Abstract
Bicarbonate uptake is one of the early steps of capacitation, but the identification of proteins regulating anion fluxes remains elusive. The aim of this study is to investigate the role of sperm solute carrier 4 (SLC4) A1 (spAE1) in the capacitation process. The expression, location, and tyrosine-phosphorylation (Tyr-P) level of spAE1 were assessed. Thereby, it was found that 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS), an SLC4 family channel blocker, inhibited capacitation in a dose-dependent manner by decreasing acrosome reaction (ARC% 24.5 ± 3.3 vs. 64.9 ± 4.3, p < 0.05) and increasing the percentage of not viable cells (NVC%), comparable to the inhibition by I-172, a cystic fibrosis transmembrane conductance regulator (CFTR) blocker (AR% 30.5 ± 4.4 and NVC% 18.6 ± 2.2). When used in combination, a synergistic inhibitory effect was observed with a remarkable increase of the percentage of NVC (45.3 ± 4.1, p < 0.001). spAE1 was identified in sperm membrane as a substrate for Tyr-protein kinases Lyn and Syk, which were identified as both soluble and membrane-bound pools. spAE1-Tyr-P level increased in the apical region of sperm under capacitating conditions and was negatively affected by I-172 or DIDS, and, to a far greater extent, by a combination of both. In conclusion, we demonstrated that spAE1 is expressed in sperm membranes and it is phosphorylated by Syk, but above all by Lyn on Tyr359, which are involved in sperm viability and capacitation.
Highlights
Capacitation, a critical step in the maturation of sperm is characterized by a series of biochemical and physiological changes both in the head and in the tail of sperm to gain the ability to fertilize oocyte. a few controversies over as yet unknown mechanisms remain to be addressed, the increase in intracellular bicarbonate (HCO3−) is widely accepted as one of the first and essential events taking place during capacitation [1].HCO3− uptake induces the activation of the atypical soluble adenylyl cyclase (ADCY10) which, in turn, leads to cAMP synthesis and the activation of protein kinase A (PKA) [2]
To study the involvement of solute carrier 4 (SLC4) family channels in sperm capacitation and functioning, we compared the effects of 4,4 -diisothiocyano-2,2 -stilbenedisulfonic acid (DIDS), a bifunctional inhibitor of the SLC4 anion exchanger family containing two isothiocyano groups known to alkylate lysine residues of eAE1 [25,26] to those of Inh-172 (I-172), a CFTR blocker [10,13]
All of the SLC4 family members AE1-3 are sensitive to DIDS, which is due to the sequence homology (53–56%) that these latter channels share [25], 70–100 μM DIDS is sufficient to block AE1 [26,27] as compared to the concentrations five [27,28] to 10 times higher [29] required to inhibit the other members of this channel family
Summary
HCO3− uptake induces the activation of the atypical soluble adenylyl cyclase (ADCY10) which, in turn, leads to cAMP synthesis and the activation of protein kinase A (PKA) [2]. This signaling pathway initiates downstream events such as plasma membrane hyperpolarization and cytoplasm alkalinization [1,3]. The membrane HCO3− transporters- are traditionally classed into two main protein families, solute carrier 26 (SLC26), acting as anion exchangers, and solute carrier 4 (SLC4), acting as either exchangers or cotransporters [4,5], to which the cystic fibrosis transmembrane conductance regulator (CFTR) can be added, this latter functioning as an ATP-gated anion channel [6]. CFTR, SLC26 with its members SLC26A3, SLC26A6, and SLC26A8, as well as SLC4, represented by the member SLC4A1, have been demonstrated to be expressed in mature spermatozoa [1,7,8,9,10,11]
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