The Study Of Bisphenol AF On Blood-testis Barrier And Spermatogenesis In Mouse

Environmental pollution an inevitable socio-ecological challenge behind our society development posing profound influences on living organisms and even on the whole ecological chain has been in the limelight of public concerns.Being an important system other than for passing down genetic information to next generation,reproductive system also as one of the most sensitive systems to environmental pollution.Human reproduction problems will ultimately cause slow or negative population growth and birth defects and severe socio-economic outcomes.As to animal husbandry,reproduction problems in animals will definitely affect economic benefits.Therefore,deciphering the of environmental pollution from molecular levels is of great significance for improving and enhancing animal fertility.Bisphenol AF,a fluoride homologous of bisphenol A,has been widely used in food packaging and pharmaceutical intermediates and other industrial productions and thereby has raised public concerns about its bio-safety when being released into environment during the process of production and use.BPAF has been ubiquitously detected in ambient medium,consumer products,food,and even human body fluids,suggesting the universality of its existence.However,the comprehensive assessment of the potential reproductive health risks when exposing to BPAF from dietary and non-dietary sources remains lacking.Accumulating evidence demonstrated that BPAF exerts higher endocrinological effects than BPA in estrogenic,anti-estrogenic and anti-androgenic activities.While BPAF could be categorized as teratogenic,neuropathic,developmental and reproductive toxicities,its impact on male fertility remains elusive.In this study,by establishing BPAF subacute poisoning model,Kun Ming male mice were randomly divided into four groups(n=9)and orally exposure to BPAF for 28 consecutive days at dose of 5,20,and 50 mg/kg(body weight)or only corn oil(vehicle control),respectively.The body weight of mice was monitored twice a week,and mice were anesthetized and sacrificed after the last exposure for further analysis.Testes and epididymides were then isolated immediately and weighted to calculate the absolute and relative weights.By detecting reproductive toxicity indexes such as sperm viability and quality,testosterone levels in serum and BTB integrity to evaluate the reproductive toxicity of BPAF and decipher the molecular mechanism.The main results are as follow:(1)Male mice were orally exposed to BPAF at the dose of 5,20,and 50 mg/kg(body weight)or equal volume of corn oil(vehicle control),respectively,by gavage for28 consecutive days prior to evaluating its effects on male fertility.BPAF has no effects on gross animal growth and development,testicular and epididymal weight/body weight ratio,albeit the mice with comparable seminiferous tubules structure and cauda epididymis morphology observed in corn oil treated reference mice.Notably,the serumal of testosterone level was significantly decreased after BPAF exposure.Moreover,a precipitous decline of 30.9% and 44.6% in sperm quantity was seen in 20 mg/kg/d and 50mg/kg/d BPAF exposed mice,respectively.(2)The intracellular ROS level and concentration of [Ca2+]i in sperm were robustly elevated upon exposure to higher concentration,50 mg/kg/d,of BPAF.Of note,the abundance of the antioxidant enzyme PRDX5 and SDHB was conspicuously decreased while somehow the expression of the antioxidant enzyme GPX4 was increased upon BPAF(20 mg/kg/d and 50 mg/kg/d)exposure.In addition,the potentiated DNA damage indicated by the increased expression of ?H2AX was observed especially in 50 mg/kg/d BPAF exposed sperms.Meanwhile,the membrane integrity of acrosome was disrupted by BPAF exposure.These evidence pinpoints that BPAF is detrimental to sperm likely by attenuating its ROS scavenging system,which results in the existence of overwhelming intracellular ROS that induces DNA damage and ultimately thereby,triggering the production of abnormal sperms.(3)BPAF exposure(20 mg/kg/d and 50 mg/kg/d)altered post-translational modifications,up-regulation of SUMO1-ylation and down-regulation of ubiquitination and SUMO2/3-ylation,in sperm.Moreover,H3K27me3,a canonical epigenetical marker for transcription repression,was significantly increased in sperm by 20 mg/kg/d and 50mg/kg/d BPAF exposure implies BPAF is epigenetically toxic.(4)Unlike the control testis,in which the biotin signals were excluded from seminiferous tubules and only be visible exterior of the basement membrane,the biotin signals however penetrated into the inner cell layers of seminiferous tubules when exposing males to a dose starting from 20 mg/kg/d of BPAF,implicating BPAF compromise the BTB integrity.Significantly,the steep decreased expression of TJ protein Zonula Occludens-1(ZO-1),GJ protein p-Connexin 43 and actin bunding protein Palladin observed in testis from 50 mg/kg/d BPAF exposed males further confirmed the compromised BTB integrity by BPAF.(5)To decipher the underlying molecular mechanisms by which BPAF compromises BTB functionality,we cultured and treated primary Sertoli cell with 0,5,25,50 ?M of BPAF(without cytotoxicity)for in depth cellular analysis.The TJ barrier integrity of primary Sertoli cell revealed by the decreased TER was obviously disrupted by 50 ?M of BPAF treatment,which was further supported by the decreased expressions of several TJ regulators ZO-1,FAK,p-Connexin 43,and Palladin in Sertoli cells.(6)Notably,exposure to 20 mg/kg/d and 50 mg/kg/d BPAF lead to the hyperactivation of MAPK signaling in testis indicated by the elevated level of phosphorylated ERK1/2.To further verify that the activated MAPK signaling was a causative of BTB dysfunctionality,the cultured Sertoli cell was pretreated with 10 ?M U0126 which is a specific MEK1/2 inhibitor for 1h prior to 50 ?M BPAF treatment.The results shown that the compromised tight junction permeability(TER value),disrupted cytoskeleton architecture as wells as the downregulation of FAK and Palladin in primary cultured Sertoli cells induced by BPAF were ameliorated by U0126 treatment.In summary,our study demonstrates that BPAF is deleterious for testicular functionality and perturbs cytoskeleton architecture and BTB integrity in Sertoli cells partially by activating ERK/MAPK signaling and thereby,compromising spermatogenesis.Our study will provide important clues not only for the comprehensive understanding of the biosafety of Bisphenols but also for the mitigation and prevention of potential impairments caused by BPA and its substitutes.