Differences and Similarities: The Richness of Comparative Sperm Physiology

Abstract

Species preservation depends on the success of fertilization. Sperm are uniquely equipped to fulfill this task, and, although several mechanisms are conserved among species, striking functional differences have evolved to contend with particular sperm-egg environmental characteristics. This review highlights similarities and differences in sperm strategies, with examples within internal and external fertilizers, pointing out unresolved issues.

Introduction

Fertilization is a fundamental and complex process required to produce a unique individual resulting from the fusion of two haploid gametes, the male sperm and the female oocyte/egg (137). Depending on the species, fertilization occurs in very diverse milieus. For instance, marine invertebrates and fish spawn their gametes into sea or fresh water, whereas mammals, being internal fertilizers, release their sperm into the female genital tract. Despite these differences, all sperm must find the egg; therefore, the signals that lead sperm to the female gamete have evolved to contend with the species particular environments, paths, and cues, with some being specific and others shared (27, 61, 83).

Notably, sperm from different species come in astoundingly diverse forms. The more typical sperm consists of head, midpiece, and flagellum (see FIGURE 1). The haploid genetic material of the male gamete is contained in the head; the propelling apparatus (the axoneme), together with mitochondria and glycolytic enzymes, are in the flagellum (6). It can be hypothesized that an ancestral sperm displayed these characteristics, although evolution then took many paths, leading to almost endless diversity (36, 48, 85). For instance, the largest sperm is from the fruit fly Drosophila bifurca with a 57-mm length, 20 times the size of the adult (98). Not only gamete size has varied enormously in sexually reproducing organisms; sperm components have done so too. Both plants (77) and the Mastotermes darwiniensis termite can display multi-flagellate sperm, with this latter species having up to 100 flagella (7). Sperm from other species have lost their mitochondria (7) or their flagellum (6, 85, 113).

FIGURE 1.

Comparative sperm morphology of two external and two internal fertilization organisms

In general, sperm morphology can be described in two cellular regions: head and flagellum. Sperm heads contain the nucleus (in purple), where hyper-compacted DNA is stored. In addition, the sea urchin, human, and mouse sperm head presents a specialized organelle called the acrosome (red), which acts as an acidic Ca²⁺ store and is indispensable for fertilization. Sperm head shape and size varies among species; the zebrafish sperm head is rounded, that of sea urchin is elongated, human sperm possess an oval head, and the mouse sperm head is larger and hooked. Flagella size and area distribution are also species-specific. The zebrafish and sea urchin midpiece display small areas close to the sperm neck, whereas in human and mouse sperm they are more prominent. Mitochondria (pink) are present in the midpiece of human and mouse sperm, whereas a single one is inside the head in sea urchin sperm. The axoneme, the sperm propelling apparatus (green line), is composed of a central pair of microtubules surrounded by nine doublets of microtubules and dynein ATPases (inset), which produce the flagellar beat. Scale bar: 5 μm.

The great diversity in sperm can be explained by different fertilizing environments (FIGURE 2) that have caused an important selection pressure on the evolution of sperm form and function (36, 41, 48, 77). Sperm competition has been found to be one of the key forces influencing sperm phenotype. The evolution of small sperm in high numbers was predicted by theory as a consequence of sperm competition (96). On the other hand, the hypothesis that sperm with longer tails swim faster and/or have longer-lasting energy reserves has received experimental support (reviewed in Ref. 41). In this respect, in some squids, small males called Sneakers produce larger spermatozoa than bigger males, although both sperm types swim with the same velocity (58). In this species, only large sperm show chemotaxis toward acid; consequently, only the larger sneaker sperm keep swarming in response to CO₂ they themselves produce (50).

FIGURE 2.

Subcellular distribution and role of sperm channels and receptors in two external and two internal fertilization organisms

Sperm physiological functions required for fertilization are accomplished by different ion channels, enzymes, and transporters in a species-specific manner. For instance, whereas sea urchin sperm motility is enhanced by speract (a chemoattractant) binding to its receptor expressed in flagellum, in human sperm probably progesterone modulates sperm motility via the ABHD2 receptor (progesterone receptor with lipase activity) in the principal piece of its flagellum. In contrast, in mouse sperm, ABDH2 receptor is located in the head, where its physiological role is unknown. At present, ABDH2 receptor has not been reported either in zebrafish or sea urchin sperm. Sperm plasma membrane hyperpolarization is a consequence of tetraKCNG K⁺ channel activation located in sea urchin flagellum, whereas this process is generated by Slo3 K⁺ channel opening in both human and mouse sperm. Interestingly, the tetraKCNG K⁺ channel is expressed in the zebrafish sperm head. Alkalinization of sea urchin and mouse sperm pH_i is triggered by the activity of the hyperpolarization-sensitive sNHE, whereas in human sperm intracellular alkalinization depends on depolarization-gated Hv1 H⁺ channels. Ca²⁺ influx necessary for sperm motility depends on CatSper Ca²⁺ channels and seems to be conserved among sea urchin, mouse, and human sperm. Other proteins proposed to play a role in sperm functions are also indicated. Eggs from different species are surrounded by different protein layers (not in scale), as illustrated. ZP, zona pellucida; CC, cumulus cells.

Cell metabolism, ion transport, ligand-receptor, and protein-protein interactions are some of the essential components of the signaling processes that allow sperm to reach and fertilize the female gamete. This brief review will focus on certain shared and distinctive sperm physiological traits, arbitrarily choosing internal and external fertilizers. In particular, we will focus on those properties that offer hints on fundamental mechanisms involved in fertilization. Due to length limitations, the sperm-egg fundamental fusion events of fertilization will not be discussed. The main sperm functions considered in this review are motility regulation, maturation, and acrosome reaction. We will focus on those species presenting notable physiological differences. As part of this summary, we will also emphasize those processes in which fundamental mechanisms are conserved.

Sperm Motility Regulation

Motility is one of the fundamental functions of sperm. Although some male gametes do not have flagellum, such as Caenorhabditis elegans (nematodes) and flowering plants in eukaryotes (15), most animals possess a beating flagellum (or flagella) to advance toward the oocyte. These beating flagella contain an axoneme, the propelling machinery basically composed of a central pair of microtubules surrounded by nine doublets of microtubules (known as the 9 + 2 structure; see FIGURE 1, INSET) (56). Dynein ATPases are cytoskeletal motor proteins localized between any two microtubule doublets. These ATPases displace the microtubule doublets producing the flagellar beat by using the energy of ATP (70).

Usually, sperm are quiescent in the testis or in sperm storage organs (i.e., epididymis in mammals), and they initiate the flagellar beat as they are released [ejaculation in mammals, spawning in sea urchins, or on contact with a specific molecule from the female gametes, as in ascidians (138) and corals (84)]. Phosphorylation of axonemal proteins is a key mechanism for flagellar beat initiation in many species (57). Also, it is known that neutralization of the acidic environment (sea water or seminal vesicle fluid) is another important factor that stimulates motility, because dynein ATPase activity is highly suppressed at low pH (21, 54).

During fertilization, the sperm flagellar beating characteristics change dynamically (1, 46). Depending on the species, sperm use various mechanisms to aid them in their search for the female gamete. Among the most important are chemotaxis, thermotaxis, and rheotaxis (cell directional movement within a chemical or a temperature gradient, or against a fluid flux, respectively). Cytoplasmic Ca²⁺ ([Ca²⁺]_i) changes have been implicated in the regulation of flagellar properties involved in these mechanisms, although debate exists in the case of rheotaxis (12, 27, 62, 79, 87, 109, 139, 144). There are reported species differences even in the case of rheotaxis, since hyperactivated stallion sperm lose this response, whereas human and ram sperm do not (106).

To contend with the enormous dilution that marine invertebrates such as sea urchins and ascidians (sea squirts) undergo upon liberating their gametes into the ocean, chemotaxis enhances their encounter probability. These sperm change their swimming direction, mediated by a highly asymmetrical flagellar beat governed by a [Ca²⁺]_i increase occurring when they swim away from the chemoattractant source (44, 81, 111). Sea urchin sperm chemotaxis involves ligand binding to its receptor, stimulating cGMP synthesis (100). This nucleotide activates a K⁺-selective cyclic nucleotide-dependent channel (tetraKCNG) (37), causing a membrane potential (V_m) hyperpolarization (66) that increases intracellular pH (pH_i) (42, 90, 110) and [Ca²⁺]_i (27, 130, 136). Under certain conditions, initially the chemoattractant decreases [Ca²⁺]_i instead of increasing it (90). Curiously, in zebrafish sperm, tetraKCNG is on the head (FIGURE 2) (32), and, although it conserves four cyclic nucleotide-binding domains, its activity is independent of the nucleotides but depends on pH_i. Conversely, mammals do not possess tetraKCNG, which appears as a pseudogene in the human genome (63).

Similarly to external fertilizers, mammalian gametes must find themselves inside the female genital tract. However, the requirement for chemotaxis is still controversial (17, 61). Although progesterone has been shown to be chemotactic in vitro, its role in this process in vivo still requires definitive experiments (16, 119, 143). On the other hand, in mammalian sperm, elevated concentrations of [Ca²⁺]_i (>400 nM) induce a vigorous flagellar movement named hyperactivated motility (116, 117), which is characterized as non-progressive movement with high amplitude, asymmetry, and low frequency in a low viscous experimental medium. Hyperactivation is indispensable for in vivo fertilization promoting 1) detachment of sperm from epithelial cells at the sperm reservoir named isthmus in the oviduct, 2) progressive movement in viscous and viscoelastic fluid, and 3) penetration through the extracellular matrix of the oocyte (cumulus oophorus and zona pellucida) (27).

CatSper and Sperm Motility

In 2001, two groups independently found components of a novel, sperm-specific Ca²⁺ channel essential for mammalian hyperactivated sperm motility, named CatSper (102, 103). Currently, it is known that CatSper is a highly pH_i-dependent, voltage-gated Ca²⁺ channel (64) located on the principal piece of the mammalian sperm flagellum, forming four strait tracks (24). Unlike typical voltage-gated channels (Ca_vs) in animals, the channel pore of CatSper is composed of four different and separated proteins (CatSper1-4). All four subunits constituting the pore are essential for a functional channel (101). Furthermore, five distinct auxiliary subunits (β, γ, δ, ε, and ζ) have been so far identified in mammals (22, 23, 74, 132). Gamma, δ, and ε possess one, and β possesses two transmembrane segments with large extracellular domains, suggesting a possible role in channel regulation by extracellular ligands. Recently, CatSperζ was reported to be associated with EFCAB9, a cytoplasmic Ca²⁺-binding protein with EF hands that confers the channel pH and Ca²⁺ sensitivity (55).

Interestingly, progesterone and some prostaglandins efficiently activate CatSper in human (71, 115) and rhesus macaque sperm (118) but not in mouse sperm (up to 10 µM progesterone) (73). In human sperm ABHD2, a lipid hydrolase, localized on the flagellar principal piece, was identified as a progesterone receptor. Notably, this enzyme is found in the sperm head in mouse. Therefore, a significant difference in the regulation of CatSper is observed between human and mouse sperm (80).

Despite the primordial importance of CatSper in mammalian reproduction, nobody has succeeded in heterologously expressing the functional channel. Therefore, its biophysical properties can only be directly determined in sperm. We recently demonstrated that PKA mediates upregulation of CatSper (94). Additionally, we indirectly examined the divalent cation selectivity and permeability of CatSper by using human Ca_v3.1 mutants, which possess CatSper-like anionic amino acids in the pore region (39). Using this strategy, we predicted that CatSper has a significant Cd²⁺ permeability compared with Ca_vs and confirmed it in human spermatozoa.

sNHE as the Regulator of CatSper

Recent experimental and modeling evidence supports that CatSper mediates the Ca²⁺ influx required for sperm chemotaxis in sea urchin sperm (31, 42, 100, 110). Its activation requires a pH_i increase mediated by a sperm-specific Na⁺/H⁺ exchanger (sNHE) discovered in the 1980s by Lee and Garbers (67). sNHE is activated by V_m hyperpolarization. On the other hand, sNHE (SLC9C1) was discovered as an essential sperm protein for mouse fertilization (131). As a unique feature, this exchanger possesses a putative voltage sensor domain (VSD) and a cyclic nucleotide-binding domain (CNBD). As in the case of CatSper, functional expression of mammalian sNHE in heterologous systems so far has not been successful. However, the research group of Dr. Benjamin Kaupp in Germany achieved functional expression of the sea urchin sNHE in a heterologous system (135). They demonstrated that sea urchin sNHE is activated by V_m hyperpolarization through the VSD, confirming the observations of Lee and Garbers. sNHE is upregulated by cAMP through the CNBD by right shifting its voltage-dependence. These findings support that sNHE is a key protein in sea urchin sperm chemotaxis that mediates the pH_i-dependent CatSper activity.

Soluble Adenylyl Cyclase and Sperm Physiology

The presence of cAMP at the right time and place in cell signal transduction requires specific adenylyl cyclases. This enzyme comes in two main flavors: the G-protein-regulated transmembrane adenylyl cyclases (tmACs) and the CO₂/HCO₃⁻/pH_i, Ca²⁺, and ATP-sensing soluble adenylyl cyclase (sAC). sAC has been proposed to be a universal HCO₃⁻ sensor and is fundamental in sperm physiology (sea urchin chemotaxis, mammalian hyperactivation, and capacitation), in tune with the preponderant role of this anion (134, 145).

Distribution of CatSper, sNHE, and sAC in Eukaryotes: A Phylogenetic Perspective

Despite the fundamental function of CatSper in mammalian fertilization, this channel is heterologously distributed among eukaryotes; namely, linage-specific gene loss of this channel has been reported (14a, 14b, 14c). In animals, porifera (sponges), cnidarians (coral and sea anemone), echinoderms (sea urchin), amphioxus, and tunicates (sea squirts) possess CatSper, but most protostomes (arthropods, including insects and mollusks) do not. Heterogeneous distributions of CatSper are observed even within the vertebrates; cartilaginous fishes, some ray-finned fishes (spotted gar), the coelacanth, reptiles, and mammals conserve this channel, but jawless fish, many ray-finned fishes (zebrafish and medaka), amphibians, and most of birds have lost it (FIGURE 3A).

FIGURE 3.

Distribution of CatSper, sNHE, and sAC in Metazoa and their signaling pathway

A: distribution of genes encoding CatSper, sNHE, and sAC among the representative taxa (or species described in italic) in the Metazoa. Boxes represent the presence of genes encoding each protein: CatSper (black), sNHE (blue), and sAC (red). Clade and species underlined represent conservation of SLO3. The branching patterns do not represent the proportional evolutionary rate. B: proposed model for the involvement of these elements in CatSper regulation in Metazoa. sAC produces cAMP, which activates PKA and sNHE; the activity of the latter alkalizes the cytoplasm, which, in conjunction with PKA, promotes Ca²⁺ influx via CatSper, resulting in hyperactivation/chemotaxis.

In mouse, sAC is essential for sperm motility regulation, and this cyclase displays an interdependent structural relationship with sNHE; sperm lacking sNHE have their sAC activity severely affected (130a). Considering the functional coupling between CatSper, sNHE, and sAC in mouse (94, 103, 130a) and sea urchin (90, 135), we performed a comparative genomic analysis of these three proteins. As we expected, the analysis revealed that their distributions are positively correlated in the Metazoa (106a) (FIGURE 3A). Namely, we found many species that conserve CatSper also possess sNHE and sAC, such as mammals, coelacanth, and brachiopoda (Lingula anatine), which is the only clade of protostomia that conserves CatSper. Conversely, many species lacking CatSper do not possess sNHE nor sAC, such as amphibian, zebrafish (Danio rerio), and fruit fly (Drosophila melanogaster). Although we predominantly observed simple, all-or-none patterns, we also found three intermediate groups: 1) ones lacking CatSper but conserving sNHE and sAC like ditrysia (butterflies and moths) and mollusca; 2) ones conserving only sAC, such as chicken (Gallus gallus) and placozoa; and 3) two primitive birds, Apteryx australis and Tinamus guttatus, which conserve CatSper and sAC without sNHE. Curiously, we did not find any species that conserve only CatSper or sNHE, or that conserve CatSper and sNHE without sAC. It is worth mentioning that we found sNHE that possesses a VSD only in Metazoa. Considering that the primitive animals porifera (sponges) conserve sNHE, this exchanger is supposed to have emerged at an early stage of Metazoan evolution as a modulator of CatSper together with sAC. Therefore, we propose that the three proteins form a prototypical machinery to regulate sperm motility in Metazoa (FIGURE 3B).

The current heterogeneous distribution of the mentioned prototypical motility machinery suggests that an alternative component(s) with a functional equivalence may substitute one or more of these three proteins independently in different taxonomic groups at distinct time points in Metazoa evolution. For example, the fruit fly conserves none of the three proteins, but the TRPP2/PKD2 channel is essential for appropriate sperm flagellar motility (65, 133). We speculate that sNHE and sAC may have become unnecessary once the TRPP2/PKD2 channel substituted the function of CatSper, because the regulation mode of the TRPP2/PKD2 channel is distinct from that of CatSper. On the other hand, if CatSper is substituted by a Ca²⁺ channel that depends on pH_i and/or cAMP (PKA), sNHE and sAC could remain even after the new Ca²⁺ channel has replaced CatSper. In this context, it is interesting to identify the Ca²⁺ channel responsible for the flagellar beat regulation in the first intermediate clades, ditrysia (butterflies and moths) and mollusca, because they lost CatSper but conserved sNHE and sAC. The genome analysis of the nematode Caenorhabditis elegans also supports the idea that all these proteins became unnecessary upon the male gamete conversion into aflagellate sperm. Despite these observations, the presence of the three genes in certain species cannot be explained by conservation of the original function of these proteins.

It is noteworthy that some key amino acids in the mammalian sNHE are not conserved in different species. For example, the arginine is missing in the phosphate-binding cassette of the CNBD of human sNHE (135). Also, positively charged amino acids in the S4 segment of the VSD are replaced by neutral amino acids in most mammals (135). Related to these observations, some mammalian sperm have other ways of regulating pH_i. Particularly, human sperm possess a significant activity of voltage-gated H⁺ channels (Hv) (72), which may functionally substitute sNHE. On the other hand, two bird species, Tinamus guttatus and Apteryx australis, conserve CatSper without having the sNHE gene as an exceptional case. Considering the relevance of pH_i in regulating CatSper, understanding this matter in these birds warrants more investigation. There is another interesting question about the presence of sNHE. We found three ray-finned fishes, Lepisosteus oculatus (spotted gar), Lucio europeo, and Salmo salar, conserve sNHE (106a). Curiously, all three species reproduce in freshwater, in which the Na⁺ concentration is not high enough to increase pH_i through Na⁺/H⁺ exchange. This fact suggests that the presence of sNHE in these species is not merely to control CatSper by pH_i elevation but also to play another unveiled role.

Besides CatSper, sNHE, and sAC, another key protein involved in sperm signaling is the sperm-specific K⁺ channel SLO3, which is essential for mammalian fertility (109a). SLO3 had been considered to be restricted to mammalian expression (109a); however, we recently reported that this channel is also present in the genome of terrestrial (birds and reptiles) and aquatic (fish) vertebrates, indicating that phylogenetic profiles of sperm-specific channels are intermittent throughout metazoan evolution (FIGURE 3A) (125a). Although detailed information is currently under investigation, we speculate that one of the functions of SLO3 is to activate sNHE by V_m hyperpolarization. We hypothesize that SLO3 is a substitute for tetraKCNG in the vertebrates. Actually, there is an interesting correlation of the appearance of SLO3 and disappearance of tetraKCNG in vertebrates (32, 125a). Although most vertebrates gained SLO3 (125a) and subsequently lost tetraKCNG, including humans (63), some vertebrates, such as Lepisosteus oculatus, still conserve both SLO3 and tetraKCNG (32, 125a). In this context, coelacanth is quite unique because it conserves tetraKCNG (32) but not SLO3 (125a). This species used to possess both tetraKCNG and SLO3, and then selected tetraKCNG (32). However, the fact that there are birds conserving SLO3, but not sNHE, may indicate that sNHE activation is not the only role of SLO3. Many experiments are required to test our hypothesis.

Capacitation

Capacitation is a process through which mammalian sperm acquire the ability to transit through the female genital tract, undergo hyperactivation, and become capable of acrosome reacting and fertilizing the female gamete (5, 18, 40). In response to a seminal plasma HCO₃⁻ raise (93) and female tract composition changes, mammalian sperm modify their phosphorylation state, undergo plasma membrane cholesterol removal (129), and increase their sAC activity (13), cyclic adenosine monophosphate (cAMP) content, and PKA catalysis (126a, 127). These responses lead to elevations in their pH_i (19a, 77a, 142) and [Ca²⁺]_i (107, 112), and, as shown in several mammalian species, make their V_m more negative (hyperpolarizing) (8, 28, 141). As mentioned earlier, the flagellar beating pattern of mammalian sperm becomes hyperactivated during this process (reviewed in Ref. 116).

Recently, it was reported that, during capacitation, bovine and mouse sperm extrude cAMP through multidrug resistance-associated protein 4 (MRP4 or ABCC4). MRP4 is a nucleotide transporter involved in downregulating intracellular cAMP levels. It has been proposed that extruded cAMP functions in an autocrine fashion outside sperm. Consistent with this hypothesis, in bovine sperm, it has been shown that external cAMP addition reverses the effects of MRP4 inhibition in bovine sperm hyperactivation, tyrosine phosphorylation, and acrosome reaction. As another species-specific difference, external cAMP does not affect mouse sperm (2a).

Interestingly, stallion sperm incubated in typical capacitating medium fail to display certain changes associated with this process in other mammals, such as hyperactivated motility, increased plasma membrane fluidity, and protein tyrosine phosphorylation. Even after conditions that allow these changes were identified, acrosome reaction and fertilization were not triggered during co-incubation with oocytes. Consistently, in stallion, it has not been possible to achieve reproducible in vitro fertilization, indicating that appropriate capacitation conditions have not yet been achieved in this species. This is a clear example of species difference where a deeper understanding of capacitation is required to benefit reproductive strategies, agriculture, and our general understanding of this process (68).

Sperm Energy Metabolism and Post-Testicular Remodeling

From the site of ejaculation to the site of fertilization, sperm must travel a large distance (which varies among species). This process demands energy, and it is noteworthy how different species use alternative strategies that vary substantially. As in somatic cells, sperm use glycolysis and oxidative phosphorylation as the main metabolic routes for ATP production. For instance, sea urchin sperm have only one large mitochondrion at the base of the head where the flagellum initiates. Oxidation of fatty acids, stored in lipid bodies at the base of the mitochondrion, is the main energy source (82). Notably, mitochondrial metabolism seems to be finely regulated by pH_i changes induced by speract, a chemotactic peptide from the egg envelope (38).

In mammalian sperm, the glycolytic enzymes are attached to the fibrous sheath in the flagellar principal piece, whereas oxidative phosphorylation takes place in the mitochondria in the midpiece. An extensive debate is ongoing as to which of these metabolic routes is preferred by sperm (99). Human and rodents depend heavily on glycolysis for ATP. In contrast, in bovine, glucose has an inhibitory effect (97), and in vitro capacitation is obtained in the complete absence of glycolytic substrates.

During spermatogenesis, a large part of the spermatids’s cytosol is phagocyted by Sertoli cells, leaving only a small amount of cytosol, known as the “cytoplasmic droplet” (FIGURE 1) (25). This droplet remains attached to the midpiece, and in some species it migrates (head to annulus direction) during further epididymal sperm maturation, and, in certain cases, it detaches from sperm (26). This droplet has been considered as a sign of immaturity and implicated in sperm volume regulation. Recently, this droplet has been considered a normal organelle named Hermes body (HB), which in rat sperm contains many glycolytic enzymes (49). In mouse sperm HB, >100 proteins were detected, 70% related to energy metabolism (140). These results suggest that the HB may contribute with ATP required for sperm motility (49). Furthermore, proteins involved in trafficking, translation, and budding of transport vesicles were also found in HB. These vesicles were proposed to transport glucose transporter 3 to the HB plasma membrane, increasing its glucose uptake and HB size. More detailed experiments are needed to fully understand the functions of this organelle (4, 19, 49).

The Acrosome Reaction

Most animal sperm must undergo the acrosome reaction to successfully fertilize the oocyte/egg, although teleost (zebrafish) sperm lost the acrosome (47, 53, 137). The acrosome is a single membrane delimited vesicle overlying the nucleus in the sperm head. Its luminal pH (pHa) is acidic and constitutes an intracellular Ca²⁺ store containing hydrolytic enzymes (51, 114). It is considered to be of lysosomal-related origin (10, 34). This unique and single exocytotic reaction is required for adequate relocalization of specific sperm fusion proteins essential for gamete fusion and/or facilitation of this cell’s journey toward the egg (14, 92). The exocytotic SNARE secretory machinery involved in the acrosome reaction is similar to that of other cells (78). Depending on the species, fusion and vesiculation at single or multiple points between the outer acrosomal membrane and the overlying plasma membrane occur in a Ca²⁺ dependent manner (9, 27). This exocytotic process results in the release of the acrosomal content, which includes enzymes that modify membrane components and possibly the external matrix (137).

The nature of the ligands that trigger the acrosome reaction among different species is diverse. In marine organisms like the sea urchin, the envelope surrounding the egg (egg jelly) contains a very large fucose sulfate polymer (126). On encountering this polymer, sea urchin sperm undergo the acrosome reaction that involves actin polymerization of a finger-like acrosomal process surrounded by plasma membrane covered by bindin, the first gamete recognition protein isolated that attaches the sperm to the vitelline layer (29, 51, 124). The fucose sulfate polymer binds to its receptor (suREJ1) in the head plasma membrane, just above the acrosome (29). The REJ module is a homolog of polycystin-1 (PKD1/PC1), a protein that is mutated in autosomal-dominant polycystic kidney disease (46a), the most common monogenic human ailment (30). This binding event triggers increases in [Ca²⁺]_i, [Na⁺]_i, pH_i, cAMP, IP3, and NAADP, and changes in V_m and protein phosphorylation, among others (29a, 90, 122, 123).

In sea urchin sperm, the [Ca²⁺]_i increase associated with the acrosome reaction involves at least three channels. A fast (seconds), transitory, and dihydropyridines-sensitive channel, a second pH_i-dependent channel stimulated by alkalinization, and the IP3 receptor in the acrosome. An additional channel, possibly of the store-operated type (SOC), could also participate (43, 45, 52). Surprisingly, until now, the identities of these channels have not been properly established, and it is not known how or whether CatSper participates in the sea urchin acrosome reaction. Furthermore, how binding of the fucose sulfate polymer to suREJ1 activates Ca²⁺ influx and whether a PKD1-PKD2 complex is involved are unknown.

The zona pellucida (ZP) surrounding the mammalian egg has been thought to be the site where sperm acrosome react. However, current findings in mouse fertilization suggest that sperm undergo this reaction before binding to ZP in the upper isthmus of the oviduct (59, 114). Therefore, at present, the physiological inductors of this exocytotic reaction in mammalian sperm and their site of action, which may be diverse among different species, is not established and constitutes an area of active research. As a matter of fact, there are interesting differences regarding ligands that induce the reaction in some species but not in others. For example, ATP triggers exocytosis in human (33) and rat (120) but not in mouse sperm (104). On the other hand, progesterone that is secreted by the cumulus cells triggers the acrosome reaction in human sperm (95), although significantly higher concentrations are needed [>5 µM (121)] than those that fully activate CatSper (0.5 µM). In mouse and rat sperm, even much higher concentrations are needed (50–100 µM) (105). How progesterone induces the acrosome reaction in mammals is not fully understood.

Differences, Similarities, and Oddities of Sperm Ion Channels

It is worth noting that the sperm-specific ion channels CatSper and SLO3 are pH_i dependent, with a strength that varies with the species. Both channels are more pH_i sensitive in sperm from mouse than from human (20, 69, 75, 80, 88), and CatSper is extremely pH_i sensitive in sea urchin sperm (42, 110). Since pH_i is fundamental for sperm physiology, there are species-specific mechanisms that regulate it. Human sperm express significant amounts of functional Hv channels, whereas their sNHE activity is very low (72). In contrast, sea urchin sperm highly express sNHE (Ref. 89; reviewed in Ref. 106a). Recent studies have pointed out that the expression of somatic NHE8 isoform is essential for acrosome formation, and its absence results in infertility (91).

CatSper and Hv are weakly voltage sensitive. Depending on the species, certain channels must open [TRPV4 (86)] or close (SLO3) to promote the needed depolarization for their activation. On the other hand, hyperpolarization is fundamental in sea urchin and certain mammalian sperm (mouse and horse), but its importance is not so clear in some species, such as human.

Due to the low K⁺ selectivity of SLO3, it is still not clear whether this is the only K⁺ channel responsible for the hyperpolarization that accompanies mammalian sperm capacitation. In addition, the sensitivity to Ca²⁺ of SLO3 in humans has cast some doubt regarding the possible presence of SLO1, a Ca²⁺-dependent K⁺ channel from the SLO family in human (11, 75, 76) and in sperm (De la Vega-Beltrán JL, Orta G, Darszon A, unpublished observations).

Perspectives

Many eukaryotic genome sequences of distinct taxa have been reported in the last decade, and there is a variety of useful programs available to perform bioinformatic analyses. Making use of the current genome information, we have found sperm functional commonalities in two species belonging to distant taxa, as well as differences within two closely related taxa. Interesting questions arise from comparative genomic analysis; thus experiments addressing them in different species are necessary to better understand sperm physiology.

Recently, the role in sperm physiology of exosomes (vesicles 30–200 nm) (125) from the epididymis (epididymosomes), the prostate (prostasome) (108), and the female genital tract (oviductosomes) (2, 35), among others, has been recognized. These vesicles can transfer different proteins, RNAs, and metabolites to sperm during their journey toward the egg influencing their functional state and even transmit information beyond fertilization (60). Future studies should unravel the possibly important functional implications of these vesicles. In this regard, caution should be taken from experiments using epididymal sperm that will not be exposed to other exosomes.

A surprising wealth of differences has emerged in recent years regarding the signaling pathways involved in the conversations sperm from different species must conduct in search of the female gamete. This astonishment possibly arises from our limited consideration of the full and unique physiological setting that is required for fertilization, which we need to understand much better. External fertilizers have to deal with the particular physicochemical characteristics of the media into which the gametes are released and where fertilization must happen, which in itself can be quite diverse in composition, hydrodynamics, and temperature. Internal fertilizers display an amazing variety of female tract topologies with distinct cell components that determine the composition and physical properties of the fluid where sperm must travel to find the egg. In this light, it is the conserved mechanisms that are amazing; how the exquisite regulation in time and location of Ca²⁺ and H⁺ concentrations, and other second messengers orchestrate, using a varied but limited set of molecules suitable for the species-specific task, the miracle of fertilization.