Freezing–Thawing Procedures Remodel the Proteome of Ram Sperm before and after In Vitro Capacitation
Abstract
:1. Introduction
2. Results
2.1. Influence of Cryopreservation and Capacitation on Sperm Functionality
2.2. Protein Profile of Fresh and Cryopreserved Ram Sperm Incubated under Capacitating and Non-capacitating Conditions
2.3. Effect of Cryopreservation and Capacitation on Protein Changes
2.4. ELISA Analysis
2.5. Functional Analysis of Identified Proteins in Fresh and Cryopreserved Sperm Incubated under Capacitating and Non-capacitating Conditions
2.6. Different Representation of Those Biological Processes Directly or Indirectly Involved in Reproduction between Fresh and Cryopreserved Sperm Incubated under Capacitating and Non-Capacitating Conditions
3. Discussion
4. Materials and Methods
4.1. Semen Collection and Initial Evaluation
4.2. Sperm Cryopreservation and Thawing
4.3. In Vitro Sperm Capacitation
4.4. Sperm Quality Assessment
4.5. Protein Extraction
4.6. Proteomics Data Acquisition and Analysis
4.7. ELISA Validation
4.8. Gene Ontology Analysis
4.9. Statistical Analyses
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Sperm Parameters | Fresh Sperm | Cryopreserved Sperm |
---|---|---|
Total motility (%) | 44.16 ± 4.17 | 22.48 ± 3.89 * |
Progressive motility (%) | 29.48 ± 3.40 | 17.16 ± 3.25 * |
Apoptosis (%) | 12.05 ± 2.21 | 20.16 ± 1.35 * |
Mitochondrial activity (%) | 32.44 ± 2.45 | 18.64 ± 2.45 * |
ROS levels (mean fluorescence intensity) | 58.83 ± 4.32 | 86.82 ± 4.01 * |
Tyrosine phosphorylation (%) | 46.68 ± 5.07 | 57.11 ± 5.07 |
Sperm Parameters | CAP | NC |
---|---|---|
Total motility (%) | 41.69 ± 4.50 | 24.92 ± 4.50 * |
Progressive motility (%) | 28.81 ± 3.48 | 17.85 ± 3.92 * |
Apoptosis (%) | 14.84 ± 1.54 | 16.37 ± 1.54 |
Mitochondrial activity (%) | 31.68 ± 2.54 | 19.40 ± 2.67 * |
ROS levels (mean fluorescence intensity) | 56.29 ± 2.54 | 89.36 ± 3.10 * |
Tyrosine phosphorylation (%) | 64.73 ± 4.49 | 39.05 ± 5.01 * |
Accession Number | Protein Name | Gene ID | Protein Representation | Reproductive Process | Subcellular Location | |
---|---|---|---|---|---|---|
In Cryopreserved Sperm a (Fresh vs. Cryopreserved Sperm) | In CAP Conditions over Time b (0–240 min) | |||||
E5FYH0 | Testis- and ovary-specific PAZ domain containing protein 1 | TOPAZ1 | ↓ at 0 min in NC | Spermatogenesis | Cytoplasm | |
Q8SQ25 | Mannose-6-phosphate/insulin-like growth factor II receptor | M6P/IGF2R | ↑ at 0 min in NC | ↑ after 240 min in F | Sperm-oocyte interaction | Plasma membrane |
B6UV59 | Hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha | HADHA | ↑ at 0 min in NC | ↓ after 1 min in C | Metabolic process | Mitochondria |
W5PEA2 | Succinate-CoA ligase (ADP-forming) subunit beta, mitochondrial | SUCLA2 | ↑ at 15 min in CAP | Metabolic process | Mitochondria | |
W5P1S6 | Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1 | RPN1 | ↓ at 15 min in CAP | Sperm-oocyte interaction | Plasma membrane | |
W5PJ95 | Serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit A, alpha isoform | PPP2R2A | ↑ after 240 min in F | Signal transduction | Cytoplasm | |
W5PEC5 | Dolichyl-diphosphooligosaccharide-protein glycosyltransferase | LOC101123268 | ↓ at 0 min in NC | ↑ after 15 min in C | Sperm-oocyte interaction | Plasma membrane |
W5QCD4 | Ankyrin repeat-SAM-basic leucine zipper domain-containing protein 1 | ASZ1 | ↓ after 240 min in C | Spermatogenesis | Cytoplasm | |
D5K281 | ADAM metallopeptidase with thrombospondin type 1 motif 1 | ADAMTS1 | ↓ at 15 min in CAP | Spermatogenesis | Plasma membrane | |
W5QBN6 | Dihydrolipoamide cetyltransferase component of pyruvate dehydrogenase complex | DLAT | ↑ at 15 min in CAP | ↑ after 15 min in C; ↑ after 240 min in F | Metabolic process | Mitochondria |
Q8WMQ9 | Pituitary adenylate cyclase-activating polypeptide type 1 receptor hop 1 splice variant | ADCYAP1R1 | ↓ at 15 min in CAP | ↓ after 15 min in C | Sperm motility | Plasma membrane |
W5NZH7 | Serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit B | PPP2R2B | ↑ at 15 min in CAP | Cell cycle-apoptosis-stress | Mitochondria | |
Q09YI7 | Capping protein (actin filament) muscle Z-line, alpha 2, 3 prime | CAPZA2 | ↑ at 15 min in CAP | Sperm motility | Cytoskeleton | |
A0A0C5GE36 | V-kit Hardy–Zuckerman 4 feline sarcoma viral oncoprotein | KIT | ↓ at 15 min in NC | Signal transduction | Plasma membrane |
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Peris-Frau, P.; Martín-Maestro, A.; Iniesta-Cuerda, M.; Sánchez-Ajofrín, I.; Mateos-Hernández, L.; Garde, J.J.; Villar, M.; Soler, A.J. Freezing–Thawing Procedures Remodel the Proteome of Ram Sperm before and after In Vitro Capacitation. Int. J. Mol. Sci. 2019, 20, 4596. https://doi.org/10.3390/ijms20184596
Peris-Frau P, Martín-Maestro A, Iniesta-Cuerda M, Sánchez-Ajofrín I, Mateos-Hernández L, Garde JJ, Villar M, Soler AJ. Freezing–Thawing Procedures Remodel the Proteome of Ram Sperm before and after In Vitro Capacitation. International Journal of Molecular Sciences. 2019; 20(18):4596. https://doi.org/10.3390/ijms20184596
Chicago/Turabian StylePeris-Frau, Patricia, Alicia Martín-Maestro, María Iniesta-Cuerda, Irene Sánchez-Ajofrín, Lourdes Mateos-Hernández, J. Julián Garde, Margarita Villar, and Ana Josefa Soler. 2019. "Freezing–Thawing Procedures Remodel the Proteome of Ram Sperm before and after In Vitro Capacitation" International Journal of Molecular Sciences 20, no. 18: 4596. https://doi.org/10.3390/ijms20184596
APA StylePeris-Frau, P., Martín-Maestro, A., Iniesta-Cuerda, M., Sánchez-Ajofrín, I., Mateos-Hernández, L., Garde, J. J., Villar, M., & Soler, A. J. (2019). Freezing–Thawing Procedures Remodel the Proteome of Ram Sperm before and after In Vitro Capacitation. International Journal of Molecular Sciences, 20(18), 4596. https://doi.org/10.3390/ijms20184596