The purpose of the study was to investigate the proteome alterations induced by equilibration and freezing/thawing process, both in spermatozoa and extracellular fluid (ECF). The differentially expressed proteins (DEPs) were analysed by 2-dimensional difference in-gel electrophoresis (2D-DIGE) and identified with matrix-assisted laser desorption/ionization mass spectrometry (MALDI TOF/TOF). The validation of the selected DEPs was performed by immunofluorescence and western blotting.
This dataset contains 6 directories:
1. The directory ‘DIGE CyDye labeled gels’ contains pictures of fresh, equilibrated and frozen/thawed spermatozoa described as SPERM and extracellular fluid described as ECF. The directory contains 54 pictures in .tif format which are described according to samples arrangement presented in Supplementary Tables 1 and 2.
2.The directory ‘Immunofluorescence spermatozoa labeling validation’ contains six representative microphotographs in .tif format showing validation of semen smear fixation and antibody concentration. Specificity of the secondary antibody (omitting the primary antibody) is presented at Photo 1A- DAPI and Photo 1B- Cy3®-conjugated secondary antibody. Validation of semen smear fixation is presented at 2A. methanol_acetone fixation and 2B. 4% paraformaldehyde fixation. Secondary antibody dilution is presented at 3A. Cy3®-conjugated secondary antibody, dilution 1:200 and 3B. Cy3®-conjugated secondary antibody, dilution 1:300.
3. The directory ‘Supplementary Figures’ contains 4 files in .tif format.
- Supplementary Fig. 1A and 1B. 2D-DIGE gels showing the proteomic profiles of fresh (A) and frozen/thawed (B) turkey spermatozoa. Numbered protein spots in red correspond to the proteins that are more abundant, whereas numbered protein spots in green indicate proteins that are less abundant in frozen/thawed spermatozoa. Results of spot identification are presented in Supplementary Table 3.(.tif)
- Supplementary Fig. 2A and 2B. 2D-DIGE gels showing the proteomic profiles of fresh (A) and frozen/thawed (B) turkey semen extracellular fluid. Numbered protein spots in red correspond to the proteins that are more abundant in frozen/thawed ECF. Results of spot identification are presented in Supplementary Table 4.(.tif)
4. The directory ‘Supplemetary Tables’ contains 6 files, 2 in format .xlsx; 2 in format .ods and 2 in .csv format.
- Supplementary Table 1. Fresh, equilibrated and frozen/thawed semen arrangement for a 2D-DIGE experiment of turkey spermatozoa in six replicates. The internal standard was generated when equal amount of protein from each of 12 samples was combined. (.xlsx)
- Supplementary Table 2. Fresh, equilibrated and frozen/thawed semen arrangement for a 2D-DIGE experiment of turkey extracellular fluid in six replicates. The internal standard was generated when equal amount of protein from each of 12 samples was combined. (.xlsx)
- Supplementary Table 3. List of differentially expressed proteins in fresh, equilibrated and frozen/thawed turkey spermatozoa identified by MALDI TOF/TOF analysis. (.ods)
- Supplementary Table 4. List of differentially expressed proteins in fresh, equilibrated and frozen/thawed turkey extracellular fluid identified by MALDI TOF/TOF analysis. (.ods)
- Supplementary Table 5. Gene ontology analysis of DEPs identified in spermatozoa between fresh and frozen/thawed semen. Gene ontology was performed using G:Profiler with the highlight driver terms GO at an adjusted p value < 0.05. (.csv)
- Supplementary Table 6. Gene ontology analysis of DEPs identified in extracellular fluid between fresh and frozen/thawed semen. Gene ontology was performed using G:Profiler with the highlight driver terms GO at an adjusted p value < 0.05. (.csv)
5. The directory ‘Western Blot pictures’ contains pictures of blots with target protein and stain free blots in .tif format (40 pictures). Pictures show blots for spermatozoa protein validation described as SPERM and for extracellular fluid proteins validation described as ECF. The following proteins were validated for spermatozoa: acrosin (ACR), aconitate hydratase (ACO2), alpha-enolase (ENO1), glycerol-3-phosphate dehydrogenase (GPD2), triosephosphate isomerase (TPI1); and for ECF: ACR (acrosin), alpha-enolase (ENO1), phosphoglycerate kinase (PGK1), pyruvate kinase PKM (PKM), and triosephosphate isomerase (TPI1).
6. The directory 'Western Blot quantitative data' contains 14 files of raw data presenting relative intensity (arbitrary units) used for statistical analysis in .xlsx format for spermatozoa validation (SPERM ) and for extracellular fluid validation (ECF).The following proteins were validated for spermatozoa: acrosin (ACR), aconitate hydratase (ACO2), alpha-enolase (ENO1), glycerol-3-phosphate dehydrogenase (GPD2), triosephosphate isomerase (TPI1); and for ECF: ACR (acrosin), alpha-enolase (ENO1), phosphoglycerate kinase (PGK1), pyruvate kinase PKM (PKM), and triosephosphate isomerase (TPI1).
(2024-09-26)
