Mass spectrometry reveals distinct proteomic profiles in high- and low-quality stallion spermatozoa

in Reproduction
Authors:
Róisín A Griffin Priority Research Centre for Reproductive Science, University of Newcastle, New South Wales, Australia

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Aleona Swegen 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

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Mark Baker Priority Research Centre for Reproductive Science, University of Newcastle, New South Wales, Australia

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Robert John Aitken Priority Research Centre for Reproductive Science, University of Newcastle, New South Wales, Australia

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David A Skerrett-Byrne Priority Research Centre for Reproductive Science, University of Newcastle, New South Wales, Australia

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Antonio Silva Rodriguez Facility of Innovation and Analysis in Animal Source Foodstuffs, University of Extremadura, Cáceres, Spain

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Francisco E Martín-Cano Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain

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Brett Nixon 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

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Fernando J Peña Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain

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Maryse Delehedde SPQI – 4BioDx-Breeding Section, Lille, France

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Nicolas Sergeant SPQI – 4BioDx-Breeding Section, Lille, France
INSERM UMRS1172, CHU-Lille, LilNCog Research Center, University of Lille, Lille, France

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Zamira Gibb Priority Research Centre for Reproductive Science, University of Newcastle, New South Wales, Australia

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Correspondence should be addressed to R A Griffin; Email: roisin.griffin@uon.edu.au
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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.

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