The Role Of GRIM-19 In Asthenozoospermia And The Function Regulation And Molecular Mechanism In GC-2 Cell

IntroductionAsthenozoospermia(AZS),which is also known as low sperm viability,refers to the percentage of forward motility sperm in semen is lower than the normal standard value,it is a common clinical cause of male infertility.Its pathogenesis is complex,and its molecular mechanism has not been clarified.Studies have shown that chromosomal abnormalities,gene fragment deletion,hormonal endocrine disorders,reproductive tract inflammation may lead to decreased sperm cell vitality and number,resulting in the occurrence of asthenozoospermia.In recent years,with the deepening of the research,people also constantly improve the understanding of asthenozoospermia,research has shown that many protein genes,signaling pathway is closely related to the asthenozoospermia,other studies have shown that sperm apoptosis rate of asthenozoospermia was significantly higher than patients with normal,and significantly lower proliferation and migration ability than patients with normal.To explore the potential pathogenesis of asthenozoospermia has become a difficult and hot topic in male infertility.In the past few decades,the number and motility of human sperm are declining.Therefore,revealing the gene expression differences and molecular regulation mechanism of asthenozoospermia is of far-reaching significance for the comprehensive understanding and clinical treatment of asthenozoospermia.GRIM-19 is an apoptosis-related gene found by Angell et al in 2000,it is located on human chromosome 19p13.1,and widely exists in cytoplasm and nucleus of cells.It is the basic functional subunit of mitochondrial NADH dehydrogenase complex,and maintains the normal function of oxidative phosphorylation of respiratory chain in mitochondrial intima,it is involved in cell proliferation,apoptosis and regulation of various signaling pathways.It is well known that the energy supply of sperm cells is mitochondria.Mitochondria continuously produce adenosine triphosphate through glycolysis and oxidative phosphorylation to provide energy for the activities of sperm cells,ensuring a series of life activities of sperm.Therefore,once the structure or function of mitochondria is damaged,the vitality of sperm cells will undoubtedly be affected.There are many studies on GRIM-19 in tumor cells,but the role of GRIM-19 in cell functions outside tumors is different,which needs to be further studied and discussed.Previous studies have shown that the down-regulation of GRIM-19 leads to the collapse of mitochondrial membrane potential and the increase of reactive oxygen species production.These high levels of ROS can eventually cause damage to nucleic acids and proteins in the cell.Other studies have shown that GRIM-19 regulates the sensitivity of cells to external stimuli through the ERK1/2 and HIF1α pathway mediated by reactive oxygen species,thus promoting autophagy and apoptosis.ERK1/2 signaling pathway,as a part of the oxidative stress mechanism,is initiated by ROS in cells.Studies have shown that ERK1/2 signaling pathway is widely involved in the regulation of cell proliferation,apoptosis,cycle and differentiation,and is also an important signaling pathway in mammalian sperm cells,participating in the regulation of sperm flagellar movement.Our previous studies showed that the expression of GRIM-19 in ovarian granulosa cells was negatively correlated with the expression of HIF1α,the down-regulation of GRIM-19 will lead to the increase of ROS and HIF1α,and the continuous increase of ROS will activate ERK1/2 signaling pathways,resulting in the expression changes of genes related to the pathogenesis of asthenozoospermia.Our preliminary experiment confirmed that the expression of GRIM-19 was significantly reduced in the sperm of patients with asthenozoospermia.but the regulatory relationship and molecular mechanism between GRIM-19 and asthenozoospermia remain to be explored.Therefore,we plan to carry out studies from three parts to explore the expression differences and regulatory effects of GRIM-19 in asthenozoospermia,to reveal the underlying pathogenesis and molecular mechanism of male asthenozoospermia from the perspective of GRIM-19.Part ⅠObjectiveTo clarify the localization and the expression of mRNA and protein of GRIM-19 in human spermatozoa,to investigate the relationship between GRIM-19 and asthenozoospermia by the Smooth Fitting Curve.Design1.Specimen collection:Asthenozoospermia specimens were obtained from patients in the outpatient clinic of the First Affiliated Hospital of Zhengzhou University between October 2020 and June 2021,and normal controls were specimens with normal semen examination.Subjects in both groups were abstinent for 3-7 days,and semen was collected by masturbation method into sterile cups,incubated at 37℃,and liquefied completely within 60 min.40 specimens were included in each of the two groups.The diagnostic criteria of asthenozoospermia were based on the World Health Organization guidelines,which classified spermatozoa into four classes according to their viability:Class A,Class B,Class C and Class D,the value of the sum of Class A and Class B spermatozoa to the total number of spermatozoa was taken as the PR value,which is an important index to assess spermatozoa viability,and the PR value of spermatozoa in asthenozoospermia was<32%,or the viability of grade A sperm<25%.2.Immunofluorescence(IF),Reberse transcription polymerase chain reaction(RT-qPCR)and Western blotting(WB)were used to detect GRIM-19 in the sperm cells of the two groups.The distribution of GRIM-19 and the expression of mRNA and protein levels in sperm cells of the two groups were detected,and the smoothing curve was established to investigate the association between GRIM-19 and the development of asthenozoospermia.Results1.Immunofluorescence analysis showed that GRIM-19 is expressed in both asthenozoospermia group and normal group,mainly in the head of spermatozoa and the middle part where the mitochondria were located,and the fluorescence expression of GRIM-19 in spermatozoa of the normal control group seemed to be significantly higher than the asthenozoospermia group.2.The results of RT-qPCR analysis showed that the mRNA expression of GRIM-19 in the spermatozoa of the asthenozoospermia group was significantly lower than that of the normal control group compared with the normal control group,P<0.001.The smoothed fitted curve further clarified that there was a significant negative correlation between the mRNA expression level of GRIM-19 and the occurrence of asthenozoospermia(OR 0.266.95%CI=0.081～0.868;P=0.028).3.The results of Western blotting analysis showed that the protein expression of GRIM-19 in the spermatozoa of the asthenozoospermia group was significantly lower than that of the normal group,P<0.001,which was statistically different.Conclusion1.GRIM-19 was mainly expressed in the head of spermatozoa and the middle part where mitochondria are located.The mRNA and protein expression levels of GRIM-19 in the asthenozoospermia group were significantly lower than the normal control group,and the smoothing curve further clarified that there was a significant negative correlation between the two groups.2.GRIM-19 is closely related to the occurrence of asthenozoospermia,and it may be involved in the process of energy metabolism,proliferation and apoptosis of spermatozoa.Part ⅡObjectiveTo investigate the role of GRIM-19 in GC-2 spd spermatocytes on proliferation,apoptosis and migration ability,and to analyze the mechanism of GRIM-19 in the pathogenesis of asthenozoospermia.Design1.GC-2 spd spermatocytes were obtained from the ATCC Cell Bank(Manassas,Virginia,USA).The GC-2 spd spermatocytes were subjected to overexpressing and silencing of GRIM-19,the cell models were constructed,and the expression of GRIM-19 in the cell models was verified by RT-qPCR and Western blotting.2.The proliferation of GC-2 spd spermatocytes with overexpressing GRIM-19 and silencing GRIM-19 was examined by MTT assay.3.The apoptosis of GC-2 spd spermatocytes with overexpressing GRIM-19 and silencing GRIM-19 was examined by flow cytometry assay.4.The migration ability of GC-2 spd spermatocytes with overexpressing GRIM-19 and silencing GRIM-19 was examined by cell scratch assay.Results1.Western blotting showed that the protein expression of GRIM-19 was significantly higher in GC-2 spd spermatocytes with overexpressing GRIM-19 compared with the control group,and the difference was statistically significant,P<0.01.Compared with the control group,the protein expression of GRIM-19 was significantly lower in GC-2 spd spermatocytes silenced with GRIM-19,and the difference was statistically significant,P<0.001.2.RT-qPCR results showed that the mRNA expression of GRIM-19 was significantly higher in GC-2 spd spermatocytes with overexpressing GRIM-19 compared with the control group,and the difference was statistically significant,P<0.001,The mRNA expression of GRIM-19 was significantly lower in GC-2 spd spermatocytes silenced with GRIM-19,and the difference was statistically significant,P<0.001.3.MTT assay results showed that compared with the control group,overexpression of GRIM-19 significantly increased the proliferation ability of GC-2 spd spermatocytes,and the proliferation were significantly higher at 12h,24h,48h and 72h,respectively,and the differences were statistically significant.Compared with the control group,silencing GRIM-19 significantly reduced the proliferation ability of GC-2 spd spermatocytes,and the proliferation were significantly lower at 12h,24h,48h and 72h,and the difference was statistically significant.4.Flow cytometry showed that the apoptosis rate of GC-2 spd spermatocytes with overexpressing GRIM-19 was reduced compared with the control group,and the difference was statistically significant,P<0.001.The apoptosis rate of GC-2 spd spermatocytes silenced with GRIM-19 was increased compared with the control group,and the difference was statistically significant,P<0.01.5.Cell scratch assay showed that the migration ability of GC-2 spd spermatocytes with overexpressing GRIM-19 was increased compared with the control group,and the difference was statistically significant,P<0.01.Compared with the control group,the migration ability of GC-2 spd spermatocytes silenced with GRIM-19 was reduced,and the difference was statistically significant,P<0.01.Conclusion1.The GC-2 spd spermatocyte model with overexpressing GRIM-19 and silencing GRIM-19 was successfully constructed.2.The proliferation and migration ability of GC-2 spd spermatocytes with overexpressing GRIM-19 were increased,while the apoptosis rate was reduced.3.The proliferation and migration ability of GC-2 spd spermatocytes with silencing GRIM-19 were reduced,while the apoptosis rate was increased.4.GRIM-19 promoted cell proliferation and migration ability in GC-2 spd spermatocytes and inhibited the apoptotic of GC-2 spd spermatocytes,it may be related to the pathogenesis of asthenozoospermia.Part ⅢObjectiveTo investigate whether GRIM-19 can affect the expression of ERK1/2 in GC-2 spd spermatocytes,and explore the regulatory role of ERK1/2 signaling pathway on proliferation,apoptosis and migration in GC-2 spd spermatocytes,and to analyze the regulatory mechanism of GRIM-19 in asthenozoospermia disease through ERK1/2 signaling pathway.Design1.Western blotting verified the expression level of p-ERKl/2 in overexpressing GRIM-19 and silencing GRIM-19 of GC-2 spd spermatocytes.2.GC-2 spd spermatocyte models with silencing of p-ERK1/2,silencing of GRIM-19,silencing of GRIM-19 with silencing of p-ERK1/2 were established,and verified the expression of GRIM-19 and p-ERK1/2 in each cell model by Western blotting assay.3.The proliferation ability of GC-2 spd spermatocytes with silencing of p-ERK1/2,silencing of GRIM-19,and silencing of GRIM-19 with silencing of p-ERK1/2 was examined by MTT assay.4.The apoptosis ability of GC-2 spd spermatocytes with silencing of p-ERK1/2,silencing of GRIM-19,and silencing of GRIM-19 with silencing of p-ERK1/2 was examined by flow cytometry.5.The migration ability of GC-2 spd spermatocytes with silencing of p-ERK1/2,silencing of GRIM-19,and silencing of GRIM-19 with silencing of p-ERK1/2 was examined by cell scratching.6.The expression of HIF-1α,c-Caspase 3 and COX-2 proteins,which are closely related to the development of asthenozoospermia,were examined in GC-2 spd spermatocytes with silencing of p-ERK1/2,silencing of GRIM-19,and silencing of GRIM-19 with silencing of p-ERK1/2 using Western blotting assay.Results1.Compared with the control group,the protein expression of GRIM-19 was significantly higher in GC-2 spd spermatocytes with overexpressing GRIM-19,P<0.001,while the protein expression of p-ERK1/2 was significantly lower,P<0.01.Compared with the control group,the protein expression of GRIM-19 in GC-2 spd spermatocytes with GRIM-19 silencing was significantly reduced,P<0.01,while the protein expression of p-ERK1/2 was significantly increased,P<0.01.2.The results of western blotting showed that the protein expression of p-ERK1/2 in GC-2 spd spermatocytes silenced with p-ERK1/2 was significantly decreased compared with the control group,P<0.001,the protein expression of p-ERK1/2 in GC-2 spd spermatocytes silenced with GRIM-19 was significantly increased compared with the control group,P<0.01,compared with silencing GRIM-19,the protein expression of p-ERK1/2 was significantly lower in silencing GRIM-19 with silencing p-ERK1/2,P<0.001.3.Compared with the control group,silencing GRIM-19 significantly decreased the proliferation ability of GC-2 spd spermatocytes,P<0.05,silencing p-ERKl/2 significantly increased the proliferation ability of GC-2 spd spermatocytes,P<0.05,and further silencing p-ERK1/2 expression in GC-2 spd spermatocytes with silencing GRIM-19 resulted in a significant increase in proliferation compared with silencing GRIM-19,P<0.05.4.Compared with the control group,silencing GRIM-19 caused a significant increase in apoptosis in GC-2 spd spermatocytes,P<0.01,silencing p-ERK1/2 caused a decrease in apoptosis in GC-2 spd cells,P<0.001,and further silencing p-ERK1/2 expression in GC-2 spd spermatocytes with silencing GRIM-19 resulted in a significant reduced in apoptosis compared with silencing GRIM-19,P<0.001.5.Compared with the control group,silencing GRIM-19 significantly reduced the migration ability of GC-2 spd spermatocytes,P<0.001,silencing p-ERK1/2 significantly increased the migration ability of GC-2 spd spermatocytes,P<0.01,and further silencing p-ERK1/2 expression in GC-2 spd spermatocytes with silencing GRIM-19 resulted in a significant increase in migration compared with silencing GRIM-19,P<0.01.6.Compared with the control group,the expression of HIF-1α,c-Caspase 3 and COX-2 proteins was significantly decreased in GC-2 spd spermatocytes silenced with p-ERK1/2,P<0.05,the expression of HIF-1α,c-Caspase 3 and COX-2 proteins was increased in GC-2 spd spermatocytes silenced with GRIM-19,P<0.05,while the expression of HIF-1α,c-Caspase 3 and COX-2 proteins was significantly reduced in GC-2 spd spermatocytes silencing p-ERK1/2 with silencing GRIM-19 compared with silencing GRIM-19,P<0.05.Conclusion1.GRIM-19 can regulate the protein expression of p-ERK1/2 in GC-2 spd spermatocytes.2.GC-2 spd spermatocytes models with silencing of p-ERK1/2,silencing of GRIM-19 and silencing of GRIM-19 with silencing of p-ERK1/2 were successfully constructed.3.Silencing p-ERK1/2 can increase the proliferation ability,decrease the apoptosis and increase the migration ability of GC-2 spd cells.4.Silencing of p-ERK1/2 expression in GC-2 spd spermatocytes silenced with GRIM-19 prevented the reduced proliferation,increased apoptosis and reduced migratory capacity of GC-2 spd spermatocytes induced by silencing GRIM-19.5.GRIM-19 can inhibit the protein expression of HIF-1α,c-Caspase 3 and COX-2 in GC-2 spd spermatocytes through the ERK1/2 pathway,and it is involved in the disease of asthenozoospermia.