Role Of Endoplasmic Reticulum Stress In Pubertal Fluoride Exposure Impaired Testis Development And Spermatogenesis In Mice

Objective To explore the role of endoplasmic reticulum stress in pubertal fluoride exposure impaired testis development and spermatogenesis in mice.Methods This study was divided into two parts: in vivo and in vitro experiments.In vivo,we randomized 36 healthy ICR male mice into three groups: the control group,25mg/L and 50 mg/L fluoride exposure groups.The mice in the control group were given ultrapure water.The mice in the fluoride exposure groups drank ultrapure water containing 25 mg/L and 50 mg/L Na F,respectively.After 70 days,all mice were sacrificed and unilateral epididymis was taken for sperm counting.Serum testosterone levels were measured using ELISA assay.H&E staining was used to observe the histological morphology of testes.TUNEL staining was used to detect apoptosis of testicular germ cells.Immunohistochemistry was used to detect the expression of GRP78.Spectrophotometry was used to detect the levels of SOD,the content of GSH and MDA.Western blot and q RT-PCR were used to detect the expression of ERS related proteins and m RNA in the testes.In vitro,Mouse GC-1 spg cells were exposed to different doses of Na F for 24 h,and the apoptosis was detected using flow cytometry.Genomic sequencing was used to detect the various functions and information pathways of organisms enriched with differentially expressed genes.In addition,we used Western blot and q RT-PCR techniques to detect the expression of ER and Calcium channel related proteins and m RNA in GC-1 spg cells.Calcium contents in cytoplasm,ER and mitochondria were detected by Fluo-4 AM,Mag-Fluo-4 AM,and Rhod-2 AM calcium ion probes,respectively.The mitochondrial membrane potential(MMP)was measured using JC-1 as a fluorescent probe.We also use DCFH-DA probes to detect cellular ROS.SOD,GSH and MDA were detected by spectrophotometry.Results In vivo experiments,sperm counts and serum testosterone levels in the Na F exposure group decreased significantly compared with those in the control group.H&E staining showed that the walls of the testicular seminiferous tubule were thinner,the surrounding interstitial cells were lost,and the proportion of seminiferous tubules in the mature stage was significantly reduced in the Na F exposure groups.TUNEL staining showed that the number of apoptotic cells and positive tubules in the seminiferous tubules increased significantly in the Na F exposure groups.Immunohistochemistry showed that the number of GRP78 positive cells increased in the Na F exposure groups.Spectrophotometry showed that the levels of SOD and the content of GSH were significantly decreased,the content of MDA was significantly increased in the Na F exposure groups.The results of q RT-PCR showed that the m RNA expression of GRP78,GRP94,CHOP,ATF-6α,ATF-4 and XBP-1 were increased.Western blot showed that the protein expressions of GRP78,p-PERK,p-IRE1α,p-e IF2α,p-JNK,XBP-1,ATF-6α,ATF-4 and CHOP were enhanced in Na F exposure groups.In vitro experiments,Na F exposure significantly reduced the viability of mouse GC-1spg cells and significantly increased the total apoptosis rate.Genome sequencing analysis showed that GO enrichment of differentially expressed genes mainly affected protein fusion and cell metabolism.The roles of these differentially expressed genes in endoplasmic reticulum stress pathways and calcium molecular pathways were also investigated by KEGG pathway enrichment analysis.The results of q RT-PCR showed that the m RNA expression levels of GRP78,GRP94,CHOP and ATF-6α,GRP75,VDAC1,MCU were significantly increased in GC-1 spg cells of mice exposed to Na F.Western Blot showed that the protein expressions of GRP78 and p-PERK,p-IRE1α,p-e IF2α,XBP-1,ATF-6α,ATF-4,CHOP and p-IP3 R,GRP75,VDAC1,MCU increased in Na F exposure groups.Moreover,the study found that Na F triggered Ca2+ transfer from endoplasmic reticulum to mitochondria,and caused mitochondrial Ca2+ overload.Further research found that Na F elevated the levels of ROS,reduced MMP and induced the release of AIF and Cyt-c from the mitochondria to the cytosol.Conclusion1.Fluoride exposure during puberty induced testicular germ cell apoptosis,impaired testicular structure and reduced sperm counts in mice.2.Fluoride exposure during puberty may activate ER stress,promote the release of calcium ions from ER,cause mitochondrial calcium overload,thereby leading to the release of apoptotic factors such as AIF and Cyt-c from mitochondria into the cytoplasm,induce testicular germ cell apoptosis and spermatogenesis impairment.