Transient receptor potential channel 4 (TRPC4) in boar sperm: Immunolocalization and functional analysis using the specific inhibitor ML204

Abstract

The TRPC4 channel is related to the movement of calcium within cells, and therefore possibly to sperm motility, capacitation and the acrosome reaction. This study investigated the presence and localization of TRPC4 channels in frozen-thawed boar sperm by direct immunofluorescence. In addition, different concentrations of the specific inhibitor ML204 were tested to evaluate its effect on post-thaw sperm motility and multiple sperm quality parameters assessed by flow cytometry, under conditions that promote motility recovery, such as caffeine supplementation or incubation in a basic extender. Our results confirmed that the presence of TRPC4 in boar sperm is independent of the use of ML204 and located in the midpiece and post-acrosomal regions. At the highest concentrations (22 µM), channel inhibition with ML204 caused a significant reduction in total and progressive motility, as well as in mitochondrial membrane potential, without a clear detrimental effect on the measured cytometry parameters. The loss of motility was exacerbated under co-incubation conditions with caffeine (2 mM), possibly due to an imbalance between calcium influx and demand via the cAMP/PKA pathway activated by this methylxanthine. Our results, however, when the thawing medium had a basic pH (8.2), motility was comparable to the control, likely because other pH-dependent channels compensated for the reduced calcium and cation influx. In conclusion, the TRPC4 channel is present in porcine sperm and appears to play an important role in regulating motility and maintaining mitochondrial membrane potential