was utilized to observe the effect of TOCP and LPC onisolated rat spermatogonial stem cells from9-day-old male Sprague-Dawley rats.2.Flow cytometry (FCM) and RT-PCR was used to investigate the impact of TOCP andLPC on the cycle of rat spermatogonial stem cells.3. Annexin V-FITC/PI doublestaining was utilized to measure the effect of TOCP and LPC on the apoptosis of ratspermatogonial stem cells.4. Western blot was used to investigate the effect of TOCPand LPC on the autophagy of rat spermatogonial stem cells.Results:1. Isolated rat spermatogonial stem cells from9-day-old maleSprague-Dawley rats were treated with different concentrations of TOCP (0mmol/L,0.25mmol/L,0.5mmol/L,1.0mmol/L) for48h, cell viability was observed withMTT assay. Compared with the control group, cell viability of rat spermatogonialstem cells was significantly inhibted by TOCP in a dose-dependent manner (P <0.05).2. Flow cytometry found that TOCP had no effect on the cycle of rat spermatogonialstem cells after the cells were treated with different concentrations of TOCP (0mmol/L,0.25mmol/L,0.5mmol/L,1.0mmol/L) for48h. RT-PCR assay showedthat there was no change in mRNA levels of P21, P27and CyclinD1betweencontrol group and TOCP-treated group.3. Annexin V-FITC/PI double stainingshowed that, compared with the control group (0mmol/L), TOCP had no significanteffect on apoptosis of rat spermatogonial stem cells.4. Compared with the controlgroup, TOCP significantly increased the protein levels of autophagic protein Atg5andBeclin1, and the proportion of LC3-II/LC3-I in rat spermatogonial stem cells.5.RT-PCR showed that TOCP significantly inhibited NTE mRNA level of rat spermatogonial stem cells in a dose-dependent manner.6. Compared with the controlgroup, LPC dramatically inhibited rat spermatogonial stem cells viability in adose-dependent manner after the cells were treated different concentrations of LPC (0μmol/L,20μmol/L,50μmol/L and100μmol/L) for48h (P <0.05).7. Flowcytometry found that LPC had no effect on the cycle of rat spermatogonial stem cellsafter the cells were treated with different concentrations of LPC (0μmol/L,20μmol/L,50μmol/L and100μmol/L) for48h. RT-PCR assay showed that there wasalso no change in mRNA levels of P21, P27and CyclinD1between control groupand LPC treated group.8. Annexin V-FITC/PI double staining showed that LPC hadno significant effect on apoptosis of rat spermatogonial stem cells.9. RT-PCR andWestern blot analysis found that there was no change in the mRNA and protein levelsof NTE in rat spermatogonial stem cells between the control group and LPC-treatedgroup.10. Compared with the control group, LPC significantly increased the proteinlevels of autophagic protein Atg5and Beclin1, and the proportion of LC3-II/LC3-I in rat spermatogonial stem cells.Conclusion:1. TOCP could induce autophagy of rat spermatogonial stem cellsand inhibit cell viability, but had no significant effect on the cycle and apoptosis of ratspermatogonial stem cells.2. LPC could induce autophagy of rat spermatogonial stemcells and inhibit cell viability, but had no dramatic effect on the cycle and apoptosis ofrat spermatogonial stem cells.3. The mRNA level of NTE in rat spermtogonial stemcells was significantly decreased by TOCP, not LPC. Taken together, TOCP might beinduce autophagy of rat spermtogonial stem cells and inhibit cell viability viaNTE/LPC signals.