Effects Of Manganese Occupational Exposure On Reproductive Function Of Male Workers And The Role Of Hypothalamic-derived PGE₂ In Its Mechanisms

Objective:Manganese?Mn?has beneficial effects on reproductive function at physiological concentrations,however,excessive long-term exposure can cause reproductive toxicity.Although significant progress has been made in reducing occupational Mn exposure,workers in some areas still experience high levels of Mn.The reproductive toxicity of Mn is mainly reported in the occupational activities,but the existing data and results are not consistent.As a heavy metal element with neurotoxicity and reproductive toxicity,Mn can be taken by astrocyte?Ast?in brain and accumulated in hypothalamus.?-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid?AMPA?receptor is a kind of ionic glutamate receptor?GluR?and could be expressed in Ast.It has been demonstrated that activated AMPA receptors can upregulate cyclooxygenase-2?COX-2?expression and stimulate secretion of prostaglandin E2?PGE₂?.PGE₂ can act on the gonadotropin-releasing hormone?GnRH?neurons through paracrine manner,which stimulate the secretion of GnRH and affect the synthesis and secretion of reproductive endocrine hormones.These suggest that Mn may be involved in the synthesis of hypothalamic-derived PGE₂ and thus regulate the secretion of GnRH and affect reproductive function.So the aim of our work is:Firstly,the effect of Mn occupational exposure on sexual function of male workers was studied.Secondly,the effect of Mn occupational exposure on reproductive endocrine hormones and semen quality of male workers was studied in a cross-sectional study.Finally,the effects of AMPA receptor on PGE₂ synthesis in Mn-induced GnRH secretion abnormality and reproductive toxicity were studied in vivo and in vitro.This study provides the direct epidemiological evidence that Mn occupational exposure can be harmful to the reproductive function of male workers,and experimental basis for exploring the mechanisms of Mn induced reproductive toxicity.Methods:Firstly,the concentrations of Mn dust in different workplaces were detected to evaluate the external Mn exposure dose of the male workers.The sexual function of 128 Mn exposure male workers and 116 non-Mn exposure male workers were investigated by the self-made questionnaires to evaluate the effect of Mn exposure on male workers'sexual function.The survey mainly contains five aspects including sexual desire,erectile ability,ejaculation,the satisfaction of the sexual life and orgasm.Secondly,the content of Mn in in urine,serum,seminal plasma and hair,reproductive hormones and semen quality were explored in 84 male workers exposure to Mn and 92 referents.Multiple linear regression analyses were used to assess the relationships.Finally,the hypothalamus Ast was cultured in vitro as the control group,Mn exposure group and NBQX intervention group.The control group and the Mn exposure group were normally cultured.NBQX intervention group was incubated with 20?mol/L NBQX 6 h in advance.Then the control group continued normally culture,Mn group were cultured with 125?mol/L?250?mol/L and 500?mol/L MnCl2 and NBQX intervention group were cultured with 500?mol/L MnCl2 for 24 h.The levels of PGE₂ in cell culture medium and the expression of GluR2 and COX-2were detected by real time RT-PCR and westem blot in order to study the effect of AMPA receptor on PGE2 synthesis in Mn-induced GnRH secretion abnormality and reproductive toxicity.Then,male castrated kunming mice were randomly divided into control group?Mn exposure group?NBQX intervention group?celecoxib intervention group and NBQX+celecoxib intervention group,with 6 rats in each group.The control group and Mn exposure group were given intraperitoneal injection of0.9%NaCl,the NBQX intervention group was given intraperitoneal injection of 22.5mg/kg of NBQX.Celecoxib intervention group was given intraperitoneal injection of30 mg/kg celecoxib,and the NBQX+celecoxib intervention group was given intraperitoneal injection of 22.5 mg/kg NBQX and 30 mg/kg celecoxib.2 h later,the control group was intraperitoneally injected with 0.9%NaCl,Mn exposed group,NBQX intervention group,celecoxib intervention group and NBQX+celecoxib intervention group were intraperitoneal injection with 50 mg/kg MnCl₂,once daily for2 weeks.The above injection doses were all 5 ml/kg.Blood was then collected from the abdominal aorta and serum GnRH and PGE₂ levels were determined.The expression of COX-2 and AMPA receptor GluR2 in the hypothalamus was determined by real time RT-PCR and westem blot.Results:1?The survey shows that the Mn concentration in part of workplaces of the enterprise exceed permissible standard.The sexual function survey showed that the prevalence of erectile dysfunction?ED?was 35.16%and 40.62%for premature ejaculation?PE?in Mn exposure group.Compared with the control group,the level of sexual desire was decreased,while the rate of the ED and PE were increased?P<0.05 or P<0.01?.What is more,there was no significant difference in satisfaction with sexual function and orgasm in the Mn exposure group.The score of sexual desire and international index of erectile function-5?IIEF-5?were decreased,while premature ejaculation diagnostic tool?PEDT?score was increased in the Mn exposure group?P<0.01?,while the difference between sexual satisfaction and orgasm score was not statistically significant.With the increase of the concentration of Mn in the working environment,the level of sexual desire,the prevalence of PE and ED of male workers exposed to Mn increased significantly?P<0.05 or P<0.01?.2?Compared with the control group,the contents of Mn in urine and seminal plasma were significantly higher,gonadotropin-releasing hormone?GnRH?levels and luteinizing hormone?LH?levels increased significantly and the levels of testosterone?TSTO?decreased significantly in Mn-exposed group?P<0.05 or P<0.01?.Multiple linear regression analysis showed that urinary Mn and seminal plasma Mn were positively correlated with serum GnRH,while negatively correlated with TSTO.There was a significant positive linear correlation between urinary Mn and LH?P<0.05 or P<0.01?.Sperm progressive motility?PR%?and total motility?PR+NP?%decreased significantly in Mn-exposure group?P<0.05 or P<0.01?.There was a significantly negative linear correlation between urinary Mn and sperm PR%and?PR+NP?%,and a negative linear correlation between seminal plasma Mn and sperm PR%?P<0.05or P<0.01?.Compared with the control group,the parameters of sperm motility related to curvilinear velocity?VCL?,straight-line?rectilinear?velocity?VSL?and average path velocity?VAP?were decreased in the Mn exposure group?P<0.01?.3?In vitro results showed that compared with the control group,with the increased of Mn exposure,the PGE₂ level in the cell culture medium were increased,Ast GluR2and COX-2 were up-regulated in Mn exposure group.However,the application of the AMPA receptor antagonist NBQX could change that?P<0.05 or P<0.01?.The results of in vivo experiments showed that compared the control group,the levels of serum GnRH and PGE₂ were significantly higher in the Mn exposed group.Hypothalamic COX-2 and GluR2 were up-regulated?P<0.01?.Compared with the Mn exposure group,hypothalamic COX-2 was significantly down-regulated,and the activation of AMPA receptor was inhibited,and the levels of serum GnRH and PGE₂were decreased in NBQX intervention group,COX-2 intervention group and especially in NBQX+celecoxib intervention group?P<0.05 or P<0.01?.Conclusion:1?Mn exposure can lead to the decrease of sexual desire and increased prevalence of ED and PE,causing sexual dysfunction,and it is related with the Mn concentration in the workplaces.2?Mn exposure was inversely associated with reproductive function of male workers,resulting in the increase of GnRH?abnormality of hormones secretion and decrease of semen quality.3?Mn may increase the levels of hypothalamic-derived PGE₂ by activating the AMPA receptor and up-regulating the expression of the COX-2,thus lead to elevated GnRH levels in serum,which may be one of the Mn-induced male reproductive toxicity mechanisms.