The Effect And Mechanism Of GRIM-19 On Mouse Sperm Quality And Testosterone Synthesis

BackgroundThe incidence of male infertility is increasing annually and has become a global health problem.Abnormal sperm parameters such as oligospermia,asthenospermia,and teratozoospermia result in male factor infertility.GRIM-19(Gene associated with retinoidinterferon-induced mortality 19)is a functional subunit of mitochondrial respiratory chain complex I,plays a role in cellular energy metabolism,and maintenance of mitochondrial membrane potential.GRIM-19 might play a dual protein function involved in cell death and mitochondrial metabolism.Mitochondri’s role as energy provider is surely fundamental for sperm motility.GRIM-19 might modulate sperm functions through mitochondria-dependent pathways.But so far,there are not many studies on the occurrence and mechanism of GRIM19 in male reproductive diseases,and we need further research to find out.Therefore,the aim of this study was to investigate the association of low levels of GRIM-19 with asthenospermia and its mechanism.Methods1.Sperm experiment:The GRIM-19 knockout model(GRIM-19+/-mouse,C57 BL/6J)was created by CRISPR/CaS9-mediated genome engineering.The effect of GRIM-19 expression on sperm quality was evaluated.Diff-Quik method was used to detect mouse sperm morphology.HE staining method was used to detect histological differences in testis and epididymis.The level of mitochondrial membrane potential(MMP),reactive oxygen species(ROS)and the apoptosis of sperm was measured by flow cytometry.2.Animal experiment:ELISA was used to detect the level of testosterone hormone in mouse serum.Immunohistochemical analysis was used to detect the expression and positioning of GRIM-19,CYP11A1 and 3β-HSD in testis.Quantitative RT-PCR and Western blot were used to verify the mRNA and protein expression of StAR,CYP11A1 and 3β-HSD.3.Primary cell experiment:Ley dig cells were isolated,cultured and identified in vitro.Quantitative RT-PCR and Western blot were used to verify the mRNA and protein expression of StAR,CYP11A1 and 3β-HSD.4.TM3 cell experiment:Low expression of GRIM-19 in TM3 cells were expressed by GRIM-19-siRNA technique.ELISA was used to detect the level of testosterone hormone in the supernatant of transfected cells.Quantitative RT-PCR and Western blot were used to verify the mRNA and protein expression of StAR,CYP11A1 and 3β-HSD.5.Mechanism research:Transfection of siRNA down-regulates the expression of GRIM19,and the changes in the expression of Notch signal-related molecular ligands mRNA are detected by RT-PCR.The siRNAs of the related molecular ligands that express changes are respectively transfected into TM3 cells,RT-PCR and Western blot were used to measure the mRNA and protein expression of StAR,CYP11A1 and 3β-HSD.Results1.The sperm count and motility of GRIM-19+/-mice was reduced,and the testicular structure was abnormal.Compared with WT mice,there was no difference in relative testicular shape and weight.Sperm count of GRIM-19+/-mice was reduced compared with WT mice(P=0.0191).The progressive motility of sperm(P<0.0001)and total motility(P=0.0397)in the GRIM-19+/-mice were lower than those of WT mice.The morphology of sperm exhibited no clear differences between the GRIM-19+/-and WT mice.Further testis histology analysis showed that the structure of the seminiferous tubules in the testis tissue of the GRIM-19+/-mice was complete,the spermatogenic cells of all levels in the lumen of the seminiferous tubules were sparsely arranged,and the intercellular space became large.The lumens of the mouse epididymis tissue were arranged tightly,and the cell morphology was normal.Flow cytometry analysis showed that compared with WT mice,the sperm MMP of GRIM-19+/-mice was lower(P=0.0015),the content of ROS was increased(P=0.0047),and apoptotic cells were increased(P=0.0258).2.The expression of testosterone and steroid synthesis-related proteins are decreased in GRIM-19+/-mice.The results of immunohistochemical staining showed that GRIM-19 was mainly distributed in the interstitium of the seminiferous tubules of the testis.The results of IHC showed that the levels of CYP11A1(P<0.0001)and 3β-HSD(P=0.0007)were reduced in the testis of GRIM-19+/-mice.Compared with the WT mice,the level of testosterone hormone in mice serum of the GRIM-19+/-mice was reduced(P=0.0104).The expression of StAR(P=0.0006),CYP11A1(P=0.0131)and 3β-HSD(P=0.0002)mRNA was reduced in GRIM-19 deficient testes(P=0.0929)compared with the control group.The protein expression of key steroidogenic proteins such as StAR(P=0.0009),CYP11A1(P=0.0246)and 3β-HSD(P=0.0128)was reduced in GRIM-19-deficient testes(P=0.0003).3.The expression of testosterone and steroid synthesis-related proteins are decreased in GRIM-19+/-mice Ley dig cells.We isolated and cultured Leydig cells in vitro,and the quantitative RT-PCR results were shown that compared with the WT mice,the mRNA expression of GRIM-19(P=0.0023),StAR(P=0.0313),CYP11A1(P=0.0194)and 3β-HSD(P=0.0742)in the GRIM-19+/-mice was reduced and 17β-HSD expression was not statistically significant.The results of Western blot detection showed that the expression of testicular biosynthesis proteins such as StAR(P=0.0023),CYP11A1(P=0.0087)and 3β-HSD(P=0.0073)was lower in the GRIM-19-deficient Leydig cells(P=0.0097)than that of the control group.4.Downregulation of GRIM-19 expression in TM3 cells reduces the expression of testosterone steroid synthesis-related proteins.The regulation was consistent with the observations in vivo.The expression of GRIM-19 was successively down-regulated by using siRNA in TM3(P=0.0065).Compared with the control group,the level of testosterone hormone in the cell supernatant of the experimental group was reduced(P=0.0197).Compared with the control group,the mRNA expression of StAR(P=0.0189),CYP11A1(P=0.0011)and 3β-HSD(P=0.0004)in the GRIM-19 siRNA group was reduced and 17βHSD expression is not statistically significant.Detected by Western blot,the expression of such as StAR(P=0.0421),CYP11A1(P=0.0092)and 3β-HSD(P=0.0482)was lower in the GRIM-19 siRNA group than that of the control group.5.Notch signaling pathway is involved in GRIM-19-mediated testosterone synthesis in TM3.We found that while down-regulating the expression of GRIM-19,the expression of Notch signaling related molecular ligands mRNA such as Notch 4(P<0.0001),Delta3(P=0.0029),Delta4(P<0.0001),Jaggedl(P=0.0006)and Jagged 2(P=0.0057)was reduced.The quantitative RT-PCR results were shown,compared with the control group,the mRNA expression of CYP11A1(P=0.0017)and 3β-HSD(P<0.0001)in the Delta3 siRNA group(P=0.0396)was reduced and the mRNA expression of StAR was not statistically significant.The results of Western blot showed that the expression of testicular biosynthesis protein such as CYP11A1(P=0.0002)and 3β-HSD(P<0.0001)was significantly lower in the Delta3 siRNA group than that of the control group and the protein expression of StAR was not statistically significant.Compared with the control group,the mRNA expression of StAR(P=0.0002),CYP11A1(P=0.0011)and 3β-HSD(P=0.0458)in the Delta4 siRNA group(P=0.0086)was reduced.Detected by Western blot，the expression of StAR(P=0.0039),CYP11A1(P=0.0495)and 3β-HSD(P=0.005)was lower in the Delta4 siRNA group than that of the control group.After Notch4，Jagged1,Jagged 2 siRNA were transfected into TM3 cells,the detection results were not statistically significant.Conclusion1.Down-regulated GRIM-19 can lead to decreased sperm count and motility.2.GRIM-19 can affect sperm motility through sperm mitochondrial membrane potential,reactive oxygen species level and apoptosis level.3.GRIM-19 may regulate the synthesis of testosterone and steroid hormones through the Notch signaling pathway to a certain extent.