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???displayArticle.abstract??? Spermatozoa are more efficient at supporting normal embryonic development than spermatids, their immature, immediate precursors. This suggests that the sperm acquires the ability to support embryonic development during spermiogenesis (spermatid to sperm maturation). Here, using Xenopus laevis as a model organism, we performed 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) and mass spectrometry analysis of differentially expressed proteins between sperm and spermatids in order to identify factors that could be responsible for the efficiency of the sperm to support embryonic development. Furthermore, benefiting from the availability of egg extracts in Xenopus, we also tested whether the chromatin of sperm could attract different egg factors compared to the chromatin of spermatids. Our analysis identified: (1) several proteins which were present exclusively in sperm; but not in spermatid nuclei and (2) numerous egg proteins binding to the sperm (but not to the spermatid chromatin) after incubation in egg extracts. Amongst these factors we identified many chromatin-associated proteins and transcriptional repressors. Presence of transcriptional repressors binding specifically to sperm chromatin could suggest its preparation for the early embryonic cell cycles, during which no transcription is observed and suggests that sperm chromatin has a unique protein composition, which facilitates the recruitment of egg chromatin remodelling factors. It is therefore likely that the acquisition of these sperm-specific factors during spermiogenesis makes the sperm chromatin suitable to interact with the maternal factors and, as a consequence, to support efficient embryonic development.
Figure 1. Experimental design for 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) analysis. (A) The same quantity of proteins isolated from sperm or spermatids was labelled with Cy5 (red) or Cy3 (green) dye; (B) After labelling, proteins were mixed and separated in the first dimension electrophoresis in the pH range, according to isoelectric points of proteins. Subsequently, the proteins were run in the second dimension electrophoresis, according to the molecular weight of proteins. These two runs allowed separation of proteins into spots of three colours: in this example the red spots were sperm-specific; the green ones spermatid-specific and the yellow ones were proteins present in both cell types.
Figure 2. 2D-DIGE electrophoresis of proteins isolated from sperm and spermatids. Proteins isolated from spermatids and sperm were labelled with Cy3 (green) and Cy5 (red) dyes, respectively, and subsequently separated in two dimensions. Examples of sperm- and spermatid-specific proteins identified are indicated with arrows: red arrows for sperm-specific proteins and green arrows for spermatid-specific proteins. (A) Laser scanned image of a gel with the proteins separated in pH range 7â11 (basic range); (B) Laser scanned image of gel with proteins separated in the pH range 3â10 (broad range).
Figure 3. Silver staining of 2D-DIGE gels for spot excision. Gels from Figure 2 were silver-stained to allow protein visualisation and spot excision. Protein examples from Figure 2 are also indicated with arrows. (A) proteins separated in pH 7â11; (B) proteins separated in pH 3â10.
Figure 4. Schematic diagram showing experimental design for mass spectrometry analysis of extract-treated sperm or spermatids. Sperm or spermatids are separately treated with egg extracts. Subsequently, sperm or spermatid chromatin is purified and chromatin-bound proteins are isolated. Isolated proteins are then subjected to 2D-DIGE and mass spectrometry identification.
Figure 5. 2D-DIGE electrophoresis of proteins from sperm and sperm-extract treated and of spermatid and spermatid-extract treated. (A) Proteins isolated from sperm (red) were run on 2-D gel together with proteins bound to the sperm chromatin after egg extract treatment (green); (B) Proteins isolated from spermatid (red) were run on 2-D gel together with proteins bound to the spermatid chromatin after egg extract treatment (green). Note the presence of numerous red or green spots on both gels (A,B) and the low number of yellow spots. The first dimension electrophoresis for both gels shown was carried in the pH 3â10 (broad range).
Figure 6. 2D-DIGE electrophoresis of sperm-extract treated and spermatid-extract treated. (A) Proteins bound to sperm chromatin after egg extract treatment (green) were run on a pH 3â10 gel together with proteins bound to spermatid chromatin after egg extract treatment (red). Examples of proteins binding specifically to sperm (green), spermatid (red) or to both types of cells (yellow) are indicated with arrows; (B) The same gel as in (A) silver-stained to allow protein spot visualisation and excision. Examples of proteins are indicated with arrows.
Figure 7. Validation of mass spectrometry results by immunoblotting. (A) Immunoblotting results on proteins isolated from sperm and spermatids after extract treatment. âspermââproteins bound to sperm chromatin after egg extract treatment; âspermatidââproteins bound to spermatid chromatin after egg extract treatment. The antibody used is indicated on the left hand side of the blot inset. 6 µg of protein lysate was loaded on each lane; (B) Quantification of the blots shown in (A). Results are shown as fold difference between the band intensity in sperm-extract treated to spermatid extract-treated. Names of the proteins are indicated below the x axis.
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