Mechanism Study Of Zearalenone Interfering Testosterone Synthesis In Goat Leydig Cells

The growth of the meat goat industry in Guizhou Province,China,has been explosive in recent years.Ensuring the reproductive performance of goats is essential for promoting the industry’s high-quality development.Zearalenone（ZEA）is a toxic secondary metabolite produced by Fusarium species.The occurrence of ZEA in feed and feed ingredients is a well-recognized problem worldwide.This mycotoxin has estrogen-like activity and acts as an endocrine-disrupting chemical.Recent studies indicate that exposure to ZEA can disrupt reproductive tract development,causing effects such as abnormalities affecting function.Thus far,many studies have focused on the toxicity of ZEA on the female reproductive system,while its mechanism of male reproductive toxicity remains unclearly demonstrated.In view of Leydig cells is the primary site of testosterone synthesis,we examined cell apoptosis,steroid hormone secretion,and metabolic pathways to identify the possible mechanism of ZEA toxicity in goat Leydig cells.The main results were summarized as follows:1:Primary culture of goat Leydig cells and establishment of in vitro model of ZEA toxicity.Goat Leydig cells were successfully isolated and primary cultured,and then treated with ZEA at different concentrations（0,5,10,20 and 30μM）.The results showed that the viability and purity of the isolated Leydig cells met the requirements and could be used for further experiments;the viability of Leydig cells decreased with the concentration of ZEA treatment,and the CC₅₀（Concentration of cytotoxicity 50%）was close to 20μM.Therefore,this concentration was selected as the dosage to establish an in vitro model of ZEA toxicity in goat Leydig cells.2:Effect of ZEA on apoptosis in goat Leydig cellsPrimary cultured goat Leydig cells from experiment 1 were used.The effect of ZEA on Leydig cells apoptosis was investigated by measuring apoptosis-related mRNA,cell healing ability,mitochondrial membrane potential,and cell cycle.The results showed that compared with control group,the mRNA abundance of Bax,Bcl-2,Caspase 3,and Caspase 9 was significantly reduced after ZEA treatment（P<0.05）;the cell scratch area was reduced,and the migration ability was decreased;the mitochondrial polarization was weakened,and the membrane potential was reduced;the cells in G1 and S phase were significantly reduced,and the proportion of cells in G2 phase was significantly increased（P<0.05）.3:Effect of ZEA on steroid hormone synthesis in goat Leydig cellsThe Effect of ZEA on steroid hormone synthesis was investigated by measuring steroid hormone synthesis-related mRNA and steroid hormone concentrations.The steroid hormone concentrations were quantified using LC-MS/MS-based targeted metabolomics approaches.The results showed that the mRNA abundance of St AR and CYP17A1 was significantly reduced,while the level of 17β-HSD was significantly elevated after ZEA treatment.The concentration of progesterone,17αhydroxyprogesterone,corticosterone,21 hydroxyprogesterone,androstenedione,and cortisone levels was elevated,while the level of dihydrotestosterone was reduced（P<0.05）.4:Metabolomic profiling of the ZEA-treated goat Leydig cellsUPLC-MS/MS-based untargeted metabolomics was applied to detect the metabolic alterations in ZEA-treated goat Leydig cells.The results showed that 52 differential metabolites were screened,including 6-aminopenicillanic acid,short-chain acylcarnitine,glycerophospholipids,glycyrrhetic acid methyl ester,4-hydroxy-1-（2-hydroxyethyl）-2,2,6,6-tetramethylpiperidine,7-alpha-hydroxy-4-cholesten-3-one,citrulline,diethanolamine,dimethyl phthalate,glutamic acid,cis-9-palmitoleic acid,aspartic acid,lactic acid,linoleic acid,N-benzyloxycarbonylglycine,pantothenate,taurine and zearalenone.Enrichment analysis showed that the ZEA significantly affects nicotine metabolism,choline metabolism,glycerophospholipid metabolism,and neurotransmitter vesicle metabolism in goat Leydig cells.Conclusion:ZEA affects the morphology of Leydig cells and disrupts cell steroid hormone synthesis.The mechanism may be related to nicotine,choline,glycerophospholipid,and neurotransmitter vesicle metabolism.