Effect Of Microcystin Lr On The Expression Of C-Jun, C-FOS Gene In The RAT Sertoli Cell And Its Mechanisms

Background and objectiveMicrocystins (MCs), generated by water cyanophyta, is a kind of singlecyclic heptapeptide compounds with biological activity. MCs has a varietyof isomers, and MC-LR is the most toxic of it. At the present time the onlycyanobacteral toxin family that have been internationally assessed for healthrisk by the WHO are the microcystins. It is widely distributed in freshwaterlakes, ponds and reservoirs, which can cause acute liver injury and are activetumour promoters. Now the MCs has been extensively researched at homeand abroad. Liver and kidney is the main target organ of MCs. The studiesabout its reproductive toxicity have reported just few, most of which ismainly about the changes of the morphology and levels of hormone. Thestudies about the toxicity of the MC-LR and its mechanism to male genitalsystem on the level of cell and gene are fewer. In this study, the primarysertoli cells from SD rat were exposed to the MC-LR, then the toxic effectsof the sertoli cells and the gene and protein expression of immediate earlyresponse gene c-fos and c-jun was detected to clarify the toxic role and mechanism of MC-LR to the male reproductive system.MethodsSertoli cells were isolated and cultured in vitro and identified byimmunofluorescence. The drug group (0.5nM, and5nM, and50nM, and500nM) and control group (0nM) were set up and administrated primarysertoli cells. The cell multiplication were detected by MTT assay after24hand48h to identify the right duration and the right concentration for thesubsequent experiments; FCM detected the apoptosis of the cell; RT-PCRassay detected the expression of mRNA; the Western-blot assay detectedthe protein expression of c-jun and c-fos. It can help analysis the toxicaleffect of MC-LR on the sertoli cells and on the expression of mRNA andprotein of c-jun and c-fos from the level of the cell and gene.ResultsThe experimental results show that, in the MTT assay, the microcystin-LReffect were detected in sertoli cells after24h and48h: after24h, it had nosignificant change in low-dose group (0.5nM,5nM) compared with thecontrol group (0nM)，but in the high dose group (50nM,500nM) theactivity of the cell decreased significantly, with statistical significance(P<0.05); after48h, it had no significant change in low-dose group (0.5nM,5nM) compared with the control group (0nM)，but in the high dose group(50nM,500nM) the activity of the cell decreased more significantly with statistical significance and the change of the group500nM has moresignificance(P <0.01). So we choose the high dose group and the controlgroup to be the right duration, and48h to be the right concentration for thesubsequent experiments.The results of the flow cytometry (FCM) showedthat the high dose group microcystin-LR can cause significant apoptosisand the change had statistically significant (P <0.05), while the low dosegroup did not change significantly. Results of RT-PCR assay showed that inhigh-dose group, c-jun and c-fos gene expression showed up-regulation,with statistical significance. Western-blot assay showed the expression ofthe protein of c-jun, of c-fos increased with statistically significant.ConclusionThe Microcystis induced cell proliferation inhibition and apoptosis inthe primary Sertoli cells of SD rat and stimulated c-fos and c-jun gene andprotein expression increased. It is suggesting that up-regulation of c-fos andc-jun change transcriptional regulation pathway and induce cell proliferationinhibition and apoptosis, eventually leading to reproductive anddevelopmental damage.