The Role And Molecular Mechanism Of Oxidative Stress In Spermatogenic Dysfunction In Varicocele

Objective:Varicocele（VC）is one of the main causes of male infertility,with an incidence of about 10%-15%in normal men and 19%-41%in infertile men.With the development of VC,reactive oxygen species（ROS）accumulate in the testicular tissue,and the testis is in a state of prolonged oxidative stress.The main objectives of this study are as follows:（1）To construct a VC rat model and analyze the transcriptome of the testis,to investigate the effects of VC on oxidative stress injury and spermatogenesis in the rat testis,and to screen the differentially expressed genes（DEGs）and pathways.（2）The oxidative stress model of mouse spermatogonia cell line GC-1 and spermatocyte cell line GC-2 was constructed to explore the role and mechanism of PARP-1 and ARL2 in VC-induced testicular injury and spermatogenesis disorder.Methods:In animal experiment,10 SD rats in the VC group and 10 SD rats in the sham-operation group were used to establish the VC model by partial ligation of the left renal vein.HE staining was used to analyze the histological changes in the testis and epididymis of VC rats,and TUNEL was used to detect the apoptosis of testicular cells.The redox status of the testis was analyzed by measuring the contents of LPO and GSH in testicular homogenate by ELISA.Total testicular RNA was extracted for RNAseq sequencing,and RNAseq data of clinical VC testis（PRJNA556935）were used for gene function enrichment.The DEGs in the sequencing results were verified by Western blot（WB）and realtime-q PCR（RT-q PCR）.In cell experiments,GC-1 and GC-2 cells were treated with H₂O₂ to construct an in vitro oxidative stress model,and CCK8,WB,RT-q PCR,immunofluorescence technology,TUNEL,si RNA interference and overexpression were used to explore the potential mechanism of PARP-1 and ARL2 in male fertility decline caused by oxidative stress in VC.Results:The diameter of the spermatic vein in the VC group was significantly higher than that in the control group（0.415±0.0081 vs.1.328±0.069,p<0.05）,and there was no significant difference in the weight of the kidneys between the two groups（1.702±0.038 vs.1.692±0.025,p>0.05）。The testicular weight ratio（3.825±0.143 vs 1.925±0.106,p<0.05）,epididymal weight ratio（2.108±0.128 vs1.318±0.033,p<0.05）and sperm count（2.05±0.521 vs 0.777±0.426,p<0.05）were significantly reducing compared with the control group.The results of HE staining showed that the spermatogenic epithelium of the VC group became thinner and arranged disorderly,there were vacuolar germinal epithelium in the lumen,and the prolonged or round spermatozoa in the lumen were significantly reduced compared with the control group.There was a significant decrease in mature sperm and an increase in apoptotic sperm in the epididymis.GO analysis of clinical RNAseq differential genes in VC testis showed that they were enriched in biological functions such as oxidative stress response,mitotic cell cycle process and hormone levels.The GO and KEGG pathway enrichment analysis of DEGs in RNAseq of VC rat testis showed that degs were enriched in cell processes such as apoptosis and lysosomes and signaling pathways such as PI3K-Akt and MAPK.The results of RT q-PCR and WB showed that the expression of PARP-1 protein in the testis of VC rats was significantly increased,and the expression of ARL2 protein was significantly decreased.After GC-2 cells were treated with H₂O₂ for 30min,WB results showed that PARP-1,PAR andγ-H2AX protein expression increased,and immunofluorescence results showed that PAR andγ-H2AX expression increased and transferred to the nucleus.Treatment of GC-2 cells with H₂O₂ for 24h resulted in a decrease in cell number and the ability to attach to the wall.Flow cytometry showed an increase in cell apoptosis,but immunofluorescence showed that PAR did not transfer to the nucleus.Treatment of GC-1 cells with H₂O₂ for 24h resulted in a decrease in cell proliferation and ARL2 protein expression.There was no significant difference in the proliferation ability of GC-1 cells after ARL2 knockdown compared with the control group,and the overexpression of ARL2 did not reverse the decrease in proliferation ability of GC-1 cells caused by H₂O₂.Conclusion:VC can cause testicular damage and spermatogenesis disorder in rats.Redox imbalance in the testis.PARP-1 may play a role in spermatogenic cell damage caused by VC,and Parthanatos is involved in the damage of spermatocytes caused by oxidative stress.The available evidence cannot prove that the reduction of ARL2 oxidative stress model of GC-1 cells induced by H₂O₂treatment related to cell proliferation ability.