Dataset Information

Mouse epididymis LC-MSMS - Male Fertility Potential Molecular Mechanisms Revealed by iTRAQ-Based Quantitative Proteomic Analysis of the Epididymis from Wip1-/- Mice

ABSTRACT: The wild-type p53-induced phosphatase 1 (WIP1) frustrated mice exhibited defects in reproductive organs. This study aimed to understand how Wip1 deficiency affects the spermatogenesis or maturation. We employed the Wip1-/- mouse model and conducted a gel-free iTRAQ LC-MS/MS based quantitative proteomics analysis of whole epididymis including the sperms and somatic tissue. A total of 8763 proteins were identified, of which 91 were significantly differentially expressed proteins (DEPs) in the Wip1 depleted mice. Four DEPs (PRM2, ODF1, PIWIL1 and KLHL10) were confirmed with western blotting. The DEPs enriched in biological process of reproduction were identified by GO analysis and further confirmed in the mouse phenotype database. Pathway analysis suggested that Smac/Diablo-mediated apoptosis pathway and SERPINA3-mediated inflammatory process might contribute to the atrophy and the marked sperm decrease in epididymis. Network analysis of productivity related DEPs revealed possible interactions that WIP1 might affect the sperm maturation by decreasing the outer dense fiber protein 1 (ODF1) and protamine-2 (PRM2), and increasing the PIWIL1/MIWI through p53. PRM2 was down-regulated and PIWIL1 was up-regulated by immunohistochemistry staining. It was further confirmed that the spermatid deficiency began from the testis observed by HE staining. Therefore, WIP1 disruption caused the deficiency of spermatogenesis potentially through regulating the expression of the above DEPs and pathways.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Epididymis

SUBMITTER: pengxia niu

LAB HEAD: Li Kui

PROVIDER: PXD010125 | Pride | 2019-01-16

REPOSITORIES: Pride

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Publications

Male Fertility Potential Molecular Mechanisms Revealed by iTRAQ-Based Quantitative Proteomic Analysis of the Epididymis from Wip1<sup>-/-</sup> Mice.

Niu Pengxia P Wei Yinghui Y Gao Qian Q Zhang Xue X Hu Yanqing Y Qiu Yiqing Y Mu Yulian Y Li Kui K

Omics : a journal of integrative biology 20190101 1

Proteomics and postgenomic technologies have found growing recent applications in reproductive biology and fertility research. Mice deficient in the wild-type p53-induced phosphatase 1 (WIP1) exhibit defects in reproductive organs. In this study, we aimed to understand how Wip1 deficiency affects spermatogenesis and sperm maturation using the Wip1<sup>-/-</sup> mouse model and gel-free isobaric tags for relative and absolute quantitation liquid chromatography-tandem mass spectrometry quantitativ ...[more]

PMID: 30629479

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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. HEE cells were cultured as described previously (Harris and Coleman 1989). RNA was isolated by Trizol extraction from three primary cultures of HEE cells. Total RNA was purified by Millipore Microcon YM-100 filter centrifugation and shipped to MoGene, MO, for gene expression analysis on Nimblegen 4 x 72K HG18 60mer arrays. This submission represents transcriptome component of study.

Project description:The role of estrogen and testosterone in the regulation of gene expression in the proximal reproductive tract is not completely understood. To address this question, mice were treated with testosterone or estradiol and RNA from the efferent ducts and caput epididymis was processed and hybridized to Affymetrix MOE 430 2.0 microarrays. Analysis of array output identified probe sets in each tissue with altered levels in hormone treated versus control animals. Hormone treatment efficacy was confirmed by determination of serum hormone levels pre- and post-treatment and observed changes in transcript levels of previously reported hormone-responsive genes. Tissue-specific hormone sensitivity was observed with 2867 and 3197 probe sets changing significantly in the efferent ducts after estrogen and testosterone treatment, respectively. In the caput epididymis, 117 and 268 probe sets changed after estrogen and testosterone treatment, respectively, demonstrating a greater response to hormone in the efferent ducts than the caput epididymis. Transcripts sharing similar profiles in the intact and hormone-treated animals compared with castrated controls were also identified. Ontological analysis of probe sets revealed a significant number of hormone-regulated transcripts encode proteins associated with lipid metabolism, transcription and steroid metabolism in both tissues. Real-time RT-PCR was employed to confirm array data and investigate other potential hormone-responsive regulators of proximal reproductive tract function. The results of this work reveal previously unknown responses to estrogen in the caput epididymis and to testosterone in the efferent ducts as well as tissue specific hormone sensitivity in the proximal reproductive tract. Adult animals were castrated or sham-castrated, allowed to recover for 14 days, and then treated with 0.015 mg estradiol (castrated), 0.015 mg testosterone propionate (castrated), or vehicle (castrated and sham-castrated as biological controls) in duplicate. Efferent duct and caput epididymis was collected from each sample and analyzed. Duplicates are included in the provided data and numbered 1 or 2 for each treatment regimen.

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 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

Mouse epididymis LC-MSMS - Male Fertility Potential Molecular Mechanisms Revealed by iTRAQ-Based Quantitative Proteomic Analysis of the Epididymis from Wip1-/- Mice

Publications

Male Fertility Potential Molecular Mechanisms Revealed by iTRAQ-Based Quantitative Proteomic Analysis of the Epididymis from Wip1<sup>-/-</sup> Mice.

Similar Datasets

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