Project description:cis-Apc+/Delta716/Smad4+/- mice spontaneously develop multiple invasive intestinal carcinomas due to loss of heterozygosity and manifest symptoms associated with cancer cachexia within four months of age. To investigate the role of the liver in cachexia pathophysiology, we compared the transcriptomes of cachexia and control mouse livers. We isolated total RNA from livers of four C57BL/6N mice and four cis-Apc+/Delta716/Smad4+/- mice. Total RNA samples were then employed to perform microarray analysis (Agilent SurePrint G3 Mouse GE 8x60K Microarray).
Project description:Mammalian embryonic stem (ES) cells and sperm exhibit unusual chromatin packaging that plays important roles in cellular function. Here, we extend a recently developed technique, based on deep paired-end sequencing of lightly digested chromatin, to assess footprints of nucleosomes and other DNA-binding proteins genome-wide in murine ES cells and sperm. In ES cells, we recover well-characterized features of chromatin such as promoter nucleosome depletion, and further identify widespread footprints of sequence-specific DNA-binding proteins such as CTCF, which we validate in knockdown studies. We document global differences in nuclease accessibility between ES cells and sperm, finding that the majority of histone retention in sperm preferentially occurs in large gene-poor genomic regions, with only a small subset of nucleosomes being retained over promoters of developmental regulators. Finally, we describe evidence that CTCF remains associated with the genome in mature sperm, where it could play a role in organizing the sperm genome. We use Micrococcal Nuclease (MNase) to map chromatin structure in mouse ES cells and sperm. Specifically, we generate paired-end deep-sequencing libraries that are able to distinguish DNA digestion products by size, thus allowing us to simultaneously map nucleosomes as well as other DNA-binding proteins such as transcription factors.
Project description:Analysis of proteins under the influence of the quorum sensing (QS) system in the nonpathogenic Agrobacterium tumefaciens strain 6N2, and the influence of the bacterial QS system in the proteome of the yeast Meyerozyma guilliermondii strain 6N.
Project description:Proteomic investigations of spermatozoa provide practical tools for distinguishing normal, functional spermatozoa from abnormal spermatozoa. Indeed, two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS) applies high-throughput industrial applications to identify sperm-specific proteins indicative of chemical exposure. As such, a direct comparison of protein expression profiles between control and exposed cells returned a set of protein markers. Because mature mammalian spermatozoa are virtually incapable of protein synthesis, the predicted protein biomarkers in spermatozoa offer considerable stability for use in clinical application. In the current study, we applied 2-DE coupled with ESI-MS/MS to investigate the modified protein profile in F1 capacitated spermatozoa due to gestational bisphenol-A (BPA) exposure to ascertain whether these proteomic modifications could explain the observed functional alterations in spermatozoa.
Project description:We reported RNA profiles of mice spermatozoa, a total of 35,288,825 reads matching 33,039 transcripts, including 27,310 coding transcripts, were obtained. RNA profiles of the spermatozoa of 9-10 weeks adult mice were sequenced by RNA-seq,using Illumina GAIIx.
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:The epithelium lining the epididymis has a pivotal role in ensuring a luminal environment that can support normal sperm maturation. Many of the individual genes that encode proteins involved in establishing the epididymal luminal fluid are well characterized. They include ion channels, ion exchangers, transporters and solute carriers. However, the molecular mechanisms that coordinate expression of these genes and modulate their activities in response to biological stimuli are less well understood. To identify cis-regulatory elements for genes expressed in human epididymis epithelial cells we generated genome-wide maps of open chromatin by DNase-seq. This analysis identified 33,542 epididymis-selective DNase I hypersensitive sites (DHS), which were not evident in five cell types of different lineages. Identification of genes with epididymis-selective DHS at their promoters revealed gene pathways that are active in immature epididymis epithelial cells. These include processes correlating with epithelial function and also others with specific roles in the epididymis including retinol metabolism and ascorbate and aldarate metabolism. Peaks of epididymis-selective chromatin were seen in the androgen receptor gene and the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which has a critical role in regulating ion transport across the epididymis epithelium. In silico prediction of transcription factor binding sites that were over-represented in epididymis-selective DHS identified epithelial transcription factors including ELF5 and ELF3, the androgen receptor, Pax2 and Sox9, as components of epididymis transcriptional networks. Active genes, which are targets of each transcription factor, reveal important biological processes in the epididymis epithelium. To identify cis-regulatory elements for genes expressed in human epididymis epithelial cells we generated genome-wide maps of open chromatin by DNase-seq.
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.