Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca2+ channel; Slo3, a K+ channel; the sperm-specific Na+/H+ exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction.

The spermatozoa acrosome reaction (AR) is essential for mammalian fertilization. Few methods allow visualization of AR in real time together with Ca²⁺ imaging. Here, we show that FM4-64, a fluorescent dye used to follow exocytosis, reliably reports AR progression induced by ionomycin and progesterone in human spermatozoa. FM4-64 clearly delimits the spermatozoa contour and reports morphological cell changes before, during, and after AR. This strategy unveiled the formation of moving tubular appendages, emerging from acrosome-reacted spermatozoa, which was confirmed by scanning electron microscopy. Alternate wavelength illumination allowed concomitant imaging of FM4-64 and Fluo-4, a Ca²⁺ indicator. These AR and intracellular Ca²⁺ ([Ca²⁺]i) recordings revealed that the presence of [Ca²⁺]i oscillations, both spontaneous and progesterone induced, prevents AR in human spermatozoa. Notably, the progesterone-induced AR is preceded by a second [Ca²⁺]i peak and ~40% of reacting spermatozoa also manifest a slow [Ca²⁺]i rise ~2 min before AR. Our findings uncover new AR features related to [Ca²⁺]i.

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.

The sperm acrosome reaction (AR), an essential event for mammalian fertilization, involves Ca permeability changes leading to exocytosis of the acrosomal vesicle. The acrosome, an intracellular Ca store whose luminal pH is acidic, contains hydrolytic enzymes. It is known that acrosomal pH (pH ) increases during capacitation and this correlates with spontaneous AR. Some AR inducers increase intracellular Ca concentration ([Ca ] ) through Ca release from internal stores, mainly the acrosome. Catsper, a sperm specific Ca channel, has been suggested to participate in the AR. Curiously, Mibefradil and NNC55-0396, two CatSper blockers, themselves elevate [Ca ] by unknown mechanisms. Here we show that these compounds, as other weak bases, can elevate pH , trigger Ca release from the acrosome, and induce the AR in both mouse and human sperm. To our surprise, μM concentrations of NNC55-0396 induced AR even in nominally Ca free media. Our findings suggest that alkalization of the acrosome is critical step for Ca release from the acrosome that leads to the acrosome reaction.

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Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca(2+)-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca(2+) dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Fertilization is a key reproductive event in which sperm and egg fuse to generate a new individual. Proper regulation of certain parameters (such as intracellular pH) is crucial for this process. Carbonic anhydrases (CAs) are among the molecular entities that control intracellular pH dynamics in most cells. Unfortunately, little is known about the function of CAs in mammalian sperm physiology. For this reason, we re-explored the expression of CAI, II, IV and XIII in human and mouse sperm. We also measured the level of CA activity, determined by mass spectrometry, and found that it is similar in non-capacitated and capacitated mouse sperm. Importantly, we found that CAII activity accounts for half of the total CA activity in capacitated mouse sperm. Using the general CA inhibitor ethoxyzolamide, we studied how CAs participate in fundamental sperm physiological processes such as motility and acrosome reaction in both species. We found that capacitated human sperm depend strongly on CA activity to support normal motility, while capacitated mouse sperm do not. Finally, we found that CA inhibition increases the acrosome reaction in capacitated human sperm, but not in capacitated mouse sperm.

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