V. Physiological Considerations

Table of Contents

• I. Introduction
• II. Origin of the Spermatozoon
• III. Epididymal Transit and Maturation
• IV. Spermatozoal Structure: Form to Function
• V. Physiological Considerations
• VI. Clinical Considerations: Semen Evaluation
• VII. Concluding Remarks

Spermatozoal Motility

Under natural conditions, regulation of spermatozoal motility occurs at three critical points: epididymal reservoir (cauda epididymis and ductus deferens)-suppression of motility; ejaculation-activation of motility; and oviductal reservoir-hyperactivation of motility. Spermatozoa in the cauda epididymis are intrinsically capable of motility,68,126 but do not exhibit motility until released from the epididymis. A threshold level of cyclic adenosine monophosphate (cAMP) is present in spermatozoa within the cauda epididymis [127]. Specific motility-inhibiting proteins have been identified in rat cauda epididymal fluid that, when removed, allow initiation of motility [128,129]. A pH-dependent inhibitory factor has been reported in bulls [130,131]. Although such inhibitory factors may exist, it is possible that sperm motility may simply be suppressed by the acidic pH of the epididymal environment. The pH of bull cauda epididymal fluid is reported to be 5.5, and the cytosolic pH of bull epididymal spermatozoa is reported to be 6.5 - 6.6 [130,132]. Caudal epididymal fluid of bulls, rams, boars, and stallions does not contain measurable quantities of bicarbonate (HCO3 -) [133], and HCO3 - is known to be a key effector of spermatozoal motility [134,135]. Bicarbonate is present at fairly high concentrations in seminal plasma and may be higher in seminal plasma of stallions than some other mammals studied [136]. Simple exposure of normal spermatozoa to seminal plasma or physiologic fluids will activate spermatozoal motility that is characterized by a moderate-amplitude and symmetrical flagellar beat leading to a forward propulsive trajectory [137,138]. This form of spermatozoal movement is defined as activated motility and is to be differentiated from hyperactivated motility discussed below. Activated motility is considered necessary for propelling sperm into the oviductal reservoir.

Figure 28. Transmission electron micrographs of spermatozoa treated with the ionic detergent, sodium codicil sulfate, from the proximal corpus epididymidis (A) and distal corpus epididymidis (B). (A) Spermatozoa from the testis to the proximal corpus epididymidis have little resistance to detergent treatment, as noted by loss of all tail components and membranes and the decondensation of the nucleus (DN). (B) Spermatozoa from the distal corpus epididymidis, through the cauda epididymidis and in the ejaculate have acquired some resistance to this detergent treatment during maturation in the epididymidis as noted by the presence of the outer membrane of mitochondria (OM), the outer dense fibers (DF) of the tail, and nucleus (N) that have not decondensed. Scale bars = 0.6 (A) and 1 µm (B) [68].

Figure 29. Drawings showing magnified views of an equine spermatozoon, represented by an uncut view (center), a mid-sagittal view (left), and a partially resected view (right). The various lengthwise divisions of the spermatozoon are represented as head, flagellum (tail), midpiece, principal piece, and end piece (end). (A) Acrosome. (B) Plasma membrane. (C) Nucleus. (D) Mitochondria. (E) Axoneme. (F) Outer dense fiber. (G) Fibrous sheath. (H) Axonemal microtubules.

Environmental cues activate spermatozoal motility though a signal transduction mechanism. Although the signaling pathway(s) of activated spermatozoal motility are not resolved completely, an ever-increasing body of information indicates that HCO3 -, calcium ions (Ca2+), and cAMP are key signaling components [112,139]. Transmembrane movement of HCO3 - into the spermatozoal cytosol is associated with an increase intracellular pH ([pH]i), leading to regulation of cAMP [140,141]. Activity of Na,K-ATPase and Na+/K+exchangers are also of central importance in regulating [pH]i and sperm motility [142-146]. Spermatozoal Na,K-ATPase is important for generation of the Na+ gradient required for ion transport. This Na+ gradient permits the Na+/K+ exchangers to catalyze the coupled exchange of extracellular Na+ for intracellular H+ [143]. A sperm-specific Na+/K+ exchanger has been localized to the principal piece and likely plays an important role in regulating [pH]i [142,145].Activation of motility requires that this exchanger is functional [145]. It is also likely that a Na+/HCO3 - cotransporter plays a role in controlling cytosolic pH (Fig. 34) [140].

Although increased alkalinization of the spermatozoal cytosol is known to activate membrane Ca2+ channels, this may be of primary importance in hyperactivation of spermatozoal motility where increased cytosolic Ca2+ is required [141,147-152]. Spermatozoal motility can be activated and maintained for a short time in Ca2+-free media for many species [126,138,153], but presence of extracellular Ca2+maximizes spermatozoal motility [154-156]. The flagellum is known to contain a variety of Ca2+ membrane transport channels, including voltage-gated, cyclic nucleotide- gated, transient receptor potential, Ca2+ release, and CatSper channels remain unknown, their mere presence suggests that they probably contribute in some manner. Sperm [Ca2+]i is also known to be reg-channels. Although the roles of some of these ulated by Ca2+-ATPases, Na+/H+ exchangers, and Ca2+/H+ exchangers [157].

Figure 30. Phase contrast (J), scanning electron (B), and transmission electron (A and C-I) micrographs of equine spermatozoa. (A) The plasma membrane (PM) encloses the entire head and tail. The head is composed of the nucleus (N), the overlying acrosome (Ac), and postacrosomal region (PR). The acrosome can be divided into the apical segment (ASA), principal segment (PSA), and equatorial segment (ESA). The inner acrosomal membrane (IAM) is in juxtaposition with the nuclear membrane (NM). The outer acrosomal membrane (OAM) fuses with the plasma membrane during the acrosome reaction to discharge the acrosomal contents and expose molecules attached to the inner acrosomal membrane. (J) The tail is composed of the middle piece (MP) that houses the mitochondria (M), the principal piece (PP) that contains the fibrous sheath (FS), and the end piece (EP). (C) The tail attaches at the implantation fossa (IF) (D-I) The distal centriole gives rise to the "9+2"-arranged microtubular complex of the axoneme, with the nine outer doublets (DIs) surrounding the central pair (CP) of microtubules. The nine dense fibers (DFs) parallel the axoneme and extend to different lengths within the principal piece. In the end piece, the axonemal doublets become disorganized and ultimately separate into 20 single microtubules (SM), but still are enclosed in the plasma membrane. The cytoplasmic droplet (CD), located at the proximal end of the middle piece in spermatozoa recovered from the equine efferent ducts (B and C) is characteristic of spermatozoa that have not yet matured in the epididymis. These cytoplasmic droplets contain remnants of spermitid cytoplasm that are not lost in the residual body on spermiation. Contents may include endoplasmic reticulum, mitochondria, and microtubules, including remnants of manchettes. Scale bars = 0.5 (A and B), 0.75 (C), 0.24 (D-I), and 1.28 µm (J) [19,26].

Figure 31. Further magnified illustrations of Fig. 29, revealing mid-sagittal and partially resected views of equine spermatozoa. (A) Plasma membrane. (B) Acrosome. (C) Outer acrosomal membrane. (D) Inner acrosomal membrane. (E) Nuclear envelope. (F) Post-acrosomal lamina. (G) Nucleus. (H) Basal lamina and underlying capitulum. (I) Proximal centriole. (J) Segmented column. (K) Outer dense fibers. (L) Outer doublets of axoneme. (M) Center pair of microtubules within the axoneme. (N) Mitochondria [14].

The signaling pathway for activated spermatozoal motility involves the cAMP-dependent protein kinase A (PKA) pathway [156,158-165]. Intracellular production of cAMP, as it relates to motility, is catalyzed by soluble adenylyl cyclase (sAC). sAC has been identified in the flagellum of spermatozoa and is required for activated motility [166] and capacitation (see below). Interestly, the cAMP-sAC-PKA pathway does not seem to be required for hyperactivated motility [150] or the acrosome reaction [166]. sAC is stimulated directly by HCO3 -[167,168] or Ca2+ [169,170], thereby catalyzing phosphorylation of PKA, which leads to phosphorylation of flagellar proteins that are central to activated motility [138,171-174]. One such protein is A kinase anchoring protein (AKAP). AKAP has been detected at the level of the axonemal central pair, the fibrous sheath (in the principal piece), and the outer dense fibers (in the middle piece) [112,138,171,175,176]. PKA is known to bind to AKAP, thus suggesting that the action of PKA can be regionalized, and its substrate specificity aided, through this anchoring mechanism [112,138,171,177,178]. Although PKA is known to be located in the vicinity of the outer dynein arms [175], the need for a localized pattern of PKA distribution remains unresolved [179].

Figure 32. Transmission electron micrograph revealing sagittal views of adjacent equine spermatozoal heads. One spermatozoon has an intact acrosome (solid arrow) and one has undergone the acrosome reaction induced by the calcium ionophore, A23187 (open arrow).

Figure 33. Magnified illustrations of cross-sections through the middle piece (midpiece) and principal piece regions of equine spermatozoa. (A) Plasma membrane. (B) Mitochondria. (C) Outer dense fiber. (D) Axonemal doublet. (E) Central pair of axonemal microtubules. (F) Sheath surrounding central pair of axonemal microtubules. (G) Radial arm. (H) Fibrous sheath. (I) Outer dynein arm. (J) Inner dynein arm. (K) Longitudinal column of fibrous sheath. (L) Connecting bridge between central pair of axonemal microtubules. (M) Nexin links. (N) Annulus.

Figure 34. A schematic model of some documented and hypothesized molecular events associated with activated and hyperactivated forms of spermatozoal motility. Please refer to the text for an explanation of these signaling pathways and for figure abbreviations.

In addition to regulating the cAMP-sAC-PKA pathway, Ca2+ affects dynein activity through direct control of the central pair of microtubules within the axoneme [180]. As Ca2+ can inhibit the activity of the axonemal central pair to regulate dynein arm function [180], excessive Ca2+ can lead to cessation of spermatozoal motility [181].

Calmodulin (CaM), a Ca2+-binding and sensor protein, is a key component of spermatozoal signaling mechanisms [181-186]. CaM associates with the outer dynein arms and the radial spokes, and may play a role in Ca2+-dependant propagation of flagellar bending [172,175]. CaM kinase II (CaMK) [151] and CaM-dependent protein phosphatase [172,187,188] are central to dynein function. One study indicated that CaM can also interact with T-type voltage-gated Ca2+ channels by a mechanism independent of PKA, CaMK, or CaM-dependent protein phosphatase [189]. In addition, CaM may be involved in regulation of adenylyl cyclase [182].

A substantial and continuous supply of energy, in the form of ATP, is required for activated spermatozoal motility, and this requirement is heightened for hyperactivated motility [150]. The mechanisms by which ATP is generated and transferred in the flagellum remain unsolved. Certainly, oxidative respiration within the mitochondria yields a plentiful supply of ATP. Some investigators propose that the ATP produced in this manner is capable of diffusing along the entire length of the flagellum in a manner suitable for initiation and maintenance of spermatozoal motility [138,190-192]. Possibly, an adenylate kinase shuttle may assist in the rapid distribution of mitochondrial-produced ATP [192]. Recently, adenylate kinases were regionalized to the principal piece of mice spermatozoa [193]. Others contend that local glycolysis within the principal piece is critical to the timely generation and distribution of ATP required for spermatozoal motility [171,194,195]. Certainly, a variety of glycolytic enzymes have been identified in association with the fibrous sheath and/or outer dense fibers [196-202], and it is well known that the glycolytic pathway to energy production is essential under anaerobic conditions. Arguments in favor of oxidative res-piration and of glycolysis exist for generation and supply of ATP for spermatozoal motility. A sperm-specific glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), has been localized to the principal piece, and targeted deletion of this enzyme is reported to lead to a severe reduction in motility of mouse spermatozoa [194]. In contrast, another study revealed that chemical inhibition of GAPDHs did not impact spermatozoal motility or ATP concentration [192,203]. At present, it seems possible that either oxidative respiration or glycolysis, or both, can support the ATP generation and availability needed to drive spermatozoal motility. The length of the principal piece in stallions is similar to that in bulls, boars, rams, and dogs. It is also considerably shorter than the principal piece of mice, rats, hamsters, and guinea pigs, where many experimental studies are conducted [113,192]. This may explain some of the inconsistencies among laboratories.

The axoneme, dynein arms, outer dense fibers, mitochondrial sheath, and fibrous sheath are often portrayed as the fundamental elements of the flagellum. Although each is vital to flagellar function, one must also be aware that these elements are embedded in a network of other molecules that are equally important. Bending of the flagellum is caused by reciprocal sliding between doublet microtubules within the axoneme. The dynein arms are permanently attached to one doublet microtubule with arms extending to intermittently engage an adjacent doublet microtubule. The dynein arms are situated at 24-(outer dynein arms) to 96-nm (inner dynein arms) intervals along the entire length of the axoneme [204,205]. When the cross-bridges created by the dynein arms are complete between neighboring doublets, the corresponding microtubules are prevented from sliding. Dynein is high molecular weight ATPase, so the dynein arms have the capacity to transform chemical energy into unidirectional mechanical force. Each dynein arm has three ATP-sensitive binding sites, and all sites must bind with ATP for the dynein arm to detach from the microtubule [206], i.e., the crossbridge attachment-detachment cycle is dependent on binding and hydrolysis of ATP. The conformational change in the dynein arms results in the force generation required for microtubule sliding. The microtubules of the axoneme enhance the rate of ADP release after the dephosphorylation to aid the reactivation step [207]. The nexin arms also undergo periods of displacement to allow sliding of microtubules [137]. The basic mechanism is similar to that of muscle myosin; however, the loss and rebinding of ATP products occurs at two to three orders of magnitude faster for dynein than for myosin [208]. Bending of the tail occurs during axonemal sliding because the microtubules are fixed at the base of the flagellum [209]. The passive elastic nature of the overlying outer dense fibers and fibrous sheath permits the flagellum to bend, and also provides elastic recoil [210,211].

Bending of the flagellum requires a patterned activation of microtubule sliding around the circumference, and along the length, of the cylindrical array of microtubules that compose the axoneme. This dynein-generated activity is regulated by the central pair of microtubules and their associated structures, collectively called the central apparatus. The asymmetry of components in the central apparatus forms the basis of a "timing device" that creates the spatially timed sliding of various microtubules [180,212]. As such, the central apparatus both constrains and activates the dynein arms through communication through the radial spokes [137,213].

Spermatozoal Metabolism

Spermatozoa require a constant supply of energy for maintenance of cellular order and functions needed for survival. This energy requirement increases significantly with the onset of activated motility [214,215], and becomes even more pronounced when hyperactivated motility is initiated [150]. Approximately 500 common metabolic reactions are known to occur in most somatic cells, many of which require energy [216]. A spermatozoon is a "stripped-down" cell that is designed expressly for propagation of genes, and it is one of the smallest cells in the body, but it accomplishes its mission at a distance far from its origin in the epididymis. While spermatozoa require a relatively small amount of energy for "housekeeping" purposes [197,214], a plentiful supply of substrate is required to provide a spermatozoon with the stamina required for this arduous journey. Spermatozoa are also capable of prolonged survival both within and outside the body. Exogenously derived nutrients are needed to fulfill the energy demands of these very metabolically active cells. These nutrients (or substrates) are metabolized intracellularly, resulting in the release of chemical bond energy. Useable energy is made available for cellular processes primarily in the form of the activated carrier molecule, ATP, with other macromolecules such as NADH, NADPH, FADH2, and acetyl CoA also providing vital high-energy intracellular linkages [216,217].

Spermatozoa possess the metabolic machinery required for glycolysis, the citric acid cycle, and oxidative physphorylation [218]. Glycolysis occurs in the cytosol and has both an anaerobic and aerobic mode [219], whereas the citric acid cycle and oxidative phosphorylation are strictly aerobic and proceed within the mitochondria A variety of substrates can be garnered for energy extraction, including monosaccharides, pyruvic acid, lactic acid, and even fatty acids and amino acids, the last four of which require an aerobic environment. Sorbitol, a sugar alcohol obtained by reduction of glucose through the polyol pathway, is thought to serve as a substrate for metabolism [218]. Although the seminal plasma of stallions does contain this product,136 stallion spermatozoa seem to be incapable of metabolizing it to produce ATP [218,220].

Generally speaking, glycolysis is considered the "backbone" of the energy-procurement pathways because it can proceed in situations of low oxygen tension and its product, pyruvate, can be further catabolized to CO2 and H2O by oxidative respiration. Efficiency of energy production is lowest in the glycolysis pathway, where one molecule of a monosaccharide can generate a net increase of two molecules of ATP. Conversely, complete oxidation of glucose through the citric acid cycle and oxidative phosphorylation generates an additional 34 molecules of ATP per monosaccharide molecule. Despite the increased efficiency of oxidative metabolism, a monosaccharide cannot directly enter the citric acid cycle, but must first be metabolized to pyruvate; hence, the need for glycolysis as the first stage of energy production where monosaccharides are concerned [216].

Glycolysis involves a sequence of 10 separate reactions, the first of which is catalyzed by hexokinase in an ATP-consuming manner. This enzyme can phosphorylate several types of monosaccharides, including glucose, fructose, mannose, and galactose. The affinity of hexokinase for glucose is reported to be 20 times higher than its affinity for fructose [221]. Another account indicated that, in mammalian spermatozoa, glucose is used in preference to fructose, but that mannose has the highest affinity of the three monosaccharides to hexokinase [218]. Of interest, a study involving astroglial cells revealed that mannose-phosphorylating ability is only 40% of glucose-phosphorylating ability [222].

Fructose was identified as the "seminal sugar" in 1946 [223]. Subsequent studies have revealed that the concentration of this sugar is high in seminal plasma of bulls, rams, and man (range of 40 - 1000 mg/100 ml) but extremely low (<1 mg/100 ml) in seminal plasma of stallions [218]. Interestingly, another report indicated that fructose concentration in stallion semen ranged from 7 to 11 mg/100 ml [224]. Seminal plasma concentrations of some other metabolizable substrates, such as citric acid, lactic acid, and pyruvic acid (used exclusively by oxidative respiration), are also much lower in stallion seminal plasma than that of bulls, rams, or man [218]. Bull and ram spermatozoa use fructose at a rate of 2 mg/109 spermatozoa/h under anaerobic conditions at 37°C, whereas boar spermatozoa use fructose at a rate that is 10 times lower [218]. One report suggests that stallion spermatozoa have a limited capacity to use fructose [220]. Interestingly, fructose is not found in seminal plasma of dogs and glucose is used preferentially to fructose by dog spermatozoa; yet, fructose maintains higher spermatozoal motility than does glucose under in vitro storage conditions [225]. Heterogeneity may also exist among motile dog spermatozoa, with subpopulations experiencing increased responsiveness to both fructose and glucose [226].

Either glucose or fructose can increase motility and ATP concentration of human spermatozoa [227]. One study with human semen revealed that spermatozoal motility was maximized in medium containing 1 mM glucose or 15 mM fructose; however, motility was further enhanced when both monosaccharides were provided [228]. Conversely, in vitro fertilization in mice and rats is not attainable when fructose is the only available substrate but high when glucose is included in the media [229-232]. Other studies certainly support the importance of glucose in gamete interaction [233-235].

Transport of monosaccharides across the plasma membrane requires the presence of carrier proteins and is an ATP-dependent process. One report involving bull spermatozoa indicated that the glucose transport protein has only a limited ability to transport fructose and that another carrier protein may be the primary means by which fructose gains access into the cytosol [214,236]. This carrier protein may be absent in stallion spermatozoa, thus explaining why fructose may not be a primary, or even a suitable, energy source in this species [214].

The extent to which spermatozoa use glycolysis or oxidative respiration for generation of energy varies considerably among mammalian species and probably with the profile of spermatozoal motility, and environmental conditions [214,218]. Few studies have been directed toward stallion spermatozoa so much of the information must be extrapolated from information garnered from other species. This approach may be precarious, however, because of the known species variation in spermatozoal metabolic preferences. One report suggests that bull and ram spermatozoa use the glyocolytic pathway at a much higher rate than stallion spermatozoa and that stallion spermatozoa rely more heavily on aerobic metabolism for energy production [214,220]. Others report that ram and bull spermatozoa oxidize acetic acid in preference to fructose and glucose [218]. Human, boar, and mouse spermatozoa seem to obtain a high proportion of their ATP by glycolysis [237-239]. Although more intensive study with stallion spermatozoa is required to more fully elucidate metabolic pathway preferences, glycolytic breakdown of glucose is currently considered to be a major source of ATP production [214].

The minimal oxygen tension required to maintain a linear rate of O2 uptake by rabbit spermatozoa has been reported to be ≈10 mmHg [218]. As such, the luminal environments of the uterus and oviduct are thought to be sufficiently high in oxygen content to allow aerobic respiration to proceed [218,240,241]. Storage of spermatozoa outside the body cavity, however, can impact availability of oxygen and metabolic processes. If raw or extended semen is left undisturbed in a laboratory setting, use of dissolved O2 by aerobic respiration leads to depletion of O2 and the need to resort to glycolysis for meeting energy demands [214]. For oxidative metabolism, bull and ram spermatozoa use O2 at a rate of ≈10 - 20 µl/h/108 spermatozoa at 37°C [218]. A potential drawback of oxidative respiration is the production of reactive oxygen species that can lead to a disruption of ATP production, lipid peroxidation, or other cellular mechanisms that result in spermatozoal dysfunction [242-246].

Metabolism of endogenous substrates by spermatozoa for the production of energy is thought to be minimal, possibly representing 10% or less of total energy production, based on calorimetric and carbon balance techniques [247]. For bull spermatozoa, endogenous metabolism occurs at a constant rate at storage temperatures between 20°C and 35°C [248].

Spermatozoal capability for glycogen synthesis and storage was once thought to be nonexistent. Recent reports, however, indicate that spermatozoa of dogs, boars, rams, and horses do contain glycogen, as well as the enzymatic machinery required for its production [192,249,250].

The pentose phosphate pathway does not seem to be directly involved with spermatozoal motility [218], but may be central to sperm-oocyte interactions [251], possibly through production of NADPH and the resultant generation of radical oxygen species required for sperm-oocyte fusion [252]. Furthermore, the pentose phosphate pathway may aid in protection against oxidative injury through generation of NADPH, which is required to return expended glutathione peroxidase to its reduced (protective) form (see Reactive Oxygen Species) [253,254].

Reactive Oxygen Species

A discussion directed at reactive oxygen species (ROS) at this point in the text seems appropriate because of the pathologic consequences that ROS can have on spermatozoal motility and spermatozoal metabolism. Reactive oxygen species are products derived from the reduction of (i.e., addition of electrons to) diatomic oxygen (O2) and include radicals and other reactive products. Radicals are atomic or molecular species that have unpaired electrons in their orbits, thus making them quite unstable, i.e., highly reactive and likely to participate in a variety of chemical reactions to displace, receive, or share electrons so that they may once again become stable [255]. Other O2 derivatives are not radicals but are highly reactive; hence, these products are collectively termed ROS. Examples of ROS (where • implies the presence of an unpaired electron) include superoxide radical (O2 -•), hydroxyl radical (OH•), hydroperoxyl radical (HO2 •), peroxyl radical (RO2 •), alkoxyl radical (RO•), hydrogen peroxide (H2O2), and subclasses of ROS that contain reactive nitrogen or chlorine species, such as nitric oxide (NO•), peroxynitrite (ONOO-; where NO• + O2 -• → ONOO-), or hypochlorous acid (HOCl) [255]. ROS may assume physiologic roles, as discussed under Capacitation; however, their presence can also lead to spermatozoal demise [66,256-260].

Spermatozoa are exposed to ROS that are derived both intracellularly and extracellularly. Production of superoxide occurs continuously within the mitochondrial electron transport chain, where O2 acts as an electron carrier during oxidative respiration. A molecule of O2must pick up a total of four electrons to form water (i.e.,O2 + 4H+ + 4e- → 2 H2O). During this process, the reactive oxygen forms are generally caged within the respiratory enzyme complexes. A small percentage of the superoxide produced does not stay within the mitochondrial respiratory chain but escapes to exert actions on other cellular components, including lipids, sugars, proteins, and nucleic acids [255,258,261-264].

Recently, presumed non-mitochondrial production of O2 -• has been shown to occur in spermatozoa of both men [265] and stallions [266]. Evidence is mounting that spermatozoa contain NADPH oxidase that catalyzes the formation of superoxide, presumably for physiologic reasons [267-270], although some workers conclude that spermatozoal-derived NADPH oxidase activity is insignificant [254,271,272]. An aromatic amino acid oxidase, released from dead spermatozoa or present in seminal plasma, has also been reported to generate production of H2O2 production in semen [273-276].

Generation of ROS is known to be more pronounced in morphologically abnormal spermatozoa, a feature that can be associated with retention of excessive amounts of residual cytoplasm [258,277-281]. Damaged or morphologically abnormal equine spermatozoa are known to generate more ROS than do morphologically normal sperm [282].

Neutrophils are known to be a primary exogenous source of ROS in semen of men, where a relatively high concentration of leukocytes is common place [283,284]. Although spiking equine semen with activated neutrophils can lead to reduced motility [285], contamination of equine semen with sufficient neutrophils to affect spermatozoal motility is quite uncommon.

Another source of ROS is the uterine environment, such as the ROS generated from estrogen-induced uterine NADPH oxidase of the endometrial epithelium [276,286]. Cycle-regulated synthesis of uterine NO has also been described [287,288]. Leukocytes of mares produce H2O2 in response to spermatozoa or bacteria, and this activity is heightened by leukocyte exposure to seminal plasma [289], suggesting a means for uterine clearance of bacteria and spermatozoa. Others, however, report a protective effect of seminal plasma against ROS [280,290,291]. Seminal plasma of some subfertile men is known to possess reduced antioxidant capacity [292,293].

Unsaturated lipids are quite susceptible to peroxidative injury when exposed to various ROS, and mammalian spermatozoa are especially susceptible because their plasma membranes are rich in polyunsaturated fatty acids [242,245,257,294-299]. A recent report indicates that the spermatozoa of individual men vary in their membranous unsaturated fatty acid content and that a superabundance of polyunsaturated fatty acids predisposes spermatozoa to oxidative injury [300]. Peroxidative injury has been thought to adversely affect membrane fluidity [258], although some work suggests that the oxidative action may not occur through lipid peroxidation [301]. Oxidation may directly affect proteins and membrane permeabilization (possibly through oxidation of sulphydryl groups on enzymes and membrane proteins), as opposed to disturbing lipid fluidity [302]. Others have described an effect of ROS on the sperm axoneme and ATP generation that can lead to an irreversible loss of motility [303,304].

In stallion spermatozoa, a ROS-associated reduction in motility was not associated with a detectable increase in lipid peroxidation or a decrease in viability or mitochondrial membrane potential [260]. This finding suggests that ROS-related effects on equine spermatozoa may occur through a mechanism unrelated to lipid peroxidation and also indicates that motility may be a more sensitive indicator of ROS-related injury than the other experimental endpoints, i.e., viability, mitochondrial membrane potential, and lipid peroxidation. Others report that lipid peroxidation is a good predictor of sperm motility and consider a lipid peroxidation assay to be a potential clinical test [305].

The mechanisms of ROS-induced damage to DNA have been evaluated intently [306]. Sperm DNA is known to be susceptible to oxidative injury, resulting in reduced fertility and perhaps even pregnancy loss or a variety of pathologic entities in off spring [307]. Aberrant DNA repair by spermatozoa is thought to increase the mutagenic load of any resulting conceptus, thereby leading to post-fertilization crises [281,308]. Mitochondrial DNA is more susceptible to H2O2-induced damage than is nuclear DNA, possibly making it a good marker of oxidative injury [309]. Spermatozoa of stallions are susceptible to ROS-induced DNA fragmentation [310].

Protection against the effects of ROS in spermatozoa is afforded by an assortment of scavenging molecules, including three enzyme systems: (1) superoxide dismutase (SOD), (2) catalase (CAT), and (3) the glutathione peroxidase system (GPX), as indicated by the following reactions:

O2 -• + O2 -•+ 2H+ → H2O2 + O2   (1)

                               SOD

2 H202 → 2 H2O + O2  (2)

            CAT

2 GSH + H2O2 → GSSG + 2 H2O  (3)

where GSH represents monomeric glutathione and GSSG represents oxidized glutathione disulfide.

Glutathione reductase (GRD) reduces GSSG to GSH -to complete the cycle as follows:

GSSG + NADPH + H+ → 2 GSH + NADP+

                                 GRD

Superoxide dismutase is considered to be prevalent in spermatozoa of many species [261,311-313] and rapidly reduces O2 -• to H2O2. The direct cellular action of O2 -• is thought to be minimal, with its major effects produced indirectly through conversion to H2O2 [243,256,260,274,294,301,307,310,314] or other radicals [255]. Spermatozoa of stallions are thought to have limited intrinsic SOD, with SOD activity derived primarily from the seminal plasma through adsorption to the plasma membrane [315].

Glutathione peroxidase has also been identified in spermatozoa or seminal plasma [311],[313,316] and is considered to impart protection against oxidative injury incurred by H2O2 [316,317]; however, a primary action of glutathione may be protection against peroxyl radicals of polyunsaturated fatty acids (RO2 •) through conversion to relatively inert hydroxyl fatty acids [313,318]. Glutathione also plays a structural role, as shown by sulfhydryl oxidation and cross-linking associated with nuclear condensation and assembly of the midpiece [317,319]. Glutathione is present in stallion semen [315,320], but the source in semen seems to be primarily of seminal plasma origin [315].

Spermatozoa from most species contain little to no catalase [243,245,321-324]; however, seminal plasma is generally high in catalase activity [67]. The amount of catalase in rabbit semen is genetically influenced (estimated heritability value of 0.48) [325]. Stallion semen contains catalase activity [311,326] that is primarily derived from prostatic secretions [326]. Several studies have shown that addition of catalase to semen of various species will help neutralize the detrimental effects of H2O2 on spermatozoa after in vitro storage [260,285,290,301,310,327-330]. Similarly, oviductal fluids may contain catalase in sufficient quantity to protect sperm from oxidative injury [330,331]. Surprisingly, stallion semen with higher initial activity of CAT, SOD, and GSH did not lead to improved retention of spermatozoal motility or membrane integrity after cooled storage [311]. Addition of CAT to extended equine semen did not improve maintenance of motility after cooled storage [332]. Similarly, addition of CAT, SOD, or GSH, ascorbic acid, or α-tocopherol to semen extender did not improve post-thaw quality of stallion spermatozoa [333]. Studies with ram spermatozoa suggest that CAT, SOD, and GSH are ineffective at preventing acute peroxidative injury to lipids [302,334], and incorporation of CAT in extenders for sex-sorted or non-sorted semen does not improve post-thaw quality of ram spermatozoa [335].

Overall, spermatozoa have limited endogenous CAT, SOD, and GPX activity; therefore, spermatozoa depend on extracellular availability of these enzymes to counter oxidative stress, namely from seminal plasma. Seminal plasma contains these enzymes, in addition to a host of other free radical scavengers [276]. Molecules with antioxidant activity include, but are not limited to, ascorbic acid [336,337], α-tocopherol [246,334,336,338-340], taurine [341,342], hypotaurine [342], butylated hydroxyanisole (BHA; an antioxidant used for long-term preservation of food, cosmetics, and pharmaceuticals) [246,334,339], albumin [261,342,343], cysteine [344,345], lipoic acid [346-348], xanthurenic acid [349-351], carnitine [352,353], ergothioneine [354], and pyruvate [349,355]. Many of these have been used as dietary supplements, or as direct semen treatments, with mixed results [267,299,332,340,356-358].

As the end product of glycolysis, pyruvate serves as an important substrate for oxidative phosphorylation, but when present at relatively high concentrations, pyruvate can also exhibit antioxidant properties [355,359]. Addition of pyruvate to milk-based extender at a concentration of 2 mM is reported to improve equine spermatozoal motility after cooled storage [349].

Presently, there is much controversy over types, doses, and delivery methods for antioxidants as they relate to semen processing. Further study is required to determine whether antioxidant therapy will maximize spermatozoal function, especially after cooled or frozen storage.

Spermatozoal Transport

Spermatozoa are virtually immotile in the epididymis but develop motility (or, more precisely, activated motility) on ejaculation. Deposition of spermatozoa is intrauterine when artificial insemination is used, and a large portion of an ejaculate is deposited directly into the uterine body at the time of natural coitus if the mare’s cervix is dilated at the time of breeding and if the stallion does not dismount prematurely during the ejaculatory process. After intrauterine deposition of semen, the spermatozoa are rapidly transported to the oviduct, where a spermatozoal reservoir forms and the spermatozoa gain fertilizing potential before interaction with the vestments of the oocyte near the ampullar-isthmic junction.

Spermatozoal migration to the oviducts is dependent, to a large part, on uterine contractions [360]. The effect of insemination volume on the frequency of these uterine contractions seems variable [361,362 but, within the range tested (5 - 50 × 106 spermatozoa/ml), spermatozoal concentration had no apparent effect on spermatozoal numbers recovered from mare oviducts at 4 hours after insemination [363].

The time required for spermatozoa to gain access into the oviduct has not been studied extensively. Spermatozoa have been detected in the oviducts as early as 2 h after insemination, based on recovery of spermatozoa in abattoir specimens [364]. Sufficient spermatozoa to establish pregnancy may be transported into the oviduct as early as 30 min after insemination, based on extensive lavage of the uterus post-insemination with an iodine-based solution to immobilize intrauterine spermatozoa. Pregnancy rates are maximized by delaying the uterine lavage until 4 h after insemination [365]. Location of intrauterine insemination seems to have a significant impact on the spermatozoal number that gains access into the oviduct. In one study, a higher number of spermatozoa were recovered from the ipsilateral oviduct after deep uterine-horn insemination than resulted from uterine body insemination. In the same study, the number of oviductal spermatozoa recovered from reproductively normal mares and mares susceptible to post-mating endometritis were similar after artificial insemination of 500 million total spermatozoa [366]. Others have found more spermatozoa, and a higher percentage of motile spermatozoa, in the isthmic oviductal region of reproductively normal mares than in mares susceptible to chronic uterine infection [367]. The same investigators reported more spermatozoa in the oviducts when mares received semen from a fertile stallion as opposed to a subfertile stallion [367]. Another group reported an effect of stallion on spermatozoal numbers in recovered oviducts of mares after artificial insemination, although the fertility or semen quality of these stallions was not provided [368].

Administration of oxytocin to mares immediately after insemination did not improve pregnancy rates in mares bred by either fertile or subfertile stallions [369]. This may be because of a directional pattern of luminal flow toward the cervix after oxytocin administration, as occurs naturally during the expulsive stage of labor (i.e., uterine evacuation) [370,371]. It is interesting to note, however, that dynamic scintigraphic analysis of radiolabeled spermatozoa in the mare uterus revealed radioactivity in the tips of the uterine horns as early as 8 min after uterine body insemination (5-ml volume) [360]. In this study, uterine contractions did not propagate spermatozoa in one direction only; rather, movement continued in both tubal and cervical directions. Similarly, ultrasonographic evaluation of the uterus after natural mating of mares revealed similar patterns of uterine contractions in cervio-tubular and tubo-cervical directions [362]. The peristaltic contractions were increased in frequency, amplitude, and duration in comparison with the pre-coital findings. It seems that bidirectional myogenic activity in the early period after insemination assists spermatozoal transport to the utero-tubal junction, and it is possible that this mechanism is impeded by administration of supraphysiologic doses of oxytocin or other drugs with oxytocic properties. Studies in pigs support this line of thought, because intrauterine infusion of cloprostenol before insemination reduced oviductal recovery rates of spermatozoa and increased cervical reflux of inseminates [372]. In another report involving pigs, spiking a semen dose with 10 IU oxytocin reduced fertilization rate, whereas IV administration of the same dose at 5 min after insemination improved fertilization rate [373]. The addition of another uterotonic agent, prostaglandin F2α, to extended semen (final concentration of 125 µg/ml) did not improve the pregnancy rate in a small group of mares [374].

The involvement of other factors that may impact spermatozoal transport in horses requires further examination. As an example, studies involving pigs revealed that the mere presence of a boar (i.e., non-tactile presence) could increase peripheral plasma oxytocinconcentration in the sow [375], and uterine activity after boar exposure was most enhanced in sows that initially had a reduced frequency of uterine contractions [376,377]. Similar studies involving horses are contradictory, with some reports of increased myometrial activity and plasma oxytocin concentrations after simple stallion exposure [378-382], and another report indicating no increase in uterine contractions until mechanical stimulation of the vagina and cervix occurs [383].

It seems logical that seminal plasma contributes to uterine transport of spermatozoa after natural cover, because it provides a medium to aid peristaltic movement of spermatozoa toward the oviducts. Mann et al. [384] showed the presence of two chemical markers of seminal plasma in the uterine horns at 50 min after mating, with concentrations similar to those detected in fresh ejaculates. These data are consistent with the concept that most of the postcoital fluid in the uterus is derived from the seminal plasma. Using the same chemical markers, these scientists also showed that seminal plasma may also gain access into the oviducts. The physiologic significance of seminal plasma in spermatozoal transport extends beyond that of a simple vehicle. Seminal plasma contains a rich assortment of both organic and inorganic constituents [136]. Prostaglandins have been identified in relatively high concentration in the seminal plasma of humans, monkeys, and the great apes, whereas the concentration in seminal plasma of stallions is miniscule [136]. Of interest, the seminal plasma of boars contains an appreciable concentration of estrogens (up to 12 µg per ejaculate), and this steroid is thought to stimulate myometrial contractions by inducing the release of PGF2α from the endometrium [376,385]. Spiking boar semen with estradiol-17β, however, did not improve pregnancy rate or fetal number after artificial insemination of gilts [386]. We are unaware of reports relating to estrogens in seminal plasma of stallions.

Seminal plasma is a well-known modulator of spermatozoon-induced uterine inflammation, a feature that likely plays an integral role in removal of spermatozoa from the uterus [387,388]. Troedsson et al. [388] reported that some proteins in equine seminal plasma can protect viable, but not damaged, spermatozoa from binding to neutrophils. Although this finding may not impact spermatozoal transport at the first breeding of an estrous cycle, it could affect spermatozoal transport mechanisms if a second insemination were placed in an inflamed uterus induced by the first insemination. Furthermore, it could potentially impact semen transport in mares bred for the first time in an estrous cycle if the mare had a pre-existing acute form of endometritis. To that effect, pregnancy rates in mares were dramatically increased on the second insemination of an estrous cycle (i.e., 12 h after insemination of 1 × 109 killed spermatozoa in semen extender or infusion of semen extender only to induce an endometritis) when the inseminate contained washed spermatozoa in seminal plasma (17/22; 77%) compared to washed spermatozoa in semen extender only (1/22; 5%) [389]. Clement et al. [390] reported embryo-recovery statistics for mares inseminated two to three times in an estrous cycle (at 2-day intervals) until ovulation occurred, using different stallions for each insemination. For mares inseminated twice in an estrous cycle, 14 of 17 embryos recovered resulted from the first insemination (82%), whereas when mares were inseminated three times in an estrous cycle, only 1 of 6 embryos recovered resulted from the first insemination (17%). Therefore, the first insemination was not uniformly the most fertile. This study was not designed to evaluate the effect of seminal plasma on pregnancy rates, and separation of insemination times by 48 h may have resulted in a uterine environment that was not as hostile as that reported by Troedsson et al.389 Seminal plasma concentration of inseminates was not reported in this study but is presumed to be >5%. In a study reported by Metcalf [391], mares were inseminated with frozen-thawed semen from two different stallions during an estrous cycle at a 4-to 10-h interval, followed by parentage determination of the resulting foals. Of nine foals born, four were the result of the first insemination (44%) and five were the result of the second insemination (56%). Although the second inseminate likely contained some seminal plasma, the amount would have been relatively low if the semen was processed for freezing in a standardized manner. The seminal plasma concentration of the frozen semen was not provided in the report. Based on the combined data from these three reports, it would appear that seminal plasma does provide some protective effect for spermatozoa entering an inflamed uterine environment, but the seminal plasma concentration in the inseminate can be quite low and still achieve this effect.

Regardless of the method(s) used to promote uterine transport of equine spermatozoa, in the end, only a small fraction of inseminated sperm reach the oviductal luminae. In one report, only 0.0006 - 0.0007% of inseminated sperm was recovered from oviductal flushings of mares at 18 h after intrauterine insemination [366].

Barring oviductal occlusion, deposition of spermatozoa in the uterine body of mares leads to similar spermatozoal numbers entering either oviduct [366,368]. This finding suggests that chemotactic effects are not present to attract uterine spermato-zoa to the oviduct ipsilateral to a periovulatory follicle, nor is a signal elicited to induce differential control of the oviductal papillae. The process of spermatozoal passage into the oviduct, however, does not seem to be a passive one, i.e., controlled only through the reproductive tract of the female. Studies to date suggest that a dynamic interaction occurs among spermatozoa, reproductive fluids, and the epithelial surface of the utero-tubal junction (UTJ) and caudal isthmus of the oviduct [392]. Based on studies with rats, it seems that only motile spermatozoa can negotiate passage through the UTJ [393]. Spermatozoa tend to associate closely with the epithelium while traversing through the oviductal papilla of the mare [392,394], a phenomenon also observed with other species [395]. Cross-talk between spermatozoa and the epithelium is further manifested by studies with transgenic mice lacking a gene for two different spermatozoal surface proteins. In these males, all functional characteristics of the spermatozoa appear normal, except that spermatozoal migration into the oviducts is hampered [396,397].

A preferential selection process occurs for the few spermatozoa that successfully pass through the UTJ into the protective confines of the caudal isthmus. Scott et al. [394] reported that >90% of spermatozoa visualized by scanning electron microscopy at the UTJ were morphologically normal, even when inseminates contained a high percentage of spermatozoa with morphologic abnormalities. The most common morphologic abnormality noted in the bound spermatozoa was a proximal cytoplasmic droplet. All of the spermatozoa were noted to have normal head morphology. A high incidence of normal morphologic features in oviductal spermatozoa has also been reported in cattle [398,399], even when the inseminate contained a high proportion of spermatozoa with abnormal heads [399]. A recent report conveyed that cytoplasmic droplets in boar spermatozoa may impede binding to the oviductal epithelium under in vitro conditions [400]. Thomas et al. [401] showed that co-culture of equine spermatozoa with oviductal epithelial cell monolayers yielded higher percentages of bound morphologically normal and motile spermatozoa than were present in the neat semen. When using oviductal explants to assess sperm- oviductal interactions, this team of investigators also found that more equine spermatozoa were bound to isthmic explants than to ampullar implants and that more spermatozoa bound to explants procured during the periovulatory period compared with luteal phase of the estrous cycle [402].

The caudal isthmus is generally considered to be the reservoir site for oviductal spermatozoa [367], although the UTJ also appears to harbor a considerable number of spermatozoa [403]. The effect of the harsh post-inseminate conditions within the uterus on these spermatozoa requires further study. The existence of a spermatozoal reservoir in the mare is further supported by the documentation in the literature that spermatozoa of some fertile stallions can persist in the mare for up to 6 days before ovulation, yet result in establishment of pregnancy [404,405]. The mechanism of spermatozoal attachment to the oviductal epithelial cells has received considerable study and seems to consist of a specific sperm-ligand interaction involving glycoconjugates (i.e., lectin-like molecules on the surface of spermatozoa interacting with carbohydrate-containing moieties on the surface of oviductal epithelial cells) [406-412]. Recently, galactose-binding proteins were characterized on equine spermatozoa that may prove to be involved in the sperm-oviductal binding mechanism [413].

This intimate oviductal cell contact with spermatozoa in the oviductal reservoir seems to play two divergent roles: assisting with the final maturational events of spermatozoa that must occur before fertilization of an oocyte [414-416], and also maintaining spermatozoa in a viable quiescent state to allow for an extended storage period [417-420]. Binding of equine spermatozoa to oviductal epithelial cells under in-vitro culture conditions results in both a quantitative and a qualitative change in protein synthesis and secretion by the epithelial cells [421]. Purified oviductal glycoprotein and polypeptides secreted by oviductal epithelial cells have been shown to have positive effect on spermatozoal capacitation, including sperm-oocyte interactions [422,423]. Peripheral proteins of the oviductal membrane seem to contribute to maintenance of boar spermatozoal viability [424]. Stallion spermatozoa that are bound to oviductal epithelial cells in culture exhibit flagellar motion for up to 4 days, with gradual release of spermatozoa during that time frame [425]. The oviductal support system and gradual spermatozoal-release pattern are consistent with the concept of ensuring that appropriately prepared spermatozoa are available to "greet" an ovulated oocyte soon after its arrival in the oviduct.

Spermatozoa attached to the isthmic epithelium will subsequently detach and traverse the oviduct to the vicinity of an oocyte. The mechanism for detachment is speculative but likely involves acquisition of hyperactivated spermatozoal motility to break the connection with the oviductal epithelial cells [426]. The lectin-like molecules on the spermatozoal surface responsible for specific binding with the oviductal cells may also be released during the capacitation process, thereby assisting with spermatozoal detachment [427]. Detachment of spermatozoa probably yields cells that are both hypermotile and primed for spermatozoon-oocyte interaction.

Spermatozoal Capacitation

In mammals, freshly ejaculated spermatozoa are not immediately capable of fertilizing an oocyte. Early studies showed that spermatozoa require residence time in the female reproductive tract to gain this capability [428,429], later termed capacitation [430]. Subsequent studies have revealed that similar changes are required by spermatozoa subjected to fertilization by in vitro methods (i.e., conventional in vitro fertilization [IVF]), as reviewed by Yanagimachi [431]. As such, capacitation is considered to be an absolute requirement of mammalian spermatozoa destined to fertilize an oocyte without mechanical assistance (i.e., by intracytoplasmic sperm injection [ICSI]). The process involves a series of preparative biochemical and biophysical changes in the spermatozoon that prime it to respond to signals originating from the oocyte and its surrounding cumulus. If capacitation is incomplete, spermatozoa are unable to penetrate the cumulus matrix [432,433], and have greatly reduced ability to penetrate the zona pellucida, undergo a zona-induced acrosome reaction, and fuse with the oolema [434,435].

Figure 35. Examples of traditional and alternative biomarkers for assessing spermatozoal capacitation.

Two early laboratory hallmarks for verifying completion of capacitation were acquisition of a hyper-activated pattern of motility and induciblity of the acrosome reaction by various stimulants (Fig. 35). Although capacitation has been defined by some as the changes that render a spermatozoon capable of undergoing the acrosome reaction [436-439], spermatozoa are also known to exhibit a hyperactivated state of motility when exposed to capacitating conditions [440-442]. The term was originally defined as the physiological changes of the spermatozoa in the female genital tract before they are capable of penetrating and fertilizing eggs [443]; thus, hyperactivated motility and the acrosome reaction would both be considered components of capacitation, based on its original meaning. Even though the acrosome reaction [116,444] and hyperactivated motility [441,445-448] are important for fertilization, the kinetic and temporal relationships of these two features are not well understood [449,450]. The signaling pathways for hyper-activated motility and the preparative changes required for the acrosome reaction seem to be divergent [150,171,441,451,452]. Interestingly, human spermatozoa that undergo hyperactivated motility are predominantly cells with normal morphology [453]. In recent years, a variety of molecular markers have been identified that allow more critical evaluation of the molecular events that emerge during capacitation (Fig. 35) [454-477]. Unfortunately, many of these assays can be technically challenging or have not become feasible for clinical use [478].

Neither the precise signaling mechanisms that govern the attainment of capacitation, nor the exact cellular characteristics of a capacitated spermatozoon, are fully elucidated despite considerable study in this area over the past few decades. Nonetheless, some key signaling pathways and cellular events have been identified and include surface (membrane) alterations, cytoskeletal modifications, influx and efflux of cytosolic constituents, and a myriad of enzymatic processes [479]. The reader is directed to several reviews on this topic [431,[451,452,479-490].

Figure 36. A schematic model of some documented and hypothesized molecular events associated with spermatozoal capacitation. Please refer to the text for an explanation of these signaling pathways and for figure abbreviations.

Interestingly, mature spermatozoa (i.e., those in the caudae epididymis or in ejaculated semen) seem to be pre-programmed to undergo capacitation, and the process can be induced by a variety of signals, as opposed to a specific molecular event. This is exemplified by the fact that in vitro culture of spermatozoa in many different media types can evoke capacitation. It seems that nature has provided spermatozoa with a broad array of capacitation induction alternatives to ensure that they may retain fertilizing potential even if some evokers are rendered nonfunctional. Figure 36 provides the reader with some of the proposed signal transduction pathways for spermatozoal capacitation. Although the literature is replete with potential activation mechanisms, in our view, those presented in this figure represent an accurate reflection of the most widely accepted signaling pathways. Species differences are known to exist in these regulatory pathways, even among eutherian mammals. Few in-depth studies have been conducted with stallion spermatozoa, so much of our predictions must be extrapolated from studies performed with other species. Conclusions drawn from work in non-equids may be misleading and thereby require verification through experiments involving stallion spermatozoa. It is also important to point out that ejaculates contain a heterogenous population of spermatozoa, and the cells do not undergo capacitation in a highly synchronous fashion. An advantage gained by such an arrangement is the continuous replenishment of capacitating spermatozoa available in the oviduct to await exposure to an ovulated oocyte, because spermatozoal lifespan is shortened appreciably by the capacitation process.

Spermatozoa are bathed in epididymal and ejaculatory fluids that contain "decapacitation factors" [491] that become adsorbed to the surface of the plasma membrane [450,492-496]. Although these factors and their regulatory roles have not been fully elucidated, they aid in suppressing premature initiation of the capacitation process during spermatozoal passage to the isthmic portion of the oviduct[479]. Desorption of these extracellular factors may unmask some key signaling mechanisms for capacitation [497,498]. A cysteine-rich protein family, termed CRISP, associate with spermatozoa in the epididymis as well as through the seminal plasma [104,499-504]. These proteins have been shown to inhibit tyrosine phosphorylation and capacitation of spermatozoa [505], possibly through their ability to block ion channels within the plasma membrane [104].

Alterations in membrane-associated cholesterol, and cytosolic concentrations of bicarbonate and calcium ions ([HCO3 -]i and [Ca2+]i, respectively) seem to be central evokers of capacitation in spermatozoa studied under in vitro conditions [141,183,454,458,470,488,506-523, and the same likely applies in vivo. Efflux of cholesterol from the plasma membrane requires the presence of sterol acceptors in the milieu, such as albumin or lipoproteins [483,497,508,514,523]. Under laboratory conditions, bovine serum albumin [483,514,523] or β-methyl cyclodextrin [524-526] are generally used to achieve this effect. The net result of cholesterol efflux seems to be destabilization and increased fluidity of the plasma membrane, combined with externalization of key surface receptors [478,508,515,527,528]. The cholesterol efflux is hypothesized to result in an increased permeability of the membrane to HCO3- and Ca2+ [520]. Both of these ions are thought to enter the cytosol from the extracellular space through ion-specific channels [529,530]. A variety of channels, transporters, and stores exist in spermatozoa that aid in regulation of [Ca2+]i [480,529]. Voltage-dependent Ca2+ channels (VDCCs) exist in the plasma membrane of spermatozoa, and membrane depolarization mediates Ca2+ entry through these channels [529,531]. Additionally, an increase in cytosolic pH ([pH]i) markedly increases the activity of VDCC [532]. Internalization of HCO3 - has been shown to further enhance Ca2+ entry through a PKA-dependent mechanism [533,534]. Interestingly, Ca2+-Pi symporters [535,536] and Na+-HCO3 -co-transporters [140] are also known to exist in mammalian spermatozoal membranes and are thought to be involved in the capacitation process. Plasma membrane Ca2+ ATPase (PMCA), an enzyme pump that extrudes Ca2+, is activated by the presence of decapacitation factors. In their absence, PMCA activity is decreased, resulting in a net increase in [Ca2+]i [537-539]. A family of transmembrane Ca2+-selective ion channels, called CatSpers, has been identified in recent years. These channels are thought to play an important role in both activated (non-capacitated) and hyperactivated (capacitated) motility [540,541]. CatSper2 channels have been localized to the flagellum [542], and CatSper1 channels have been detected in the principal piece of the flagellum [543].

Receptors for adenosine, calcitonin, and fertilization promoting peptide (seminal plasma constituents) have been identified in the plasma membrane of spermatozoa and seem to play a modulating role in capacitation through regulation of membrane-associated adenylyl cyclase (mAC). These molecules activate mAC in uncapacitated spermatozoa but down-regulate mAC in capacitated cells, possibly to prevent "overcapacitation" of cells [544]. Another method of preventing overcapacitation of spermatozoa is the polymerization of actin [545]. These molecules have been identified in many areas of the spermatozoon, including the peri-acrosomal space, the equatorial and post-acrosomal regions, and the flagellum [546-548]. Actin polymerization is a phospholipase D (PLD)-dependent event, and this enzyme is regulated by both PKA and protein kinase C (PKC) [549,550]. The formation of a filamentous form of actin in the peri-acrosomal space may prevent premature fusion of outer acrosomal and overlying plasma membranes when the properties of these two membranes become more fusogenic during the capacitation process. The actin must de-polymerize for the acrosome reaction to occur [545].

An increase in [HCO3 -]i and [Ca2+]i activate a soluble cytosolic form of adenylyl cyclase (sAC), leading to elevated intracellular concentration of cAMP [167,463,551]. This nucleotide subsequently activates cAMP-dependent PKA activity, eventually leading to an up-regulation in protein tyrosine phosphorylation [520]. A large number of proteins are known to be typrosine phosphorylated during capacitation, but the roles of many remain unclear [552]. Indeed, tyrosine phosphorylation of spermatozoal proteins seems to be crucial to acquisition of a capacitated state [489]. Tyrosine phosphorylation of head proteins occurs during capacitation, but this activity is even more pronounced in the flagellum [477,553], thereby suggesting an importance of these proteins in hyperactivation [554]. Two members of a tyrosine-containing family of proteins, termed AKAPs, are localized in the fibrous sheath and seem to tether PKA and various other signaling enzymes of the flagellum. These AKAP members are phosphorylated during capacitation and are thought to provide regional control of signal transduction [187,523,554,555]. Similarly, a calcium-binding protein, CABYR, has been localized to the fibrous sheath and is known to be tyrosine phosphorylated during capacitation [556]. Phospholipid hydroperoxide glutathione peroxidase (PHGPx) has been identified in the mitochondrial capsule of the spermatozoal midpiece, and tyrosine phosphorylation of this enzyme during capacitation may play a role in hyperactivated motility [452]. The phosphotyrosine protein, valosin-containing protein, is known to redistribute from the neck region to the anterior head region and may be involved with the acrosome reaction [552]. Other phosphotyrosine proteins may be involved with recognition of the zona pellucida and sperm-zona binding [557]. Another spermatozoon-specific enzyme, scramblase, may be activated through the PKA-tyrosine phosphorylation pathway, leading to a collapse of the asymmetric bilayer distribution of phospholipids, lipid packing disorders, and phospholipase activation [481,513,557-559]. Some workers consider the HCO3 --cAMP-PKAtyrosine phosphorylation (scramblase) pathway to precede an efflux of cholesterol from the plasma membrane [472,481,513,557-560]. Flippases, floppases, and an amnophospholipid transporter localized to the acrosomal region of mouse spermatozoa are thought to be important in maintaining the lipid asymmetry of the lipid bilayers before capacitation by establishing higher concentrations of phosphatidylserine and phosphatidylethanolamine in the inner leaflet and higher concentrations of sphingomyelin and phosphatidylcholine in the outer leaflet [561].

Another probable pathway to tyrosine phosphorylation involves formation of ROS that trigger downstream events that lead to capacitation. The effects of superoxide anion (O2 -) and H2O2 on spermatozoal capacitation were first described 15 yr ago [562,563]. More recently, NO- has been shown to regulate capacitation [564,565]. These findings have stimulated intensive studies regarding the physiologic roles of ROS in this process [266,268,272,485,566-576]. Current evidence supports the concept that ROS induce the cAMP-mediated tyrosine-phosphorylation cascade [577], but additional routes of action are also quite likely [485,578].

As indicated above, the plasma membrane undergoes considerable remodeling during capacitation, with an appreciable efflux of sterols and transverse asymmetry of phospholipids. These events increase the fluidity of the membrane, thereby allowing horizontal redistribution of membrane molecules. Recent studies have revealed that lipid rafts participate in this redistribution process [579]. These cholesterol-rich microdomains are laden with signaling molecules that are known to be involved with capacitation and zona binding [580,581]. The capacitation process can increase the number of membrane rafts, as well as their affinity for zona binding [582]. Actin polymerization may play a role in association/dissociation of the membrane rafts [583]. This has become an intense area of study, as these signal-carrying lipid rafts appear to play an increasingly important role in capacitation and spermatozoon-oocyte interaction [584,585].

Figure 37. A schematic model of some documented and hypothesized molecular events associated with the spermatozoal acrosome reaction. Please refer to the text for an explanation of these signaling pathways and for figure abbreviations.

The list of other potential endogenous signaling mechanisms is extensive, including involvement of mitogen-activated protein kinase (MAPK) [566], extracellular signal-regulated kinases (ERKs) [586], calmodulin-dependent kinases [182,587], protein tyrosine K (PTK) [485,588], PKC [549], Na+-Ca2+ exchangers [589-591], Na+/K+ase,482 KATP channels [592], and the renin-angiotensin system [593-596]. This list is not meant to be all inclusive, but to show that capacitation involves an extremely complex series of molecular events. Future studies will more clearly define the precise molecules and mechanisms that control capacitation, including spatial, temporal, and kinetic patterns.

Acrosome Reaction

Regulated acrosomal exocytosis is a fundamental element of fertilization and might be a more appropriate defining term than "acrosome reaction" [116,597,598]. It must be preceded by a complex series of events, including spermatozoal capacitation, followed by spermatozoon-oocyte recognition, spermatozoon-zona binding, and elicitation of signaling pathways [431,599-601]. An excellent review of the acrosome reaction was recently provided by Bailey [554], and the reader is referred to this source for an in-depth account of the biochemical mechanisms involved in the process. Other resources are also available for those that desire a thorough grasp of the subject [116,444,451,481,484,602]. Capacitation primes a spermatozoon for the acrosome reaction through alterations in the membrane lipids; membrane hyperpolarization; relocation, aggregation, and externalization of membrane-signaling molecules; and changes in cytosolic and cytoskeletal components. A trigger, however, is required for induction of the acrosome reaction, and most experimental evidence suggests that the zona pellucida provides the inciting factor [602-604]. Spermatozoal interaction with the zona seems to involve a precise species-specific receptor-ligand interaction, although the exact identity of the spermatozoal receptor remains elusive. Evidence abounds, however, that designated glycoproteins of the zona pellucida, termed ZP3, are responsible for initiating the signaling cascade(s) that lead(s) to the acrosome reaction [1,444,601,603,605-611]. The exact signal transduction pathway(s) for acquisition of the acrosome reaction is (are) not fully understood, but decades of intensive study have yielded much enlightening information on the subject. Figure 37conceptualizes possible pathways involved. As with capacitation, it is quite possible that more than one reaction cascade can elicit the acrosome reaction.

The zona pellucida (ZP) is an extracellular glycoprotein matrix surrounding the oocyte. In most mammalian species studied, excluding humans [612,613], the matrix is composed of only three glycoproteins, typically termed ZP1, ZP2, and ZP3 [609,611,614]. The peptide component of these glycoproteins seems to be well conserved across species, but post-translational modifications (i.e., glycosylation) lead to species heterogeneity and hence species-specific binding affinity [615-619]. The interactions seems to involve recognition of specific carbohydrate moieties present on the polypeptide backbone of ZP3 by complementary zona-binding proteins of the plasma membrane of the spermatozoon [601,615,620,621]. In the mouse, β1,4-galactosyltransferase I (GalT I) is the putative receptor on the spermatozoal surface for a glycoside ligand of ZP3, the binding of which leads to induction of the acrosome reaction [621-627]. A complex of spermatozoal surface molecules is likely involved in spermatozoon-zona interaction [628]. Whereas ZP3 can be considered the most likely signaling molecule of the ZP for induction of the acrosome reaction, various experiments also suggest that ZP2 may be involved in the anchoring of acrosome-reacted spermatozoa to the zona [605,620].The ZP2 and ZP3 exist as heterodimers in filamentous arrangement cross-linked by ZP1, so it seems a fitting motif for integrated roles of ZP2 and ZP3 in spermatozoon-oocyte interaction [444,606,629-631]. As evidence that the intricacies of spermatozoon-zona interactions are not yet resolved, a recent report revealed that, in bovine spermatozoa, glycosylation of ZP3 may not be an absolute requirement for induction of the acrosome reaction [632].

Extensive literature indicates that elevated intracellular Ca2+ is a steadfast requirement of the acrosome reaction. Spermatozoa use a range of intracellular messengers for various functions. Cytosolic Ca2+ is certainly a key regulator in these processes and is subject to complex and sophisticated spatio-temporal control so that Ca2+-sensitive responses can be activated discretely within various compartments of the cell [529,633]. A capacitated spermatozoon has an increased [Ca2+]i [484], created primarily by influx of transients through voltage-operated Ca2+ channels in the plasma membrane. Exposure of spermatozoa to solubilized ZP has been shown to result in a further increase in [Ca2+]i transients. Although intracellular Ca2+ stores are thought to be minimal, the acrosome may serve as an intracellular Ca2+ depot that is subsequently mobilized during the acrosomal reaction cascade [484,634]. The efflux of such intracellular stores may then activate store-operated channels (SOCs) in the plasma membrane [529], probably transient receptor potential (TRP) channels [635], thus creating the sustained rise in intracellular Ca2+ that is required to drive the acrosome reaction [636-638]. The mechanism of TRP channel activation is unresolved, but both depletion of Ca2+ from internal stores and direct receptor activation have been proposed methods of activation [636,639]. The TRP channels have been expressed in both the head and flagellar regions of spermatozoa, suggesting that they may play a role in both the acrosome reaction and in hyper-activated spermatozoal motility [640].

Receptor-activated Gi proteins seem to play a central role in the acrosome reaction, as shown by their activation by ZP3-GalT-I binding [641] and their inactivation by pertussis toxin (which inactivates Gi proteins) [642,643]. Activation of Gi proteins leads to a rise in [Ca2+]i[642]. Spermatozoon-ZP3 adhesion directly evokes Ca2+ influx through low-voltage-activated calcium channel (T-type channel) activation in membranes. Hyperpolarization of the membrane potential, as occurs during capacitation, is thought to release the channels from inactivation in order that they may respond effectively to a stimulus derived from the zona pellucida [462,480,592,644], i.e., membrane potential changes are translated into intracellular Ca2+ signals [531]. A ZP-mediated increase in membrane adenylyl cyclase (mAC) has also been reported to occur through Gi protein activation [645], presumably resulting in the increase in cAMP that is known to occur after spermatozoal exposure to the ZP [645,646]. Evidence for a cAMP/ PKA-mediated acrosome reaction exists after Ca2+ entry into the cell [647,648], but little is known about the role of this pathway in the acrosome reaction. Breitbart and coworkers [634,649,650] suggested that PKA may activate a voltage-dependent channel in the outer acrosomal membrane that leads to release of Ca2+ stores from the acrosome. Zona-induced acrosomal exocytosis can be blocked by an inhibitor of PKA [651].

The phospholipase (PLC) family seems to be central to the events leading to the acrosome reaction, and these enzymes have been localized to the spermatozoal head region [652]. Activation of PLC requires Ca2+ [604,653,654], but at relatively low concentrations [451], as might be produced by the pathways in the preceding paragraph. Zona-mediated co-activation of membrane-bound PLCβ1 (through a Gi-protein-coupled receptor) and membrane-bound PLCγ (through a tyrosine kinase [TK] receptor) has been proposed [634,652,655]. Activation of PLC is thought to promote the acrosome reaction through a couple of routes. First, PLC activates hydrolysis of phosphatidyl inositol 4,5-biphosphate (PIP2) within the membrane, resulting in production of two active intracellular messengers: diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). DAG mediates the activation of PKC, and IP3 releases Ca2+ from intracellular stores, i.e., the acrosome [451,655]. Activation of PLC may also lead to disassembly of F-actin filaments that are formed between the outer acrosomal membrane and overlying plasma membrane during capacitation, presumably to separate these increasingly fusogenic membranes [484,634,656]. PIP2 is purported to inhibit actin-severing proteins. Another member of the PLC family, PLCδ4, plays a central role in the acrosome reaction. This enzyme seems to be important for mobilizing Ca2+ stores in the acrosome and also activating SOC in the plasma membrane, thereby creating a sustained increase in cytosolic Ca2+ concentration [484,634,655-659]. The IP3 receptor and PLCδ4 have been localized to the acrosome [655,657] and both probably play a role in TRP-channel regulation of Ca2+ entry into the cell [635,660-664].

The role of PKC, as a signaling pathway in the acrosome reaction, remains somewhat unclear. The PKC activity of spermatozoa is dramatically lower than that detected in some other tissues [665-667], but nonetheless, has been identified in various regions of the spermatozoa [666,668]. Certainly, DAG, produced through the activity of PLC, is known to be a potent activator of PKC [665,669], possibly leading to opening of a membrane [634,649,650]. Ca2+ channel in the plasma DAG is also a membrane destabilizing molecule that increases membrane fusibility [451,670,671]. Experimentation has revealed that phosphorylation of spermatozoal proteins occurs on spermatozoal stimulation by progesterone, a known inducer of the acrosome reaction, and the activity can be mimicked with various activators of PKC and inhibited by various inhibitors of PKC [672]. Synaptogagmins are a family of calcium-binding transmembrane proteins purported to be calcium sensors responsible for triggering exocytosis in various cells [673,674]. Synaptotagmin VI has been localized to the outer acrosomal membrane of human spermatozoa [675]. Recently, PKC-mediated phosphorylation of spermatozoal synaptogagmin VI has been shown [672]. These workers proposed a model by which PKC has a regulatory role in synaptotagmin function through phosphorylation of synaptotagmin C2 domains [672,673]; hence, PKC may play a vital role in controlling the molecular machinery that mediates membrane fusion (discussed below).

Phospholipase A2 (PLA2) is recognized as an important enzyme for the acrosome reaction. Certainly, indirect evidence suggests that treatment of spermatozoa with PLA2 inhibitors will impede this event [676-679]. Both Ca2+ and DAG (in the presence of Ca2+) are capable of activating PLA2 [654,680-683]. The resulting enzymatic action is deacylation of fatty acids from phospholipids, producing fatty acids (such as arachidonic acid and lyophospholipids). A key target is phosphatidylcholine, yielding lysophosphatidylcholine. This "metabolite" is a likely contributor to the acrosome reaction, because it is known to trigger the reaction in capacitated spermatozoa [684-687]. Exposure of capacitated spermatozoa to solubilized zona results in increased metabolism of phosphatidylcholine to arachidonic acid and lysophosphatidylcholine, resulting in a high proportion of acrosome-reacted cells. The PLA activation is regulated by a signal transduction pathway involving Gi protein and DAG [682]. Lysophosphatidylcholine, or similar metabolites of PLA2 activity, seem to be important to the final stages of cAMP-mediated acrosomal exostosis [688].

Regulated exocytosis, as it relates to membrane fusion at neural synapses, has been studied extensively [689]. Expanding these studies to spermatozoa has revealed that a similar mechanism of action occurs between the outer acrosomal and plasma membranes [672,690-692], culminating in release of acrosomal contents and exposure of previously hidden domains on the inner acrosomal membrane. Studies to date indicate that the machinery required for this exocytotic event (which requires the finely regulated merger and fusion of two membranes) includes several core components: (1) soluble N-ethylmalameide-sensitive factor attachment protein receptor (SNARE) proteins; (2) N-ethylmalameide-sensitive factor (NSF); (3) soluble NSF attachment protein (α-SNAP); (4) Rab-GTPase; (5) synaptotagmin; and (6) calcium ions. This basic machinery is considered obligate for acrosomal exocytosis [690,693]. Of interest, the SNARE proteins, NSF, and synaptotagmin have recently been identified immunocytochemically in stallion spermatozoa, with predominant staining in the regions of the acrosomal cap and equatorial segment [694].

The SNARE proteins are functionally classified as v-SNAREs and t-SNAREs, and membrane fusion is executed when v-SNAREs on the acrosomal (i.e., vesicle) membrane pair the complementary t-SNARES on the plasma (i.e., target) membrane to form trans-SNARE complexes or SNAREpins. Both v-and t-SNARE proteins are thought to be secured to their respective membranes by hydrophobic anchors that span both leaflets of the lipid bilayers [689,695,696].

NSF and its co-factor, α-SNAP, are cytosolic proteins that act cooperatively to regulate SNARE protein function. The α-SNAPs bind to the SNARE protein so as to correctly position NSF, and are responsible for stimulation of the ATPase activity of NSF that dissociates trans-SNARE complexes [697-701]. This is a mechanism for recycling of components of the fusion machinery in other cell types, but would not seem necessary in the acrosome, where the reaction is a one-time event. Recent work with spermatozoa indicate that they also serve essential pre-fusion roles [691,692], probably by inducing conformational changes in the SNARE proteins complexes, i.e., catalyzing disassembly of an inactive cis SNARE complexes, to allow re-association as active trans complexes [691]. The NSF has also been reported to act in a pre-fusion step in non-spermatozoal cells by catalyzing SNARE protein-induced membrane rearrangement that is more suitable for Ca2+-mediated fusion [702].

Rabs are a family of proteins that are included in the Ras superfamily of G proteins. They are anchored to the acrosomal membrane, a process augmented by cholesterol efflux [703], and, on activation (through guanosine triphosphate [GTP binding]), can interact with Rab effector molecules on the plasma membrane to form an initial physical link, allowing the v-SNARE and t-SNARE proteins to interact to form a trans-SNARE complex [689,704,705]. The assembly of the trans-SNARE complex is thought to generate sufficient energy to overcome the repulsive forces generated by the polar head groups of the two membranes [689]. In essence, the acrosome becomes tethered to the plasma membrane. It is possible that that the filamentous F-actin interspersed between the outer acrosomal and plasma membranes could dampen the repulsive forces [451]. Epac, a known guanine nucleotide exchange factor (i.e., catalyzes replacement of GDP with GTP on Rab proteins) is activated by cAMP, resulting in induction of the acrosome reaction [706]; hence, another signaling cascade enters the mix of possible regulatory mechanisms for the acrosome reaction.

Ca2+-binding synaptotagmin, also a major Ca2+ sensor, is central to both the spatial and the kinetic elements of the fusion process. Synaptotagmin is known to have direct interaction with t-SNARE and SNAP proteins, a feature augmented by Ca2+, and may actually assist in SNARE complex formation [673,707]. This protein is a large transmembrane protein whose cytosolic component is primarily C2 domains, similar to that of pKC isoforms [673]. In other cell types, the protein is thought to facilitate docking of secretory vesicles close to Ca2+ channels where they might be exposed to high local concentrations of this ion [673,708,709]. As mentioned above, studies with spermatozoa indicate that synaptotagmin may also inhibit spontaneous fusion, through regulation by PKC. This control is likely to be driven by [Ca2+]i.

Without doubt, Ca2+ is the driving force for the final stages of acrosomal preparedness for reaction, from Rab3 activation through final pore formation [691]. It seems that Ca2+ is maintained in locally low concentrations during SNARE complex formation, possibly through synaptotagmin control. When trans SNARE complexes are fully assembled for the final fusion event, synaptotagmin likely triggers release of Ca2+ [673,691].Studies involving non-spermatozoal cells reveal that the docking mechanism pulls the pre-fusion membranes to within a distance of 2 - 3 Å [691,710]. Subsequent bridging of adjacent phosphate head groups by Ca2+ allows regional intermembranous displacement of water (i.e., by calcium hydration) so that the resulting anhydrous complex is amenable to lipid mixing [691,710-712]. The regions of fusion are restricted by the circular array of SNARE complexes [691,711]. Similar mechanisms probably exist for spermatozoa because the basic machinery is similar to that of somatic secretory cells.

As with capacitation, the precise pathways involved in the acrosome reaction are not fully elucidated despite considerable study in this area, and species differences are likely. The potential for involvement of signaling pathways and endogenous molecules, other than those listed above, is exemplified by the potential role of angiotensin II in the acrosome reaction of stallion spermatozoa [593,594].

Hyperactivated Motility

A hyperactivated form of motility is required to free the spermatozoa from the oviductal reservoir (through spermatozoal release from the oviductal epithelium and subsequent passage through the mucinous environment of the oviduct) and to penetrate the cumulus matrix and zona pellucida surrounding the oocyte [149,152,441,445]. It is of interest that hyperactivated spermatozoa tend to have normal morphologic characteristics [453]. When observed in aqueous media, the motility pattern of a hyperactivated spermatozoon is characterized by a high-amplitude, often asymmetric, whiplash flagellar beat pattern, leading to a circular or non-progressive trajectory [149,152,441]. Studies have revealed that this flagellar wave form actually improves the progressivity of spermatozoa over that of activated motility when spermatozoa are exposed to viscoelastic conditions such as those that exist in the oviduct [441,713].

Activated motility and hyperactivated motility may require different environmental signals. Presumably, a signal for hyperactivated motility is elicited within the oviduct under natural conditions to initiate the event at a time that is conducive to fertilization [441]. Although the precise signal(s) for initiation of hyperactivation remain(s) unsolved, it is possible that chemotactic and/or thermotactic factors serve in this capacity [714]. It is also possible that spermatozoal exposure to alkalinizing conditions, as exists in the oviduct and above that which initiates activated motility, is the primary initiator of hyperactivated motility [150,152,715,716].

The signaling pathway is also dissimilar between activated and hyperactivated forms of motility. Activated motility primarily uses the cAMP-sAC-PKA pathway and requires a relatively low concentration of Ca2+ for phosphorylation of flagellar proteins (Fig. 34). Conversely, hyperactivated motility seems to require an elevated concentration of Ca2+ [148,164,717-719], and no cAMP [149,150] or sAC [166].

The precise mechanisms controlling hyperactivated motility are subject to continued study, but an increasing body of literature suggests that an increase in extracellular pH leads to an increase in [pH]i, thereby potentiating the action of Ca2+ channels [147,467,716,718,720]. The most likely mechanism for an increase in cytosolic Ca2+ is through CatSper channels and through release from internal stores. Four CatSperm channels (CatSper1 - 4) have been localized to the principal piece, and they seem to have a physical interaction [716]. One report suggests that all four CatSper channels must be operational for hyperactivation to occur [716]; however, others provide evidence that this may not be the case [721]. CatSper1 may serve as an internal pH sensor, as evidenced by the composition of its N terminus [147,543]. The redundant nuclear envelope (RNE) at the base of the flagellum has been identified as a possible internal store for Ca2+ . This structure represents the remnants of the nuclear membrane following spermiogenesis. The RNE is reported to contain IP3 receptor-gated Ca2+ channels that are thought to release Ca2+ stores and regulate hyper-activated motility independent of similar channels in the acrosome [719,722]. Because the RNE is located at the base of the flagellum, where flagellar bending is propagated, it seems a logical location for an internal Ca2+ store [138,721]. A specific activator of PKC has been shown to induce hyperactivated motility in human spermatozoa [723]; thus, a PKC signal transduction pathway may also be important to stimulation of hyperactivated motility. Although Ca2+ is known to affect the bending pattern of the flagellum and added Ca2+ is required for hyperactivated motility, the mechanism by which Ca2+ controls this event remains unknown.

The signaling pathways and cellular events leading to hyperactivated motility and to the acrosome reaction are different, and the events of each can occur independently [138,148]. Nonetheless, internal alkalinization seems to be a common inciter for the two events. The pathways, although not the same, achieve the critical spermatozoal priming required for interaction with the oocyte. Because capacitation was originally defined with this endpoint in mind, it seems appropriate to consider hyperactivated motility as a component of the capacitation process. It has been quite difficult, however, to understand the interplay of the various components of capacitation. Indeed, the various events of the capacitation process are not tightly coupled. Penetration of zona-free hamster eggs by human spermatozoa is maximal at 18 h under in vitro conditions, whereas changes in tyrosine phosphorylation occur after 1 - 2 h, and hyperactivation is maximal after 3h of incubation [187,449,598,724,725]. Furthermore, different groups have shown variability among men in spermatozoal capacitation time [598,726,727]. An uncoupling of the acrosome reaction and hyperactivated spermatozoal motility has also been shown in hamsters and bulls [506,719,728]. Indeed, we now know a great deal about the molecular control of various facets of the capacitation process, but many more discoveries are needed to uncover the secrets of the spermatozoon.

Sperm-Oocyte Interaction

Although spermatozoal capacitation is not a site-specific phenomenon and can be induced in a variety of artificial media, the caudal segment of the oviduct seems to be the principle location for spermatozoal capacitation and storage under in vivo conditions [436]. Interactions with an ovulated oocyte, however, require spermatozoal migration to the ampullar region of the oviduct, and only a small percentage of spermatozoa that gain access into the oviduct will eventually arrive at this fertilization site [436]. Such spermatozoa are thought to have achieved full fertilizing potential. The precise mechanisms by which spermatozoa migrate to the ampullar region of the oviduct remain speculative, but contractile movements of the oviduct and hyperactivated spermatozoal motility are thought to play key roles in this migratory phase. Evidence is mounting that chemotactic factors are also important to directional migration of oviductal spermatozoa [729-741]. Some investigators assert that chemotactic responsiveness is also a part of the capacitation process [729,730]. Spermatozoa possess specific chemotactic receptors [478]. Olfactory receptors are considered to represent the largest family of genes in the human genome, with up to 1000 members [742,743], and odorant receptors have been identified, and genes encoding for olfactory receptors have been expressed, in the testes of mammals [744-750]. Olfactory receptor genes have even been identified in the primordial germ cells of fetuses [751]. Odorant receptors have been localized to the flagellar base and proximal midpiece of spermatozoa [752,753], suggesting that odorant-like molecules emanating from the female reproductive tract might induce hyperactivated spermatozoal motility, possibly by release of Ca2+ from internal stores [152,747] or another signal-transduction pathway [754]. Spermatozoa do not seem to acquire a capacitated state, complete with chemotactic competence, in a synchronous manner; rather, a small percentage of a spermatozoal population is thought to achieve this potential at any given time. This is thought to serve as a safeguard to ensure that capacitated spermatozoa, which are short-lived, are available over a protracted period for prompt interaction with an ovulated oocyte [729,741].

Oriented migration of spermatozoa to follicular factors has been shown under in vitro conditions [714,741,755], and progesterone is thought to be an important mediator of this event [714,739]. Follicular fluid is not thought to pass down the oviduct to the caudal isthmic region, however, and migrating capacitated sperm can be detected in the more proximal regions of the oviduct before ovulation [738]. Studies involving oocyte transfer in horses have also shown that fertilization can occur when oocytes are transferred to the oviduct contralateral to an ovary containing a preovulatory follicle or when transferred to oviducts of non-cyclic mares supplemented with exogenous steroids [756]. One working hypothesis is that spermatozoa are transported to the general site of fertilization by thermotaxis and contractions of the oviduct and, when in the direct vicinity of the oocyte, are directed by chemoattractants secreted by the cumulus cells [714,757,758]. Certainly, studies regarding spermatozoal chemotaxis remain scanty at this juncture, but continued study may divulge a specific role of chemoattractants in spermatozoal migration patterns.

Before a spermatozoon can interact directly with the oocyte, it must first negotiate passage through the two sizable extracellular matrices which surround the oocyte, i.e., the cumulus complex and the zona pellucida. An excellent description of the three-dimensional structure of the zona pellucida was recently provided [759]. Acquisition of hyper-activated motility and translocation/exposure of glycosylphosphatidylinositol-anchored surface hyaluronidase (termed PH-20) likely permit penetration of the cumulus matrix [760-762]. Binding of acrosome intact spermatozoa to the zona pellucida through species-specific carbohydrate moieties of the zona pellucida and corresponding receptors on the spermatozoal surface lead to induction of the acrosome reaction [605,609,610,763-766]. An assortment of spermatozoal surface lectins and glycoenzymes have been shown to have zona-binding ability [767-774]. Previous studies involving mouse spermatozoa suggest that the binding of the spermatozoal surface receptor, GalT-I, to specific oligosaccharide moieties of ZP3 on the zona pellucida is important to induction of the acrosome reaction [608,775]. More recent work, however, indicates that other mechanisms may be responsible for initial adhesion of spermatozoa to the zona pellucida [775-777]. A newly voiced theory is that the zona pellucida acts as a spermatozoal scaffold, with ZP2 acting to orchestrate zona permissibility to zona penetration by a spermatozoon, and initial zona penetration by the spermatozoon activating the acrosome reaction through "mechanosensory" signals [778]. One report involving horses suggests that spermatozoal surface galactosyltransferase (GalT) and zona glycoprotein ZPC (probably similar to ZP3 in the mouse) are not required for spermatozoal binding to the zona pellucida [779]. Acquisition of the acrosome reaction was not an experimental endpoint in this study, so it remains possible that a GalT-ZPC-independent mechanism is responsible for initial adhesion, but that a GalT-ZPC-dependent interaction may be required for the acrosome reaction. Passage through the cumulus may also displace the surface-associated sperm glycoproteins, glycodelin-A and glycodelin-F, to promote spermatozoal-zona binding [780]. Not surprisingly, the zona pellucida of ovulated oocytes is also known to contain products secreted by the oviducts that may play a role in initial sperm adhesion [781,782]. Further research may lead to identification of a multitude of receptors that are involved with spermatozoal-zona binding.

Figure 38. Conceptualization of spermatozoon-oocyte interactions. (A) Spermatozoon negotiates passage through the intercellular matrix of the cumulus oophorus. (B) Initial adhesion of the equatorial region of the spermatozoon with the zona pellucida. (C) Induction of the acrosome reaction. (D) Spermatozoal penetration of the zona pellucida at an oblique angle, with entry into the perivitelline space. (E) fusion of the post-acrosomal spermatozoal membrane with the microvillar region of the oolemma. (F) Establishment of a gateway for spermatozoal nucleus entry into the cytoplasm of the oocyte, as well as entry of spermatozoon cytosolic molecules (presumably spermatozoon-specific phospholipase C) that serve as a signaling mechanism for activation of the oocyte and exocytosis of oocyte cortical granules. Modified from Primakoff et al. 2002 and Rubinstein et al. 2006 [760,789].

The cutting thrust issued by hyperactivated spermatozoa [783], possibly combined with externalization/ release of proteases and glycosidases within the acrosome [598,760,784,785], allow spermatozoal penetration of the zona pellucida. Studies involving hamster spermatozoa indicate that the process of spermatozoal adhesion to, and penetration of, the zona pellucida requires only 10 - 15 min [598,786,787]. Once situated within the perivitelline space, the spermatozoon binds to, and fuses with, the egg plasma membrane (or oolemma), leading to internalization of the sperm haploid chromosomal complement by the oocyte. Only spermatozoa that have undergone an acrosome reaction are thought to be capable of fusion with the plasma membrane of the oocyte [788], and this binding/fusion process involves specific domains of the post-equitorial region of the sperm head and the microvillar region of the oolemma (Fig. 38) [760,789]. The specific sperm proteins that are involved with spermatozoal-oocyte binding remains an enigma, although several candidate proteins have been identified [504,760,789,790].

A family of sperm-associated cysteine-rich secretory proteins (CRISPs) have been characterized in many mammalian species. These proteins are synthesized and secreted by the epididymis and associate with the spermatozoal membrane, most prominently in the distal corpus and cauda epididymis [504,791]. CRISPs have been localized to the equatorial region of capacitated and acrosome-reacted spermatozoa [792]. Interestingly, stallion spermatozoa possess an abundance of CRISP, with largest amounts detected in ejaculated spermatozoa [503]. Three members of the CRISP family have been identified in the testis, epididymis, ampullae, or seminal vesicles of stallions [499,503]. A portion of CRISP is tightly associated with the spermatozoa and is localized to the post-acrosomal and equitorial regions of the sperm head, as well as the midpiece [503]. The regionalized distribution of the protein hints to a potential role in spermatozoon-oocyte interaction; however, complementary molecules have not been characterized on the oocyte membrane of any species studied [504]. In addition, the lack of hydrophobic domains in this molecule suggests that it may not be directly involved in membrane fusion events [598,790].

The spermatozoal ADAM (A Disintegrin And Metalloprotease) family of proteins (including fertilin α [ADAM1], fertilin β [ADAM2], and cyritestin [ADAM3]) have been implicated in spermatozoon-oocyte binding [504,760] and are known to possess fusogenic potential [793]. Nonetheless, gene disruption data do not corroborate an essential role of these proteins in fertilization [760,794]. The protein, Izumo, has withstood the spatio-temporal, immunological, and gene-knockout tests required to consider it as a probable spermatozoal candidate in gamete fusion [789,795]. Treatment of spermatozoa with antibodies to integrins or osteopontin also reduce spermatozoon-oocyte binding and fertilization in vitro [796]. Recently, proteomic analysis of the spermatozoal membrane regions involved in spermatozoon-oocyte interactions has uncovered an abundance of proteins that could be participants, including some previously uncharacterized proteins [797].

A tetraspanin protein, CD9, has been implicated as a very viable candidate oocyte protein [608,760,788,789,798], for spermatozoal adhesion and fusion, although additional proteins might also be involved [798]. The microvilli of the oolemma seem to be key to spermatozoon-oocyte fusion, and CD9 is enriched in the microvillar portion of the oolemma [799]. In addition, CD9 may be involved in coordination of microvillar shape and distribution along the oolemma [799]. Membrane lipid molecules are also of fundamental importance to spermatozoon-oocyte fusion [800]. In addition, polymerization of actin filaments may be critical to spermatozoal incorporation into the oocyte cytoplasm, decondensation of the nucleus, and activation of the oocyte block to polyspermy [545,801-803].

Entry of the sperm into the ooplasm elicits an initial increase in intracellular Ca2+ concentration, followed by repetitive Ca2+ oscillations. This signaling mechanism induces exocytosis of oocyte cortical granules (to prevent polyspermy); release of the oocyte from meiotic arrest; pronuclear formation; mediation of genomic union; progression into mitosis with reorganization of both nuclear and cytoskeletal components; and stimulation of oocyte mitochondrial respiration [804-810]. The factor responsible for this activation is derived from the spermatozoal cytosol, and a spermatozoon-specific PLC is considered to be the most likely candidate molecule [811-813]. In a manner similar to that of the acrosome reaction, PLC might activate hydrolysis of phosphatidyl inositol 4,5-biphosphate (PIP2) within the membrane, resulting in production of inositol 1,4,5-triphosphate (IP3), which, in turn, could mediate release of Ca2+ from intracellular (i.e., endoplasmic reticulum) stores [814].

Internal energy generation is required for spermatozoon-oocyte interactions. In the mouse, spermatozoon-oocyte fusion is inhibited in the absence of glucose [233]. In this species, glucose seems to mediate tyrosine phosphorylation of proteins in midpiece-specific sites [234]. Sperm entry into the mouse oocyte is characterized by pentose phosphate pathway activity and redox regulation, in addition to glycolysis [251,252]. Interestingly, oocytes are incapable of metabolizing glucose; thus, pyruvate or cumulus cells (which convert glucose or lactate to pyruvate) must be present in IVF medium [598,815].