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

Abstract

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.

Melatonin affects the motility and adhesiveness of in vitro capacitated boar spermatozoa via a mechanism that does not depend on intracellular ROS levels

“…This feature is different from other species, such as the horse, in which ROS production is more intense (see Gibb & Aitken, 2016; as a review). In fact, the antioxidant effect of melatonin in species such as the horse is not only related to a direct action on the oxidative potential but also to intracellular Na + concentrations, which also affect the overall redox status (Ortega Ferrusola et al, 2017). In fact, the antioxidant effect of melatonin in species such as the horse is not only related to a direct action on the oxidative potential but also to intracellular Na + concentrations, which also affect the overall redox status (Ortega Ferrusola et al, 2017).…”

“…As a consequence, in species such as equine, bovine and human, melatonin has a clear antioxidant effect and affects membrane lipid peroxidation and ROS levels (Gadella et al, 2008;Rao & Gangadharan, 2008;Du Plessis et al, 2010;Jang et al, 2010;Da Silva et al, 2011;Najafi et al, 2018). In fact, the antioxidant effect of melatonin in species such as the horse is not only related to a direct action on the oxidative potential but also to intracellular Na + concentrations, which also affect the overall redox status (Ortega Ferrusola et al, 2017). Taking all of these data into consideration, an expected antioxidant effect of melatonin on boar spermatozoa should be very subtle, if detectable.…”

Flow cytometry analysis of spermatozoa: Is it time for flow spermetry?

“…The use of flow cytometry allows determining, at the single cell level, many different characteristics of the spermatozoa. Recently, changes in the potential of the membrane have been studied in relation to capacitation and cryopreservation (Escoffier et al., ; Ortega Ferrusola et al., ). These changes relate to modifications in intracellular Na+ that also can be monitored using flow cytometry.…”

Rosiglitazone in the thawing medium improves mitochondrial function in stallion spermatozoa through Akt phosphorylation and reduction of caspase 3

“…These facts have important implications for the selection of more fertile spermatozoa [37] and sperm conservation [17, 48–50]. The thawed spermatozoa are characterized by compromised mitochondrial function [8, 14] and an unstable redox status, leading rapidly to oxidative stress [1, 14, 51]. We aimed to induce metabolic flexibility to improve the functionality of thawed spermatozoa, a strategy that has proven successful in the conservation in stallion spermatozoa in refrigeration [21].…”

“…In brief, osmotic shock induces membrane and mitochondrial damage [11], the mitochondrial damage causes impairment of redox regulation, leading to lipid, protein and DNA modifications in the spermatozoa, resulting in decreased motility and viability [8, 12, 13]. Other changes recently described in relation to cryopreservation include increased intracellular Na+ and membrane depolarization due to the compromised functionality of the Na+-K+ATPase pump [14]. One key protein related to sperm survival is the Akt or protein kinase B.…”