Hippocampal Gene Expression During The Estrous Cycle And Hippocampal Transcripts In Sex Difference In Animal Models Of Depression

Estrogen(E2)modulates a wide range of neuronal functions in the brain such as spine formation,synaptic plasticity,and neurotransmission.The hippocampus,a major target of estrogen,plays an important role in learning and memory.The effects of E2 on the hippocampal cell signaling pathways,epigenetic processes and synthesis of local proteins are mediated via intracellular estrogen receptors(ERs)and membrane-bound ERs such as the G-protein-coupled estrogen receptor(GPER).Alterations in dendritic spines number,size and shape in the neurons are associated with psychiatric diseases.E2 replacement increases spine density in the hippocampal CA1 pyramidal neurons,enhances long-term potentiation(LTP)and affects the hippocampus-dependent learning and memory tasks in ovariectomized(OVX)female rats.Dendritic spines and synapse numbers in hippocampal neurons of female rats cyclically fluctuate with the fluctuation of E2 levels across the estrus cycle,but the key genes responsible for these fluctuations are still unknown.To identify these key genes,the hippocampal transcriptome was explored via RNA-sequencing(RNA-seq)at the proestrus(PE)and estrus(ES)stages in female rats.At standard two fold-change selection criteria,37 differentially expressed genes(DEGs)were found in PE vs.ES groups(FDR adjusted p-value(q)<0.05).RNA-seq identified important transcriptional changes which were confirmed by quantitative real-time PCR(qRT-PCR).To gain insight into the functions of the identified DEGs,the E2-regulated genes were annotated by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes database(KEGG).Based on GO and KEGG pathways,we found that the identified DEGs of the PE vs.ES stages were involved in regulating neurite outgrowth,nerve regeneration process,spine development,synapse formation,synaptic plasticity,neuroprotection,neurotransmission,maintaining normal cognitive process,enhancing learning and memory,neural resilience against aging,oligodendrocyte maturation and myelination,mediating retinoid signaling,and AKT/ERK signaling pathways.The results of the gene expression profiles at the PE vs.ES stages showed that females in PE stage exhibited transcriptomic reorganization under the control of E2 to upregulate expression of the neuroactive ligand receptor,tight junctional protein complex,cell adhesion molecules,and proteins involved in digestion and absorption process via genomic way.The pathways such as PI3K-Akt signaling pathway,MAPK signaling pathway,Jak-STAT signaling pathway,and neurotrophin signaling pathway were enriched at the PE stage suggesting that the gene expression profile in these pathways is important for rapid non-genomic effects of E2 on hippocampus.Females in ES stage showed upregulation of genes involved in immunity,signal transduction,myelination and cellular development,highlighting the importance of these processes at low E2 levels.Ovariectomized(OVX)model and E2 replacement is frequently used in animal research as a suitable model for studying menopause and E2 therapy effects.The next two important questions were:how to verify the DEGS identified in the PE vs ES stages with the OVX model and what would be the profile of hippocampal gene expression in female rats devoid of circulating E2.These questions were addressed by treating OVX rats with exogenous E2 at different time points to develop the PE and ES regimen in the OVX model.The E2-responsive transcripts identified at the PE vs.ES stages were validated in OVX rats receiving E2 replacement via RNA-seq,qRT-PCR,and western blot.RNA-seq identified 187 DEGs which were significantly regulated by E2.These DEGs were further studied by GO and KEGG pathways.OVX-regulated DEGs were involved in transcription factor signaling,lipid metabolic process,neuroactive ligand receptor interactions,cell adhesion process,cell differentiation,immune response,growth,and development.OVX plus E2 regulated DEGs were involved in signal transduction process,regulating neurite outgrowth,spine development,synapse formation,synaptic plasticity,neuroprotection,neurotransmission,nerve regeneration process,maintaining normal cognitive process,enhancing learning and memory,immune system,aging,neuropeptide signaling,axon myelination,and ECM formation.Moreover,E2 and subsequent treatments with isoform specific E2 receptor agonist ERa(PPT)and ER?(DPN)regulated the expression of K1,Kcnj13,F5,and Sostdcl genes via ERa and ER?.E2 and these two isoform specific agonists differentially activate ERa,ER?,and GPER to regulate mRNA expression of these genes in the hippocampus.The distinct effects of PPT and DPN administration on mRNA levels suggest that the desired outcome of E2 on the expression of these genes is controlled by specific ERs.In humans,depression affects both women and men but women have twice the incidence of depression.Although a number of studies report sex differences in stress responses,it remains unclear which animal models of depression can better mimic the sex difference in human depression.To address this question,stress and depression related DEGs were selected from the RNA-seq data of estrous cycle,OVX and OVX plus E2 groups.These DEGs were studied in subchronic variable stress(SCVS)and chronic unpredictable mild stress(CUMS)animal models of depression to evaluate the susceptibility versus resilient phenotypes in male and female rats.To explore the underlying mechanisms of sex difference in depression,qRT-PCR was used to examine and compare the mRNA levels of various transcripts(Htr7,Egr2,HDAC2,Robo2,Sgk2,Otx2,Pth2r,and Npas4)in SCVS and CUMS models.The results showed that SCVS induced depression-like behaviors in female rats only;CUMS induced depression-like behaviors in male rats only.SCVS significantly altered the mRNA levels of Npas4,Otx2,Robo2,Pth2r,and Sgk2 in the female hippocampus only,and HDAC2 in the male hippocampus.CUMS significantly changed the mRNA levels of one transcript(Robo2)in the female hippocampus only when compared with SCVS.This is the first time,we showed that SCVS could be used to mimic the sex difference in depression by studying sex differences in depression-like behaviors induced by SCVS in rats.SCVS-induced depressive-like behavior in female rats may be associated with transcriptional activation of the above genes.The findings of this research can help in future study to understand the higher rate of depression in women than men.