The Effects Of Bisphenol A Exposure On Meiosis In Spermatocyte And Its Mechanisms

Background：Bisphenol A (BPA) is a well-known endocrine-disrupting chemical (EDC) that hasreceived particular attention because of its widespread distribution in humans. Due to itschemical similarity to estradiol, the possible reproductive toxicity of BPA has alreadylargely been evaluated. Certain studies have reported that BPA may cause meioticabnormalities in C. elegans and female mice. However, whether environmental exposure toBPA may induce reproductive disorders is still controversial, little is known about the effectof BPA on meiosis in adult males, and evidence is lacking with regard to the mechanismsinvolved.Methods：①9-week-old male Wistar rats were exposed to BPA by gavage at20μg/kg bodyweight (bw)/day. After60consecutive days, we use hematoxylin and eosin (H&E) stainingfor histological examination, Periodic acid schiff-hematoxylin (PAS-H) staining was usedto analyze the stage of seminiferous epithelium, meiotic chromosomal spread technique wasused to show the process of meiosis, immunofluorescence against SCP3, γH2AX， thephosphatidylinositol3-kinase-related protein kinase (ATM), checkpoint kinase1(CHK1)and DAPI was also performed.②We conducted a60days exposure procedure in which BPA at0,2,20or200μg/kgbw/d was given by gavage to9weeks old Wistar rats, administration of17β-estradiol (E2)at10μg/kg/d was used to mimic the estrogenic effects. ICI182780was used to antagonizeestrogen receptor (ER) signaling. After treatments, sperm parameters, sperm apoptosis andsex hormones were analyzed, the stages of seminiferous epithelium were evaluated by PAS-Hstaining, the progress of meiosis was studied by flow cytometry or immunofluorescenceagainst SCP3, γH2AX，ATM and DAPI, extra meiotic DNA strand breaks (mDSBs) weredetected by comet assay, germ cell apoptosis was evaluated as evaluated by terminal dUTPnick-end labeling (TUNEL) assay and western blot for caspase3. ③Adult male Sprague-Dawley rats were orally administered BPA at a dose of200mg/kg bw/day for10consecutive days with or without melatonin pretreatment. Thethiobarbituric acid reactive substances (TBARS) level and superoxide dismutase (SOD)activity in the testes were evaluated. Subsequently, their spermatocytes were isolated, andDNA damage was assessed using an alkaline comet assay and the meiotic spread method.Results：①We found that BPA significantly increased the proportion of stage VII seminiferousepithelium and decreased the proportion of stage VIII. Consequently, spermiation wasinhibited and that spermatogenesis was disrupted. Further investigation revealed that BPAexposure delayed meiosis initiation in the early meiotic stage and induced the accumulationof chromosomal abnormalities and mDSBs in the late meiotic stage. The latter eventsubsequently activated ATM signals.②Treatments with200μg/kg bw/day of BPA and E2significantly decreased spermcounts and inhibited spermiation, characterized by an increase in stage VII and decrease instage VIII in the seminiferous epithelium. This was concomitant with a disruption in theprogression of meiosis I and the persistent mDSBs in pachytene spermatocytes; and ATMand CHK2signal pathway was also activated; eventually germ cell apoptosis was triggered.Using ER antagonist ICI182780, we determined that ER signaling mediated BPA-inducedmeiotic disruption and reproductive impairment.③Short term exposure of BPA administration with high dose did not significantlyaffect the weights of rats and their reproductive organs, and no alteration in sperm countwas found. However, we demonstrated that BPA administration induced a significantincrease in TBARS levels and a decrease in SOD activity that were concomitant with anincrease in DNA migration within male germ cells and γH2AX foci formation on theautosomes of pachytene spermatocytes. Furthermore, a decrease in the proportion of4C-cells was observed. These BPA effects were significantly alleviated by melatoninpretreatment. Nevertheless, the genotoxic effects of BPA were not accompanied byapoptosis in germ cells and morphological changes in the testes.Conclusion：In summary, our results suggest that long-term exposure to BPA at environmentallyrelevant dose may lead to continuous meiotic abnormalities and ultimately put mammalian reproductive health at risk. Moreover, ER signaling-mediated meiotic disruption may be amajor contributor to the molecular events leading to BPA-related meiotic abnormalities.These rodent data support the growing association between BPA exposure and the rapidincrease in the incidence of male reproductive disorders. Furthermore, the protective role ofmelatonin on BPA induced DNA damage accumulation in germ cells indicate that melatoninmay be a promising pharmacological candidate for preventing the potential genotoxicityand reproductive toxicity of BPAfollowing occupational or environmental exposure.