The Role Of Rna Methylation Recognition Protein YTHDC2 In Regulating CCNB In Manganese-induced Spermatogenesis Disorders

Objective:With the development of industrialization,7%of men in the world suffer from infertility,making it the focus of public attention.The influencing factors of male infertility are many,and environmental pollution is one of the important influencing factors.There are many types of environmental pollutants that cause reproductive dysfunction,and heavy metals are one of them Manganese?Mn?,as a kind of heavy metal,is an essential trace element of the human body and participates in various functional activities of the human body.However,excessive exposure of manganese in the environment will also affect the male reproductive system.Studies have shown that manganese can accumulate in the testis through the blood-testis barrier,thereby damaging the male reproductive system.Spermatogenesis is the basis for the production of hundreds of millions of sperm per day throughout the lifetime of mammalian testes.It includes the four stages of spermatogonial stem cell self-renewal,mitosis of spermatogonia,meiosis of spermatocytes,and sperm deformation.Any disorder in the link will lead to spermatogenesis.The change of the cell cycle phase can precisely regulate the spermatogenesis process.Its cyclin B1?CCNB1?and cyclin B2?CCNB2?are regulatory subunits,which promote the orderly progress of each phase of the cell cycle.Spermatogenesis disorder is an important cause of male infertility.Exploring the regulation mechanism of spermatogenesis from different levels is of great significance to effectively prevent male infertility.The m⁶A recognition protein YTHDC2 is indispensable for spermatogenesis in mice,but its function and mechanism of action in spermatogenesis are unclear.Therefore,this study explores the role of m6A recognition protein YTHDC2 in manganese-induced spermatogenesis disorders,and provides a theoretical basis for further studying the toxicological mechanism of manganese on the male reproductive system.Methods:1.In this study,the seminal plasma of 28 male workers exposed to Mn and 31non-Mn exposed male workers were examined for their sperm quality.ICP-MS was used to detect the manganese content in seminal plasma.Real-time RT-PCR was used to detect YTHDC2,CCNB1 and CCNB2 mRNA expression in seminal plasma.2.For animal experiments,32 Kunming male mice?25±2g?from 3 to 4 weeks were randomly divided into 4 groups.Control group,low,medium and high dose manganese group,mice in each group were injected intraperitoneally with saline,12.5 mg/kg MnCl₂,25 mg/kg MnCl₂ and 50 mg/kg MnCl₂.For 2 weeks,the testes were sacrificed after 35days of continued feeding.Sperm count and morphology were observed under a microscope of epididymis.Testicular tissues were prepared for paraffin sections,and HE morphology and spermatogenesis were observed by HE staining.The mouse testis tissue RNA and protein were taken for the detection of YTHDC2,CCNB1 and CCNB2 mRNA expression and protein level.3.For cell experiments,spermatogonia?GC-1 spg?cells were treated with MnCl2 at final concentrations of 0?mol/L,100?mol/L,200?mol/L,and 400?mol/L for 24 hours.Cell morphology of each dose group was observed using an inverted microscope.and CCK8 were used to detect cell viability.GC-1 spg cell RNA and protein were extracted for detection of YTHDC2,CCNB1 and CCNB2 mRNA expression and protein level.The cell cycle of GC-1 spg cells was detected by flow cytometry.The liposome transfection method was used to construct YTHDC2overexpressing GC-1 spg cells.At the same time,GFP-labeled unloaded liposomes were transfected as a negative control.48 hours after transfection,the fluorescence intensity was observed with an inverted fluorescence microscope and Real-time RT-PCR and Western blotting were used to detect the mRNA expression and protein level of YTHDC2 to evaluate cell transfection efficiency.A control group and a 400?mol/L MnCl2 group were set for each of the negative control cells and the YTHDC2overexpressing cells,and RNA and protein of each group of cells were extracted for detection of CCNB1 and CCNB2 mRNA expression and protein levels.The cell cycle of GC-1 spg cells was detected by flow cytometry.Result:1.Compared with the control group,the total number of sperm and the sperm concentration of male workers in the manganese-exposed group decreased significantly?P<0.05 or P<0.01?.At the same time,the sperm total motility?PR+NP?%and forward The percentage of progressive motility?PR?decreased significantly?P<0.05 or P<0.01?.The content of Mn in seminal plasma increased significantly?P<0.01?.The mRNA expression of YTHDC2 in seminal plasma decreased significantly?P<0.05?.2.The results of animal experiments,compared with the control group,with the increase of manganese dose,the HE results showed that the testicular tissue morphology changed significantly,the number of mature sperm in the official cavity decreased significantly,the sperm number of mice decreased significantly,and the rate of sperm abnormalities increased significantly.The mRNA expression and protein level of YTHDC2 in testis decreased significantly and showed a dose-dependent trend.3.The results of cell experiments showed that,compared with the control group,with the increase of manganese dose,GC-1 spg cells shrank and deformed,their adhesion decreased,the number of cells gradually decreased,and the cell viability gradually decreased.The mRNA expression and protein level of YTHDC2 gradually decreased and showed a dose-dependent trend.The results of flow cytometry showed that compared with the control group,in the 200 and 400?mol/L MnCl₂ group,the S-phase cell index decreased significantly?P<0.01?,and the G2/M-phase cell index increased significantly?P<0.01?,There was no significant change in G0/G1 cell index.4.Pathway and Go analysis of the database studied by Wojtas MN and others found that CCNB pathway may be a downstream molecule regulated by YTHDC2,and found that Mn can cause decline of CCNB1 and CCNB2 in GC-1 spg cells,mouse testes and human seminal plasma.5.The results of liposome transfection of YTHDC2 gene showed that the transfection efficiency of immunofluorescence detection was more than 90%.Flow cytometry results showed that compared with the OE-NC group,the cell cycle G2/M phase cell index in the 400?mol/L MnCl₂ group of OE-NC and the 400?mol/L MnCl₂ group of OE-YTHDC2increased significantly?P<0.01?;Compared with the 400?mol/L MnCl2 group of OE-NC,the cell cycle G2/M cell index in the 400?mol/L MnCl2 group of OE-YTHDC2decreased significantly?P<0.01?.Compared with the OE-NC group,the mRNA expression and protein level of CCNB1 in the 400?mol/L MnCl2 group of OE-NC and the 400?mol/L MnCl2 group of OE-YTHDC2 were significantly decreased,while the mRNA expression and protein level of CCNB2 in the OE-YTHDC2 group were significantly increased?P<0.01?;Compared with the 400?mol/L MnCl2 group of OE-NC,the mRNA expression and protein level of CCNB1 in the 400?mol/L MnCl2 group of OE-YTHDC2 did not change significantly,while the mRNA expression and protein level of CCNB2 increased significantly?P<0.01?.Conclusion:1.Occupational manganese exposure can lead to an increase in manganese content in male seminal plasma,resulting in a decrease in sperm concentration and total sperm count,which in turn can result in poor semen quality.2.Manganese exposure can cause damage to the reproductive system of male mice and impair spermatogenesis.3.Manganese can inhibit the expression of CCNB2 by down-regulating YTHDC2,so that the spermatogonia cell cycle is blocked at the G2/M phase,which in turn interferes with the spermatogenesis process.