Fshr Deficiency Induces Depression-like Behaviors In Female Mice

Background:Follicle stimulating hormone(FSH),a gonadotropin synthesized and secreted by the basophilic cells in anterior pituitary gland,regulates growth,development,pubertal maturation and reproductive processes.The serum FSH levels change during the women's lifespan and are associated with female physiological periods,including puberty,pregnancy,puerperium and menopause transition.During the puberty,the rising serum levels of FSH and LH stimulate follicular development and maturation,and enhance estrogen secretion,thereby promote female pubertal maturation.During the pregnancy and puerperium,FSH and other reproductive hormones levels change significantly to induce maternal organs' adaptive responses for guarantying the growth and development of fetus and infants.During the menopause transition,declining estrogen levels and rising FSH levels are associated with multiple menopausal diseases.Dramatic changes in FSH and other reproductive hormone levels,during these periods mentioned above,are associated with rising depression prevalence in susceptible women,suggesting the pathophysiologic relevance of these reproductive hormones in mood regulation and depression pathogenesis.Therefore,understanding the neuroregulatory effects of reproductive hormones is crucial for the prevention of and specific therapeutic approaches of depression.FSH,as an important sex hormone,change significantly during these female physiological periods.However,whether and how FSH is associated with depression is still unclear and worthwhile researching.Follicle stimulating hormone receptor(FSHR)expressed in multiple extragonadal organs,including bone,liver,fat and brain.The FSH extragonadal function has been widely researched.Bone mass is increased and osteoclastic resorption is decreased in Fsh?-/-and Fshr-/-mice.FSH couples the FSHR in Osteoclasts and their precursors,and then activates MEK/Erk,NF-?B,and Akt pathway to enhance osteoclast formation and function.FSH couples the FSHR in liver to active Gi2?/?-arrestin-2/Akt pathway,suppress the binding of FoxO1 and SREBP-2 promoter,promoting the SEREBP-2 transcription and increasing cholesterol content of liver cells.FSH activates FSHR of fat cells and increases cAMP-response-element-binding protein phosphorylation,promoting lipid droplet formation and lipid biosynthesis.FSH couples the FSHR in umbilical vascular endothelial cell,activates FSHR/GaS/cAMP/PKA and PI3K/Akt/mTOR/NF-KB pathways,therefore upregulates VCAM-1(vascular cell adhesion molecule-1)expression and accelerates Atherogenesis.Thus,blocking FSH signaling is considered a novel therapeutic approach and strategy for menopausal osteoporosis,obesity and atherogenesis.While the susceptible women during menopausal transition are usually troubled by mental disturbance,the effect of blocking FSH signaling on central nervous system is worthwhile to be researched.Recent study has confirmed that the neurons in hippocampus and cerebellum not only synthesize FSH,but also express FSHR via in situ hybridization and immunohistochemistry staining.These results imply that FSH signaling may have a biologic function.Moreover,whether blocking FSH signaling for treating menopausal osteoporosis,obesity and atherogenesis induces side effect in central nervous system is still unclear.Therefore,study on the effect of FSH signaling in central nervous system,particularly in mood regulation,is of great biologic and clinical value.Pathophysiology of depression involves multiple aspects including monoamine system,hypothalamic-pituitary-adrenal axis,inflammation,neuroplasticity and neurogenesis,structural and functional brain changes,gene,environmental milieu.Oxidative stress,the imbalance between ROS production and endogenous antioxidant systems,plays a pivotal role in the pathogenesis of depression.Glutathione(GSH),an endogenous antioxidant,is decreased in the brains of depressed patients and stress-induced rats.NAC,the precursor of GSH and ROS scavenger,attenuates the depression-like behavior of diabetic rats and stress induced depressive mice.Moreover,FSH stimulates glutathione synthesis to increase antioxidant and anti-apoptosis defense in rat/mouse ovarian follicles.Whether FSH mediates the redox-optimized ROS balance in central nervous system and is involved in the pathogenesis of depression is worthwhile researching.In our study,we evaluated the changes in mice behaviors,redox-optimized ROS balance and gene expression profile in absence of FSHR,and the effect of FSH on ROS production of N2a cells,investigating the neuroregulatory effects of FSH signaling and the role of FSH in depression pathogenesis.Our study provides new insights for depression mechanism and therapy,and experimental evidence for extragonadal function of FSH signaling.Objectives:The main purpose of this study was to evaluate the effect of FSH signaling in mood regulation,from the aspects of not only behavioral and molecular changes in vivo,but also the molecular expression of key genes in redox-oxidative balance in vitro,aid in the understanding of association between mood regulation and reproductive hormones.Methods:1.Animal modelEight-week-old female Fshr+/+and Fshr-/-mice were exposed to ovariectomy to rule out the effect of estrogen.Group assignments according to experimental designs:(1)Fshr+/+group and Fshr-/-group,(2)Fshr+/++saline group,Fshr-/-+saline group,Fshr+/++NAC group,Fshr-/-+ NAC group.2.Determination of FSHR in mood-mediating brain regions using mmunofluorescence staining.3.Mice behavior testsFshr+/+mice and Fshr-/-mice were subjected to forced swim test(FST),elevated plus maze(EPM),tail suspension test(TST),sucrose preference test,marris water maze(MWM),open field test(OFT)and rotarod test to evaluate the behavioral changes in the absence of FSHR.4.The oxidative stress evaluation of brainsWe used GSH assay kit to evaluate GSH/GSSG ratio,which stands for the oxidative stress level,in brains of Fshr+/+mice and Fshr-/-mice.5.Evaluation of changes in brain gene expression profileWe evaluated the changes in genes expression profile using RNA sequencing of brains from Fshr+/++and Fshr-/-mice and verified multiple genes expression via real time PCR.6.Serum reproductive hormone levels evaluationThe concentrations of FSH,LH,estradiol and testosterone in the serum were measured using ELISA kits according to manufacturer's instructions.7.Cell cultureN2a cells were routinely maintained in high-glucose DMEM supplemented with 10%fetal bovine serum(FBS).Group assignments according to experimental designs:control group(PBS 3h),H2O2 treatment group(PBS 2h+200?M H2O2 1h,FSH+H2O2 treatment group(FSH 50ng/mL 2h+200?M H2O2 1h).8.Cellular oxidative stress level evaluationChanges in cellular oxidative stress levels induced by FSH addition were measured by Mitosox.9.Evaluate the expression of key genes regulating GSH synthesis and transitionWestern Blotting was used to evaluate the protein expression of GCLm,GCLc and G6PD.10.Software SPSS 20.0 was used to analyze the experimental data.Values were presented as mean ± standard deviation.The differences between two groups were determined by Student's t-test.Differences among multiple groups were detected using one-way ANOVA.P<0.05 was considered statistically significant.Results:1.FSHR was present in multiple mood-mediating brain regions.The immunofluorescence results showed that FSHR expressed in multiple mood-mediated regions,including the hippocampus,cortex,nucleus accumbens,amygdala,and prefrontal cortex,not in the bed nucleus of the stria terminalis or lateral habenula.Moreover,FSHR is co-expressed with NeuN(the marker of Neuron),but not with GFAP(the marker of astrocytes),demonstrating that FSHR expressed in neurons but not in astrocytes.2.Fshr-/-mice displayed more severe depression-like behaviors.Fshr-/-mice exhibited a longer immobility time than control Fshr+/+ mice in the FST,indicating increased vulnerability to depression and be despaired in stress environment.No differences were found in the sucrose preference test,EPM,TST and MWM.3.FSHR deficiency increases cellular oxidative stress.Fshr knockout mice displayed more severe oxidative stress,as the GSH/GSSG ratio in the whole brains of Fshr-/-mice was much lower than that of Fshr+/+mice.The similar results were also found in multiple mood-mediating regions,including hippocampus,cortex,prefrontal cortex and amygdala.4.ROS scavenger NAC attenuates the depression-like behaviors and decreases the cellular oxidative stress level in Fshr-/-mice.Fshr+/+mice and Fshr-/-mice were treated with NAC(150 mg·kg-1·d-1)and saline i.p.,and then subjected to behavioral test and tested by GSH assay.Fshr+/++saline VS Fshr-/-+saline VS Fshr+/++NAC VS Fshr-/-+NAC:immobility time in FST:8.70±10.0s VS 25.86±17.74s VS 4.82±5.26s VS 8.20±6.77s,P<0.05;GSH/GSSG ratio in brains:1.41±0.37 VS 0.36±0.23 VS 1.60±0.31 VS 1.58±0.41,P<0.05.These results demonstrated that Oxidative stress plays a pivotal role in the depression-like behaviors of Fshr-/-mice.5.FSH treatment attenuated the increasement of ROS production induced by H2O2,and decreased the cellular oxidative stress level.Compared to cells without treatments(control group),H2O2 addition increased the Mitosox fluorescence intensity,which reflected cellular ROS levels.However,FSH treatment reversed 'the increased Mitosox fluorescence intensity induced by H2O2 addition,suggesting that FSH protects the N2a cells from cellular oxidative stress.Mitosox fluorescence intensity of control group VS H2O2 group VS FSH+H2O2 group:3.38±0.87 VS 10.83 ±2.48 VS 5.23±2.42,P<0.05.6.FSH regulates the protein expression level of GCLm and G6PD,which is the key gene in GSH synthesis and transition.Western Blotting revealed that FSH treatment(50ng/mL or 100ng/mL)upregulated the protein expression level of GCLm and G6PD compared to control group.Consistently,the protein expression levels of GCLm and G6PD in the brain of Fshr/-mice was lower than that of Fshr+/+ +mice.7.Fshr ablation altered gene expression profile of brain.Unsupervised hierarchical clustering analysis indicated that the gene expression profiles of Fshr-/-mice were clustered together and separated from Fshr+/++mice representing different expression states between Fshr-/-mice and Fshr+/++mice.Real time PCR verified the differences in multiple genes expression between two groups,suggesting that FSHR ablation induce gene expression profile changes significantly in brains.Conclusion:1.Fshr ablation induces depression-like behavior of female mice.2.ROS scavenger attenuates the depression-like behavior of FSHR knockout mice.Redox-oxidative imbalance plays a crucial role in the pathogenesis of depression-like behavior in the absence of FSHR.3.FSH signaling is involved in the GSH synthesis and transition,and participates in the regulation of redox-oxidative imbalance.4.Although blocking FSH signaling is considered a novel therapeutic avenue and strategy for treating menopause associated diseases by clinicians and pharmacologists,the side effect in central nervous systems should be paid attention and be studied in further researches.