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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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.

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