Expression Of Melatonin In Testis Of Rooster And Its Effect On Apoptosis Of Leydig Cells

Melatonin is an endogenous substance that can be synthesized and secreted by the body itself.Previous studies on its expression and action on gonads have focused on mammals such as humans and rats.However,its effect on the expression of avian gonads and its effect on apoptosis and apoptosis of gonadal cells has rarely been studied.This study focuses on clarifying the expression pattern of melatonin in the testis during growth and sexual maturation and its effect on Leydig cell apoptosis.Quantitative PCR(qRT-PCR),Western blot,immunohistochemistry and other molecular biological methods were used to detect and localize the melatonin receptors(MEL-1A,MEL-1B and MEL-1C)and synthetase(AANAT and ASMT)in the testis at different stages of development.Expression;using MTT,flow cytometry to detect the effect of melatonin on testicular stromal cell apoptosis,the results are as follows:1.qRT-PCR and Western blot were used to detect the expression of MEL-1A,MEL-1B,MEL-1C,AANAT and ASMT in testicular tissue of different ages(12,18 and 24 weeks old).With the increase of age,the expressions of MEL-1A,MEL-1B and AANAT mRNA and protein were significantly decreased and the expression of MEL-1C increased first and then decreased,the expression of ASMT increased significantly.Immunohistochemical method was used to locate the above five types of proteins.Proteins all expressed in Leydig cells.2.Percoll method separation and purification of chicken Leydig cells,the purity of up to 90%.The cells were treated with 0,0.1,1,10,100 ng / ml melanin for 12,24,36,48 h respectively.The cell growth status was observed by MTT assay and Select 36 h as the best processing time.Cells were treated with 0,0.1,1,10,and 100 ng/ml melatonin for 36 h,and apoptosis was detected by flow cytometry.Melatonin can significantly reduce the level of apoptosis.Quantitative RT-PCR was used to detect the expression of Bax,caspase-3 and Bcl-2 after melatonin was added.Melatonin significantly reduced the expression of Bax and Caspase-3,and significantly increased Bcl-2 expression level.The results of this study indicate that melatonin receptors and synthetases have different expression patterns at different stages of development in chicken testes.Melatonin can reduce the apoptosis of Leydig cells.