The landscape of mouse epididymal cells defined by the single-cell RNA-Seq
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE151998 | GEO | 2021/06/02
REPOSITORIES: GEO
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Project description:Spermatozoa released from the testis are unable to fertilize an egg without a coordinated process of maturation in the lumen of the epididymis. Relatively little is known about the molecular events that integrate this critical progression along the male genital ducts in man. Here we use single cell RNA-sequencing to construct an atlas of the human proximal epididymis. We find that the cystic fibrosis transmembrane conductance regulator (CFTR), which is pivotal in normal epididymis fluid transport, is most abundant in surface epithelial cells in the efferent ducts and in rare clear cells in the caput epididymis, suggesting region-specific functional properties. We reveal transcriptional signatures for multiple cell clusters, which identify the individual roles of principal, apical, narrow, basal, clear, halo and stromal cells in the epididymis. A marked cell-type-specific distribution of function is seen along the duct with local specialization of individual cell types integrating processes of sperm maturation.
2020-08-25 | GSE148963 | GEO
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.
2016-01-04 | E-GEOD-72986 | biostudies-arrayexpress
Project description:The small RNA payload of mammalian sperm undergoes dramatic remodeling during development, as several waves of microRNAs and tRNA fragments are shipped to sperm during post-testicular maturation in the epididymis. Here, we take advantage of this developmental process to probe the function of the sperm RNA payload in preimplantation development. We generated zygotes via intracytoplasmic sperm injection (ICSI) using sperm obtained from the proximal (caput) vs. distal (cauda) epididymis, then characterized development of the resulting embryos. Embryos generated using caput sperm significantly overexpress multiple regulatory factors throughout preimplantation development, and subsequently implant inefficiently and fail soon after implantation. Remarkably, microinjection of purified cauda-specific small RNAs into caput-derived embryos not only completely rescued preimplantation molecular defects, but also suppressed the postimplantation embryonic lethality phenotype. These findings reveal an essential role for small RNA remodeling during post-testicular maturation of mammalian sperm, and identify a specific preimplantation gene expression program responsive to sperm-delivered microRNAs.
2018-07-25 | GSE111864 | GEO
Project description:Epididymosomes are small membrane vesicles (50-500nm) secreted by epididymal epithelial cells and involved in post-testicular sperm maturation. While their role in protein transfer to the sperm membrane is well acknowledged, we unveil here their capacity to vehicle microRNAs (miRNAs), which are potent regulators of post-transcriptional gene expression. Using a global microarray approach, we showed that epididymosomes providing from two discrete bovine epididymal regions (caput and cauda) possess distinct miRNA signatures. In addition, we established that miRNA repertoires contained in epididymosomes differ from those of their parent epithelial cells, suggesting that miRNA populations released from the cells may be selectively sorted. Binding of DilC12-labeled epididymosomes to primary cultured epididymal cells was measured by flow cytometry and indicated that epididymosomes from the median caput and their miRNA content may be incorporated into distal caput epithelial cells. Overall, these findings reveal that distinct miRNA repertoires are released into the intraluminal fluid in a region-specific manner and could be involved in a novel mechanism of intercellular communication throughout the epididymis via epididymosomes. We determined the miRNA profiles of epididymosomes isolated by perfusion from caput and cauda regions of the epididymis and compared it with the miRNA content of epididymal epithelial cells from the same regions.
2013-07-01 | E-GEOD-44365 | biostudies-arrayexpress
Project description:In total, 40,623 cells were detected and further clustered into 8 identified cell populations. Focused analyses revealed the subpopulations of principal cells, basal cells, clear/narrow cells, and halo/T cells. Notably, two subtypes of principal cells, the Prc7 and Prc8 subpopulations were enriched as stereocilia-like cells according to GO analysis. Further analysis demonstrated the spatially specific pattern of the DEGs in each cell cluster. Unexpectedly, the abundance of mitochondria and mitochondrial transcription (MT) was found to be higher in the corpus and cauda epididymis than in the caput epididymis by scRNA-seq, immunostaining, and qPCR validation. In addition, the spatio-temporal profile of the DEGs from the P42 and P56 epididymis, including transiting spermatozoa, was depicted.
2021-04-12 | GSE159713 | GEO
Project description: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.
2024-09-01 | GSE269443 | GEO
Project description:Previously we have reported that cysteine rich secretory protein 2 (CRISP2) is involved in high molecular weight complexes in boar spermatozoa under a native state. In the current study, CRISP2 interacting partners were identified using proteomic technology and their interaction was investigated under non-capacitating conditions, after the induction of in vitro sperm capacitation and then subsequently during an ionophore A23187 induced acrosome reaction. Blue native gel electrophoresis and native immunoblots revealed that CRISP2 was present within a ~150 kDa protein complex under all three conditions. Interrogation of CRISP2-immunoreactive bands from blue native gels as well as CRISP2 immunoprecipitated products using mass spectrometry consistently revealed that acrosin and acrosin binding protein (ACRBP) were among the most abundant proteins and present under the three conditions. Co-immunoprecipitation and immunoblotting indicated that CRISP2 interacted with pro-acrosin (~53 kDa) and ACRBP under all three conditions and additionally to acrosin (~35 kDa) after capacitation and the acrosome reaction. The colocalization of these interacting proteins with CRISP2 was assessed via in situ proximity ligation assays. The colocalization signal of CRISP2 and acrosin in the acrosome seemed dispersed after capacitation but was consistently present in the sperm tail under all conditions. The fluorescent foci of CRISP2 and ACRBP colocalization appeared to be redistributed within the sperm head from the anterior acrosome to the post acrosomal sheath region upon capacitation. These results suggest that CRISP2 may act as a scaffold for protein complexes formation to ensure the correct positioning of proteins required for the acrosome reaction and zona pellucida penetration.
2023-03-09 | PXD037181 | Pride
Project description:The male gamete is not completely mature after ejaculation and requires further events in the female genital tract to acquire fertilizing ability, including the processes of capacitation and acrosome reaction. In order to shed light on dynamic protein changes experienced by the sperm cell in preparation for fertilization, a comprehensive quantitative proteomic profiling based on isotopic peptide labeling and liquid chromatography followed by tandem mass spectrometry was performed on spermatozoa from three donors of proven fertility under three sequential conditions: purification with density gradient centrifugation, incubation with capacitation medium, and induction of acrosome reaction by the exposure to the calcium ionophore A23187. After applying strict selection criteria for peptide quantification and for statistical analyses, 36 proteins with significant changes in their relative abundance within sperm protein extracts were detected. Moreover, the presence of peptide residues potentially harboring sites for post-translational modification was revealed, suggesting that protein modification may be an important mechanism in sperm maturation. In this regard, increased levels of proteins mainly involved in motility and signaling, both regulated by protein modifiers, were detected in sperm lysates following incubation with capacitation medium. In contrast, less abundant proteins in acrosome-reacted cell lysates did not contain potentially modifiable residues, suggesting the possibility that all those proteins might be relocated or released during the process. Protein-protein interaction analysis revealed a subset of proteins potentially involved in sperm maturation, including ERLIN2, GGH, TMED 10, and ATP synthases. These results contribute to the current knowledge of the molecular basis of human fertilization and propose new candidates to be validated as modulators of male fertility.
2019-12-17 | PXD014871 | Pride
Project description:Between the testes and the vas deferens lies the epididymis, a highly convoluted tubule whose unique environmental characteristics are crucial for the functional maturation of spermatozoa. Within the epididymal epithelium, the secretory system releases small non-coding RNA molecules (sncRNAs) and proteins housed within extracellular vesicles (epididymosomes) that are destined for delivery to recipient sperm cells which play key roles in fertility success and act as major conduits of epigenetic information delivered to the oocyte. The epithelial cells of the epididymis have proven to be highly sensitive to environmental stressors which can influence the sperm epigenome. Utilizing a label-free mass spectrometry proteomic approach we sought to characterize an immortalized mouse caput epididymal epithelial cell line (mECap18) and sequenced >5,100 proteins. When compared to a previous in-vivo mouse caput epithelial proteomic profile, a significant overlap (>75%) and proportionally similar patterns of protein classification were observed. Furthermore, key pathways associated with protein synthesis (e.g. EIF2 signaling) and cellular protection in the male reproductive tract (e.g. sirtuin signaling) were enriched in both proteomes. Leveraging this comparison supports mECap18 cells as a promising in-vitro model for recapitulating the in-vivo environment, providing a platform for testing therapeutic invention.
2023-09-29 | PXD042082 | Pride
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.
2016-01-04 | GSE72986 | GEO