The inhibition of spermatic cystine/glutamate antiporter xCT (SLC7A11) influences the ability of cryopreserved stallion sperm to bind to heterologous zonae pellucidae.
Abstract: Sperm are redox-regulated cells, and deregulation of their redox status is considered to affect male fertility and to reduce their fertilizing ability following biotechnological procedures, such as cryopreservation. Cystine (CysS), after incorporation in sperm via SLC7A11 antiporter, has been demonstrated to increase intracellular GSH content, the most important non enzymatic antioxidant. This study was aimed at investigating the role of SLC7A11 antiporter on frozen-thawed stallion sperm ability to respond to in vitro capacitating environment after post-thaw incubation with CysS and/or Sulfasalazine (SS), a specific inhibitor of SLC7A11 antiporter. Viability, motility, immunolocalization of tyrosine phosphorylated proteins and the ability to bind to heterologous zonae pellucidae were evaluated. Thawed sperm from seven stallions (2 ejaculates/stallion) was washed and resuspended in Tyrodes media; each thawed ejaculate was divided in Control (CTR) and 3 samples supplemented with: 0.5 mM Cystine (CysS), 500 μM Sulfasalazine (SS) and 0.5 mM CysS + 500 μM SS (CysS + SS). After 1 h of incubation at 37 °C, samples were washed twice, resuspended in capacitating BWW medium and incubated at 38 °C under 5% CO. After 30 and 60 min, sperm motility, viability and tyrosine phosphorylated protein immunolocalization, used as capacitation status index, were evaluated. After 30 min of capacitation, 4 × 10 sperm were co-incubated with denuded pig oocytes in capacitation medium for 30 min for the heterologous binding assay. None of the sperm parameters studied (motility, viability and tyrosine phosphorylation) showed any difference respective to control. The number of sperm bound per oocyte (mean ± SEM) tended to increase in CysS group (44.0 ± 12.3) respect CTR (40.8 ± 10.8) while decreased in SS group (32.4 ± 7.8) (p < 0.01). Moreover, CysS + SS group showed a lower binding rate (32.0 ± 10.0) compared to CysS (p < 0.001). Our results suggest that CysS supplementation of thawed stallion sperm can influence their ability to bind to heterologous zona pellucidae as the inhibition of CysS incorporation by SLC7A11 reduced the number of sperm bound per oocyte. This effect does not seem to be ascribed to a modification of sperm motility, membrane integrity and tyrosine phosphorylation.
Copyright © 2021 Elsevier Inc. All rights reserved.
Publication Date: 2021-03-09 PubMed ID: 33743505DOI: 10.1016/j.theriogenology.2021.03.002Google Scholar: Lookup
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- Journal Article
Summary
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This research investigates the effect of a specific antiporter (SLC7A11) on the capability of frozen-thawed stallion sperm to respond to changes in a lab environment. It found that the uptake of Cystine (a compound gained via the SLC7A11 antiporter) can influence sperm’s ability to bind to a specific part of the egg, the zona pellucida. Sperm motility, membrane integrity, and other physiological features do not seem to change due to this process.
Concepts and Methods
- The study revolves around the understanding of an antiporter named SLC7A11. Antiporters are a type of protein that help transport certain substances across cell membranes.
- Cystine, a non-enzymatic antioxidant, plays a major role in the study. It is incorporated into the sperm via the SLC7A11 antiporter and has been shown to increase the content of Glutathione (GSH), another antioxidant, inside the cell.
- Sulfasalazine (SS) is introduced as a specific inhibitor of the SLC7A11 antiporter. It is used to understand the influence of the lack of Cystine within the sperm.
- The research inspected the ability of previously frozen-thawed stallion sperm to respond to a lab-created environment conducive for sperm activation (capacitation). Properties such as viability, motility, and patterns of phosphorylation were evaluated.
Results and Observations
- The research consisted of multiple experiments involving the sperm samples from seven stallions.
- Each sample was divided into a control group and three other groups – one supplemented with Cystine, one with Sulfasalazine, and a third with both compounds.
- None of the parameters scrutinized, including motility and viability, showed any remarkable difference among the different groups of sperm.
- However, the number of sperm capable of binding to the zona pellucida of a pig’s oocyte (egg) is observed to increase in the Cystine group, compared to the control, while decreasing in the Sulfasalazine group.
- Samples treated with both Cystine and Sulfasalazine showed a lower sperm binding rate compared with the Cystine alone group.
Conclusions
- The data suggest that the uptake of Cystine might influence the ability of stallion sperm (that have been previously frozen and then thawed) to bind to heterologous zonae pellucidae.
- Interestingly, inhibiting this uptake with Sulfasalazine appears to reduce the number of sperm binding per oocyte, indicating the influence of the SLC7A11 antiporter in this process.
- The effect doesn’t seem to result from changes in motility, membrane integrity, or the phosphorylation status of the sperm.
Cite This Article
APA
Ortiz-Rodriguez JM, Nerozzi C, Bucci D, Mislei B, Mari G, Tamanini C, Peña FJ, Spinaci M, Galeati G.
(2021).
The inhibition of spermatic cystine/glutamate antiporter xCT (SLC7A11) influences the ability of cryopreserved stallion sperm to bind to heterologous zonae pellucidae.
Theriogenology, 167, 24-31.
https://doi.org/10.1016/j.theriogenology.2021.03.002 Publication
Researcher Affiliations
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Caceres, Spain.
- Department of Veterinary Medical Sciences (DIMEVET), University of Bologna, Bologna, Italy.
- Department of Veterinary Medical Sciences (DIMEVET), University of Bologna, Bologna, Italy.
- National Institute of Artificial Insemination (AUB-INFA), University of Bologna, Bologna, Italy.
- Department of Veterinary Medical Sciences (DIMEVET), University of Bologna, Bologna, Italy; National Institute of Artificial Insemination (AUB-INFA), University of Bologna, Bologna, Italy.
- Department of Veterinary Medical Sciences (DIMEVET), University of Bologna, Bologna, Italy.
- Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Caceres, Spain.
- Department of Veterinary Medical Sciences (DIMEVET), University of Bologna, Bologna, Italy. Electronic address: marcella.spinaci@unibo.it.
- Department of Veterinary Medical Sciences (DIMEVET), University of Bologna, Bologna, Italy.
MeSH Terms
- Amino Acid Transport System y+ / antagonists & inhibitors
- Animals
- Antiporters
- Cryopreservation / veterinary
- Cystine / metabolism
- Glutamic Acid
- Horses
- Male
- Semen Preservation / veterinary
- Sperm Motility
- Spermatozoa / metabolism
- Swine
Conflict of Interest Statement
Declaration of competing interest None of the authors have conflict of interest to declare.
Citations
This article has been cited 3 times.- Bucci D, Spinaci M, Bustamante-Filho IC, Nesci S. The sperm mitochondria: clues and challenges.. Anim Reprod 2022;19(4):e20220131.
- Su Y, Liu Z, Xie K, Ren Y, Li C, Chen W. Ferroptosis: A Novel Type of Cell Death in Male Reproduction.. Genes (Basel) 2022 Dec 23;14(1).
- Wang Y, Liu T, Li X, Sheng H, Ma X, Hao L. Ferroptosis-Inducing Nanomedicine for Cancer Therapy.. Front Pharmacol 2021;12:735965.
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