Abstract
Little data exist about the physiological role of ion channels during the freeze–thaw process in mammalian sperm. Herein, we determined the relevance of potassium channels, including SLO1, and of voltage-gated proton channels (HVCN1) during mammalian sperm cryopreservation, using the pig as a model and through the addition of specific blockers (TEA: tetraethyl ammonium chloride, PAX: paxilline or 2-GBI: 2-guanidino benzimidazole) to the cryoprotective media at either 15 °C or 5 °C. Sperm quality of the control and blocked samples was performed at 30- and 240-min post-thaw, by assessing sperm motility and kinematics, plasma and acrosome membrane integrity, membrane lipid disorder, intracellular calcium levels, mitochondrial membrane potential, and intracellular O2−⁻ and H2O2 levels. General blockade of K+ channels by TEA and specific blockade of SLO1 channels by PAX did not result in alterations in sperm quality after thawing as compared to control samples. In contrast, HVCN1-blocking with 2-GBI led to a significant decrease in post-thaw sperm quality as compared to the control, despite intracellular O2−⁻ and H2O2 levels in 2-GBI blocked samples being lower than in the control and in TEA- and PAX-blocked samples. We can thus conclude that HVCN1 channels are related to mammalian sperm cryotolerance and have an essential role during cryopreservation. In contrast, potassium channels do not seem to play such an instrumental role.
Highlights
Ion channels are essential for sperm physiology during spermatogenesis, epididymal maturation, and storage, transit throughout the female reproductive tract and interaction with the oocyte [1]
In Experiment 1, sperm samples were cryopreserved in the presence of tetraethyl ammonium chloride (TEA), PAX, or 2-GBI blockers which were added to LEY medium at 15 ◦ C
Post-thaw sperm viability, expressed as the percentage of spermatozoa with an intact plasma membrane, did not differ significantly between the control and samples blocked with TEA and PAX in Experiment 1 (P > 0.05) and Experiment 2 (P > 0.05)
Summary
Ion channels are essential for sperm physiology during spermatogenesis, epididymal maturation, and storage, transit throughout the female reproductive tract and interaction with the oocyte [1]. Since the first identification of cation sperm channels (CatSper) in the plasma membrane of mouse spermatozoa in 2001 [2], several studies have investigated different ion channels and their relevance for sperm function (reviewed in [3]). These studies were usually performed in mouse and human sperm and focused on the physiological role of these ion channels during sperm capacitation. Cryopreservation is the most efficient method for long-term storage of sperm In humans, it is crucial for fertility preservation strategies [4,5], and in domestic animals it is frequently used as a technique to accelerate the rate of genetic improvement [6]. Great variability in sperm cryotolerance has been observed between breeds, between individuals of the same breed, and even between ejaculates from the same animal [7,10,15]
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