RESUMO
Introduction: Mammalian sperm motility is facilitated by flagellar beating, which depends on active ion movement through ion channels and their regulation. Prunus japonica Thunb., also known as oriental bush cherry, is a widely used traditional medicinal plant. However, its significance in improving fertility and sperm quality has not been fully elucidated yet. One of our previous reports revealed that P. japonica seed extract (PJE) can improve human sperm motility through intracellular pH modulation. Aim of the study: The present study was designed to investigate the effects of PJE on boar spermatozoa and potential underlying mechanisms. Materials and methods: Sperm motility changes were examined using a computer-assisted sperm analysis (CASA) system under both capacitated and non-capacitated conditions. Intracellular calcium concentration was measured using either confocal microscopy or a fluorescent microplate reader with Fluo-4AM calcium fluorescent dye. Sperm capacitation-related proteins were analyzed using western blotting. Results: A significant increase in rapid motility, velocity, and linear displacement of sperm was observed in PJE-treated capacitated boar sperm, whereas the effect was insignificant in the non-capacitated counterparts. Intracellular calcium levels were significantly elevated upon PJE treatment (20-100 µg/L) in a concentration-dependent manner. The increase in intracellular calcium levels was inhibited when the sperm were treated with a CatSper (cation channel of sperm) channel inhibitor, 10 µM Mibefradil, indicating the involvement of the ion channel in the PJE modulatory mechanism. In addition, western blotting revealed an increased level of protein phosphorylation (p-tyrosine and p-PKA), which is a hallmark of sperm capacitation. Conclusions: PJE treatment resulted in a combination of increased motility, intracellular calcium concentration, and capacitation, thereby indicating its potential to ameliorate sperm motility parameters and induce capacitation of boar spermatozoa as a result of intracellular calcium elevation via the CatSper channel. Our observations further elaborate ion channel-related underlying mechanisms and show putative implications of the seed extract of traditionally used P. japonica Thunb. in ameliorating sperm quality.
RESUMO
Prunus japonica var. nakaii is used in traditional Korean medicine to treat various conditions; however, it has not been investigated for treating male infertility. In this study, we investigated the in vitro effects of the ethanolic extract of P. japonica seeds on human sperm motility and identified its mechanism of action. Eleven male volunteers were selected, and the effects of the extract on human spermatozoa were assessed through a computer-assisted semen analysis. The P. japonica seed extract increased the percentage of total and progressive motility of spermatozoa. To understand the mechanism of action, we monitored intracellular alkalization using flow cytometry and obtained electrophysiological recordings of human voltage-gated proton channels hHv1 that were overexpressed in HEK-293 cells. The extract shifted the activation curves in a concentration-dependent manner. Two major constituents of the extract, linoleic acid and oleic acid, exhibited proton channel activity. Our in vitro experiments suggested that P. japonica seed extract could be potentially used to rescue sperm motility in idiopathic infertility patients via pharmacological modulation of the proton channels during capacitation. Therefore, our results indicate the therapeutic potential of P. japonica seed extract for treating male infertility.