Activation Of P53/BAX-PUMA Signaling Pathway Mediated Sleep Deprivation Induced Ovarian Dysfunction And The Related Protection Strategy

ObjectivesSleep deprivation,which comes from shift work,long-distance travel,or a busy lifestyle,has become a common situation under modern society and has a huge impact on our health.The health hazards caused by sleep deprivation in women have significant manifestations in the reproductive system,including menstrual cycle disorders,reproductive dysfunction and so on.However,the molecular mechanism of female reproductive dysfunction caused by sleep loss has not been fully revealed.Ovary is a unique organ in the female reproductive system,which has both reproductive and endocrine functions.Supported by the key military scientific research project,this thesis carried out the study on the molecular mechanism of reproductive dysfunction in female mice caused by sleep deprivation.Based on the clues revealed by the mechanism study,we also designed the strategy to protect against the damage in female reproductive system,thus providing the new theoretical and technical support for the health maintenance of female reproductive system under the condition of sleep deprivation.ContentsFirst of all,we investigated the level of melatonin secreted by the suprachiasmatic nuclei(SCN)in the hypothalamus and the expression levels of the circadian clock genes in the hypothalamus and ovary of female mice under sleep deprivation to explore whether sleep deprivation affected the circadian rhythm of the female mice.Secondly,we determined the expression levels of hormones secreted by the pituitary gland and ovary with or without sleep deprivation.At the same time,the estrous cycle length,follicular development and the apoptosis of granulosa cells(GC)were also detected under the condition of sleep deprivation.Then,the potential mechanism of female reproductive dysfunction caused by sleep deprivation was explored.Finally,according to the key signal molecules involving in regulating female reproductive dysfunction,the protective strategy was designed and its effectiveness was determined.MethodsFemale mice were subjected to sleep deprivation by sleep deprivation apparatus,and the secretion levels of melatonin and hormones were detected by Enzyme-Linked Immunosorbent Assay(ELISA).The expression levels of circadian clock genes in hypothalamus and ovary of female mice before and after sleep deprivation were measured by Western Blot assay.The changes of estrous cycle were examined by vaginal cytological smear for 20 days.The follicular development of female mice was observed by hematoxylin-eosin staining.The apoptosis of ovarian granulosa cells was examined by TUNEL staining.Then,we detected the expression level of tumor suppressor gene p53 and its phosphorylated and acetylated proteins(p-p53-Ser6,p-p53-Ser15,p-p53-Ser37,p-p53-Ser392,ac-p53-Lys305,ac-p53-Lys370),small ubiquitin modification related factors(SUMO1,SUM02/3,PIAS1,PIASy),and transcriptional target of p53(BAX,PUMA,NOXA,BID,Bcl-2,Bcl-xL)by Western blotting assay.mRNA levels of p53,SUMO2 and SUMO3 were determined by RTPCR assay.The binding of p53 and SUMO2/3 was detected by co-immunoprecipitation assay.p53 siRNA was transfected into primary ovarian granulosa cells to determine the relationship between p53 and its target gene;Tauroursodeoxycholic acid(TUDCA)was injected into the tail vein of mice to observe the regulatory effect of TUDCA on ovarian dysfunction induced by sleep deprivation and the related mechanism.ResultsFirstly,to explore the effects of sleep deprivation on circadian rhythm in the female mice,we detected the changes of serum melatonin level,which is a humoral signal indicating circadian rhythm.The results showed that the oscillatory expression of melatonin in the serum of female mice disappeared after sleep deprivation.Secondly,we analyzed the changes of circadian clock gene expression in hypothalamus and ovary,two major components of HPO axis.We found significant changes on the expression of the circadian clock gene PER2 and/or REV-ERBα in hypothalamus and ovary.Then we explored the effect of sleep deprivation on the reproductive function of female mice.To this end,several indexes of reproductive function of female mice were determined before and after sleep deprivation.Then we found that sleep deprivation induced disorder of estrogen and progesterone expression in ovary and prolonged the estrous cycle of female mice.Furthermore,the follicular development was abnormal,and the atresia rate increased after sleep deprivation.Finally,we observed the apoptosis of granulosa cells in the ovary of female rice,and the results indicated that the apoptosis of granulosa cells increased significantly after sleep deprivation.The above results indicated that sleep deprivation can induce circadian rhythm disorder,abnormal clock genes expression in the HPO axis,hormone secretion disorder and ovarian dysfunction in female mice.To clarify the potential mechanism of ovarian dysfunction induced by sleep deprivation in female mice,we examined the expression level of p53 and its post-translational modification,which acts as the key signal events mediating cell apoptosis.The results showed that sleep deprivation induced a significant increase in the expression level of p53 in the ovary,accompanied by an increase in SUMO modification.Subsequently,we detected the expression changes of anti-apoptotic and pro-apoptotic target genes downstream of p53,and found that the expression levels of pro-apoptotic proteins BAX and PUMA were up-regulated.In addition,transfection of p53 siRNA into the primary ovarian granulosa cells could downregulate the mRNA expression of BAX and PUMA.These results suggested that the activation of p53/BAX-PUMA pathway may play an important role in ovarian dysfunction induced by sleep deprivation.In order to further clarify the above conclusions,we selected TUDCA,an inhibitor targeting BAX,to intervene the signaling event in sleep deprived mice.Then the improvement of ovarian dysfunction induced by sleep deprivation was analyzed.As expected,compared with the female mice treated by sleep deprivation,sleep deprivation female mice pretreated with TUDCA showed the recovered estrous cycle length,normal follicular atresia rate and decreased granulosa cell apoptosis in the ovary.The above results suggested that the activation of p53/BAX-PUMA pathway can mediate the ovarian dysfunction induced by sleep deprivation,and the protective strategy targeting this pathway can effectively alleviate the harm on female reproductive function caused by sleep deprivation.Most importantly,TUDCA has the development value of protecting female reproductive function under certain stress conditions.ConclusionsTo sum up,the results of this study revealed that sleep deprivation induced circadian rhythm disorder,abnormal expression of hypothalamic and ovarian circadian clock genes expression,and disorder of estrogen and progesterone expression in ovary of female mice.At the same time,prolonged estrous cycle and increased follicular atresia and ovarian granulosa cells apoptosis were observed.At the molecular level,binding between p53 and SUMO2/3 in ovarian granulosa cells led to the up-regulation of the pro-apoptotic transcriptional target of p53,BAX and PUMA,resulting in apoptosis of ovarian granulosa cells and reproductive dysfunction of female mice.TUDCA targeting p53/BAX-PUMA pathway can antagonize the reproductive dysfunction induced by sleep deprivation in female mice.