Mechanism Of Melatonin Inhibiting Effect On Testosterone Synthesis In Rooster Leydig Cells

Melatonin(MT)is a neuroendocrine hormone secreted by the pineal gland,which regulates hypothalamic-pituitary-testicular axis activity,thereby regulating steroid hormone secretion and affecting male reproductive performance.Leydig cells are the main steroid hormone secretion in the testis.Studies on mammals such as rats,mice and sheep have shown that melatonin can directly act on leydig cells to affect testosterone secretion.In this study,chicken leydig cells were cultured in vitro and treated with different concentrations of MT for 36 h,ELISA kit was used to detect testosterone secretion,and real-time PCR and Western blot were also used to detect the expression of steroid hormone synthase(StAR,CyP11a1 and 3β-HSD).To investigate the role of melatonin receptor in testosterone secretion by interfering with melatonin receptor expression;finally,by activating or inhibiting cAMP/PKA/CREB signaling pathway,to determine whether this pathway is involved in melatonin inhibition of chicken leydig cells.The results of testosterone secretion studies are as follows:1.21-week-old white-feathering cocks were selected and the leydig cells was extracted from the testes for identification.After the cell growth was stable,different concentrations(0,0.1,1,10,100 μg/mL)of MT were added for 36 h and the testosterone secretion was detected by the ELISA Kit.StAR,CyP11a1 and 3β-HSD was also detected by real-time PCR and Western blot analysis.Our results showed that 1,10,100 μg/mL MT significantly inhibited testosterone secretion(P<0.05),and 1 μg/mL MT had the strongest inhibitory effect(P<0.01).Also,results from Real-time PCR and Western blot analysis also showed that MT significantly inhibited the expression of StAR,CyP11a1 and 3β-HSD mRNA and protein(P<0.01).2.In order to clarify whether MEL-1A and MEL-1C are involved in the inhibition of testosterone synthesis by MEL,the expression of MEL-1A and MEL-1C in chicken leydig cells was knocked down by RNAi technology.The experiment was divided into CTR,NC,and siRNA and in the MT and MT+ siRNA groups,the carrier was transfected into chicken leydig cells for 24 h,and 1 μg/mL MT was added for 36 h;MEL-1B specific receptor antagonist(4-PPDOT)was used to identify MT.The effects of-1B were divided into CTR,MT and MT+PPDOT(1,5,10 μM)groups;the antagonists were pretreated for 30 min and treated with MT for 36 h.The interference efficiency was verified by real-time PCR and Western blot analysis,and the amount of testosterone was detected by ELISA Kit.The results showed that MEL-1A knockdown and MEL-1B antagonism could attenuate the inhibitory effect of MT on testosterone(P<0.05),and the receptor MEL-1C was not knocked down after RNAi technology.Inhibition of MT on testosterone inhibition suggests that MT inhibits the secretion of chicken leydig cells through membrane receptors MEL-1A and MEL-1B,whereas MEL-1C is not involved.3.Cells were also treated with 1 μg/mL MT for 0,10,20 and 30 min,and cAMP content was determined by ELISA Kit.The results showed that MT treatment for 10 min significantly inhibited cAMP content(P<0.05).Also,the cells were treated with 1 μg/mL MEL for 0,15,30 and 45 min,and the phosphorylation level of CREB was detected by Western blot.The results showed that MT treatment for 30 min significantly inhibited CREB phosphorylation(P<0.05).After pre-treatment of cells with cAMP/PKA/CREB signaling pathway inhibitor,MEL was added for 30 min and our results showed that cAMP inhibitor and melatonin could reduce the inhibitory effect of melatonin on CREB phosphorylation(P<0.05),and PKA inhibitor and MEL co-treatment had the same effect.The MEL treatment was extended to 36 h,and the testosterone secretion was measured by ELISA Kit.The results showed that cAMP inhibitor and melatonin could reduce the inhibitory effect of MT on testosterone(P<0.05).The results of real-time PCR showed that the pretreatment of cAMP and PKA inhibitors attenuated the inhibitory effect of MT on the expression of StAR,CyP11a1 and 3β-HSD mRNA(P<0.05),pretreatment with cAMP activator or PKA activator or CREB.Expression and treatment with MT showed that cAMP,PKA activator and CREB overexpression vector could attenuate the inhibitory effect of MT on CREB phosphorylation and testosterone secretion(P<0.05),accompanied by MT.The inhibition of StAR,CyP11a1 and 3β-HSD mRNA expression was attenuated(P<0.05).In summary,MT significantly inhibits testosterone secretion and expression of key synthetase in chicken leydig cells with 1 μg/mL MT was revealed as being the most potent inhibitor.Also,MT passage through membrane receptors MEL-1A and MEL-1B triggered the inhibition of testosterone synthesis and the cAMP/PKA/CREB signaling pathway was observed to be involved in this process.