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Depletion of thiols leads to redox deregulation, production of 4-hydroxinonenal and sperm senescence: a possible role for GSH regulation in spermatozoa†.
Abstract: We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation of stallion sperm functionality. To test this hypothesis, we initially investigated the relationship between sperm function and GSH content, revealing highly significant correlations between GSH, sperm viability, motility, and velocity parameters (P < 0.001). Furthermore, the deleterious effects of GSH depletion using menadione and 1,3 dimethoxy 1,4, naphtoquinone (DMNQ) were able to be prevented by the addition of cysteine, but no other antioxidant. Pre-incubation with cysteine prevented menadione and DMNQ induced damage to sperm membranes after 1 h (P < 0.001; P < 0.05) and after 3 h of incubation (P < 0.001, P < 0.05). Pre-incubation with cysteine ameliorated both the menadione- and DMNQ-induced increase in 4-hydroxynonenal (P < 0.001). As cysteine is a precursor of GSH, we hypothesized that stallion spermatozoa are able to synthesize this tripeptide using exogenous cysteine. To test this hypothesis, we investigated the presence of two enzymes required to synthesize GSH (GSH and GCLC) and using western blotting and immunocytochemistry we detected both enzymes in stallion spermatozoa. The inhibition of GCLC reduced the recovery of GSH by addition of cysteine after depletion, suggesting that stallion spermatozoa may use exogenous cysteine to regulate GSH. Other findings supporting this hypothesis were changes in sperm functionality after BSO treatment and changes in GSH and GSSG validated using HPLC-MS, showing that BSO prevented the increase in GSH in the presence of cysteine, although important stallion to stallion variability occurred and suggested differences in expression of glutamate cysteine ligase. Mean concentration of GSH in stallion spermatozoa was 8.2 ± 2.1 μM/109 spermatozoa, well above the nanomolar ranges per billion spermatozoa reported for other mammals.
© The Author(s) 2018. Published by Oxford University Press on behalf of Society for the Study of Reproduction.
Publication Date: 2018-11-13 PubMed ID: 30418487DOI: 10.1093/biolre/ioy241Google 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
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
The research suggests that the compound glutathione (GSH) plays a crucial role in maintaining the functionality of stallion sperm. Experimental findings indicate a substantial correlation between GSH content, and sperm viability, motion, and speed. Alterations to GSH levels significantly disrupted sperm function, but this could be counteracted by cysteine, a predecessor to GSH. The study also demonstrated that stallion sperm could potentially create GSH from external cysteine sources.
Relationship of GSH and Sperm Function
- The researchers proposed that thiols, specifically glutathione (GSH), are vital for controlling the functionality of stallion sperm.
- They examined the correlation between GSH content and sperm function such as vitality, motility, and velocity parameters. The result disclosed a very notable correlation between GSH and these parameters.
Effects of Depleting GSH
- Depleting GSH using substances like menadione and 1,3 dimethoxy 1,4, naphtoquinone (DMNQ) had harmful effects on the sperm. However, these damaging results were preventable by adding cysteine, an antioxidant and precursor of GSH.
- Using cysteine prior to the induction of these substances prevented damage to sperm membranes after 1 and 3 hours of incubation. Furthermore, it alleviated the increase in 4-hydroxynonenal induced by menadione and DMNQ.
The Role of Cysteine and Enzymes in GSH Production
- The study hypothesized that stallion sperm could generate GSH using external cysteine sources. They investigated two critical enzymes for GSH synthesis, GSH and GCLC, and found evidence of both within stallion sperm.
- The inhibition of GCLC led to reduced recovery of GSH by the addition of cysteine after depletion, suggesting that stallion sperm could use external cysteine to control GSH.
Changes in Sperm Functionality
- Alterations in sperm function occurred after BSO treatment, along with variations in GSH and GSSG levels, confirming that BSO blocked the GSH increase in the presence of cysteine.
- However, there were significant differences between individual stallions, implying variations in the expression of glutamate cysteine ligase, an enzyme required in the production of GSH.
Concentration of GSH in Stallion Sperm
- The average concentration of GSH in stallion sperm was identified to be 8.2 ± 2.1 μM/109 spermatozoa, which is considerably higher than per billion spermatozoa measurements reported for other mammals, indicating a loftier level of GSH in stallion sperm.
Cite This Article
APA
Ortega-Ferrusola C, Martin Muñoz P, Ortiz-Rodriguez JM, Anel-López L, Balao da Silva C, Álvarez M, de Paz P, Tapia JA, Anel L, Silva-Rodríguez A, Aitken RJ, Gil MC, Gibb Z, Peña FJ.
(2018).
Depletion of thiols leads to redox deregulation, production of 4-hydroxinonenal and sperm senescence: a possible role for GSH regulation in spermatozoa†.
Biol Reprod, 100(4), 1090-1107.
https://doi.org/10.1093/biolre/ioy241 Publication
Researcher Affiliations
- Reproduction and Obstetrics Department of Animal Medicine and Surgery, University of León, 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.
- Reproduction and Obstetrics Department of Animal Medicine and Surgery, University of León, Spain.
- Escola Superior Agraria de Elvas, Elvas, Portugal.
- Reproduction and Obstetrics Department of Animal Medicine and Surgery, University of León, Spain.
- Reproduction and Obstetrics Department of Animal Medicine and Surgery, University of León, Spain.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
- Reproduction and Obstetrics Department of Animal Medicine and Surgery, University of León, Spain.
- Facility of Innovation and Analysis in Animal Source Foodstuffs, University of Extremadura, Cáceres, Spain.
- 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 Center in Reproductive Science, School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales, Australia.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
MeSH Terms
- Aldehydes / metabolism
- Aldehydes / pharmacology
- Animals
- Cellular Senescence / drug effects
- Glutathione / metabolism
- Glutathione / physiology
- Horses
- Lipid Peroxidation / drug effects
- Male
- Oxidation-Reduction
- Reactive Oxygen Species / metabolism
- Semen Analysis
- Semen Preservation
- Sperm Motility
- Spermatozoa / chemistry
- Spermatozoa / drug effects
- Spermatozoa / metabolism
- Spermatozoa / physiology
- Sulfhydryl Compounds / metabolism
Citations
This article has been cited 11 times.- Akhtar MF, Ma Q, Li Y, Chai W, Zhang Z, Li L, Wang C. Effect of Sperm Cryopreservation in Farm Animals Using Nanotechnology. Animals (Basel) 2022 Sep 2;12(17).
- Yánez-Ortiz I, Catalán J, Delgado-Bermúdez A, Carluccio A, Miró J, Yeste M. Addition of Reduced Glutathione (GSH) to Freezing Medium Reduces Intracellular ROS Levels in Donkey Sperm. Vet Sci 2021 Dec 2;8(12).
- Zhu Z, Zeng Y, Zeng W. Cysteine improves boar sperm quality via glutathione biosynthesis during the liquid storage. Anim Biosci 2022 Feb;35(2):166-176.
- Peña FJ, O'Flaherty C, Ortiz Rodríguez JM, Martín Cano FE, Gaitskell-Phillips GL, Gil MC, Ortega Ferrusola C. Redox Regulation and Oxidative Stress: The Particular Case of the Stallion Spermatozoa. Antioxidants (Basel) 2019 Nov 19;8(11).
- Ortiz-Rodriguez JM, Balao da Silva C, Masot J, Redondo E, Gazquez A, Tapia JA, Gil C, Ortega-Ferrusola C, Peña FJ. Rosiglitazone in the thawing medium improves mitochondrial function in stallion spermatozoa through regulating Akt phosphorylation and reduction of caspase 3. PLoS One 2019;14(7):e0211994.
- Zayed M, Abdelrazek M, Jeong BH. Ferroptosis in veterinary medicine: mechanisms, therapies, and unmet challenges. Vet Q 2025 Dec;45(1):2569558.
- Soria-Meneses PJ, Jurado-Campos A, Rubio de Juan A, Montoro Angulo V, Soler AJ, Garde JJ, Ramón Fernández M, Fernández-Santos MDR. Effects of long-term cryopreservation on sperm quality and oxidative stress in Manchega rams (Ovis aries). Reproduction 2025 Oct 1;170(4).
- Wang Z, Gu H, Zhu C, Wang Y, Liu H, Song W, Tao Z, Xu W, Zhang S, Li H. Optimization of Duck Semen Freezing Procedure and Regulation of Oxidative Stress. Animals (Basel) 2025 Aug 6;15(15).
- Onochie C, Evi K, O'Flaherty C. Role of Redox-Induced Protein Modifications in Spermatozoa in Health and Disease. Antioxidants (Basel) 2025 Jun 12;14(6).
- Becerro-Rey L, Martín-Cano FE, Silva-Rodríguez A, Ortega-Ferrusola C, da Silva-Álvarez E, Ortiz-Placín C, Tapia JA, Gil MC, Peña FJ. Stallion spermatozoa express LDH isoforms A, B, and C, with LDHC playing a crucial role in sustaining sperm viability. Reproduction 2025 Jul 1;170(1).
- 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).
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