Mechanism Of Aging-mediated ER Stress Regulating Testosterone Synthesis Through Circadian Clockwork In Mouse Leydig Cells

In mammals,the circadian clock system encompasses nearly all organs,tissues,and cells.Various diurnal changes in physiological functions and behaviors,and release of endocrine hormones,are driven by endogenous circadian clockwork in a cycle of nearly 24hours.Circadian clock system is closely related to gametogenesis and endocrine regulation in animals,however,the molecular mechanism of circadian clock regulates testosterone synthesis in mouse Leydig cells has not been reported yet.As a large membrane-bound organelle involved in protein synthesis,folding,secretion and storage of Ca²⁺in eukaryotic cells,endoplasmic reticulum?ER?plays an important role in maintaining cell homeostasis and normal function.ER stress impairs core circadian clockwork via unfolded protein response?UPR?signaling regulates miR 211,and directly suppresses both Bmal1 and Clock levers.There is a significantly decreasing serum testosterone concentration detrend and high lever of ER stress in aging male mammals.These research progress suggested that ER stress-circadian clock pathway may be involved in testosterone synthesis of aging mice,the specific regulatory mechanism needs further study.In this study,Bmal1 knockout mice,NIH3T3 cells and mouse Leydig cells were selected to detect the mechanism of aging-mediated ER stress regulating testosterone synthesis through circadian clockwork by immunofluorescence staining,real-time biofluorescence detection system,qPCR,Western blotting,ELISA and RNAi.The results obtained are as follows:1.A certain number of Bmal1^-/-homozygotes were obtained by mating and breeding from Bmal1^+/-heterozygote mice.The rhythmic expression and distribution of Bmal1 gene and protein were not detected in Bmal1^-/-mice,proved that Bmal1 was completely knocked out in Bmal1^-/-mice.2.BMAL1 protein rhythmically expressed and distributed in mouse Leydig cells;expression pattern of circadian clock and steroidogenic-related genes were rhythmic in mouse Leydig cells;serum testosterone content significantly decreased,BMAL1 and steroid synthesis protein STAR decreased,and circadian clock,steroidogenic-related and nuclear receptor genes related to testosterone synthesis were also decreased in Bmal1^-/-mice;statistical analysis of bioinformatics showed that there are some E-box and RORE modules in the promoter regions of steroidogenic-related genes,suggesting that endogenous clock genes were involved in a series of physiological activities in mouse testis and Leydig cells,thus affecting the secretion of testosterone.3.60 nM Thapsigargin?Tg?and 60 ng/mL Tunicamycin?Tm?induced ER stress response in NIH3T3 cells,inhibited the rhythmic oscillation of Bmal1-Luc,inhibited the expression of BMAL1 protein,inhibited the expression of circadian clock and clock-controlled gene transcription level;ER stress inhibitor 4-phenylbutyric acid?4-PBA?can alleviate the Tg/Tm induced ER stress in NIH3T3 cells,and partially restored the expression of Bmal1-Luc rhythmic oscillation and clock genes transcription level,while interfering with ATF4?rather than ATF6?can partially rescued the inhibition of clock gene transcription and the destruction of Bmal1-Luc oscillation by Tg induced ER stress.The results demonstrate that ER stress activation impairs the cellular circadian clockwork via an ATF4 signaling axis to regulate various physiological processes.4.The serum testosterone concentration decreased in aging mice,ER stress proteins and genes increased,and circadian clock and steroidogenic-related genes decreased in aging mice and mouse Leydig cells?TM3?senescence cells,suggesting that there is a negative correlation between ER stress and the circadian clock system in aging mice Leydig cells,thus affecting the secretion of testosterone.5.60 nM Tg and 60 ng/mL Tm induced ER stress response in TM3 cells,inhibited the rhythmic oscillation of Per2-dLuc,inhibited the expression of BMAL1 protein,inhibited the expression of circadian clock,clock-controlled and steroidogenic-related genes transcription level;ER stress inhibitor 4-PBA can alleviate the Tg induced ER stress in TM3 cells,and partially restored the expression of Per2-dLuc rhythmic oscillation and clock and steroidogenic-related genes transcription level;Tg-induced ER stress inhibited testosterone secretion,circadian clock,steroidogenic-related and nuclear receptor genes related to testosterone synthesis were also decreased in mouse Leydig tumor cells?MLTC1?and primary mouse Leydig cells?MLC?.These results suggest that ER stress affects testosterone secretion by disturbing the clockwork in mouse Leydig cells.In conclusion,the testosterone concentration is regulated by circadian clock system in mice,and activation of ER stress destroys the circadian clockwork.Aging mice undergo reduced testosterone,accompanied with ER stress and circadian clock disorder.This discovery perfects a signaling pathway that ER stress affects the circadian clockwork and regulates testosterone secretion in mouse Leydig cells,explaining the pathogenic mechanism of reduced testosterone secretion in aging male animals.