Reproductive Toxicity And Molecular Mechanism Of Fluoride Exposure In Male Mice Under Different Dietary Calcium Levels

Fluorine(F)is an essential trace element for the human body.Excessive accumulation of fluorine causes systemic physiological and pathological changes in the body,which is called endemic fluorosis.Dental fluorosis and skeletal fluorosis are the main symptoms of bone-related organ damage caused by exposure to fluoride.Exposure to fluoride can also cause damage to non-skeletal organs,such as the brain,kidneys,and liver.Studies have shown that excessive intake of fluoride can penetrate the blood-testis/epididymal barrier,accumulate in the reproductive organs,and affect the normal physiological functions of the male reproductive system.In recent years,the impact of fluorosis on the male reproductive system has become a research hotspot in endemic fluorosis.Ca²⁺is an important"second messenger"in the body's cells,and it is also the key to Ca²⁺signal transduction to complete a series of physiological functions.It has been reported in the literature that calcium supplementation can reduce the absorption and toxicity of fluorine in the body,and reduce the rate of cell apoptosis.However,there has been no systematic report about the effect of fluorine exposure on male reproduction toxicity and molecular mechanisms under different calcium levels.120 first-weaned ICR male mice were selected in this experiment.After being adapted to the breeding environment for 7 days,they were divided into 6 groups according to the random number table of body weight.After 3 and 6 months of fluoride exposure,observe the mouse dental fluorosis,and detect the blood/urine fluoride,blood/urine calcium content,and measure the free radical NO content of testicular cells,NOS and antioxidant enzyme CAT activity,and the male Reproductive endocrine T,LH and FSH levels;observe testicular cell morphology and sperm ultrastructure;observe testicular cell apoptosis rate and Ca²⁺level,test testicular cell membrane L-type calcium channel Cav1.2,endoplasmic reticulum stress partner molecule GRP78,Endoplasmic reticulum stress and apoptosis pathway signaling molecules IRE1,TRAF2,ASK1,JNK,Caspase-3 gene/protein expression level,it is the first time to study the effect of fluoride exposure on male reproductive toxicity and molecular mechanism from the endoplasmic reticulum pathway system.Provide basic information for exploring economical and effective intervention methods of fluoride-induced male reproductive toxicity and perfecting the theory of"fluoride-induced calcium contradictory diseases".The results of the study are as follows:(1)Replication of subchronic and chronic fluorosis models in miceCompared with group C,the symptoms of dental fluorosis in the sub-chronic and chronic fluoride exposure groups were obvious,and the urinary fluoride content extremely significantly increased(P<0.01).The results showed that the sub-chronic and chronic fluoride exposure models in this experiment were successfully replicated.(2)The statistical results of the detection of NO content,NOS and antioxidant enzyme CAT activity in mouse testis tissueCompared with group F,CAT activity in testis tissue of mice in subchronic/chronic fluoride exposure LCa+F group was significantly or extremely significantly decreased(P<0.05 or P<0.01),and NOS activity and NO content were significantly increased(P<0.05).The CAT activity in the testis tissue of HCa+F mice was significantly or extremely significantly increased(P<0.05 or P<0.01),and the NOS activity and NO content in the testis tissue of the HCa+F mice were significantly or extremely significantly decreased(P<0.05 or P<0.01).(3)The statistical results of the detection of serum testosterone,luteinizing hormone,and follicle stimulating hormone levels in miceCompared with group F,the serum testosterone levels of mice in the HCa+F group with chronic fluorine exposure were significantly increased(P<0.05),and the levels of luteinizing hormone and follicle stimulating hormone were significantly decreased(P<0.05).(4)Observation of the morphological structure of mouse testis cellsCompared with group F,the structure of spermatogonia at all levels in LCa+F group increased and the number of sperm further decreased,while the number of spermatogonia,mesenchymal cells,spermatocytes and sperm in HCa+F group increased.(5)Observation results of ultrastructure of mature sperm in mouse epididymisObservation of the head structure of mature sperm in mouse epididymis showed that compared with group F,the overall structure of sperm in the LCa+F group was incomplete,the acrosome was partially missing,and the double-layer membrane was broken;while the sperm morphology and structure of the HCa+F group were relatively normal,the top body is clearly visible,and the double layer membrane is relatively complete.Observation of the longitudinal section structure of the middle section of the mature sperm tail in the mouse epididymis showed that compared with the F group,the mitochondria in the LCa+F group were incomplete,arranged loosely,mitochondria were missing,and the double-layer membrane was shed;while the mitochondria in the HCa+F group had regular morphology and size evenly,the arrangement is slightly loose.Observation of the cross-sectional structure of the middle section of the mature sperm tail in the mouse epididymis showed that compared with the F group,the mitochondria of the LCa+F group were missing,the mitochondria and the cell membrane gap were significantly widened,the boundary between the sperm was blurred,and the mitochondria of the HCa+F group were more blurred.The structure is complete and the cell membrane is clearly visible.(6)Test results of cell apoptosis in mouse testesCompared with the F group,the number of testicular cell apoptosis in the subchronic and chronic fluoride exposure LCa+F mice was extremely significantly increased(P<0.01),while the number of testicular cell apoptosis in the subchronic and chronic fluoride exposure HCa+F mice was extremely significantly decreased(P<0.01).(7)Test results of Ca²⁺level in mouse testis cellsCompared with the F group,the intracellular Ca²⁺level in the testis cells of the subchronic and chronic fluorine exposure mice in the LCa+F group was significantly increased(P<0.05),and the Ca²⁺level in the testis cells of the HCa+F group was significantly or extremely significantly decreased(P<0.05 or P<0.01).(8)Statistic results of Cav1.2 mRNA expression and endoplasmic reticulum apoptosis signaling pathway molecules mRNA in mouse testis cell membraneCompared with group F,the expression level of GRP78 mRNA in testicular cells of mice in subchronic fluorine exposure LCa+F group was significantly increased(P<0.05),and the expression level of ASK1 and TRAF2 mRNA was significantly decreased(P<0.05).Mice in HCa+F group testicular cell membrane Cav1.2 and testicular cells GRP78,JNK,Caspase-3 mRNA expression levels were significantly or extremely significantly decreased(P<0.05 or P<0.01),IRE1,ASK1,TRAF2mRNA expression levels were significantly or extremely significantly decreased(P<0.05 or P<0.01).Chronic fluoride exposure LCa+F mice testicular cell membrane Cav1.2 and testicular cell GRP78,JNK,Caspase-3 mRNA expression levels were significantly increased(P<0.05),IRE1,ASK1,TRAF2 mRNA expression levels were significantly or extremely significantly decreased(P<0.05 or P<0.01),the expression levels of GRP78,JNK,and Caspase-3 mRNA in the testis cells of mice in the HCa+F group were significantly or extremely significantly decreased(P<0.05 or P<0.01),and the expression levels of IRE1,ASK1,and TRAF2 significantly or extremely significantly increased(P<0.05 or P<0.01).(9)Statistic results of Cav1.2 protein expression and endoplasmic reticulum apoptosis signaling pathway molecules protein in mouse testesCompared with group F,the expression levels of JNK and Caspase-3 protein in testicular cells of mice in subchronic fluorine exposure LCa+F group were significantly or extremely increased(P<0.05 or P<0.01),and the protein expression levels of IRE1,ASK1,and TRAF2 were significantly Or extremely significantly decreased(P<0.05 or P<0.01),HCa+F mice testicular cell membrane Cav1.2 and testicular cell GRP78,JNK,Caspase-3 protein expression levels significantly or extremely significantly decreased(P<0.05 or P<0.01),the protein expression levels of IRE1,ASK1,and TRAF2 significantly or extremely significantly increased(P<0.05 or P<0.01).Chronic fluoride exposure LCa+F mice testicular cell membrane Cav1.2 and testicular cells JNK,Caspase-3 protein expression levels were significantly or extremely significantly increased(P<0.05 or P<0.01),IRE1,TRAF2 protein expression levels were extremely significantly decreased(P<0.01),the expression levels of Cav1.2,GRP78,and JNK protein in the testicular cell membrane of mice in the HCa+F group were significantly decreased(P<0.05),and the expression levels of IRE1,ASK1,and TRAF2 protein were significantly or extremely significantly increased(P<0.05)or P<0.01).In summary,the reproductive toxicity and molecular mechanism of fluoride exposure in male mice may be:subchronic and chronic fluoride exposure increase the content of free radicals in testicular cells,decrease the antioxidant capacity,and testicular cell L-type calcium channel Cav1.2 Increased gene/protein expression levels increase the influx of extracellular Ca²⁺,resulting in excessive endoplasmic reticulum stress.The endoplasmic reticulum chaperone GRP78 gene/protein of testicular cells is significantly increased,and the endoplasmic reticulum pathway apoptosis signaling pathway molecule IRE1,ASK1 and TRAF2 genes/proteins were significantly decreased,and downstream apoptosis signaling molecules JNK,Caspase-3genes/proteins were significantly increased,and the testicular cell apoptosis was abnormally enhanced,leading to endocrine disorders and ultimately damaging the male reproductive system.Dietary high calcium(2%)can significantly reverse the abnormal antioxidant capacity caused by fluorine exposure,intracellular calcium overload and abnormal expression of testicular cell membrane,endoplasmic reticulum chaperone molecules and apoptosis regulatory signal molecules,and reduce the rate of testicular cell apoptosis.So as to antagonize the reproductive damage of male mice caused by fluorine exposure.Dietary high calcium(2%)may be a cost-effective anti-fluoride agent.