The Effects And Mechanism Of Zearalenone On Testosterone Secretion In Mouse Leydig Cells In Vitro

To explore the molecular mechanism of the inhibition of zearalenone(ZEA) to testosterone(T) biosynthesis in Leydig cells, the inhibition of ZEA to T secretion in Leydig cells in vitro, the effect of ZEA on the factors of T secretion pathway, the mitochondria damage of ZEA to Leydig cells and the action sites of ZEA in Leydig cells were detected.Primary Leydig cells in vivo were taken to determine the testosterone level(hCG or cAMP stimulated) and cAMP level(hCG stimulated) after exposed to0,1,5,10,20μg/mL ZEA for12h or to2.5μg/mL for1,6,12,24h by ELISA, determined binding activity of LH/hCG receptor by Chemiluminescence Immunoassay(CLIA), as well as determined the protein levels of LH/hCG receptor, estrogen receptor and steroidogenic enzymes by Western Blot, tested the mRNA level of steroidogenic enzymes by real-time PCR, and determined the mitochondrial membrane potential by flow cytometry. Cells were taken to determined the ultrastructural damage by transmission electron microscopy and determined the orphan nuclear receptor Nur77protein expression level by Western blot after incubated with lower concentration(5μg/mL) and higher concentration(20μg/mL) of ZEA for12h or with2.5μg/mL of ZEA for shorter time(6h) and longer time(24h).The result showed that in hCG environment long time and high dose ZEA exposure inhibited T secretion extremely significantly (P<0.01).However, in cAMP environment every dosage and time exposure by ZEA reduced T secretion significantly(P<0.05), and the time-or concentration-effect relationship was obvious. Every exposure condition effected neither LH/hCG receptor binding activity nor protein expression obviously(P>0.05), and low concentration and long time ZEA exposure could increase the intercellular cAMP level significantly(P<0.05). The expression of steroidogenic enzymes were disturbed by ZEA both in concentration group and time group. The expression level of StAR increased in dosage group and there is an obviously trend(P<0.05), but there was no obvious change in time group(P>0.05). The expression level of3β-HSD decreased in1h group, and increased in others time groups. The expression level of P450scc, P450c17al and170-HSD decreased at different extent and decreased obviously compared with the control group(P<0.05). There was a decreased trend for mitochondrial membrane potential after exposed to ZEA, and10μg/mL,20μg/mL group and6h,12h,24h group were significantly decreased compared to control group. Oppositely, Estrogen receptor protein expression wasn’t affected by ZEA obviously(P>0.05). ZEA exposure damaged the Leydig cells internal structure, induced mitochondrial vacuolization and the endoplasmic reticulum injury, higher dosage group was much more seriously than lower dosage group, the same in longer time group and shorter time group. Nur77expression was inhibited by ZEA both at higher or lower dosage as well as short time groups.These results indicates that ZEA may bind to membrane ER(mER), disturb the balance of PDEs and ACs to increase cAMP level and StAR mRNA expression.The binding nuclear ER(nER) reduces Nur77protein expression level, then inhibits steroidogenic enzyme. The crosstalk of the two pathways(mER pathway and nER pathway) leads to the result of androgen biosynthesis inhibition.