Human spermatozoa possess a calcium‐dependent chloride channel that may participate in the acrosomal reaction

Abstract

Key points• Ion channels participate in crucial sperm functions such as motility, capacitation and the acrosome reaction.• Chloride, the main anion in physiological solutions, is deeply involved in sperm physiology.• We implemented a modified perforated patch-clamp strategy to obtain whole cell recordings sealing on the head of mature human spermatozoa to investigate their ion channels.• This work presents the first evidence for the presence of calcium-dependent chloride channels (CaCCs) in human spermatozoa; they could be constituted by TMEM16.• The CaCCs play an important role in the physiology of human spermatozoa and participate in the acrosome reaction.Abstract Motility, maturation and the acrosome reaction (AR) are fundamental functions of mammalian spermatozoa. While travelling through the female reproductive tract, spermatozoa must mature through a process named capacitation, so that they can reach the egg and undergo the AR, an exocytotic event necessary to fertilize the egg. Though Cl − is important for sperm capacitation and for the AR, not much is known about the molecular identity of the Cl − transporters involved in these processes. We implemented a modified perforated patch-clamp strategy to obtain whole cell recordings sealing on the head of mature human spermatozoa. Our whole cell recordings revealed the presence of a Ca 2+ -dependent Cl − current. The biophysical characteristics of this current and its sensitivity to niflumic acid (NFA) and 4,4 -diisothiocyano-2,2 -stilbene disulphonic acid (DIDIS) are consistent with those displayed by the Ca 2+ -dependent Cl − channel from the anoctamin family (TMEM16). Whole cell patch clamp recordings in the cytoplasmic droplet of human spermatozoa corroborated the presence of these currents, which were sensitive to NFA and to a small molecule TMEM16A inhibitor (TMEM16A inh , an aminophenylthiazole). Importantly, the human sperm AR induced by a recombinant human glycoprotein from the zona pellucida, rhZP3, displayed a similar sensitivity to NFA, DIDS and TMEM16A inh as the sperm Ca 2+ -dependent Cl − currents. Abbreviations AR, acrosome reaction; CaCC, calcium-dependent chloride channel; cAMP, adenosine 3 ,5 -cyclic monophosphate; DIDS, 4,4 -diisothiocyano-2,2 -stilbene disulphonic acid; NFA, niflumic acid; rhZP3, recombinant human glycoprotein from the zona pellucida number 3; TMEM16A, transmembrane protein 16A, also called anoctamin-1; TMEM16A inh , a small molecule TMEM16A inhibitor, ZP, zona pellucida.

Flagellar ion channels of sperm: similarities and differences between species

“…Human spermatozoa, however, do not show the same I ATP which indicates that P2X2 ion channels might not be expressed in human sperm cells ( [153] and unpublished data from our lab). Calciumactivated chloride channels (anoctamins) have been recently found in human sperm [154], but not in mouse [30], as well as aquaporins, water channels that are required for sperm osmoadaptation [155]. Several members of the transient receptor potential (TRP) ion channel family [156][157][158] were thought to function in mammalian sperm cells.…”

Role of Ion Channels in the Sperm Acrosome Reaction

“…Thereafter, it was reported that other anion channel blockers such as 1,9-dideoxyforskolin (DDF) potently inhibited the AR . Ca 2+ -dependent whole cell Cl À currents were recorded by Orta et al (2012) patch clamping human sperm in the head. The authors proposed that TMEM16A may contribute to these currents and also that sperm CaCCs may participate in the rhZP3-induced AR.…”

Cl− Channels and Transporters in Sperm Physiology

“…NFA also partially inhibited a Ca 2+ -induced hyperpolarization partially driven by Cl − in mouse spermatozoa (Espinosa et al 1998 ). Anion channel blockers such as NFA, for example, DIDS, also inhibit the mouse and human sperm AR, as well as Cl − channels detected in these cells (Espinosa and Darszon 1995 ;Espinosa et al 1998 ;Figueiras-Fierro et al 2013 ;Orta et al 2012 ).…”

“…In spermatozoa, as in other cells, Cl − is the main anion and is involved in volume regulation and osmotic stress protection (Cooper and Yeung 2007 ;Furst et al 2002 ;Yeung et al 2005 ). Capacitation, a maturational process and the AR, a unique exocytotic event essential for fertilization, both discussed next, are signifi cantly affected in mouse and human spermatozoa when external Cl − concentrations are lowered Figueiras-Fierro et al 2013 ;Orta et al 2012 ;Wertheimer et al 2008 ;Yeung and Cooper 2008 ). Although these fi ndings suggest that Cl − plays a relevant role in sperm physiology, not much is known about its transport across the membrane of this fundamental cell.…”