Melatonin Inhibits Testosterone Synthesis In Roosters Leydig Cells By Lipid Droplet Lipolysis

Leydig cells,as an important component of testicular cells,can synthesize testosterone by free cholesterol.Testosterone is involved in regulating important physiological processes,such as spermatogenesis and maintaining the second sex.Melatonin,secreted by pineal gland,affects the circadian rhythm,immune regulation,temperature regulation and endocrine regulation of animals,and has antioxidant and DNA repair effects.Lipid droplets are intracellular organelles,which can not only maintain the normal life activities of cells,but also provide raw materials for cell synthesis of steroids through lipolysis.Lipolysis of lipid droplets by structural protein perilipin 1(PLIN1)on lipid droplet surface and regulation of lipase hormone-sensitive lipase(HSL)and triacylglycerol hydrolase(ATGL).In our previous study,we found that 1 ng/m L melatonin could inhibit the expression of key steroidogenic enzyme through c AMP/protein kinase A(PKA)pathway in rooster Leydig cells after 36 h treatment,thereby inhibiting the synthesis of testosterone in rooster Leydig cells.Some studies have also pointed out that the lipolysis process is regulated by c AMP/PKA pathway,but the mechanism whether melatonin can regulate PLIN1 through c AMP/PKA pathway to inhibit testosterone synthesis in rooster Leydig cells by lipolysis is unclear.In order to clarify the effect of melatonin on testosterone synthesis in rooster Leydig cells through lipid droplet lipolysis,we used primary isolated and cultured rooster Leydig cells as the research object,detected the effects of melatonin on lipid droplets and free cholesterol content,lipolysis-related enzyme expression and testosterone level in rooster Leydig cells by using enzyme-linked immunosorbent assay(ELISA),fluorescence staining,free cholesterol kit,real-time fluorescence quantitative PCR(q RT-PCR)and western blot.to Subsequently,the effects of melatonin on lipid droplets and free cholesterol content,lipase expression and testosterone level of rooster Leydig cells were detected after activation of c AMP/PKA pathway by using c AMP activator Forskolin and PKA activator 8-Bromo-c AMP.Finally,the effects of melatonin on lipid droplets and free cholesterol content,lipolysis-related gene expression and testosterone level of rooster Leydig cells was detected after PLIN1 interference.The results are as follows:1.Compared with the control group,melatonin treatment could reduced testosterone level,free cholesterol content,HSL and ATGL expression levels,and increased lipid droplet content and PLIN1 expression levels;2.Compared with the single melatonin treatment group,the c AMP/PKA pathway activated by c AMP activator Forskolin and PKA activator8-Bromo-c AMP weakened the inhibitory effect of melatonin on testosterone synthesis in rooster Leydig cells,increased the content of free cholesterol,HSL and ATGL expression levels,and reduced the content of lipid droplets and the expression level of PLIN1;3.Compared with the NC group,the interference PLIN1 could increased the testosterone level,free cholesterol content,HSL and ATGL expression levels,the lipid droplet content was reduced in rooster Leydig cells.4.Compared with the NC and melatonin co-treatment group,the interference PLIN1 and melatonin co-treatment group weakened the inhibitory effect of melatonin on testosterone synthesis in rooster Leydig cells,increased free cholesterol content,HSL and ATGL expression levels,and reduced lipid droplet content.The results showed that melatonin could promote the expression of PLIN1 through c AMP/PKA pathway,inhibit the expression of HSL and ATGL,and inhibit the lipolysis of rooster Leydig cells,thereby reducing the content of intracellular free cholesterol and inhibiting the synthesis of testosterone.