Glyphosate-induced Oxidative Stress Contributes To Male Reproductive Toxicity In Rats

As a broad-spectrum herbicide,glyphosate(GLY)-based herbicides have been becoming the most used herbicide around the globe.The excessive use of GLY leads to increasing residues in the ecological environments,posing a threat to public health.It is clear that GLY has toxic effect on multiple organs and systems in animals,and its impacts on male reproductive health have attracted increasing attention.The male reproductive toxicity of GLY has only been reported from epidemiology and simple animal experiments.However,the mechanisms of GLY-induced male reproductive toxicity have not been systematically and deeply deciphered.Therefore,taking oxidative stress as the perspective point,this study employed rats as research subjects,in vivo and in vitro experiments,to further reveal the mechanisms of GLY-induced male reproductive toxicity in terms of testicular GLY residues,gut microbiota dysbiosis,blood-testis barrier damage,and autophagy.1.Effects of GLY exposure on male reproductive health in ratsA total of 108 4-week-old specific pathogen-free(SPF)Sprague Dawley male rats(body weight 112.4 ± 6.7g)were randomly divided into 3 groups(36 rats per group)after 1-week acclimation,including the control group,low-dose group,and high-dose group.Rats in each group were fed the commercial chow evenly supplemented with 0,10 and 250 mg/kg GLY,respectively.Rats was allowed to be free access to sterilized feed and water.The experiment period spanned for four months.Serum and different organs(testis,epididymis,liver,kidney,spleen,and brain)of 18 rats in each group were collected at 2 and 4 months,respectively,to explore the effects of GLY on reproductive health of rats.Compared with the control,the average daily gain and organ coefficient of rats exposed to GLY had no significant changes,but the average diet consumption of rats in the high-dose exposure group was dramatically more than that in the control group.GLY levels in the testis were determined using GC-MS/MS assay after exposure to GLY for 4 months and the residues of the three groups were 0,0.002 ± 0.001 and 0.016 ± 0.006 ?g/g,separately.Morphological results of testis and epididymis showed that compared with the control group,spermatogenic cells in seminiferous tubules were arranged loosely and vacuolated increasingly in the low-dose exposed rats for both 2 months and 4 months.In the high-dose exposed rats for 2 months and4 months,the seminiferous tubules were atrophied and deformed,the thickness of the seminiferous tubules became thinner,the space between adjacent seminiferous tubules was increased,and spermatogenic cells were arranged loosely,even dropped off,and vacuolated increasingly.Whereas,there were no obvious lesions in the epididymis during the whole experiment.Computer-assisted sperm analysis(CASA)showed that GLY decreased sperm quality in rats after exposure for either 2 months or 4 months,mainly manifesting the decreased sperm concentration,motility,rapid progressive and rapid velocity,as well as VCL,VSL,VAP,ALH,BCF,and LIN.After GLY exposure for either 2 months or 4 months,serum levels of testosterone,luteinizing hormone,follicle-stimulating hormone,and inhibin-B were decreased significantly,while serum levels of estrogen were increased dramatically.Finally,we measured the changes of parameters related to oxidative stress and found that the contents of ROS in the testis and MDA in the serum were increased in a dose-dependent manner,while the levels of TAOC,SOD,CAT,and GPx were decreased dramatically.The results of animal experiments indicate that GLY interferes with sex hormone levels and causes testicular oxidative damage,resulting in decreased sperm quality and affecting male reproductive health.2.GLY-induced gut microbiota dysbiosis facilitates testicular oxidative damage in ratsWhile GLY enters into the intestinal tract(mainly accumulating in the small intestine)through contaminated food and water,interfering with the innate gut microbiota,so we wonder whether the changes of intestinal microbiota affect reproductive health.Therefore,this part of the study selected rats exposed to GLY for 2 months as research candidates to probe the correlation between the change of gut microbiota and male reproductive health.First,16 S r DNA sequencing of gut microbiota of 5 rats randomly selected from the control group and the high-dose treatment group,respectively,was analyzed and the results showed that the diversity of gut microbiota from GLY-treated rats was significantly changed.The relative abundance of Firmicutes was notably declined,while the relative abundance of Bacteroidetes and Tenericutes was dramatically increased.Then,we analyzed the predominant19 families with the abundance greater than 1% and found Prevotellaceae and Bacteroidaceae,belonging to Bacteroidetes phyla,were shown to be the most obviously increased families.At the genus level,the relative abundances of Lactobacillus and Enterorhabdus were notably decreased after GLY treatment,while the relative abundances of Bacteroides and Prevotella?1were significantly increased.Thereafter,we analyzed the correlation between the changes of Bacteroides and Prevotella?1 and sperm quality,and found that both the combined relative abundance of these two genera and the relative abundance of any single genus were negatively correlated with sperm motility and rapid velocity.Subsequently,we probed the causes of sperm quality caused by the changes of the two gut bacteria genera.The results showed that the increase of the two genera can drive the migration of gut-derived Th17 cells to testis,stimulating the production of testicular IL-17 A and activating the IL-17 A signaling pathway in the testis.The activation of IL-17A/TNF-? signaling pathway in turn triggered oxidative stress in rat testicular tissue,resulting in sperm quality decrease.In summary,GLY induces dysbiosis of special gut microbiota to activate the IL-17 A signaling pathway in the testis,inducing oxidative stress and ultimately leading to decreased sperm quality.3.GLY damages blood-testis barrier via NOX1-triggered oxidative stress in ratsIn vivo experiments revealed that GLY induced testicular oxidative stress in rats.To further explore GLY-induced oxidative stress resulting in male reproductive toxicity,this part of the study selected the rats exposed to GLY for 4 months and primary rat Sertoli cells as research candidates to clarify how GLY damaged blood-testis barrier via oxidative stress in rats.First,we observed the integrity of blood-testis barrier using biotin trace assay and transmission electron microscope(TEM)and the results indicated that biotin was penetrated into spermatogenic tubules in both low-and high-dose of GLY exposure for 4 months(red fluorescence),which was in agreement with the results of TEM showing that the ultrastructure of blood-testis barrier was blurred and broken.q PCR and western blotting assays further confirmed that GLY significantly decreased the expression of key genes and proteins.These findings indicated that GLY damaged the integrity of blood-testis barrier.Next,to explore the possible contributors,we analyzed the transcription changes of testis after GLY exposure and RNA-Seq analysis showed that GLY significantly up-regulated the expression of NOX1,which catalyzes the generation of ROS by single electron transfer from NADPH to O2.These suggested that GLY may damage the integrity of blood-testis barrier through NOX1-triggered oxidative stress.To confirm this potential mechanism,we generated primary rat Sertoli cells model and found that 10 ?M GLY treatment for 24 h induced ROS over-production and significantly decreased antioxidant enzymes SOD2,CAT,and GPx1.Whereas,co-treatment 1m M NAC(a ROS scavenger)dramatically reduced GLY-induced ROS generation,as well as,reversed the expression of antioxidant enzymes and key proteins involving blood-testis barrier,suggesting that GLY-induced oxidative stress is a key factor to destroy blood-testis barrier.Subsequently,si-NOX1 assay verified that down-regulation of NOX1 reduced ROS levels and recovered the decreased levels of antioxidant enzymes,thereby improving the expression of key proteins related to blood-testis barrier damage induced by GLY.Using molecular docking and luciferase reporter assays,GLY can directly bind to ER-? to form four hydrogen bonds with Pro399 and Lys401(binding energy:-1.81 kcal/mol)and activate ER-? transcriptional activity.ER-? activation upregulated NOX1 expression,which leads to damage of the bloodtestis barrier.These results indicate that GLY directly binds to ER-? to mediate the expression of NOX1,which then induces oxidative stress and damages blood-testis barrier,finally causing male reproductive dysfunction in rats.4.GLY-induced oxidative stress leads to autophagy disorder in rat testisPrevious studies have found that GLY can directly or indirectly affect Sertoli cells,inducing oxidative stress.As a group of key cells consisting of blood-testis barrier and regulating spermatogenesis,autophagy is essential for many normal functions of Sertoli cells.Therefore,to clarify the mechanisms of autophagy in GLY-induced destruction of blood-testis barrier,this part of the study selected the rats exposed to GLY for 4 months and primary rat Sertoli cells as research candidates to explore the effects of GLY-induced oxidative stress on autophagy in rats.First,through transmission electron microscope observation,GLY induced the formation and accumulation of autophagosomes in Sertoli cells of testis.Meanwhile,10 ?M GLY exposure for 24 h increased the expression of LC3-?and the accumulation of autophagosome in rat primary Sertoli cells.At the same time,we confirmed that the GLY activated autophagy flux in primary Sertoli cells after exposure for 24 h.Additionally,GLY exposure upregulates the expression of CTSB,CTSD,LAMP1,and LAMP2 in both testis and primary Sertoli cells,suggesting that GLY enhanced lysosome function.Finally,co-treating ROS scavenger NAC and GLY down-regulated the expression of LC3-? in protein levels and of CTSB,CTSD,LAMP1 and LAMP2 in m RNA levels.These results suggest that GLY-induced oxidative stress mediates autophagy activation in Sertoli cells.In conclusion,the conclusions were obtained:(1)Through an established GLY-exposed male rat model,GLY interferes with gut microbiota and directly enters testicular tissue,causing male reproductive health.(2)GLY-induced specific gut microbiota dysregulation negatively correlated with sperm quality.(3)GLY directly binds to ER-? and activates its transcriptional activity,which causes NOX1 expression,causing ROS accumulation and subsequently damaging blood-testis barrier.(4)GLY-induced oxidative stress mediates the enhancement of autophagy flux in rat Sertoli cells.This study demonstrates the mechanism of GLY-induced male reproductive toxicity and uncovers potential targets for preventing and treating GLYinduced male reproductive toxicity,which is of great significance to protect animal and human reproductive health and provides theoretical guidance for public health...