The inhibition of spermatic cystine/glutamate antiporter xCT (SLC7A11) influences the ability of cryopreserved stallion sperm to bind to heterologous zonae pellucidae.

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% CO2. 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 × 105 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.

Stallion spermatozoa surviving freezing and thawing experience membrane depolarization and increased intracellular Na.

In order to gain insight of the modifications that freezing and thawing cause to the surviving population of spermatozoa, changes in the potential of the plasma membrane (Em) and intracellular Na+ content of stallion spermatozoa were investigated using flow cytometry. Moreover, caspase 3 activity was also investigated and the functionality of the Na+ -K+ ATPase pump was investigated before and after freezing and thawing. Cryopreservation caused a significant (p < 0.001) increase in the subpopulation of spermatozoa with depolarized sperm membranes, concomitantly with an increase (p < 0.05) in intracellular Na+ . These changes occurred in relation to activation of caspase 3 (p < 0.001). Cryopreservation reduced the activity of the Na-K+ pump and inhibition of the Na+ -K+ ATPase pump with ouabain-induced caspase 3 activation. It is concluded that inactivation of Na+ -K+ ATPase occurs during cryopreservation, an inhibition that could play a role explaining the accelerated senescence of the surviving population of spermatozoa.