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Extracellular vesicles are involved in the paracrine communication between epithelial cells in different regions of the domestic cat epididymis

ABSTRACT: Sperm maturation depends on exposure to specific microenvironments within the different segments of the epididymis, but mechanisms underlying how these microenvironments are produced or maintained are not well understood. We hypothesize that epididymal extracellular vesicles (EVs) could play a role in this process, and therefore set out to test whether the EVs from different regions of the epididymis can serve as a form of paracrine communication between epithelial cells. Domestic cat tissues were used to develop a reproducible in vitro culture system for corpus epididymis explants that were then exposed to EVs collected from upstream (i.e., caput) segments. The impacts of different culture or exposure conditions were compared by analyzing the morphology, apoptosis, transcriptional activity, and gene expression in the explants. Here, we report the development of the first in vitro culture system for epididymal tissue explants in the domestic cat model. Using this system, we found that EVs from the caput segment have a significant effect on the transcriptional profile of tissue from the corpus segment (1,233 differentially expressed genes due to EV supplementation). Of note, expression of genes associated with regulation of epithelial cell differentiation and cytokine signaling in the epididymis were regulated by the presence of EVs. Together, our findings comprise the first report of paracrine control of segmental gene regulation by epididymal EVs in any species. These results contribute to a better understanding of epididymis biology and could lead to techniques to enhance or suppress male fertility.

ORGANISM(S): Felis catus

PROVIDER: GSE269443 | GEO | 2024/09/01

REPOSITORIES: GEO

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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:Study question: Can region-specific transcriptional profiling of the epididymis from fertile and sub-fertile bulls predict the aetiology of fertility/subfertility in males? Summary answer:The highly regulated gene expression along the bovine epididymis is affected by the fertility status of bulls used for artificial insemination. What is known already: In mammals, sperm maturation and storage occur in the epididymis. Each epididymal segment has his own transcriptomic signature that modulates the intraluminal composition that governs sequential modifications of the maturing male gamete. Main results and the role of chance: Hierarchical clustering and Principal Component Analysis revealed a clear separation between caput, corpus and cauda epididymides. Some transcripts characterize a particular anatomical segment, whereas others are expressed in two out of three epididymal segments. GO analysis allowed deduction of specific functions played by each epididymal segment. The transcriptional profiles between fertile vs. sub-fertile conditions clustered most closely in the corpus and cauda segments, whereas the profiles in the caput segment were distinct between fertile and sub-fertile bulls. Of the differently expressed genes, ten (including AKAP4, SMCP, SPATA3, TCP11, ODF1, CTCFL, SPATA18, ADAM28, SORD and FAM161A) were found to exert functions related to reproductive systems and 5 genes (including DEAD, CYST11, DEFB119, DEFB124 and MX1) were found to be associated with the defence response. Limitations, reasons for caution: Further work is required to correlate these modulations of epididymal functions with sperm fertilizing ability in order to understand the aetiology of certain cases of idiopathic infertility in livestock and men. Wider implications of the findings: As fertility can be quantified in bulls used for artificial insemination, this species is a unique model to aid in the understanding of male fertility/subfertility in man.Our data provide a molecular characterization that will facilitate advances in understanding the involvement of epididymalphysiology in sub/infertility aetiology.

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:Purpose: To compare the transcriptome profiles (RNA-seq) of cultured human epididymis cells and tissue from the caput, corpus and cauda regions of the human epididymis. Methods: Human epididymis tissue was obtained with Institutional Review Board approval from 3 patients (UC05, UC06, UC09, range: 22 - 36 years) undergoing inguinal radical orchiectomy for a clinical diagnosis of testicular cancer. None of the epididymides had extension of the testicular cancer. The three anatomical regions: caput, corpus and cauda, were separated and segments of each snap frozen. Adult human epididymis epithelial (HEE) cultures were also established from tissue. RNA was extracted from both tissue and cultured HEE cells and RNA-seq libraries prepared (TruSeq RNA Sample Preparation Kit v2, Low-Throughput protocol, Illumina). Libraries were sequenced on Illumina HiSeq2500 machines. Data were analyzed using TopHat and Cufflinks. Results: Libraries generated ~19-39 million reads per library from the cells (95-99% mapping to the human genome) and ~14-39 million reads from the tissue samples (84-99% mapped). Raw reads were aligned to the genome with Tophat and gene expression values were processed using Cufflinks as Fragments Per Kilobase per Million mapped fragments (FPKM). FPKM values were subject to principle component analysis, which revealed that though caput, corpus and cauda cell samples respectively from UC05, UC06 and UC09 clustered together. RNA-seq data from the 3 biological replicas (UC05, UC06 and UC09) of caput, corpus and cauda were pooled for further analysis. Cufflinks was used to determine differentially expressed genes (DEGs) between caput, corpus and cauda cells, combined from the 3 donors. The gene expression profiles of corpus and cauda are remarkably similar and both differ from the caput to a similar degree. We identified ~40 genes differentially expressed between corpus and cauda and more than 1600 DEGs between caput and cauda. The DEGs for each comparison (caput and corpus/cauda) were analysed using a gene ontology process enrichment analysis (DAVID, Huang et al., NAR 2009;37:1-13, Huang et al., 2009 Nat Prot 4:44-57). Conclusions: Here we describe an in depth analysis of the gene expression repertoire of primary cultures of epithelial cells and intact tissues from each region of the adult human epididymis. These data will be valuable to decipher pathways of normal epididymis function and aspects of epididymis disease that cause male infertility. RNA-seq was performed on libraries generated from caput, corpus and cauda-derived cultured cells (passage 2 or 3) from 3 donors and on caput, corpus and cauda tissue from 2 of the same donors. Donor age range: 22 - 36 years.

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Extracellular vesicles are involved in the paracrine communication between epithelial cells in different regions of the domestic cat epididymis

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