Ca²⁺ signaling in mammalian spermatozoa

Highlights

• Ca²⁺ is essential ion in spermatozoa for successful fertilization.

• Intracellular [Ca²⁺] in sperm is precisely regulated by various systems.

• Sperm motility is regulated by intracellular [Ca²⁺].

• Sperm capacitation occurs in the female reproductive tract, is regulated by [Ca²⁺] including the development of hyperactivated motility and actin polymerization.

• The occurrence of the acrosome reaction depends upon high increase in intracellular [Ca].

Abstract

Calcium is an essential ion which regulates sperm motility, capacitation and the acrosome reaction (AR), three processes necessary for successful fertilization. The AR enables the spermatozoon to penetrate into the egg. In order to undergo the AR, the spermatozoon must reside in the female reproductive tract for several hours, during which a series of biochemical transformations takes place, collectively called capacitation. An early event in capacitation is relatively small elevation of intracellular Ca²⁺ (in the nM range) and bicarbonate, which collectively activate the soluble adenylyl cyclase to produce cyclic-AMP; c-AMP activates protein kinase A (PKA), leading to indirect tyrosine phosphorylation of proteins. During capacitation, there is an increase in the membrane-bound phospholipase C (PLC) which is activated prior to the AR by relatively high increase in intracellular Ca²⁺ (in the μM range). PLC catalyzes the hydrolysis of phosphatidyl-inositol-4,5-bisphosphate (PIP₂) to diacylglycerol and inositol-trisphosphate (IP₃), leading to activation of protein kinase C (PKC) and the IP₃-receptor. PKC activates a Ca²⁺- channel in the plasma membrane, and IP₃ activates the Ca²⁺- channel in the outer acrosomal membrane, leading to Ca²⁺ depletion from the acrosome. As a result, the plasma-membrane store-operated Ca²⁺ channel (SOCC) is activated to increase cytosolic Ca²⁺ concentration, enabling completion of the acrosome reaction. The hydrolysis of PIP₂ by PLC results in the release and activation of PIP₂-bound gelsolin, leading to F-actin dispersion, an essential step prior to the AR. Ca²⁺ is also involved in the regulation of sperm motility. During capacitation, the sperm develops a unique motility pattern called hyper-activated motility (HAM) which is essential for successful fertilization. The main Ca²⁺-channel that mediates HAM is the sperm-specific CatSper located in the sperm tail.

Introduction

Ca²⁺-signaling is a key regulator of post-translational modification in living cells. Although translational activity occurs during sperm capacitation (Gur and Breitbart, 2006), sperm functions are mainly regulated by post-translational processes. Calcium is essential ion which regulates sperm motility, capacitation and the acrosome reaction (AR), three processes necessary for successful fertilization. Ca²⁺-signaling plays a pivotal role in controlling sperm motility, capacitation and the acrosome reaction, three essential functions for successful fertilization (Darszon et al., 2011; Publicover et al., 2007). Intracellular Ca²⁺ concentrations are carefully regulated in cells. The plasma membrane and subcellular compartments such as the acrosome, the redundant-nuclear-envelope (RNE) and the mitochondria, tightly control Ca²⁺ entry, secretion and mobilization within the sperm. Along the sperm tail, which is very long and narrow, Ca²⁺ signaling must be coordinated by highly organized molecules. To allow fertilization, these Ca²⁺-signaling molecules are organized in a way that enables them to regulate progressive motility, as well as the development of flagellar asymmetric motility pattern called hyperactivated motility (HAM) during sperm capacitation.

Several processes regulate intracellular Ca²⁺-concentrations ([Ca²⁺] _i), at the levels of influx, efflux, and intracellular stores. Ca²⁺-influx is mainly controlled by the sperm specific Ca²⁺-channel CatSper (Ren et al., 2001) and by other Ca²⁺ channels (Cohen et al., 2014; Florman et al., 2008). Ca²⁺- efflux is regulated by the plasma membrane Ca²⁺-ATPase (Bradley and Forrester, 1980a, Bradley and Forrester, 1980b; Breitbart et al., 1983) and the Na⁺/Ca²⁺ exchanger (Bradley and Forrester, 1980a, Bradley and Forrester, 1980b; Rufo et al., 1984). In addition, intracellular Ca²⁺-mobilization is regulated by Ca2⁺-ATPase and IP₃-R located in the outer acrosomal membrane, and active in Ca²⁺-influx and efflux into/from the acrosome (Dragileva et al., 1999; Walensky and Snyder, 1995). The sperm mitochondria are also active in Ca²⁺ uptake and release (Breitbart et al., 1996; Drago et al., 2011; Pizzo et al., 2012). An additional third Ca²⁺ store is the RNE, which contains IP₃R (Ho and Suarez, 2001a, Ho and Suarez, 2001b, 2003), is localized in the sperm neck, and involved in Ca²⁺-mobilization. Altogether, [Ca²⁺]_i is regulated by fluxes through the plasma membrane and via mobilization by the acrosome, the mitochondria and the RNE.

Thus, in order to achieve sperm motility, capacitation, and the AR at the right time and location, intracellular Ca²⁺ concentrations must be precisely regulated. In this review we summarize the sperm systems that regulate intracellular [Ca²⁺]_i in spermatozoa.

Intracellular Ca²⁺ concentrations ([Ca²⁺]_i) are regulated by Ca²⁺-pumps (Ca²⁺-ATPases), Na⁺/Ca²⁺-exchangers, and Ca²⁺-channels in the plasma membrane and in intracellular organelles including the acrosome, the RNE, and the mitochondria. The systems that cause an increase in cytosolic [Ca²⁺]_i include Ca²⁺-channels in the plasma membrane, inositol –triphosphate- receptor (IP₃R) in the outer-acrosomal membrane (Walensky and Snyder, 1995), IP₃R and ryanodine receptor (RyR) in the RNE (Harper et al., 2004; Trevino et al., 1998), and the Ca²⁺ efflux system in the mitochondria (Breitbart et al., 1996). Ca²⁺-channels in the plasma membrane include 6 types of the voltage-gated channels Ca_v; T-type TRPC (transient-receptor-potential-channel) of the types 1, 2, 3, 4, and 6; CNG (cyclic-nucleotide-gated) channels of the types A3 and B1 (Felix, 2005) and CatSper types 1–4 (Felix, 2005). TRPV1, 4 and 8 were found in human sperm (De Blas et al., 2009; De Toni et al., 2016; Kumar et al., 2016). Another channel in the plasma membrane is SOCC (store-operated-Ca-channel), activated as a result of Ca²⁺ efflux from the acrosome via activation of IP₃R by IP₃ generated from PIP₂ hydrolysis by Ca-dependent phospholipase C activity (Dragileva et al., 1999; Fukami et al., 2003).

The systems that decrease cytosolic [Ca²⁺]_i include the Ca²⁺-ATPase (Breitbart et al., 1983) and Na⁺/Ca²⁺-exchanger (Babcock and Pfeiffer, 1987) of the plasma membrane, Ca²⁺-ATPase of the outer acrosomal membrane and the RNE (Lawson et al., 2007), and Ca²⁺-transporter of the mitochondria (Babcock et al., 1976).

Ca²⁺ has an essential role in regulating sperm motility. After leaving epididymis, sperm acquire progressive motility regulated by Ca²⁺. One of the most important roles of Ca²⁺ in human sperm motility is the activation of the sAC to produce cAMP. Moreover, the inhibition of Ca²⁺ signaling is associated with male sub-fertility (Espino et al., 2009). Human sperm are maintained in a basal motility state in the caudal portion of the epididymis and vas deferens by low resting Ca²⁺ concentration. However, in the female reproductive tract, Ca²⁺ concentration increases to induce capacitation-dependent HAM. The female reproductive system controls the increase of Ca²⁺ concentration in the sperm through signals that are regulated in different parts of the reproductive tract, and at different phases of the menstrual cycle. Experimental evidence suggests that HAM is regulated by IP₃R-gated intracellular Ca²⁺ store (RNE) located in the neck region of the sperm (Ho and Suarez, 2001a, Ho and Suarez, 2001b). In addition to its roles in capacitation and the AR, the Ca²⁺-channel CatSper plays an essential role in motility by controlling calcium influx (Ren et al., 2001) (see Fig. 3). Hyper-activated motility requires CatSper, a sperm-specific flagellar- Ca²⁺-channel, which is also important for protein-tyrosine phosphorylation in sperm capacitation. Male mice lacking CatSper are infertile as are human males with loss-of-function mutations (Luo et al., 2019; Ren et al., 2001). Nevertheless, a recent study showed that human sperm rotational motion and rheotaxis are independent of CatSper activity (Schiffer et al., 2020).

The mammalian CatSper complex is comprised of nine different subunits: four alpha pore-forming subunits CatSper 1 to 4 and five auxiliary subunits – CatSper-β, γ, δ, ε, and ζ, all of which are sperm-specific trans-membrane proteins (Carlson et al., 2005; Chung et al., 2011; Liu et al., 2007; Lobley et al., 2003; Ren et al., 2001; Strunker et al., 2011; Wang et al., 2009). CatSper is activated by intracellular alkalization (Kirichok et al., 2006). Although intracellular alkalization activates the mouse CatSper, this increase in pH is not sufficient for opening of human sperm CatSper (Kirichok and Lishko, 2011; Lishko et al., 2010, 2011; Shiba et al., 2006). Also, progesterone activates human but not mouse CatSper (Lishko et al., 2011; Strunker et al., 2011) via binding to serine hydrolase, ABHD2 (α/β hydrolase domain-containing protein2) (Mannowetz et al., 2017; Miller et al., 2016). In human sperm, the H⁺-channel H_v1 is the dominant proton extrusion pathway activated by membrane depolarization (Lishko et al., 2010). H_v1 does not exist in mouse sperm, and intracellular alkalization probably occurs through the activity of the sperm specific Na⁺/H⁺-exchanger (sNHE) (Wang et al., 2009). It was also suggested that SLO3 K⁺ -channels control calcium entry through CatSper (Chavez et al., 2014). These authors show that during sperm capacitation, SLO3 hyperpolarizes the sperm and promotes rise in intracellular pH suggesting a possible mechanisms for CatSper activation. A recent publication suggests that PKA-dependent phosphorylation regulates [Ca²⁺]_i homeostasis by activating CatSper channel complexes (Orta et al., 2018). We recently showed that PKA-dependent HAM is stimulated by adding low concentrations (10 μM) of 8Br-cAMP or treatment with the cAMP-phosphodiesterase inhibitor IBMX (Allouche-Fitoussi et al., 2018). Male mice lacking CatSper genes are infertile (Chung et al., 2011; Qi et al., 2007; Ren et al., 2001), as are human males with loss-of-function mutations (Avenarius et al., 2009; Avidan et al., 2003; Smith et al., 2013).

Elevation of [IP₃]_i causes the release of Ca²⁺ from the acrosome and from the RNE which, in turn, modulates sperm motility by inducing flagellar beat asymmetry, probably involved in sperm chemotactic behavior (Cook et al., 1994; Publicover et al., 2007). In bulls, boar and mice, HAM can be induced in uncapacitated sperm by activating IP₃R using thimerosal (Marquez et al., 2007; Otsuka and Harayama, 2017). Under physiological capacitation conditions, IP₃R of RNE and of the outer acrosomal membrane, are activated by IP₃ generated from the hydrolysis of PIP₂ by the Ca²⁺-activated phospholipase C, causing Ca²⁺ release from the RNE and the acrosome, promoting the development of HAM. In human sperm, RyR mediates recruitment of cytoplasmic Ca²⁺ from RNE in a manner of Ca²⁺-induced-Ca²⁺-release which regulates the development of HAM (Harper et al., 2004). It was also suggested that TRPC3 is present in the midpiece of boar sperm, and is involved in the occurrence of HAM (Otsuka and Harayama, 2017).

Calcium/calmodulin and calmodulin-kinase II stimulate HAM (Ignotz and Suarez, 2005) and mediate F-actin formation (Shabtay and Breitbart, 2016) in bull sperm. Moreover, formation of F-actin during human sperm capacitation induces HAM (Itach et al., 2012). These results indicate that Ca²⁺-dependent F-actin formation during sperm capacitation induces the development of HAM. Reports from additional species have proposed the participation of several hyperactivation-associated molecules activated by [Ca²⁺] elevation, including calcineurin (Miyata et al., 2015) and calaxin (Inaba, 2015; Mizuno et al., 2012; Shojima et al., 2018). Interestingly, treatment of infertile CatSper, Adcy10 or SLO3 KO mice with Ca²⁺-ionophore, rescues the ability of these sperm to undergo HAM and to fertilize oocytes (Navarrete et al., 2016), suggesting the importance of the increase in cytoplasmic Ca²⁺ for HAM stimulation and fertility.

Ca_v channels are expressed in sperm tail and may participate in motility regulation. Ca_v3 channels are present in mouse and human sperm, and compounds known to inhibit them cause a small decrease in sperm motility (Trevino et al., 2004). Mouse sperm devoid of the α_1E subunit of Ca_v2.3 show aberrant sperm motility (Sakata et al., 2002). Capacitative Ca²⁺ channels (TRPCs) are also expressed in human sperm and these proteins are also involved in motility regulation (Castellano et al., 2003). Chemotaxis in response to progesterone is mediated by Ca²⁺ mobilization from intracellular Ca-stores followed by TRPC activation (Teves et al., 2009). Cyclic nucleotide-gated Ca²⁺-channel is involved in bovine sperm motility (Wiesner et al., 1998). Inhibition of the Na⁺/Ca²⁺-exchanger caused an increase in [Ca²⁺]_i leading to human sperm motility inhibition (Shiba et al., 2006).

[Ca²⁺]_i is slightly increased during mammalian sperm capacitation but still kept low in the nM range (Cohen et al., 2014; Luque et al., 2018). Human sperm possess a depolarization-activated H⁺ conductance via H_v1 channel, which is activated during capacitation and induces Ca²⁺ influx through the sperm-specific CatSper channel (Miller et al., 2018; Zhao et al., 2018). The transient receptor potential TRPV4 is proposed to carry a depolarizing Na⁺ conductance that facilitates CatSper/H_v1 activity (Mundt et al., 2018). One of the first events in sperm capacitation is the activation of the soluble-adenylyl-cyclase (sAC) activated by bicarbonate and Ca²⁺ (Chen et al., 2000) (see Fig. 1). Ca²⁺ causes an increase in the affinity of ATP for sAC leading to increased sensitivity to bicarbonate (Zippin et al., 2013). The resulting increase in cellular cAMP activates protein kinase A (PKA) leading to an increase in tyrosine phosphorylation of proteins (pTyr) (Visconti et al., 1995). Interestingly, incubation of human sperm under capacitation conditions containing reduced concentrations of extracellular Ca²⁺ induces an increase in pTyr (Aitken and Baker, 2002; Leclerc and Goupil, 2002; Marin-Briggiler et al., 2003). Moreover, human sperm incubated in medium without added Ca²⁺ revealed an increase in pTyr but did not modify PKA-mediated phosphorylation (Battistone et al., 2014). The Battistone group reported the presence of Ca²⁺-dependent nonreceptor proline-rich tyrosine kinase 2 (Pyk2) in sperm. A role for Pyk2 in the regulation of [Ca²⁺]_i during capacitation via modulation of pH-induced CatSper activation was also suggested (Brukman et al., 2019). We demonstrated the presence of Pyk2 in bovine sperm activated by CaMKII (Ca²⁺-calmodulin kinase II) and phosphorylates PI3K (phosphatidyl-inositol-3-kinase) on tyrosine (Rotfeld et al., 2014) (see Fig. 1). However, another study in mouse sperm argues against a role of Pyk2 in protein tyrosine phosphorylation suggesting that the tyrosine kinase FER mediates tyrosine phosphorylation during capacitation (Alvau et al., 2016). These authors also suggested that the increase in protein tyrosine phosphorylation during capacitation is not essential for fertility, in contradiction to the current conception of sperm capacitation. Thus, the mechanism by which the serine/threonine kinase PKA mediates protein tyrosine phosphorylation and whether this phosphorylation is essential for sperm capacitation is still an open question.

The CaMKII cascade mediates the actin-polymerization process during sperm capacitation (Shabtay and Breitbart, 2016), an essential process for preventing spontaneous-AR (Shabtay and Breitbart, 2016) and for successful fertilization (Brener et al., 2003; Itach et al., 2012) (see Fig. 1). CaMKII is activated by Ca²⁺-bound calmodulin, and thus Ca²⁺-regulated CaMKII leads to actin polymerization during capacitation. Actin polymerization in sperm capacitation mediates the development of HAM (Itach et al., 2012), and CaM antagonists have been shown to inhibit HAM (Battistone et al., 2014). It was also shown that interaction between CaMKII and the protein MUPP1 prevents spontaneous AR (Ackermann et al., 2009).

Ca²⁺ has been attributed both positive and negative roles in the regulation of PKA/pTyr in mouse sperm capacitation (Navarrete et al., 2015). It was shown that extracellular [Ca²⁺] in the mM range stimulates PKA/pTyr, with no effect at 50–100 μM Ca²⁺; chelating extracellular Ca²⁺ by EGTA caused a strong increase in PKA/pTyr activities, but fertilization did not occur in the presence of EGTA. These authors suggested that the Ca²⁺-dependent phosphatase activity of calcineurin is affected by changes in [Ca²⁺]_i, in which relatively high or very low [Ca²⁺]_i inactivates this phosphatase, but at [Ca²⁺] of 50–100 μM the phosphatase is active and causes dephosphorylation of proteins. Interestingly, in contrast to the mouse model, in human (Battistone et al., 2014; Carrera et al., 1996) and horse (Gonzalez-Fernandez et al., 2013) sperm incubated in media with no addition of Ca²⁺ undergo pTyr. These results suggest species-specific differences regarding the [Ca²⁺]_i essential for phosphatase activity.

CABYR is another Ca²⁺-binding phosphorylation-regulated protein that undergoes tyrosine and serine/threonine phosphorylation during capacitation (Naaby-Hansen et al., 2002). CABYR was found in the principal piece of the sperm tail and gains Ca²⁺-binding ability after phosphorylation during capacitation (Naaby-Hansen et al., 2002). A study in human sperm suggests that the development of HAM does not directly depend on activity of CatSper, a sperm-specific calcium channel, but on the release of Ca²⁺ from the RNE localized in the sperm neck (Alasmari et al., 2013). Interestingly, a recent study suggested that infertile men with complete disruption of CatSper 2 exhibit normal semen parameters, but show impaired egg penetration, are deficient in HAM, and do not respond to progesterone, a known activator of CatSper (Luo et al., 2019). HAM is essential for fertilization since it enables the capacitated sperm to detach from the oviductal epithelium, and subsequently helps to penetrate the vestments of the egg (Ardon et al., 2016; Li et al., 2015). HAM is initiated by intra-flagellar alkalization (Marquez and Suarez, 2007), membrane depolarization (Kirichok et al., 2006), and an increase in [Ca²⁺]_i (Suarez et al., 1993). Bicarbonate, an essential component of capacitation medium, activates sAC (Chen et al., 2000) to produce cAMP and activation of PKA. Moreover, bicarbonate raises pHi to activate CatSper channels (Kirichok et al., 2006). Interestingly, pTyr is further enhanced in CatSper 1 null sperm, suggesting that disruption of CatSper channel deregulates capacitation-dependent protein tyrosine phosphorylation (Chung et al., 2014).

It was recently shown that PKA-dependent phosphorylation regulates [Ca²⁺]_i by activating the CatSper channel (Orta et al., 2018). However, a recent study in human sperm show that CatSper is neither activated by intracellular cAMP directly nor indirectly by the cAMP/PKA-signaling pathway (Wang et al., 2020). These authors also show that non-physiological concentrations of cAMP and membrane-permeable cAMP analogs activate CatSper from outside the cell via unknown extracellular site. These interesting data should be supported by other studies. We recently showed CatSper-dependent stimulation of HAM in human sperm by adding low concentrations (10 μM) 8Br-cAMP (Allouche-Fitoussi et al., 2018). In human sperm, extracellular Ca²⁺ negatively modulates pTyr during capacitation (Luconi et al., 1996). Changes in [Ca²⁺]_i affect pTyr, and proper motility requires a spatially localized network of CatSper and pTyr systems (Chung et al., 2014). Accordingly, only sperm with intact CatSper that organize time-dependent and spatially specific pTyr successfully develop HAM (Ardon et al., 2016; Chung et al., 2014; Li et al., 2015). This is consistent with the finding that only a subpopulation of sperm exhibits HAM upon capacitation (Buffone et al., 2009; Goodson et al., 2011; Kulan and Shivaji, 2001).

Actin polymerization (F-actin formation) occurs during mammalian sperm capacitation. Prior to the acrosome reaction F-actin is dispersed to allow the interaction between the outer acrosomal membrane and the overlying plasma membrane (Brener et al., 2003; Spungin et al., 1995) (see Fig. 1, Fig. 2). The increase in [Ca²⁺]i prior to the AR activates the actin-severing proteins gelsolin and cofilin, resulting in F-actin depolymerization (Finkelstein et al., 2010; Megnagi et al., 2015). To allow F-actin formation, gelsolin should be inactive during capacitation, therefore [Ca²⁺]i should be kept relatively low. Sperm membrane potential is hyperpolarized during capacitation to −80mV, which shifts voltage-activated channels into a closed state (Arnoult et al., 1999). Formation of F-actin in the sperm head prevents premature AR (Spungin et al., 1995), and F-actin in the tail leads to hyperactivated motility (Itach et al., 2012). During capacitation, gelsolin and cofilin translocate from the tail to the head (Finkelstein et al., 2010; Megnagi et al., 2015) which is a prerequisite for the occurrence of the AR. The activity of CatSper (Allouche-Fitoussi et al., 2018; Ardon et al., 2016; Chung et al., 2014; Li et al., 2015) and actin polymerization (Itach et al., 2012) in the tail during capacitation is essential for the development of HAM (see Fig. 3). The increase of [Ca²⁺] in the tail due to CatSper activation does not depolymerize F-actin, since the levels of gelsolin and cofilin in the tail are low due to their translocation to the head. HAM is significantly enhanced in bovine sperm by up to 0.4 μM Ca²⁺ but at 1 mM Ca²⁺, motility is suppressed (Ho et al., 2002), indicating that a relatively low [Ca²⁺] concentration is necessary to achieve HAM; however, at high [Ca²⁺] concentrations, HAM is inhibited. Working with demembranated mammalian sperm revealed beat activation of the tail at 30 nM Ca²⁺and HAM is initiated at 100 nM Ca²⁺ (Ho and Suarez, 2001a, Ho and Suarez, 2001b). In more recent study with mouse sperm, it was shown that [Ca²⁺]_i must decrease below a threshold to facilitate flagellar beat (Sanchez-Cardenas et al., 2018).

The acrosomal reaction (AR) in mammalian sperm is a highly-regulated process essential for sperm penetration into the egg. It is widely accepted that physiological AR occurs as a result of the interaction of intact sperm with the egg zona-pellucida (ZP), although it has been suggested that fertilizing mouse sperm can initiate their AR before contact with the ZP (Jin et al., 2011). The AR is a Ca²⁺-regulated exocytosis process composed of a series of molecular events leading to the fusion between the outer acrosomal membrane and the overlying plasma membrane (Barros et al., 1967). It is important to distinguish between spontaneous-AR (sAR), which is negatively correlated with fertilization ability, and induced-AR (iAR) which is essential for successful fertilization. Under in vitro capacitation conditions, some of the sperm cells undergo sAR, resulting in a decrease in fertilization rate (Wiser et al., 2014). Moreover, only intact sperm, and not acrosome reacted ones, exhibit chemotactic motility towards the egg (Guidobaldi et al., 2017) suggesting that sAR occurs in vivo. The cells that have intact acrosome at the end of the incubation are capacitated sperm that can undergo iAR upon addition of isolated zona-pellucida or Ca²⁺-ionophore, which causes an increase in intracellular Ca²⁺ and pH.

Actin polymerization is one of the capacitation-dependent mechanisms that protect the sperm from undergoing sAR (Shabtay and Breitbart, 2016). In other words, actin polymerization is associated with reduced sAR. In more recent studies, it was suggested that pH-dependent Ca²⁺ oscillations prevent premature AR (Mata-Martinez et al., 2018; Sanchez-Cardenas et al., 2014). In addition, it was shown that ~30% of human sperm display spontaneous Ca²⁺ oscillations correlated with absence of AR, suggesting another mechanism to prevent premature AR. It was also suggested that protein-protein interaction between the Ca²⁺-sensor protein synaptotagmin (Koh and Bellen, 2003) and the SNARE-associated (soluble N-ethylmaleimide-sensitive attachment protein receptor), complexin (Brose, 2008; Brukman et al., 2019; McMahon et al., 1995) maintain the fusion machinery at an intermediate pre-fusion stage (Sudhof and Rothman, 2009), thereby preventing spontaneous AR (Weber et al., 1998). The fusion machinery in the AR consists of SNAREs which form a membrane –trafficking complex composed of vesicular synaptobrevin (v-SNARE) and plasma membrane syntaxin and SNAP25 (t-SNAREs) (Jahn and Scheller, 2006; Lang and Jahn, 2008; Mayorga et al., 2020).

The interaction between capacitated sperm and the egg zona-pellucida induces elevation in sperm [Ca²⁺]_i resulting in the AR (Florman et al., 2008; Publicover et al., 2007). Several studies suggested that the first transient [Ca²⁺]_i increase is mediated by T-type Ca²⁺-channels (Arnoult et al., 1996; Jagannathan et al., 2002; Lievano et al., 1996), which leads to a second enhancement of [Ca²⁺]_i resulting in AR (Darszon et al., 2005; Dragileva et al., 1999; Gonzalez-Martinez et al., 2001; Jungnickel et al., 2001; Llanos, 1998; Meizel and Turner, 1993; O'Toole et al., 2000; Rossato et al., 2001). Working with isolated membranes, we found that phospholipase C (PLC) requires 2 μM Ca²⁺ for half maximal activity, while 80 μM Ca²⁺ is needed for F-actin release from the plasma membrane, which is essential to achieve AR (Spungin and Breitbart, 1996). Thus, the first Ca²⁺ increase activates PLC resulting in PIP₂- hydrolysis to inositol-1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG) (see Fig. 2). IP₃ activates IP₃R of the outer acrosomal membrane which causes Ca²⁺ secretion from the acrosome resulting in the activation of SOCC in plasma membrane (Walensky and Snyder, 1995). It is well established that SOCC is activated after depletion of Ca²⁺ in intracellular Ca²⁺ stores like the acrosome (Dragileva et al., 1999; Fukami et al., 2003; O'Toole et al., 2000). As a result, a sustained [Ca²⁺]_i increase occurs triggering the AR. Notably, in PLC_δ4 null mice, there is a reduction in Ca²⁺ influx via SOCC (Fukami et al., 2003). Sperm express epidermal-growth-factor-receptor (EGFR) and AR can be induced by EGF, indicating that PLC_γ is required for the AR (Etkovitz et al., 2009; Lax et al., 1994). In addition, we showed that α7-nicotinic-acetyl-choline –receptor(α7nAChR) is a potential sperm receptor that can be activated by the egg ZP to induce Src-family-kinase (SFK)-dependent EGFR activation and Ca²⁺-influx leading to AR (Jaldety et al., 2012). It has been shown by others that α7nAChR is involved in ZP induced acrosome reaction (Bray et al., 2002; Son and Meizel, 2003) and α7 null sperm have impaired motility (Bray et al., 2005). EGF signaling was shown to be an important pathway for fertilization (Peddinti et al., 2008). Interestingly, micromolar levels of Zn²⁺ were shown to activate EGFR during capacitation, which is mediated by the trans-membrane adenylyl cyclase (tmAC), PKA, and the tyrosine kinase Src (Michailov et al., 2014).

Moreover, the addition of Zn²⁺ to capacitated sperm causes further stimulation of EGFR and PI3K phosphorylation/activation, leading to the AR (Michailov et al., 2014). Interestingly, stimulation of AR by Zn²⁺ may also occur in absence of Ca²⁺ in the incubation medium, and is dependent on tmAC activity, indicating the involvement of the GPCR (G-protein-coupled-receptor), GPR39 (Michailov et al., 2014). Thus, Zn²⁺ induces the AR, through the following suggested cascade: Zn²⁺->GPR39->PKA->Src->EGFR->PI3K followed by PLC and then PKC activation (Michailov et al., 2014). SFK participates in sperm capacitation, Ca²⁺ fluxes, tyrosine phosphorylation and the AR (Varano et al., 2008). SFK phosphorylates the actin severing protein, gelsolin, on Tyr-438, which enhances its binding to PIP₂ and inhibits its activity during capacitation (Finkelstein et al., 2010). The inhibition of gelsolin allows the production of F-actin during capacitation. Prior to the AR, the increase in [Ca²⁺]_i activates PLC to hydrolyze PIP₂; as a result, phospho-gelsolin is released to the cytosol and undergoes dephosphorylation and activation by Ca²⁺ (Finkelstein et al., 2010) (see Fig. 2).

The outer acrosomal membrane possesses a Ca²⁺-pump (Ca²⁺-ATPase) (Rossato et al., 2001) and its inhibition by thapsigargin leads to a decrease in intra-acrosomal [Ca²⁺] resulting in SOCC activation and the AR (Dragileva et al., 1999). Entry of Ca²⁺ in mouse sperm through SOCC is initiated by ZP3 (O'Toole et al., 2000). Intra-acrosomal Ca²⁺ is secreted through IP₃-R, thus the formation of IP₃ just before the AR keeps the IP₃-dependent- Ca²⁺ channel inactive during capacitation, ensuring Ca²⁺ elevation inside the acrosome during capacitation. The sharp increase of cytosolic [Ca²⁺] prior to the AR activates actin-severing proteins including gelsolin and cofilin, which disperse the F-actin barrier intervening between the outer acrosomal and the overlying plasma membrane, enabling their fusion and completing the acrosome reaction (Finkelstein et al., 2010; Megnagi et al., 2015; Spungin et al., 1995). Gelsolin is inactivated during capacitation due to its binding to PIP₂ and phosphorylation on tyrosine −438 (Finkelstein et al., 2010). Prior to the AR, the elevation in [Ca²⁺]i activates PLC, which hydrolyzes PIP₂ resulting in gelsolin release and tyrosine dephosphorylation (Finkelstein et al., 2010). This rise in Ca²⁺ activates closed gelsolin to its open/activated form. The hydrolysis of PIP₂ by PLC produces DAG, and together with the increase in [Ca²⁺]i, activates the Ca²⁺-dependent protein kinase Cα (PKCα) which participates in the AR (Breitbart et al., 1992; Rotem et al., 1992). The sperm plasma membrane contains PKC-activated Ca²⁺-channel involved in the AR (Spungin and Breitbart, 1996; Spungin et al., 1995). In addition, extra-cellular ATP activates sperm P2-purinoceptor leading to [Ca²⁺]_i elevation and PKCα-dependent AR (Luria et al., 2002). ERK1/2 also participates in the AR through elevation of [Ca²⁺]_i (Jaldety and Breitbart, 2015). The Ca²⁺ channel Ca_v2.3 participates in the occurrence of AR in mouse sperm (Cohen et al., 2014). Ca²⁺-mediated induction of AMP-activated protein kinase (AMPK) phosphorylation was blocked by SOCC inhibition (Nguyen et al., 2016). Ca²⁺ entry via SOCC appears to be the most likely pathway for AMPK activation, and for energy requiring sperm functions such as motility and the acrosome reaction (Nguyen et al., 2016). Although it is not located in the sperm's head, CatSper also appears to be involved in the acrosome reaction by increasing Ca²⁺ concentration in the tail, leading to Ca²⁺ increase in the head in unknown mechanism. In human sperm, progesterone induces Ca²⁺ entry via CatSper and thus promotes the AR (Baron et al., 2016; Itzhakov et al., 2019; Lishko et al., 2011). Progesterone activates CatSper indirectly by elimination of the CatSper inhibitor 2-arachidonoylglycerol (2-AG) (Miller et al., 2016) by the α/β-hydrolase domain containing protein 2 (ABHD2) which hydrolyzes 2-AG.

Ca²⁺-induced AR requires activation of the cAMP-dependent-EPAC-mediated pathway (Branham et al., 2006). EPAC (exchange protein directly activated by cAMP) is a Rap-specific guanine-nucleotide exchange factor, which was shown to mobilize Ca²⁺ from the sperm acrosome prior to the AR (Ruete et al., 2014). Interestingly, we recently showed that AR induced by inhibition of PKA is mediated by EPAC but not by CatSper, whereas AR induced by the Ca²⁺-pump inhibitor thapsigargin, is mediated by EPAC and by CatSper, as well (Itzhakov et al., 2019). Thapsigargin treatment results in Ca²⁺-mobilization from the acrosome, which is mediated by EPAC. Moreover, inhibition of the outer acrosomal Ca²⁺-pump, leads to SOCC activation (Dragileva et al., 1999) and likely to CatSper activation, as well, resulting in AR. Interestingly, as mentioned above, a study in human sperm suggests that the development of HAM does not directly depend on CatSper activity, but on the release of Ca²⁺ from the RNE localized in the sperm neck (Alasmari et al., 2013). This study supports the involvement of CatSper in Ca²⁺ release from the acrosome, which leads to AR (Itzhakov et al., 2019).