| Part Ⅰ.The association between semen selenium and semen quality ObjectiveTo investigate the association between semen selenium content and semen quality,and to provide an epidemiological reference basis for clinical prevention and treatment of male infertility.MethodsA hospital-based case-control study method was conducted,with the case group derived from 262 cases of abnormal semen quality who visited a 3A hospital in Shenzhen for semen examination from September 2016 to March 2018,and the control group based on 262 cases of normal semen quality matched for age ± 0 years and abstinence time ± 0 days in the case group,and all study subjects received their informed consent.Based on semen quality abnormal parameters,the case groups were divided into oligospermia group,asthenospermia group,and oligozoospermia group.The basic information,life habits,disease history and other factors of the study subjects were investigated through self-designed questionnaires;semen samples were collected,and the computer-assisted sperm analysis(CASA)system was used to detect the routine indexes of semen quality;semen selenium concentration was detected by inductively coupled plasma mass spectrometry(ICP-MS),and the correlation between semen selenium concentration and semen quality was analyzed using conditional logistic regression models.ResultsIn the total case-control population,sperm concentration,total sperm count,progressive motility rate,total motility rate,and selenium concentration were higher in the control group than in the case group;however,semen selenium concentration in the asthenospermia group did not differ from its matched control group.In the logistic regression model,after adjusting for BMI,education level,smoking status and alcohol consumption frequency,an increase in semen selenium concentration was associated with a decreased risk of abnormal semen quality(P trend=0.025);with an increase in semen selenium grade concentration,the risk of developing in the oligospermia group showed a decreasing trend(P=0.001),and the risk of developing in the oligozoospermia group.With the increase of semen selenium grade concentration,the risk of developing in the asthenospermia group showed an increasing trend,and the risk of developing in the Q4 group was 2.72 times higher than that in the Q1 group(OR=2.72,95%CI=1.25～5.92),but the difference was not statistically significant(P trend=0.135).Since fewer cases were found in the oligospermia group,a linear regression model was further used to analyze the association between semen selenium concentration and sperm concentration and total sperm count,which was consistent with the logistic regression model results.ConclusionAn increase in semen selenium concentration was positively correlated with an increase in sperm concentration and total sperm count.Selenium is beneficial in reducing the risk of developing abnormal semen quality.Part Ⅱ.GPx4-mediated mechanism study of selenium and GC2-spd cells apoptosis ObjectiveTo investigate the effect of selenium on apoptosis of mouse testicular spermatocytes(GC2-spd cells)and the potential mediating role of selenoprotein GPx4.To provide new molecular targets for the prevention and treatment of male infertility.MethodsGC2-spd cells were used to study the effect of selenium on their apoptosis.According to the sequence of mouse GPx4 gene,the recombinant adenovirus suppression vector(sh RNA-GPx4,fluorescent protein GFP as control gene)and overexpression vector(ORF-GPx4,fluorescent protein Mcherry as control gene)were amplified and transfected with GC2-spd cells.For GC2-spd cells and adenovirustransfected cells,the cells were cultured using serum-free medium for 24 h to simulate selenium deficiency,and then the cells were intervened with the optimal concentration of selenium(0.5 μM sodium selenite,5.0 μM selenomethionine)and excess concentration of selenium(5.0 μM sodium selenite,50.0 μM selenomethionine)for 24 h,respectively.The cell proliferation activity of each group was compared.Malondialdehyde(MDA)content and 15-lipoxygenase(lox15)gene expression level were detected to respond to the degree of cellular lipid peroxidation.Apoptosis level was detected by flow cytometry,GPx4 protein expression was detected by Western Blot,and the expression levels of apoptosis-related genes was detected by quantitative realtime fluorescence PCR(q RT-PCR).Results1.In GC2-spd cells,selenium deficiency resulted in decreased cell proliferation activity,decreased GPx4 protein expression,decreased percentage of viable cells,increased percentage of early apoptosis,and increased MDA content.And in moderate supplementation with 0.5 μM sodium selenite or 5.0 μM selenomethionine,the proliferative activity,percentage of living cells,and GPx4 protein expression of GC2-spd cells were significantly higher and the percentage of early apoptosis and MDA content were significantly lower compared with the selenium-deficient group.However,the excessive supplementation with 5.0 μM sodium selenite led to a decrease in cell proliferation activity and GPx4 protein expression protein expression.2.In GC2-spd cells with adenoviral inhibition of GPx4 gene,the control group of sh RNA-GPx4 cells showed decreased proliferative activity,increased percentage of early apoptosis,increased expression levels of apoptosis related genes caspase3 and bax,and increased lox15 gene and MDA content compared with the control group of GFP cells.When sh RNA-GPx4 cells were moderately supplemented with 0.5 μM sodium selenite or 5.0 μM selenomethionine,the percentage of early apoptosis was decreased,caspase3 and bax gene expression was decreased and MDA content was reduced compared with the selenium-deficient group.3.In GC2-spd cells with adenoviral overexpression of GPx4 gene,the control group of ORF-GPx4 cells showed a decreased percentage of early apoptosis,increased cell proliferation activity and decreased MDA content compared with the control group of Mcherry cells.The MDA content in the selenium-deficient group of ORF-GPx4 cells was significantly lower than that in the selenium-deficient group of Mcherry cells.ConclusionGPx4 was involved in regulating lipid peroxidation and apoptosis in GC2-spd cells.Selenium mediates through GPx4 to reduce the degree of lipid peroxidation,promote cell proliferation activity and protect GC2-spd cells from apoptosis. |
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