Proteomic profiling of stallion spermatozoa suggests changes in sperm metabolism and compromised redox regulation after cryopreservation.
Abstract: Proteomic technologies allow the detection of thousands of proteins at the same time, being a powerful technique to reveal molecular regulatory mechanisms in spermatozoa and also sperm damage linked to low fertility or specific biotechnologies. Modifications induced by the cryopreservation in the stallion sperm proteome were studied using UHPLC/MS/MS. Ejaculates from fertile stallions were collected and split in two subsamples, one was investigated as fresh (control) samples, and the other aliquot frozen and thawed using standard procedures and investigated as frozen thawed subsamples. UHPLC/MS/MS was used to study the sperm proteome under these two distinct conditions and bioinformatic enrichment analysis conducted. Gene Ontology (GO) and pathway enrichment analysis were performed revealing dramatic changes as consequence of cryopreservation. The terms oxidative phosphorylation, mitochondrial ATP synthesis coupled electron transport and electron transport chain were significantly enriched in fresh samples (P = 5.50 × 10, 4.26 × 10 and 7.26 × 10 respectively), while were not significantly enriched in frozen thawed samples (P = 1). The GO terms oxidation reduction process and oxidoreductase activity were enriched in fresh samples and the enrichment was reduced in frozen thawed samples (1.40 × 10, 1.69 × 10 versus 1.13 × 10 and 2-86 × 10 respectively). Reactome pathways (using human orthologs) significantly enriched in fresh sperm were TCA cycle and respiratory electron transport (P = 1.867 × 10), Respiratory electron transport ATP synthesis by chemiosmosis coupling (P = 2.124 × 10), Citric acid cycle (TCA cycle)(P = 8.395 × 10) Pyruvate metabolism and TCA cycle (P = 3.380 × 10), Respiratory electron transport (P = 2.764 × 10) and Beta oxidation of laurolyl-CoA to decanoyl CoA-CoA (P = 1.854 × 10) none of these pathways were enriched in thawed samples (P = 1). We have provided the first detailed study on how the cryopreservation process impacts the stallion sperm proteome. Our findings identify the metabolic proteome and redoxome as the two key groups of proteins affected by the procedure. SIGNIFICANCE: In the present manuscript we investigated how the cryopreservation of stallion spermatozoa impacts the proteome of these cells. This procedure is routinely used in horse breeding and has a major impact in the industry, facilitating the trade of genetic material. This is still a suboptimal biotechnology, with numerous unresolved problems. The limited knowledge of the molecular insults occurring during cryopreservation is behind these problems. The application and development of proteomics to the spermatozoa, allow to obtain valuable information of the specific mechanisms affected by the procedure. In this paper, we report that cryopreservation impacts numerous proteins involved in metabolism regulation (mainly mitochondrial proteins involved in the TCA cycle, and oxidative phosphorylation) and also affects proteins with oxidoreductase activity. Moreover, specific proteins involved in the sperm-oocyte interaction are also affected by the procedure. The information gathered in this study, opens interesting questions and offer new lines of research for the improvement of the technology focusing the targets here identified, and the specific steps in the procedure (cooling, toxicity of antioxidants etc.) to be modified to reduce the damage.
Copyright © 2020 Elsevier B.V. All rights reserved.
Publication Date: 2020-04-02 PubMed ID: 32247875DOI: 10.1016/j.jprot.2020.103765Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research investigates the impact of the cryopreservation process on stallion spermatozoa. Using proteomic technologies, the study found that cryopreservation significantly affects the metabolic function and redox regulation of the spermatozoa.
Research Method
- Proteomics technologies were used to detect changes in the protein composition within the stallion spermatozoa caused by the cryopreservation process.
- Ejaculates from fertile stallions were divided into two samples – one fresh and the other frozen and thawed using standard procedures.
- Ultra-high-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) was used to study the sperm proteome under the two distinct conditions.
- Gene ontology (GO) and pathway enrichment analysis were performed to understand the changes in protein makeup due to the cryopreservation.
Findings
- The study highlighted significant changes in sperm metabolism and redox regulation that occur due to the process of cryopreservation.
- Fresh sperm samples showed significant enrichment in pathways related to oxidative functions – oxidative phosphorylation, mitochondrial ATP synthesis coupled electron transport, and electron transport chain.
- This enrichment was notably absent in frozen thawed samples, suggesting an interference with these vital metabolic functions because of the cryopreservation process.
- Furthermore, oxidation reduction processes and oxidoreductase activity were less prevalent in the cryopreserved sperm samples, indicating compromised redox regulation.
Significance and Implications
- This research is first of its kind to provide detailed insights on the changes that occur in the proteome of stallion spermatozoa due to cryopreservation.
- The findings have major implications for the horse breeding industry where cryopreservation of spermatozoa is a common practice yet continues to face unresolved challenges, predominantly due to the lack of understanding of the molecular processes affected by cryopreservation.
- These results can be used to generate new research avenues to improve the cryopreservation technology. The study has highlighted clear target areas associated with metabolism and redox regulation that can be tuned to minimize damage during the freezing process.
- Additionally, these insights can guide the refinement of steps in the cryopreservation procedure, such as the cooling process and antitoxidant use, to reduce cell damage and improve fertility outcomes.
Cite This Article
APA
Martín-Cano FE, Gaitskell-Phillips G, Ortiz-Rodríguez JM, Silva-Rodríguez A, Román Á, Rojo-Domínguez P, Alonso-Rodríguez E, Tapia JA, Gil MC, Ortega-Ferrusola C, Peña FJ.
(2020).
Proteomic profiling of stallion spermatozoa suggests changes in sperm metabolism and compromised redox regulation after cryopreservation.
J Proteomics, 221, 103765.
https://doi.org/10.1016/j.jprot.2020.103765 Publication
Researcher Affiliations
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
- Facility of Innovation and Analysis in Animal Source Foodstuffs, University of Extremadura, Cáceres, Spain.
- Department of Biochemistry and Molecular Biology, University of Extremadura, Badajoz, Spain.
- Department of Physiology, University of Extremadura, Cáceres, Spain.
- Department of Physiology, University of Extremadura, Cáceres, Spain.
- Department of Physiology, University of Extremadura, Cáceres, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain. Electronic address: fjuanpvega@unex.es.
MeSH Terms
- Animals
- Cryopreservation
- Horses
- Humans
- Male
- Oxidation-Reduction
- Proteomics
- Semen Preservation
- Sperm Motility
- Spermatozoa
- Tandem Mass Spectrometry
Conflict of Interest Statement
Declaration of Competing Interest The authors declare that there are no conflicts of interest that could be perceived to prejudice the reported research.
Citations
This article has been cited 16 times.- Horta Remedios M, Liang W, González LN, Li V, Da Ros VG, Cohen DJ, Zaremberg V. Ether lipids and a peroxisomal riddle in sperm. Front Cell Dev Biol 2023;11:1166232.
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