Manganese Caused Reproductive Toxicity Of Male Mice Involving Activation Of GnRH Secretion By Prostaglandin E₂ Receptors In Hypothalamus

Objective:Manganese（Mn）is ubiquitous in the natural environment.Although Mn is an essential trace element in human body,exposure to excessive Mn can cause damage to the body,mainly in occupational chronic Mn poisoning.The reproductive toxicity of Mn is significant.Abnormal secretion of sex hormones is one of the important causes of reproductive damage caused by Mn exposure.Gonadotropin releasing hormone（GnRH）is a neuropeptide that plays an important role in the neuromodulation of vertebrate reproduction.Astrocytes can indirectly regulate the secretion of gonadotropin-releasing hormone by the binding of paracrine Prostaglandin E₂（PGE₂）specifically to the receptors EP1 and EP2 on GnRH neurons.There have been studies on the abnormal secretion of GnRH caused by Mn exposure,but the specific mechanism has not been reported in detail.The purpose of this study was to investigate the effects of Mn exposure on the reproductive system of male mice and to clarify the role of PGE₂ in the abnormal secretion of GnRH in the hypothalamus caused by Mn exposure,and provide experimental evidence for further study on the mechanism of action and prevention strategy of Mn toxicity.Methods:Take 64 Kunming male mice（25±2g）.They were randomly divided into 8groups.They were normal saline（0.9%）control group,low,medium and high dose manganese groups,EP1 receptor antagonist group（SC-51322+50 mg/kg MnCl₂）,and EP2 receptor antagonist group（AH6809+50 mg）./kg MnCl₂),DMSO solvent control group（DMSO+50 mg/kg MnCl₂）,DMSO control group,intraperitoneal injection of manganese for 2 weeks,twice daily antagonist intervention.Take 36 Kunming male mice（25±2g）.The mice underwent bilateral orchiectomy on aseptic conditions,and intramuscular injection of gentamicin 50,000U/（kg/d）on the day of surgery and postoperatively.After the wound was completely healed,it was randomly divided into 6 groups.They were saline（0.9%）control group,high dose Mn group,EP1 receptor antagonist group（SC-51322+50 mg/kg MnCl₂）,and EP2 receptor antagonist group（AH6809+50 mg/kg MnCl₂）.The DMSO solvent control group（DMSO+50 mg/kg MnCl₂）and the DMSO control group were intraperitoneally injected with manganese for 2 weeks,and the antagonist intervention was given twice a week.After 24 hours of the last Mn-treated,the serum levels of GnRH,FSH,LH,T,and PGE₂ were measured by ELISA.Microscopic observation of sperm count and sperm deformity morphology.We extraction of mouse hypothalamic RNA and detection mRNA levels of PGE₂ receptor EP1,EP2 in the hypothalamus.We extraction of mouse hypothalamic protein for PGE₂ receptor EP1,EP2 protein to detect protein levels.Paraffin sections were prepared from mouse hypothalamus and testis tissues for colocalization detection of hypothalamic PGE₂ receptors EP1,EP2 and GnRH neurons and testicular HE staining.Result:Compared with the control group,with the increase of the dose of Mn,the sperm count of the mice decreased significantly,the deformed spermatozoa appeared,the serum testosterone level decreased significantly,but the testis showed no obvious histomorphological changes,serum GnRH,FSH,LH,PGE₂ levels Gradually,the level of PGE₂ receptor EP1,EP2 protein and mRNA in the hypothalamus increased significantly.After bilateral orchiectomy,the results of each index were basically the same as those before castration,and the levels of serum GnRH,FSH and LH gradually increased.The levels of PGE₂ receptor EP1,EP2 protein and mRNA in the hypothalamus were significantly increased.After EP1 and EP2 receptor antagonists were intervened respectively,serum testosterone levels increased significantly,sperm count increased,GnRH,FSH,LH serum hormone levels were significantly lower than those of Mn-dynched groups,PGE₂ hormone levels and Mn-stained groups.Compared to the increase.The levels of EP1,EP2 protein and mRNA decreased.The results of immunofluorescence double staining showed that the expression of EP1 and GnRH was significantly increased compared with the control group with the increase of the dose of Mn.The expression of EP1 protein in the EP1 antagonist intervention group was significantly lower than that of the high Mn-treated group.There was no significant difference in the expression levels of EP2 and GnRH compared with the control group.There was no decreased in the expression of EP2 protein in the antagonist group compared with the high Mn-treated group.Conclusion:Manganese exposure can regulate GnRH secretion by affecting PGE₂receptors EP1 and EP2 in the hypothalamus,causing reproductive damage.