Dataset Information

Phosphoproteomic analysis of the adaption of epididymal epithelial cells to corticosterone challenge

ABSTRACT: Background: The epididymis has long been of interest owing to its role in promoting the functional maturation of the male germline. More recent evidence has also implicated the epididymis as an important sensory tissue responsible for remodeling of the sperm epigenome, both under physiological conditions and in response to diverse forms of environmental stress. Despite this knowledge, the intricacies of the molecular pathways involved in regulating the adaptation of epididymal tissue to paternal stressors remains to be fully resolved. Objective: The overall objective of this study was to investigate the direct impact of corticosterone challenge on a tractable epididymal epithelial cell line (i.e., mECap18 cells) in terms of driving adaptation of the cellular proteome and phosphoproteome signaling networks. Materials and methods: The newly developed phosphoproteomic platform EasyPhos coupled with sequencing via an Orbitrap Exploris 480 mass spectrometer was applied to survey global changes in the mECap18 cell (phospho)proteome resulting from sub-chronic (10-day) corticosterone challenge.Results: The imposed corticosterone exposure regimen elicited relatively subtle modifications of the global mECap18 proteome [i.e., only 73/4171 (~1.8%) proteins displayed altered abundance]. By contrast, ~15% of the mECap18 phosphoproteome was substantially altered following corticosterone challenge. In-silico analysis of the corresponding parent proteins revealed an activation of pathways linked to DNA damage repair and oxidative stress responses as well as a reciprocal inhibition of pathways associated with organismal death. Corticosterone challenge also induced the phosphorylation of several proteins linked to the biogenesis of microRNAs. Accordingly, orthogonal validation strategies confirmed an increased in DNA damage, which was ameliorated upon selective kinase inhibition, and an altered abundance profile of a subset of microRNAs in corticosterone-treated cells. Conclusions: Together, these data confirm that epididymal epithelial cells are reactive to corticosterone challenge and that their response is tightly coupled to the opposing action of cellular kinases and phosphatases.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Epithelial Cell

SUBMITTER: David Skerrett-Byrne

LAB HEAD: Brett Nixon

PROVIDER: PXD028789 | Pride | 2024-04-08

REPOSITORIES: Pride

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Publications

Phosphoproteomic analysis of the adaption of epididymal epithelial cells to corticosterone challenge.

Skerrett-Byrne David A DA Stanger Simone J SJ Trigg Natalie A NA Anderson Amanda L AL Sipilä Petra P Bernstein Ilana R IR Lord Tessa T Schjenken John E JE Murray Heather C HC Verrills Nicole M NM Dun Matthew D MD Pang Terence Y TY Nixon Brett B

Andrology 20240404

<h4>Background</h4>The epididymis has long been of interest owing to its role in promoting the functional maturation of the male germline. More recent evidence has also implicated the epididymis as an important sensory tissue responsible for remodeling of the sperm epigenome, both under physiological conditions and in response to diverse forms of environmental stress. Despite this knowledge, the intricacies of the molecular pathways involved in regulating the adaptation of epididymal tissue to p ...[more]

PMID: 38576152

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Project description:Mammalian spermatozoa acquire their fertilizing ability during epididymal transit. Gene expression patterns along the epididymis are established by specific transcription factor networks that coordinate region-specific functions. The epididymis is usually divided into 3 segments: caput, corpus, and cauda. The human epididymis anatomy does not allow clear distinction between these three segments. To determine to which extent gene expression is segmented along the human epididymis, transcriptome profiling was performed on 8 distinct epididymal regions from 3 donors. Microarray analysis was performed on a Gene Chip Human Clariom S (Affymetrix®) array representing 337 100 transcriptional variants encoded by 20 800 genes. Proximal segments 1 to 3 were distinguishable from the distal epididymal segments (4 to 8) as shown by unsupervised Principal Component Analysis. Transcripts from each segment with differentially expressed genes (DEGs) > 2-fold change and FDR < 0.05 were clustered in relation to their intensity profiles. While no DEGs were detected between segments 1–3 corresponding to the efferent ducts, 1140 DEGs were detected between efferent ducts (1–3) and the epididymis (4–8), 400 between caput (4–6) vs. corpus/cauda (7–8) and none between corpus (7) and cauda (8). Gene Ontology annotation revealed that up-regulated DEGs in the efferent ducts (1–3) were predominantly related to cilium assembly/movement and cell differentiation. The biological process terms fertilization, defense and immune responses were associated with caput epididymis (4–6) while spermatogenesis and protein binding were found all along the epididymis (4–8). In conclusion, the proximal human epididymis is exclusively occupied by efferent ducts with a distinct DEG profile compared with the downstream epididymal segments. Moreover, gene expression profiling revealed two regions in the human epididymis; the caput and the distal corpus/cauda region. Taken together, analysis of the human epididymal transcriptome reveals a limited DEG profile.

Project description:BACKGROUND: The molecular mechanisms resulting in the establishment of regionalized gene expression in the human epididymis involve numerous and complex regulatory elements that have not yet been fully elucidated. Interestingly, more than 200 microRNAs (miRNAs) have been identified in the human epididymis that could be involved in the regulation of mRNA stability and post-transcriptionnal expression in this organ. METHODS: Using a global miRNA microarray approach, we investigated the correlation between the miRNA signature and the gene expression profile found in three distinct regions (caput, corpus and cauda) of human epididymides from 3 donors. In silico prediction of transcript miRNA targets was performed in silico using TargetScan and Miranda software’s. To experimentally verify some of these data, FHCE1 immortalized epididymal cell lines were cotransfected with mimics microRNAs and plasmid constructs containing the 3’UTR of target genes downstream of the luciferase gene. RESULTS: We identified 35 miRNAs differentially expressed between the different segments of the epididymis (fold change > 2, P < 0.01). Among these miRNAs, miR-890, miR-892a, miR-892b, miR-891a, miR-891b belonging to the same cluster located on the X chromosome and specifically expressed in epididymal tissues, are significantly more expressed in the corpus and cauda regions compared to the caput. Interestingly, a strong negative correlation (r= -0,89, P <0.001) was found between the pattern of expression of miR-892b and its potential mRNA target Esrrg (Estrogen Related Receptor Gamma), with miR-145 and Cldn10 mRNA (r= -0,92, P<0.001) and with miR-145 and Cftr mRNA (r= -0,69, P<0.05). We confirmed that miR-145 and miR-892b inhibit the expression of the luciferase reporter via Cldn10 and Esrrg 3’ UTRs respectively, whereas miR-145 does not affect the expression of luciferase after its co-transfection with the plasmid containing the Cftr 3’UTR. CONCLUSION: Overall, our study shows that the expression of miRNAs is segmented along the different regions of the human epididymal duct and correlates with the pattern of target gene expression in these regions. Therefore, epididymal miRNAs may be in charge of the maintenance of gene expression profile in the epididymis that dictate segment-specific secretion of proteins and establish physiological compartments that directly or indirectly affect sperm maturation and fertility. Normal human epididymides from three donors of 26–50 years of age, were collected and dissected into 3 anatomical regions (caput, corpus and cauda).A cross-comparison between the miRNA signature found in different segments was performed using GeneChip miRNA Array 1.0 (Affimetrix, Santa Clara, CA).

Project description:The functional maturation of mammalian spermatozoa is accomplished as the cells descend through the highly specialized microenvironment of the epididymis. This dynamic environment is, in turn, created by the combined secretory and absorptive activity of the surrounding epithelium and displays an extraordinary level of regionalization. Although the regulatory network responsible for spatial coordination of epididymal function remains unclear, recent evidence has highlighted a novel role for the RNA interference pathway. Indeed, as noncanonical regulators of gene expression, small noncoding RNAs have emerged as key elements of the circuitry involved in regulating epididymal function and hence sperm maturation. Herein we have employed next generation sequencing technology to profile the genome-wide miRNA signatures of mouse epididymal cells and characterize segmental patterns of expression. An impressive profile of some 370 miRNAs were detected in the mouse epididymis, with a subset of these specifically identified within the epithelial cells that line the tubule (218). A majority of the latter miRNAs (75%) were detected at equivalent levels along the entire length of the mouse epididymis. We did however identify a small cohort of miRNAs that displayed highly regionalized patterns of expression, including miR-204-5p and miR-196b-5p, which were down- and up-regulated by approximately 39- and 45-fold between the caput/caudal regions, respectively. In addition we identified 79 miRNAs (representing ~ 21% of all miRNAs) as displaying conserved expression within all regions of the mouse, rat and human epididymal tissue. These included 8/14 members of let-7 family of miRNAs that have been widely implicated in the control of androgen signaling and the repression of cell proliferation and oncogenic pathways. Overall these data provide novel insights into the sophistication of the miRNA network that regulates the function of the male reproductive tract. Examination of the microRNA expression profile in the whole mouse epididymis and mouse epididymal epithelial cells using next generation sequencing in duplicate.

Dataset Information

Phosphoproteomic analysis of the adaption of epididymal epithelial cells to corticosterone challenge

Publications

Phosphoproteomic analysis of the adaption of epididymal epithelial cells to corticosterone challenge.

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