To Explore The Mechanism Of Melatonin Inhibition The Rhythm Of Testosterone Secretion In TM3 Cells

Objective:To explore the role of clock gene Bmall in inhibition of testosterone synthesis by melatonin in Leydig cells.Methods:1.Cell growth kineticsCells in logarithmic growth were inoculated and counted continuously for 7 days to calculate the doubling time.2.Dexamethasone synchronization on cell cycleCells were cultured in serum free medium for 20 hours with dexamethasone of 100 nmol/L, and were analyzed 4,8,16,32 hours after the treatment for cell cycle by flowcytometry.3.Detection of gene expression rhythmGene expressions were detected by RT-PCR in cells of 0,4,8,12,16,20,24 hours after the melatonin treatment.4. Gene expression after Bmall gene InterferenceThe Bmall gene was knock-down by liposome transfection of RNAi plasmid, and the genes expressions were detected by RT-PCR and Western Blot.Results:1.The set-up of synchronizing cell modelThe cell doubling time was 22.53+/-2.84 h. No obvious change was found in cell cycle after dexamethasone treatment. The expression of Bmall and Star gene was rhythmic, with peaks at ZT 16 and 12 and troughs at ZT4.2. Changes in testosterone level and gene expression after Melatonin treatmentThe testosterone levels of melatonin treatment group were significantly lower than the control group, with a reduced amplitude of circadian rhythm. The expressions of Bmall, Star and ROR alpha genes were also changed in a similar way of testosterone.3. Changes in testosterone and gene expression in Bmall gene interference cellsIn Bmall gene interference cells, melatonin treatment resulted in an increase in testosterone concentration, and an advance of peak phase of the Star gene expression rhythm.Conclusions:1. A cell model of rhythmic Bmall gene expression was established in leydig cells.2. Melatonin treatment decreased testosterone levels, possibly by regulation of rhythmic expression of the Bmall and Star gene.