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Mass spectrometry reveals distinct proteomic profiles in high- and low-quality stallion spermatozoa.
Abstract: The horse breeding industry relies upon optimal stallion fertility. Conventional sperm assessments provide limited information regarding ejaculate quality and are not individually predictive of fertilizing potential. The aim of this study was to harness mass spectrometry to compare the proteomic profiles of high- and low-quality stallion spermatozoa, with the ultimate goal of identifying fertility biomarker candidates. Extended stallion semen (n = 12) was fractionated using Percoll density gradients to isolate low-quality and high-quality sperm populations. Motility and morphological assessments were carried out, and proteomic analyses was conducted using UHPLC-MS/MS. High-quality spermatozoa recorded higher total (95.2 ± 0.52% vs 70.6 ± 4.20%; P ≤ 0.001) and progressive motilities (43.4 ± 3.42% vs 27.3 ± 4.32%; P ≤ 0.05), and a higher proportion of morphologically normal cells (50.2 ± 4.34% vs 38.8 ± 2.72%; P ≤ 0.05). In total, 1069 proteins were quantified by UHPLC-MS/MS, of which 22 proteins were significantly more abundant in the high-quality sperm population (P ≤ 0.05). A-kinase anchor protein 4 (AKAP4) and Hexokinase 1 (HK1) were considered possible biomarker candidates and their differential expression was confirmed by immunoblot. Protein expression was significantly correlated with total (AKAP4 R2 = 0.38, P ≤ 0.01; HK1 R2 = 0.46, P ≤ 0.001) and progressive motilities (AKAP4 R 2 = 0.51, P ≤ 0.001; HK1 R2 = 0.55, P ≤ 0.01), percentage rapid (AKAP4 R2 = 0.29, P ≤ 0.05; HK1 R2 = 0.58, P ≤ 0.001), straight-line velocity (HK1 R2 = 0.50, P ≤ 0.01) and straightness (HK1 R2 = 0.40, P ≤ 0.01). Furthermore, AKAP4 was highly susceptible to adduction by 4-hydroxynonenal (4HNE), which resulted in a global reduction in the phosphorylation profiles following capacitation. In conclusion, the proteomic profiles of high- and low-quality stallion spermatozoa differ substantially, and proteins such as AKAP4 and HK1 could serve as biomarkers of ejaculate quality.
Publication Date: 2020-08-18 PubMed ID: 32805711DOI: 10.1530/REP-20-0284Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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The research paper examines the differences between high-quality and low-quality horse sperm at the protein level, in order to identify potential markers of fertility. The study uses mass spectrometry to compare the presence and abundance of proteins, finding that proteins AKAP4 and HK1 could serve as potential indicators of sperm quality.
Research Background
- This research addresses the need for improved sperm assessments in the horse breeding industry with a focus on identifying the potential of certain proteins as fertility biomarkers.
- Traditional methods provide limited details on ejaculate quality and are not individually predictive of a stallion’s fertilizing potential.
Research Methods
- The researchers used Percoll density gradients to separate high-quality and low-quality sperm populations from stallion semen samples (n = 12).
- The team then performed motility and morphological assessments on these separated populations, noting that high-quality spermatozoa showed improved metrics in these areas.
- Using UHPLC-MS/MS, a technique involving ultra-high-performance liquid chromatography and tandem mass spectrometry, the team conducted a proteomic analysis.
Research Findings
- From the proteomic analysis, the team identified a total of 1069 proteins, 22 of which were significantly more abundant in the high-quality sperm.
- Among these 22 proteins, A-kinase anchor protein 4 (AKAP4) and Hexokinase 1 (HK1) were considered plausible biomarker candidates based on their relative abundance.
- These findings were validated through another technique called immunoblotting which confirmed their differential expression in the two sperm populations.
Research Implications
- The study correlated the presence of these proteins with essential parameters for sperm quality including total and progressive motilities, straight-line velocity, and straightness.
- Furthermore, they found AKAP4 to be highly susceptible to 4-hydroxynonenal (4HNE), an oxidative stress marker, suggesting it might play a critical role in sperm function.
- In conclusion, the study disproved the notion that sperm quality on a proteomic level was homogeneous, revealing substantial differences between high- and low-quality spermatozoa populations.
- Given these findings, proteins AKAP4 and HK1 could potentially serve as valuable markers for stallion ejaculate quality in the horse breeding industry.
Cite This Article
APA
Griffin RA, Swegen A, Baker M, Aitken RJ, Skerrett-Byrne DA, Silva Rodriguez A, Martín-Cano FE, Nixon B, Peña FJ, Delehedde M, Sergeant N, Gibb Z.
(2020).
Mass spectrometry reveals distinct proteomic profiles in high- and low-quality stallion spermatozoa.
Reproduction, 160(5), 695-707.
https://doi.org/10.1530/REP-20-0284 Publication
Researcher Affiliations
- Priority Research Centre for Reproductive Science, University of Newcastle, New South Wales, Australia.
- Priority Research Centre for Reproductive Science, University of Newcastle, New South Wales, Australia.
- Nuffield Department of Women's and Reproductive Health, University of Oxford, Oxford, UK.
- Priority Research Centre for Reproductive Science, University of Newcastle, New South Wales, Australia.
- Priority Research Centre for Reproductive Science, University of Newcastle, New South Wales, Australia.
- Priority Research Centre for Reproductive Science, University of Newcastle, New South Wales, Australia.
- Facility of Innovation and Analysis in Animal Source Foodstuffs, University of Extremadura, Cáceres, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
- Priority Research Centre for Reproductive Science, University of Newcastle, New South Wales, Australia.
- Pregnancy and Reproduction Program, Hunter Medical Research Institute, New South Wales, Australia.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
- SPQI - 4BioDx-Breeding Section, Lille, France.
- SPQI - 4BioDx-Breeding Section, Lille, France.
- INSERM UMRS1172, CHU-Lille, LilNCog Research Center, University of Lille, Lille, France.
- Priority Research Centre for Reproductive Science, University of Newcastle, New South Wales, Australia.
MeSH Terms
- Animals
- Horses
- Male
- Proteome / analysis
- Proteome / metabolism
- Semen Analysis / veterinary
- Sperm Motility
- Spermatozoa / metabolism
- Spermatozoa / physiology
- Tandem Mass Spectrometry / methods
Citations
This article has been cited 11 times.- Harris IT, Maddock C, Farnworth M, Nankervis K, Perrett J, Pyatt AZ, Blanchard RN. Temporal trends in equine sperm progressive motility: a systematic review and meta-regression. Reproduction 2023 Jun 1;165(6):M1-M10.
- Dordas-Perpinyà M, Yanez-Ortiz I, Sergeant N, Mevel V, Bruyas JF, Catalán J, Delehedde M, Briand-Amirat L, Miró J. ProAKAP4 Concentration Is Related to Sperm Motility and Motile Sperm Subpopulations in Frozen-Thawed Horse Semen. Animals (Basel) 2022 Dec 4;12(23).
- Perera TRW, Skerrett-Byrne DA, Gibb Z, Nixon B, Swegen A. The Future of Biomarkers in Veterinary Medicine: Emerging Approaches and Associated Challenges. Animals (Basel) 2022 Aug 26;12(17).
- Klein EK, Swegen A, Gunn AJ, Stephen CP, Aitken RJ, Gibb Z. The future of assessing bull fertility: Can the 'omics fields identify usable biomarkers?†. Biol Reprod 2022 May 17;106(5):854-864.
- Perrett J, Harris IT, Maddock C, Farnworth M, Pyatt AZ, Sumner RN. Systematic Analysis of Breed, Methodological, and Geographical Impact on Equine Sperm Progressive Motility. Animals (Basel) 2021 Oct 29;11(11).
- Kumaresan A, Elango K, Datta TK, Morrell JM. Cellular and Molecular Insights Into the Etiology of Subfertility/Infertility in Crossbred Bulls (Bos taurus × Bos indicus): A Review. Front Cell Dev Biol 2021;9:696637.
- Peña FJ, Martín-Cano FE, Becerro-Rey L, da Silva-Álvarez E, Gaitskell-Phillips G, Aparicio IM, Gil MC, Ortega-Ferrusola C. Redox Regulation and Glucose Metabolism in the Stallion Spermatozoa. Antioxidants (Basel) 2025 Feb 17;14(2).
- Liu S, Wang T, Liu Y, Wang S, Li F, Chen J, Hu X, Zhang M, Wang J, Li Y, James A, Hou R, Cai K. Dataset of Panda sperm proteome. Data Brief 2024 Dec;57:111052.
- Ren H, Wen X, He Q, Yi M, Dugarjaviin M, Bou G. Comparative Study on the Sperm Proteomes of Horses and Donkeys. Animals (Basel) 2024 Jul 31;14(15).
- Hernández-Avilés C, Ramírez-Agámez L, Weintraub ST, Scoggin CF, Davis BW, Raudsepp T, Varner DD, Love CC. Proteomic analysis of sperm from fertile stallions and subfertile stallions due to impaired acrosomal exocytosis. Sci Rep 2024 May 30;14(1):12446.
- Dordas-Perpinyà M, Yánez-Ortiz I, Sergeant N, Mevel V, Catalán J, Bruyas JF, Miró J, Briand-Amirat L. ProAKAP4 as Indicator of Long-Lasting Motility Marker in Post-Thaw Conditions in Stallions. Animals (Basel) 2024 Apr 23;14(9).
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