Study On The Effects And Mechanism Of Low Dose Bisphenol A On Sperm Functions In Male Mice

BackgroundBisphenol A?BPA?,as a common plasticizer,is widely used in industrial synthetic processes such as epoxy resin and polycarbonate,as well as various commonly used plastic products such as bottles,toys and so on.It can be exposed to the human body through a variety of ways such as skin contact,inhalation and ingestion.As the chemical structure of BPA is similar to that of the natural estrogen estradiol,it can also combine with estrogen receptors to exert a weak estrogenic effect,causing endocrine disorders and imbalance of hormone secretion.Particularly in the male reproductive system,its toxic damage effects are mainly concentrated in the decline of semen quality,abnormal testicular development,and reduced levels of testosterone hormones,but the related roles and mechanisms are still unclear.In earlier research,we found that the dose of BPA?5 mg/kg/d?lower than the FDA regulations resulted in impaired sperm function in mice,suggesting that it is necessary to systematically evaluate the toxic effects of low-dose BPA.In addition,we screened the signal pathway in BPA-treated mouse spermatocyte GC-2 cell.The results showed that the enriched signaling pathway was olfactory receptor signaling pathway,sterol metabolic process,toll-like receptor signaling pathways.Previous studies have shown that olfactory receptor genes may be involved in the regulation of sperm motility and sperm motility in human sperm.It is suggested that OR may mediate the process of sperm function damage induced by low-dose BPA.Therefore,this study is to analyze the adverse effects of low doses of BPA in detail,and investigated the role of olfactory receptor genes in damage of sperm function exposed to BPA.Objective 1.To investigate the effect of long-term exposure to low-dose bisphenol A on sperm function such as capacitation?chemataxis and acrosome reaction in male mice.2.To investigate the effect of Ca²⁺ regulation in sperm function damaged by low dose bisphenol A.3.To investigate the role of OR gene in sperm function damaged by low dose bisphenol A.4.To investigate the correlation between OR4E2 expression and sperm parameters in population.Methods1.32-Kunming mice were randomly divided into four groups after feeding one week: the control group?oil?,the low-dose BPA?0.03mg/kg/d?,the middle-dose BPA?0.3mg/kg/d?and the high-dose BPA?3mg/kg/d?.After intragastrical administration for 10 weeks,animals were sacrificed and testis weighed to calculate their index.2.Using Eosin Y and HE solution to detect sperm and testis morphology.3.Using the CASA system to detect the parameters of sperm motility;microfluidic chips to detect sperm chemotaxis;CTC-Hoechst to dectect sperm acrosome reaction;Fluo4 AM to detcet the level of sperm Ca²⁺concentration.4.Construction of si RNA cell model to study the role and molecular mechanism of OR receptor gene in GC-2 cells.5.Using qRT-PCR,flow cytometry,Western blot,and immunofluorescence to detcet the expression level of calcium channel related genes in mouse sperm and si RNA GC-2 cells;6.The q RT-PCR technique was used to detect the m RNA expression level of OR4E2 gene in human semen samples.The correlation between the analysis results and semen parameters was analyzed.Results:1.Compared with the control group,there was no significant difference in body weight,organ index,and testicular morphology of the mice in treatment groups;the rate of mouse sperm abnormalities increased with the increase of BPA dose,but there was no statistical difference.2.Compared with the control group,the proportion of fast progressive spermatozoa in the low and high dose groups was significantly decreased?P<0.05?, and the proportion of non-progressive spermatozoa was significantly increased?P<0.05?;There is no significant difference in slow forward and immobile sperm percentage between the control group and BPA-treated groups.3.Compared with the control group,the percentage of chemotactic spermatozoa in mouse sperm gradually decreased with the increase BPA exposure dose?P<0.01?.After progesterone stimulation,the proportion of AR-type spermatozoa decreased significantly,and the proportion of F-type and B-type increased significantly as the BPA exposure increased.4.The concentration of intracellular Ca²⁺ in mouse sperm decreased with increasing exposure dose of BPA;the level of Ca²⁺ current in sperm was also decreased after stimulation with progesterone.5.Through expression microarray screening and differential gene enrichment analysis,it was found that the olfactory receptor gene signaling pathway has a significant change in GC-2 model induced by BPA.Decrease in expression of OR genes?Olfr1277,Olfr25,Olfr1509?m RNA in mouse sperm after BPA exposure.6.After si RNA interfered with the expression of Olfr1509 in GC-2 cells,the results of flow cytometry showed that the proliferation,cycle and apoptosis of GC-2 cells did not change significantly,but its Ca²⁺concentration significantly decrease.7.The m RNA and protein expression of Catsper channel were significantly decreased after Olfr1509 gene expression was interfered.8.The expression of Catsper channel related genes in sperm were decreased after low-dose BPA exposure.9.By analyzing 122 semen samples,the correlation of semen parameters concluding semen volume,semen density,total sperm count,sperm motility,and sperm progressive movement with OR4E2 m RNA expression level was analyzed.The results showed that significant associations were observed between sperm motility and the level of OR4E2 m RNA expression?person's r=0.361,p<0.01?.Conclusions:1.There was no significant effect in testicular organ index,morphology and sperm abnormality in male mice after exposure to low-dose BPA,but it could reduce the rate of sperm capacitation,the proportion of chemotaxis sperm,the incidence of sperm acrosome reaction in mice leading sperm function damage.2.Olfr1509 may play an important role in sperm function by regulating the Ca²⁺ level and the expression of Catsper channel in male germ cells.