Epidemiological Investigation And Molecular Mechanism Of Chronic Psychological Stress-induced Male Reproductive Damage

Background:Since the 1990s,more and more researches have shown that human there is a global or regional decline in semen quality.The downward trend in semen quality is likely multifactorial,which includes genetic,environmental,social-psychological-behavioral factors.Therefore,identification of the relevant hazardous factors may promote the prevention and treatment of semen damage.With the development of society and the advancement of urbanization in China,the incidence of mental health problems is increased.Epidemiological studies reported that psychological stress（e.g.,depression and post-traumatic stress disorder）is negatively associated with human semen quality.Animal experiments also demonstrated that psychological stress induced a decrease in serum testosterone levels and caused spermatogenesis impairment in male rats or mice.However there were disadvantages in these studies which may include the volunteer bias（most epidemiological studies were performed on infertile men or other selected people）and the improper animal model use（a single predictable stress may not present actual stress faced by humans or animals in real life）.Glucocorticoids,which are a hallmark of stress,can improve adaption to environmental changes though elevating blood sugar levels.However,long-term exposure to chronic psychological stress will lead to a sustained increase in glucocorticoid levels in humans or animals.High doses of glucocorticoid exposure have been shown to have reproductive toxicity effects.Glucocorticoids inhibit testosterone synthesis by glucocorticoid receptor（GR）in Leydig cells.As GR is also expressed in early spermatogenic cells（spermatogonia and spermatocytes）,whether stress-induced spermatogenesis impairment is mediated by the direct action of glucocorticoids via GR in early spermatogenic cells is not clear and deserves further study.Therefore,we examined the associations of depression and work stress with semen quality in two independent normal populations.Meanwhile,using the unpredictable chronic mild stress（uCMS）model in rats,we explored the effects of chronic stress-induced spermatogenesis impairment in male rats.Finally,we clarified the effects and mechanism of CORT-induced proliferation inhibition and G₀/G₁ cycle arrest in spermatogonia.Content:1.Association between depressive symptoms and semen parameters in college students:Results from the MARHCS study.In 2013,the“Male Reproductive Health in Chongqing College Students（MARHCS）”cohort study was established to exam the effects of social-psychological-behavioral and environmental factors on male reproductive health.The population of present study was 587volunteers who completed the psychological questionnaire scale assessment in the baseline of the MARHCS cohort study in 2013.Depressive symptoms were measured using a Chinese version of the Self-rated Depression Scale（SDS）.Other information included age,abstinence time,physical activity,smoking,and alcohol consumption.Semen samples were collected according to the guidelines of the WHO-recommended methods.The following semen parameters were measured,including semen volume,sperm motility,sperm concentration and total sperm count.Peripheral venous blood was collected and serum sex hormone levels were measured by nuclear radioimmunoassay.This study analyzed the relationship between depression symptoms and semen quality and explored potential interaction effects of depression symptoms and physical activity on semen parameters.2.Association between work stress and semen parameters in male workers:Results from two towns in Chongqing.A total of 384 adult male volunteers were recruited between April 2014 and December2015 from eight communities in Bishan and Fengdu,two towns in Chongqing,China.Work stress was assessed by a simplified Chinese version of the Job Stress Questionnaire（JCQ）with 22 items（JCQ-22）.JCQ-22 consists of three core subscales:decision latitude（nine items）,psychological job demands（five items）,and social support（eight items）.Covariate assessment information included age,abstinence time,physical activity,smoking,and alcohol consumption.Semen samples were collected according to the guidelines of the WHO-recommended methods.The following semen parameters were measured,including semen volume,sperm motility,sperm concentration and total sperm count.The study was aimed at（i）estimating the association between work stress and semen quality and（ii）exploring the moderating effect of social support in semen parameters among Chinese male workers.3.The role and mechnism of GR in stress-induced spermatogenesis impairment in male rats.The present study used a 42-day uCMS rat model,which is a commonly used,reliable,and effective model that best simulates chronic psychological stress in humans.Male Sprague-Dawley rats were exposed to uCMS along with co-treatment of a GR antagonist RU486（1mg/kg/day）.Testicular damage was assessed by testicular pathological evaluation,epididymal sperm concentration;serum testosterone levels;testicular apoptotic cell measurements,and cell cycle progression analyses.To further explore the cells targeted by stress-induced spermatogenesis impairment,cells were counted within the spermatogenic epithelium（including Sertoli cells,spermatogonia,spermatocytes,and spermatozoa）.GR protein levels in testes were also measured.4.The role and mechnism of GILZ in glucocorticoid-induced cell cycle arrest in mouse spermatogonia cell line（GC-1）.The cytotoxic effect of CORT（corticosterone,an endogenous glucocorticoid）on the mouse spermatogonia-derived GC-1 cells was detected using measurements in cell apoptosis,cell proliferation and cell cycle arrest progression distribution.RU486 and siGILZ were used to assess the role of GR and its downstream gene GILZ in CORT-induced cell proliferation inhibition and G₀/G₁ phase cell cycle arrest in GC-1 cells.Proteins binding with GILZ were detected by immunoprecipitation and protein mass spectrometry,and GRP94 was identified as a downstream target protein of GILZ.GRP94 was blocked using BEP-NVP800 to detect whether GRP94 was involved in CORT-induced G₀/G₁ phase cell cycle arrest.The mRNA and protein levels of GRP94 in CORT-treated GC-1 cells were detected.Furthermore,after blocking GILZ expression,the synthesis and degradation rate of GRP94 protein were measured to explore whether GILZ mediated CORT-induced proteolysis of GRP94 though ubiquitination.Results:1.A total of 63 men（10.7%）were classified as potentially having clinical depression（SDS standardized score≥53）.In univariate analysis,depressed men had lower sperm concentration（66.9 vs.72.6×10⁶/mL,P=0.043）and total sperm count（241.6 vs.257.0×10⁶,P=0.024）than nondepressed men.There were no significant differences in serum reproductive hormones level between depressed individuals and nondepressed individuals（P>0.05）.In multivariate analysis,after adjusting for potential confounders,depressed men had 18.90%（95%confidence interval[CI]=1.14%–33.47%,P=0.038）lower sperm concentration and 21.84%（95%CI=3.39%–36.90%,P=0.022）lower total sperm count than nondepressed men.Further analysis revealed that there was an interaction effect between depression and physical activity on sperm concentration（P=0.033）.In conclusion,in this sample of 587 young male college students from China,depression was associated with decreased sperm concentration and total sperm count but not serum reproductive hormones levels.In addition,interaction effects were detected between depression and physical activity on sperm concentration.2.According to the cut-off of JCQ-22,a total of 88（22.9%）subjects were classified as having high work stress status.In univariate analysis,subjects with high work stress had a lower sperm concentration（36.2±43.0 vs.42.2±47.8[10⁶/mL],P=0.03）and total sperm count（133.4±192.3 vs.154.2±205.2[10⁶],P=0.03）than subjects with low work stress had.After adjusting for age,body mass index,ejaculation abstinence period,smoking,alcohol consumption,and sleep quality,subjects with high work stress had an increased risk of being classified below WHO’s thresholds for“normal”defined by sperm concentration（OR 2.14,95%CI 1.24–3.68,P=0.006）or total sperm count（OR 1.95,95%CI 1.13–3.36,P=0.02）criteria than subjects with low work stress had.The moderating effect of social support was detected.Among subjects with low social support,subjects with high work stress had an increased risk of being classified below WHO’s thresholds for“normal,”defined by sperm concentration（OR 3.46,95%CI 1.62–7.36,P=0.001）or total sperm count（OR 2.18,95%CI 1.04–4.59,P=0.04）criteria than subjects with low work stress had.However,among subjects with high social support,we found no statistically significant associations between work stress and all semen parameters（P>0.05）.These results revealed that work stress is associated with lower levels of semen quality.Social support attenuates the negative association between work stress and semen quality.3.Rats in the uCMS group had decreased levels of serum testosterone and decreased epididymal sperm concentration.The uCMS-treated rats also had decreased numbers of spermatids and increased levels of apoptotic seminiferous tubules;additionally cell cycle progression of spermatogonia was arrested at the G₀/G₁ phase.Furthermore,uCMS exposure caused an increase in serum corticosterone level and activated GR signaling in testes.RU486treatment suppressed GR signaling and alleviated the damaging effects of stress;resulting in an increased epididymal sperm concentration.Overall,this work demonstrated for the first time that the activation of GR signaling mediates stress-induced spermatogenesis impairment and that this outcome was related to cell apoptosis and cell cycle arrest in germ cells.4.Here we show that CORT induced cell proliferation inhibition,G₀/G₁ phase cell cycle arrest,and GILZ upregulation in GC-1 cells.Meanwhile,RU486 co-treatment inhibited CORT-induced GR nuclear translocation and normalizes cell cycle distributions with GILZ downregulation in GC-1 cells.Moreover,knockdown of GILZ normalized cell cycle distributions in condition of CORT exposure.Mechanistically,GILZ accelerates GRP94protein degradation via ubiquitin proteasome pathway in CORT-treated cells,and subsequently inhibits AKT1 and its downstream genes which are essential to cell cycle G₁/S transition,such as CDK4,Cyclin D1,and p-Rb.Overall,we for the first time,uncovered a role for the GILZ in regulating the proliferation and cell cycle progression in CORT-treated spermatogonia cells in vitro and identified GRP94 as a new target gene of GILZ in glucocorticoids actions.Conclusion:Depression and work stress were associated with decreased sperm concentration and total sperm count.In vivo results demonstrated that the activation of GR signaling mediated stress-induced spermatogenesis impairment in male rats and that this outcome was related to cell apoptosis and cell cycle arrest in germ cells.Mechanistically,in vitro results revealed that CORT exposure induced GR nuclear translocation and transcriptionally activates GILZ which mediates glucocorticoids-induced G₀/G₁ phase cell cycle arrest in mouse spermatogonia by targeting GRP94 protein.This study reported the damage effects and characteristics of chronic psychological stress on reproductive toxicity,and clarified the role and related mechanism of GR in stress-induced spermatogenesis impairment.This study revealed the molecular mechanism of GILZ in glucocorticoid-induced cell cycle arrest in germ cells,and may provide a novel target for drug intervention and theoretical support for chronic psychological stress-induced reproductive damage.