Effects And Mechanisms Of Reproduction Endocrine Function In Male Rats Induced By Microcysitns Exposure

Objective:Microcystins（MCs）,the secondary metabolites produced by cyanobacteria blooms,have become a global public health problem and seriously endanger human health.In recent years,it has been found that MCs can enter the organism through a variety of ways and produce hepatotoxicity,reproductive toxicity and neurotoxicity,etc.More and more evidence show that MCs can interfere with reproductive endocrine function.The purpose of this study was to establish a male Wistar rat model infected with MCs,and to investigate the effects of MCs exposure on reproductive endocrine function and its regulatory mechanism in rats.This study will provide a theoretical basis for the prevention and treatment of human reproductive toxicity caused by MCs exposure.Methods:The median lethal dose(LD₅₀)of male Wistar rats was calculated by intravenous injection（i.v.）of MC-LR.After 5 days of adaptive feeding,healthy male Wistar rats were randomly divided into low,medium and high exposure groups and control group,with exposure doses of 25μg/kg bw,50μg/kg bw and 75μg/kg bw,respectively.The control group was injected 0.9%Na Cl solution at 75μg/kg bw.All rats were injected intravenously once.After 24 h intravenously,2%pentobarbital sodium was injected intraperitoneally to anesthetize the rats.Blood was collected from the heart,serum was separated,hypothalamus,pituitary and testicular tissues were collected.Enzyme Linked Immunosorbent Assay（ELISA）was used to determine the hormone levels in serum reproductive endocrine system.Real-Time Quantitative Reverse Transcription PCR（q RT-PCR）was used to determine the expression levels of hormones,related receptors and synthetic transporters in reproductive endocrine systemResults:The LD₅₀of male Wistar rats was 100μg/kg bw with a 95%confidence interval of 70.68-131μg/kg bw after intravenous injection（i.v.）of MC-LR.Serum hormone levels:Compared with the control group,Gn RH levels in low-dose groups was significantly increased（P<0.05）,while that in medium-dose and high-dose groups were significantly decreased（P<0.05）;The levels of FSH in low dose group had no change,but increased significantly in medium and high dose groups（P<0.05）;The levels of LH increased with the increase of the dose（P<0.05）.The levels of T decreased with the increase of the exposure dose（P<0.05）.The levels of E2 in low and medium dose groups were significantly increased（P<0.05）,but no change was observed in high dose group.Gene expression of hormone-related receptors and synthetic transporter:Compared with the control group,（1）In hypothalamus,the expressions of Kiss1 and Gpr54 were significantly down-regulated（P<0.05）,while the m RNA levels of Gnrh1,Ar,Erαand Erβwere not significantly changed;（2）In pituitary,the expression of Gnrhr was significantly up-regulated in low-dose groups（P<0.05）,but no significant change was observed in medium-high dose groups（P<0.05）.The expression of Fshβwas significantly down-regulated in medium-high dose groups（P<0.05）,and the expression of Lhβwas significantly up-regulated in medium and high dose groups（P<0.05）.The expression of Erβwas significantly down-regulated in low dose group（P<0.05）,significantly up-regulated in high-dose group（P<0.05）;The m RNA levels of Ar and Erαdid not change significantly.（3）In testis,m RNA levels of Lhr,Fshr,Srb I,Tspo,Star,Cyp11a1,3βHSD and Srd5α1 were significantly down-regulated（P<0.05）.The expression of Cyp17a1 was significantly up-regulated in low-dose group（P<0.05）and down-regulated in high-dose group（P<0.05）.The expression of Cyp19a1was significantly up-regulated in the high-dose group（P<0.05）,while the expression levels of Sssbp,Ldlr and 17βHSD were not significantly changed in the low-dose,medium-dose and high-dose groups.Gene expression of testosterone synthesis related signaling pathways and transcription factors:Compared with the control group,the expression of Pka in medium-dose and high-dose groups was significantly decreased（P<0.05）;The expression of Insr and Pi3k had no difference in low-dose group,but increased in medium-dose and high-dose groups（P<0.05）.The m RNA level of Akt was significantly up-regulated in high-dose group（P<0.05）.The expression of Mkk6 was significantly down-regulated in the medium-dose group（P<0.05）,and the expression of Erk was significantly up-regulated in the high-dose group（P<0.05）,while the expressions of Mek1 and Mkk3 were significantly down-regulated（P<0.05）.There were no significant changes in Mtor,Mkk7,Jnk and P38 gene expression levels in low,medium and high dose groups.The expressions of Mef2 and Sf1 in high-dose group were significantly increased（P<0.05）;The expression of Nur77 was significantly decreased in medium-dose and high-dose groups（P<0.05）;Gata4 expression was significantly increased in medium-dose and high-dose groups（P<0.05）.Conclusion:1.After MC-LR exposure,the levels of hormones related to reproductive endocrine system in serum of rats were changed,and the transcription levels of genes related to hormone synthesis,secretion and transport were affected.Suggesting that the endocrine function of HPG axis in male rats after MC-LR exposure was disturbed,and the positive and negative feedback was unbalanced.2.The m RNA transcriptional levels of cholesterol transporter receptors（SR-BI and TSPO）in testicular tissue of rats after MC-LR exposure were decreased.Suggesting that MC-LR can not only inhibit the transport of HDL into testicular interstitial cells,but also inhibit the mutual binding of TSPO and St AR,reduce the transport rate of cholesterol into mitochondria,and reduce the synthesis of testosterone.Several key steps of steroid synthesis are inhibited after MC-LR exposure,thus affecting steroid biosynthesis.3.The m RNA transcription levels of key enzymes for testosterone synthesis（St AR,CYP11A1 and 3β-HSD）were decreased after MC-LR exposure.This study found that m RNA levels of the related signaling pathways（c AMP/PKA,PI3K/AKT,ERK MAPK signaling pathway）and transcription factors（Nur77,GATA4,SF-1,MEF2）regulating these three key enzymes also changed to varying degrees.It is speculated that testosterone synthesis is co-regulated by multiple signaling pathways and transcription factors.