Proteome and phosphoproteome of Wip1-knockout mouse testis
Ontology highlight
ABSTRACT: Spermatogenesis plays an important role in the mammalian testis, involving in the complex processes of mitosis, meiosis, and spermiogenesis. Spermatogenesis may also be disrupted in the absence of the immunological and ‘fence’ functions of the BTB, resulting in male subfertility or infertility. Mice lacking wild-type p53-induced phosphatase 1 (Wip1) display male reproductive organ defects, but the molecular mechanisms underlying these abnormalities remain unclear. We explored the function of Wip1 in spermatogenesis and fertility by examining differences in the expressed testis proteome and phosphoproteome between Wip1-deficient and wild-type mice using a proteomics approach. 90 proteins and 178 phosphoproteins were differentially regulated between these two groups of mice. These results suggested that proinflammatory cytokines may impair the blood–testis barrier dynamics by decreasing the expression of junction-associated proteins, which effect could be partially responsible for the subfertility and spermatogenesis defects in Wip1-knockout mice.
Project description:Spermatogenesis plays an important role in the mammalian testis, involving in the complex processes of mitosis, meiosis, and spermiogenesis. Spermatogenesis may also be disrupted in the absence of the immunological and ‘fence’ functions of the BTB, resulting in male subfertility or infertility. Mice lacking wild-type p53-induced phosphatase 1 (Wip1) display male reproductive organ defects, but the molecular mechanisms underlying these abnormalities remain unclear. We explored the function of Wip1 in spermatogenesis and fertility by examining differences in the expressed testis proteome and phosphoproteome between Wip1-deficient and wild-type mice using a proteomics approach. Among a total of 6872 proteins and 4280 phosphorylation sites on 1614 proteins identified in our analysis, 58 proteins and 159 phosphorylation sites on 141 proteins were differentially regulated between these two groups of mice. These results suggested that proinflammatory cytokines may impair the blood–testis barrier dynamics by decreasing the expression of junction-associated proteins, which effect could be partially responsible for the subfertility and spermatogenesis defects in Wip1-knockout mice.
Project description: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.
Project description:Long non-coding RNA (lncRNA), as diverse functional regulators, are abundantly expressed in the testis. However, lncRNAs preferentially or exclusively expressed in the testis remain largely unknown. Here, we report a testis-specific lncRNA, 1700052I22Rik, which exhibits a dynamic expression pattern during spermatogenesis. Our findings demonstrate that knockout of 1700052I22Rik in mice leads to reduced sperm counts and subfertility in males, as well as defective spermatid chromatin condensation. We further elucidate the underlying mechanism by which 1700052I22Rik modulates the translation of protamine 1 (PRM1) through interaction with Y-box binding protein 2 (YBX2). Collectively, our results uncover a crucial role for the testis-specific lncRNA 1700052I22Rik in regulating spermatid chromatin condensation in mice, providing novel insights into the functions of lncRNAs in spermatogenesis and potential targets for the diagnosis and treatment of male infertility.
Project description:Finding missing proteins (MPs) has been one of the critical missions of Chromosome-Centric Human Proteome Project (C-HPP) since 2012, twenty-five research teams from seventeen countries have been trying to search adequate and accurate evidence for MPs through various biochemical strategies. In our previous study, we found that phosphoproteomics is one pleasant means to catch low-abundance and membrane proteins which are phosphorylated. Therefore, we speculate it may be an available approach for MPs detection. In this study, kidney cancer and adjacent tissues were used for phosphoproteomics research, we totally identified 8962 proteins including 6415 phosphoproteins, and 44728 phosphosites, of which 10266 were unreported previously. Interestingly, a total of 75 MPs were found, after rigorous screening and manual checking, 26 MPs were ranked as confident MPs by the verification with the synthesized peptides and a stringent manual check of their MS2 spectra, among which 14 MPs were phosphorylated. Functional analysis for 26 MPs revealed that 16 MPs were found to be membrane proteins, 7 MPs were testis-enriched and 1 MPs were kidney-specific. Therefore we concluded that phosphoproteomics is a promising approach in digging MPs for C-HPP studies.
Project description:Spermatogenesis is a complex sperm-generating process involving the mitosis of spermatogonia, meiosis of spermatocytes and spermiogenesis of spermatids. Elucidating the phosphorylation-based regulations should advance our understanding of the underlying molecular mechanisms. Here we present an integrative study of phosphorylation events in the testis. Large-scale phosphoproteome profiling in the adult mouse testis identified 17,829 phosphorylation sites in 3,955 phosphoproteins.
Project description:Testis were obtained from 2 sibling pairs of of young male mice, 1 knock-out and 1 heterozygous for the Sirt1 allele, and used to compare global gene expression using spotted oligo microarrays (the MEEBO arrays from the Stanford microarray core- MOE series). Eighty genes were up or down regulated, where the criteria for differential expression was a greater than 2 fold change in expression level on at least 5 of the 6 sample arrays. The list of differentially expressed genes was enriched for genes involved in spermatogenesis. Keywords: spotted oligonucleotide microarray, mouse 2 pairs of Sirt1 knock-out and heterozygous testis were analyzed. The global gene expression was assessed intriplicate for each pair, including 1 dye swap.
Project description:Investigation of a new RNA-modification-based mechanism in mammalian spermatogenesis. We created a mice line devoid of testis-specific m5C RNA methyltransferase Nsun7. These mice demonstrated male infertility due to a peculiar defect in sperm tail assembly – mispositioning of the longitudinal columns of the fibrous sheath. The whole testis proteomes of wild type and Nsun7 knockout mice were obtained to study the molecular pathways affected by NSUN7.
Project description:Protein phosphatase 6 (PP6) is a member of the PP2A-like subfamily, which plays significant roles in numerous fundamental biological activities. We found that PPP6C plays important roles in male germ cells recently. Spermatogenesis is supported by the Sertoli cells in seminiferous epithelium. In this study, we crossed Ppp6cF/F mice with AMH-Cre mice to gain mutant mice with specific depletion of the Ppp6c gene in the Sertoli cells. We discovered that the PPP6C cKO male mice were absolutely infertile and germ cells were largely lost during spermatogenesis. By combing phosphoproteome with bioinformatics analysis, we showed that the phosphorylation status of β-catenin at S552 (a marker of adherens junctions) was significantly upregulated in mutant mice. Abnormal β-catenin accumulation resulted in impaired testicular junction integrity, thus led to abnormal structure and functions of BTB. Taken together, our study reveals a novel function for PPP6C in male germ cell survival and differentiation by regulating the cell-cell communication through dephosphorylating β-catenin at S552.
Project description:Spermatogenesis is a complex multi-step process involving intricate interactions between different cell types in the male testis. Disruption of these interactions results in infertility. Combination of shotgun tissue proteomics with MALDI imaging mass spectrometry is markedly potent in revealing topological maps of molecular processes within tissues. Here, we use a combinatorial approach on a characterized mouse model of hormone induced male infertility to uncover misregulated pathways. Comparative testicular proteome of wildtype and mice overexpressing human P450 aromatase (AROM+) with pathologically increased estrogen levels unravels gross dysregulation of spermatogenesis and emergence of pro-inflammatory pathways in AROM+ testis. In situ MS allowed us to localize misregulated proteins/peptides to defined regions within the testis. Results suggest that infertility is associated with substantial loss of proteomic heterogeneity, which define distinct stages of seminiferous tubuli in healthy animals. Importantly, considerable loss of mitochondrial factors, proteins associated with late stages of spermatogenesis and steroidogenic factors characterise AROM+ mice. Thus, the novel proteomic approach pinpoints in unprecedented ways the disruption of normal processes in testis and provides a signature for male infertility.
Project description:Temporal study of the first wave of spermatogenesis in juvenile mouse testis and analysis of four germ cell deficient mouse models.<br> A series of purified testis cell-types (McCarrey libraries)constituting a focussed gene set was exploited to examine gene expression in prepubertal mouse testis. Testis RNA from C57/BL6J mice at various ages post partum was compared to a common control consisting of adult (8 week) testis RNA from the same strain(also corresponding to the last time-point).This generates a time course showing the activation of genes on the array across the first wave of spermatogenesis, which can then be correlated with the appearance of specific germ cell types, allowing assignation of unknown genes to differing cell types. <br> <br> Adult testis RNA from four different genetic models of infertility (XXSxrb, mshi, Bax -/-, bs) were compared to age- and strain-matched normal control testis RNA. These models possess different cellular complements within the testis, which can be interpreted within the framework established by the first wave analysis and used to refine the assignment of genes to cell types.