DUGi

Role of non-enzymatic acetylation and NHE1 channel in sperm acrosome reaction

http://dugi.udg.edu/item/http:@@@@hdl.handle.net@@10256@@26335

With increasing interest in therapies targeting infertility, many research avenues are being probed to provide both greater understanding and feasible remedies. One of these specific avenues of interest being explored is a subset of ion channels implicated in intracellular homeostasis and pH regulation, the socalled Na+/H+ exchangers, or NHEs, specifically in this study, the isoform NHE1. While the main task of the NHE1 channel is suggested to be pH modulation, recent interest has peaked in its hypothesised role in protein acetylation, particularly non-enzymatic acetylation as directly depends on pH. Both of these NHE1-regulated phenomena have been presumed to be important factors directing pre-fecundation steps such as sperm capacitation and acrosome reaction (AR). As such, this study aims to first investigate the impact of acrosomal pH levels on non-enzymatic acetylation, specifically during the acrosomal reaction. Furthermore, by building on a recent understanding of NHE1 channel relevancy, this study also aims to determine the effects of NHE1 channel inhibition on acrosomal pH and the sperm ability to undergo AR. In this present study, we analysed non-enzymatic acetylation and NHE1 inhibition using pig as a model. Pig sperm samples underwent both capacitation and AR in vitro by incubating sperm in capacitating media and in presence of progesterone, that is an inductor of AR. Then, the effects of pH on non-enzymatic acetylation and the relevancy of NHE1 inhibition on acrosomal pH and AR during in-vitro capacitation were examined using an acetylase inhibitor cocktail and a specific NHE1 channel blocker, respectively. Non-enzymatic acetylation was stimulated incubating sperm with an acetylase inhibitor cocktail under alkaline pH (9). NHE1 was inhibited with 5-NN-dimethyl amiloride hydrochloride. Both non-enzimatic acetylation and NHE1 activity were probe with an increase in the acetylation level and pH modificatios, respectively. Samples were then analysed for acrosomal integrity, intracellular pH, and acrosomal pH by flow cytometry. Additionally, protein acetylation was evaluated via immunochemical analysis. Our results showed that acetylation plays an important role during sperm capacitation and AR, with acetylation increases dependent on acetylase action. Moreover, AR completion was found to be highly dependent on correct acetylase performance. Additionally, while the induction of non-enzymatic acetylation can provoke increases in protein acetylation, it is not capable of reestablishing baseline levels of AR attainment. Similarly, the importance of acrosomal pH was also highlighted, showcasing its essential role in correct AR completion. Finally, while NHE1 was not found to be involved in the sperm’s ability to undergo capacitation, our findings indicate that the NHE1 channel plays a specific role in controlling sperm protein acetylation through the modulation of cytoplasmic pH

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