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Sperm Chromatin Proteomics identifies conserved fertility factors

Sperm Chromatin Proteomics identifies conserved fertility factors. by D.S. Chu, H. Liu, P. Nix, T.F. Wu, E.J. Ralston, J.R. Yates III, B.J. Meyer. Outline. Background C. elegans breeding biology Basic mass spec. proteomics Paper Methods and Results Conclusions.

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Sperm Chromatin Proteomics identifies conserved fertility factors

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  1. Sperm Chromatin Proteomics identifies conserved fertility factors by D.S. Chu, H. Liu, P. Nix, T.F. Wu, E.J. Ralston, J.R. Yates III, B.J. Meyer

  2. Outline • Background • C. elegans breeding biology • Basic mass spec. proteomics • Paper • Methods and Results • Conclusions

  3. C. elegans, where men are men and women are, well… • www.wormbook.org, www.wormclassroom.org

  4. Unique chromatin remodeling during development of male germ cells • Nature 434: 583.

  5. Basic Mass Spec Proteomics • Requires effective protein prep • Effective Ionization Required for loading on Mass Spec • Genome required for comparison to Mass Spectrum Aebersold and Mann (2003) Nature

  6. Variations of Mass Spec Tech Aebersold and Mann (2003) Nature

  7. MudPIT Technology • Multidimensional Protein Identification Technology • Two Dimensional LC • Strong Cation Exchange • Reverse Phase • Tandem Mass Spec • Rough abundance estimate from spectral sampling Why do you want to use a 2D-LC? Washburn et al (2001) Nature Biotech

  8. The Goal: Identify and characterize spermatogenic chromatin-associated proteins that are important for fertility • Purification of chromatin from meiotic cells • MudPIT analysis of factors co-purifying with chromatin • Prioritization of sperm proteins based on abundance • Subtraction of oogenic and somatic chromatin proteins • Functional analysis by immunolocalization and RNAi

  9. Problems: 1. A good male is hard to find2. How to purify oocytes from a hermaphrodite

  10. Chromatin Purification 20 Strokes 100 Strokes Centrifuged and Resuspended in Tris Buffer Pelleted, Washed, and Resuspended Centrfuged, Extracted, and Washed 20% TCA Precipitated Sucrose Gradient

  11. Chromatin Purification Check What is wrong with the picture?

  12. 2D-LC-MS/MS Analysis • Precipitated proteins dissolved in “Digestion Buffer” with Lys-C and Trypsin • Six Spermatogenic and Five Oogenic Chromatins analyzed Washburn et al (2001) Nature Biotech

  13. 2D-LC-MS/MS Settings • SCX/RP Columns • Ion Trap MS • Dynamic Exclusion = detection of a single peptide mass in first MS will exclude secondary MS analysis for a set time period • Wormpep Database used to compare to spectrum

  14. Proteomic Results • 1,099 spermatogenic and 812 oogenic proteins identified • Reduced to 502 spermatogenic proteins based upon occurrence in three or more samples (88% relative mass)

  15. Comparative Analysis Is this method justified?

  16. Composition of chromatin

  17. Immunostaining Analysis • 11 candidate proteins tested for sub-cellular localization with chromatin • Four families • Glc-seven phosphatase • Sperm meosis PDZ • Histone two A sperm • Sperm Chromatin Enriched • Three others • Topo-isomerase • Germ line helicase • Holocentric chromosome-binding protein

  18. Immunostaining Subcellular Localization Previously Identified spermatogenic chormatin protein All 11 tested showed co-localization with Chromatin Figure 2

  19. HCP-4 associates with somatic and mature sperm chromatin

  20. Functional analysis by RNAi • him-8(e1489) hermaphrodites were injected w/ appropriate dsRNA, F1 progeny were assayed for number of progeny • To assess male fertility, F1 males were mated to spermatogenic-defective spe-8 dpy-4 hermaphrodites Why did they assess fertility of the offspring of the mothers that were injected with dsRNA?

  21. Spermatogenesis defects caused by RNAi of genes identified by MudPIT

  22. Functional analysis by RNAi • RNAi of 50 of 132 genes resulted in sterility or embryonic lethality • 20 of the 50 genes displayed germline cytological defects • 18 of the 20 disrupted aspects of male fertility • What about the other 82?

  23. Functional analysis, cont’d • 42 identified proteins overlap with previously-identified genes upregulated during spermatogenesis • 31% of these displayed sterility and embryonic lethality with RNAi • 90 proteins do not overlap • 41% of these displayed sterility and embryonic lethality with RNAi

  24. Relevance to mammalian fertility • 29 C. elegans proteins correspond to 19 genetically disrupted mouse homologues • 7 of the 19 disrupted homologues cause male infertility • These 7 mammalian proteins correspond to 14 worm proteins • 7 of the 14 worm proteins show spermatogenesis RNAi defect or associate with spermatogenic chromatin

  25. Similar defects caused by RNAi of Topoisomerase top-1 and SR (RNA processing regulator) protein rsp-6

  26. Summary • MudPIT and subtractive analysis identified 132 spermatogenic chromatin-associated protein • Immunolocalization confirmed that 11 of these were associated with chromatin • RNAi confirmed that at least 50 play a role in fertility • Some correspond to known mammalian fertility factors, others have not yet been characterized in mammalian fertility

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