Study On The Mechanism Of Male Reproductive Damage Induced By Triclosan Exposure Through Autophagy

BackgroundInfertility is a public health issue of widespread concern in society,which affects approximately 8-12%of couples worldwide,and 40-50%of them may be due to "male factors",One of the main reason for male infertility is the reduction of sperm count and quality.Studies have shown that many Environmental endocrine disruptors(EEDs)can cause damage to the reproductive system of humans and mice,and reduce the quality of male’s sperm.Triclosan(TCS)is a broad-spectrum antibacterial agent which is widely used in personal care products and clinical environments,and considered to be a potential EEDs by some researchers.Sperm production is a highly ordered process that relies on the balance between testicular germ cell proliferation,differentiation and death.Autophagy is a form of cell death that differs from apoptosis.Moderate autophagy promotes sperm differentiation and mature,and protects spermatogenic cells from harmful conditions,and autophagy beyond the ability of cells will induce programmed cell death.At the beginning,we selected ten pairs of sperm samples(with abnormal sperm quality in case group and with normal sperm quality in control group)for screening autophagy-related gene that expressed differently in two groups,and combined with autophagy-related gene related to the mechanism of EEDs in the reports of the literature,we finally selected CXCR4,ESR1,PTEN,LC3B Genes to study,At present,whether TCS could cause germ cell damage through autophagy has not been reported.This paper aims to investigate the possible mechanism of male germ cell injury induced by triclosan exposure through autophagyMaterials and MethodsIn the first part:screening and verification of autophagy-related genes.The subjects were selected from the men who underwent semen examination at the reproductive center of Grade-A Tertiary Hospital in Shenzhen from June 2016 to September 2017,The semen samples,the results of semen analysis and questionnaire information were collected from the object of study.Methods:1.Case-control method was used to select 10 cases of abnormal sperm quality and normal respectively,and the autophagy chip about sperm sample was conducted,Combined with the genes related to the mechanism of EEDs from the literatures.The result will reveal the differentially expressed autophagy-related gene,which may be related to TCS and lead to decreased semen quality.2.Using a case-control study method in groups,104 cases of abnormal semen quality and 250 cases of normal semen quality were selected to verify the differentially expressed genes.In the second part:Animal experiments exposed by TCS.Eight-week-old SD rats were exposed orally with TCS in the blank group,corn oil control group(10 mg/kg),low dose group(10 mg/kg),medium dose group(50 mg/kg)and high dose groups(250 mg/kg)respectively.Designed exposure dose with no observed adverse effects(NOAEL=50 mg/kg/d)for medium exposure doses.Testicular tissue damage and expression of autophagy-related genes were observed in the five experimental groups.Methods:3.HE staining and transmission electron microscope were used to observe the pathological damage of testicular tissue 4.the Screened autophagy-related genes were detected by immunohistochemistry,Western blot and PCR.Results1.In the result of autophagy gene chip,compared with the control group,among the 84 autophagy-related functional genes,there are 18 autophagy-related gene with the expression differential multiples greater than 2 and all expressions were down-regulated.Combined with literature reports related to EEDs,we finally screened CXCR4,ESR1,PTEN and LC3B four environmental-autophagy related genes.2.In the validation phase of population,the expression of ESR1 gene was significantly decreased in the case group compared with the control group(P<0.05),and CXCR4,PTEN and LC3B showed a downward trend in the case group compared with the control group,but it was not statistically significant.3.The results of HE staining showed that,testicular seminiferous tubule injury,vacuolar-like changes,sperm reduction,and there may be dose-response relationship between each dose group Transmission electron microscope revealed mature autophagosome lysosomal structure,Golgi swelling and vacuoles.4.The results of immunohistochenistry for LC3B protein showed that only a small amount of pale yellow particles in the blank group and the solvent control group,while a large number of brown-yellow particles appeared in the low,medium and high dose groups,and moved away from the seminiferous tubules with increasing dose.distributed.however CXCR4,ESR1 and PTEN protein expression did not differ between groups;Compared with blank group and the solvent control group,the results of qPCR also showed the expression of LC3B gene was increased in the all case group;besides,the results of Western blot showed the expression of LC3B-Ⅱ and LC3B-Ⅱ/Ⅰ was increased in the low and medium dose groups.Conclusions1.The low expression of the mRNA expression levels of the autophagy-related gene ESR1 in the human population may be related to abnormal semen quality.2.TCS exposure can lead to damage to the testicular seminiferous tubules,and cause the reduction of spermatogenic cells in SD rats,which may reveal TCS has a certain reproductive toxicity3.TCS exposure increased the mRNA expression levels of LC3B gene,LC3B-Ⅱprotein and LC3B-Ⅱ/Ⅰ in testis of male rats,suggesting that autophagy may be involved in the process of TCS-induced testicular tissue damage.