Project description:In this study, we conducted a comprehensive quantitative phosphoproteomic analysis of mouse epididymal sperm from different regions (caput, corpus, and cauda) to unveil the dynamics of protein phosphorylation during sperm maturation. And we performed phosphoproteomic analysis of ALK inhibitor 2 compound 18-treated and control sperm to gain further insights into the potential mechanisms by which TSSK2 regulates sperm motility.
Project description:Following their production in the testis, spermatozoa enter the epididymis to gain their motility and fertilizing abilities. This post-testicular maturation coincides with sperm epigenetic profile changes that influence the progeny outcome. While recent studies underscored the dynamics of small non-coding RNAs in the maturing spermatozoa, little is known regarding sperm methylation changes and their impact at the post-fertilization level. To map out the sperm methylome dynamics, we purified spermatozoa by FACS from the testis and the different epididymal segments (i.e. caput, corpus and cauda) of CAG/su9-DsRed2; Acr3-EGFP transgenic mice. Reduced-Representation Bisulfite Sequencing (RRBS-Seq) performed on DNA from these respective sperm populations indicated that high methylation changes were observed between spermatozoa from the caput vs. testis with 5546 entries meeting our threshold values (q value < 0.01, methylation difference above 25 %). Most of these changes were transitory during epididymal sperm maturation according to the low number of entries identified between spermatozoa from cauda vs. testis.
Project description:This study investigated the differential responses of the epididymis and testis to infection with uropathogenic Escherichia coli (UPEC) in adult mice. In contrast to the cauda epididymidis, which exhibited ongoing damage following UPEC-infection, spermatogenic disruption was reversible and the caput epididymidis was largely unaffected. The caput epididymidis is structurally and functionally very different from the cauda, whereas beta-defensins as important epididymal antimicrobial factors are highly expressed in both tissues. The transcriptome of the testis, caput, corpus and cauda epididymidis were analysed in normal, wildtype C57BL/6J mice. Distinct differences in gene signatures were observed between caput and cauda epididymidis, particularly among immune-related genes, whereas corpus and cauda were much more alike. Beta-defensins were found at high levels in both caput and cauda epididymidis, whereas other antimicrobial factors, such as Lipocalin 2 and Lypd8 were found expressed selectively high in the caput epididymidis and hence may play an important role in local protection from bacterial pathogens, including UPEC.
Project description:Purpose: The goal of this study is to detect differentially expressed genes, among Wild type caput, corpus, and cauda epididymis by RNA sequencing Methods: Caput, corpus, and cauda epidiymal mRNA profiles of 9-month-old wild-type mice were generated by deep sequencing, in triplicate, using Illumina GAIIx. Results: RNA-seq data identified transcripts differentially expressed in caput, corpus, and cauda epididymis. Conclusions: Our results show that the expression of many genes were differentially regulated in caput, corpus, and cauda epididymis.
Project description:We carried out whole genome bisulfite sequencing in seven purified sperm populations, including primary spermatocytes, early and late round spermatids, and mature sperm isolated from the caput, corpus, and cauda epididiymis, and from the vas deferens.
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: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.
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.
Project description:The epididymis can be subdivided into three regions, the caput, corpus and cauda epididymidis, that have different characteristics and functions. Goal was to determine the differential pattern of gene expression along the regions of the human epididymis. Two-condition experiment, region 1 vs region 2. Replicates: 4 biological replicates from each epididymal region.
Project description:Total RNAs were extracted from the Testis and Epididymal Caput, Corpus and Cauda tissues of 2-month and 13-month-old WT and Gpx5 KO mice (C57BL/6). The 18 - 40 nt fraction small RNAs and transcriptomes were subjected to library construction and deep sequencing, using Illumina GAIIx or Hiseq 2000.