The Capacitation-Apoptosis Highway: Oxysterols and Mammalian Sperm Function

R. John Aitken · 2011 · 111 citationsRead the paper

The notion that densely packed populations of spermatozoa generate toxicants that inhibit respiration has been around since the 1920s, when the concept of allelostasis (an increase in oxygen uptake that accompanies the dilution of sperm populations) was born [1]. It was not until 1946 that Tosic and Walton [2] identified the molecule responsible for this allelostatic effect in bull spermatozoa as hydrogen peroxide. Mammalian spermatozoa were thus the first cell type in which the cellular generation of reactive oxygen species (ROS) was biochemically confirmed, predating the discovery of this activity in leukocytes by more than a decade. Since Tosic and Walton's original report, the cellular production of ROS has been confirmed in the spermatozoa of a wide variety of mammals, including human, mouse, hamster, rat, rabbit, and horse [3]. This potential for ROS production is curious given the susceptibility of mammalian spermatozoa to oxidative stress. These cells are well known to possess high concentrations of unsaturated fatty acids, particularly docosahexaenoic acid (22:6), that are very vulnerable to attack by reactive oxygen metabolites and the consequent initiation of lipid peroxidation cascades [4]. Such peroxidative damage impacts these cells in such a way that motility, their competence for sperm-oocyte fusion, and DNA integrity become rapidly compromised [3, 4]. The spectacular ability of ROS to damage sperm function has been understood since the pioneering studies of MacLeod in the 1930s [5], Jones and Mann in the 1970s [6, 7] and Alvarez and Storey in the 1980s [8, 9]. Given this background, it is clear that if spermatozoa are such active generators of ROS, they must have a very good reason for engaging in this potentially fatal activity. Claude Gagnon and colleagues at McGill University solved this conundrum by being the first to demonstrate that sperm capacitation is an oxidative process that is heavily dependent on the active generation of ROS [10]. While several independent laboratories have confirmed this observation [11–13], the way in which oxidative stress drives spermatozoa into a capacitated state has remained largely unresolved. To date, evidence suggests that both cAMP generation [14, 15] and tyrosine phosphorylation [16, 17] are redox-regulated events associated with capacitation. However, the paper published by Brouwers et al. [18] in this issue of Biology of Reproduction raises another important feature of this process. It is well known that one of the key elements of capacitation is the removal of cholesterol from the sperm plasma membrane by albumin [19]. Such a change is thought to enhance the fluidity of the plasma membrane, promoting critical intermolecular interactions that in turn promote the development of a capacitated state. However, the mechanisms by which cholesterol is induced to leave the plasmalemma have never been satisfactorily explained. Brouwers et al. [18] have provided a possible answer. The sperm plasma membrane has been found to contain a mixture of oxysterols. The latter are oxidized derivatives of cholesterol that can move more freely out of the membrane to bind acceptor proteins, such as albumin, because they are much more hydrophilic than the parent molecule. Thus, the oxidation of cholesterol by ROS promotes capacitation by facilitating the efflux of this decapacitation factor from the sperm plasma membrane. These observations represent a significant advance in our understanding of the redox regulation of sperm capacitation, but they also raise some interesting questions that will have to be addressed in future studies. For instance, the role of desmosterol in this process remains unresolved. The chemical details of oxysterol formation indicate that cholesterol is the preferred substrate for oxidation. However, because desmosterol is as effective a decapacitation factor as cholesterol, this leaves open the question of how this particular sterol is removed from the plasma membrane if the capacitation process is to proceed [20]. Also unaddressed is the relationship between the hypothesis advanced by Brouwers et al. [18] and an earlier concept presented by Langlais et al. [21]. According to this model, cholesterol is stabilized in the sperm plasma membrane as cholesterol sulfate. As spermatozoa ascend the female reproductive tract and initiate capacitation, sterol sulfatases affect the enzymatic hydrolysis of the sulfate group, thereby increasing the pool of cholesterol available for esterification. The fatty acids required for the esterification of cholesterol are provided via their enzymatic cleavage from membrane phospholipids by phospholipase A2. One of the by-products of this process is the creation of highly unstable lysophospholipids that generate increased membrane fluidity and permeability to calcium, both of which should promote capacitation and subsequent acrosomal exocytosis. Perhaps this scheme and that proposed by Brouwers et al. [18] are not mutually exclusive, in that cholesterol might be desulfated before it becomes oxidized and/or esterified. One of the interesting features of ROS as regulators of sperm function is that they act as a two-edged sword. On the one hand, low levels of ROS are needed to promote the cholesterol oxidation and the tyrosine phosphorylation events that underpin capacitation. On the other, the overproduction of ROS can lead to a state of oxidative stress that compromises sperm function [3]. In this context, oxysterols may be regarded as efficient reporters of the oxidative stress suffered by cells during their life history and superior to conventional signatures of peroxidation, such as malondialdehyde and 4–hydroxynonenal, that tend to lose association with oxidatively damaged cells because of their relative hydrophilicity. As well as being good indicators of oxidative stress, oxysterols might also be efficient inducers of this process. Thus, oxysterol accumulation has been widely linked with the activation of apoptosis and cell death in other cell types [22]. Significantly, mitochondria appear to be key mediators of oxysterol-induced pathology via mechanisms involving stimulation of the intrinsic apoptotic cascade and activation of excessive ROS generation [23–25]. If this is the case, how can we balance these two opposing visions of oxysterol generation in spermatozoa (i.e., as mediators of sperm function on the one hand and intercessors of pathology on the other)? The first questions we might consider in this context are the identity of the ROS that stimulate cholesterol oxidation and the sites of oxysterol formation. With respect to the former, one candidate worthy of consideration is peroxynitrite. This reactive nitrogen metabolite, formed by the rapid reaction of superoxide anion (generated by the sperm mitochondria) and nitric oxide (possibly, but not necessarily, generated by nitric oxide synthase), is a well-recognized promoter of capacitation in several different mammalian species [26, 27]. Peroxynitrite is also known to activate oxysterol formation in cells, particularly 5,6beta-epoxycholesterol, one of the major cholesterol derivatives found in spermatozoa [18, 28]. As a candidate for the reactive oxygen metabolite that drives capacitation, it has much to commend it. Brouwers et al. [18] also observe that oxysterol generation is disrupted following cryopreservation, whereas lipid peroxidation is generally enhanced [29]. This, they point out, may be because cholesterol is more difficult to oxidize than highly unsaturated fatty acids, or possibly because ROS generation is a property of the sperm midpiece but cholesterol is concentrated in the plasma membrane overlying the sperm head. We might also speculate that as a result of cryopreservation, superoxide production by the sperm mitochondria continues unabated, leading to the oxidative stress associated with low-temperature storage, whereas nitric oxide generation is compromised. As a result of this change, the redox balance for the spermatozoa would be expected to shift from peroxynitrite to predominantly superoxide and (by dismutation) hydrogen peroxide, leading to oxidative stress [30] and an impaired capacity for capacitation [31]. We could further speculate that the competence to capacitate might be restored to such cells by direct exposure to peroxynitrite. This is exactly what has been reported in the literature following direct exposure of cryostored equine spermatozoa to 3-morpholinosydnonimine, an peroxynitrite donor [26]. Whether the restoration of capacitation under such circumstances involves the formation of oxysterols would, in light of the study by Brouwers et al. [18], be interesting to determine. The specific site of oxysterol formation might have a major bearing on the net biological consequence of cholesterol oxidation. Thus, in the plasma membrane, oxysterol formation promotes its egress from the cell, leading to an increase in membrane fluidity and promotion of the molecular interactions that facilitate capacitation. On the other hand, oxysterol formation within the mitochondria could lead to activation of the intrinsic apoptotic cascade and initiation of a number of cellular changes associated with sperm senescence, including enhanced mitochondrial superoxide generation, motility loss, caspase activation, and phosphatidylserine (PS) exposure [22]. Intriguingly, some of these apoptotic changes have also been associated with capacitation and invite speculation that capacitation and apoptosis may be part of a continuous process driven by peroxynitrite and oxysterol formation. In the early part of this process, the changes promoted by ROS exposure are all features of the capacitation process that might reasonably be considered peroxynitrite-dependent, including cholesterol efflux from the plasma membrane [18], PS exposure [32], presentation of zona-receptor complexes on the sperm surface [33], inactivation of tyrosine phosphatases [34], and stimulation of cAMP production [15, 35]. However, if the spermatozoa fail to find an egg, then the continued generation of this oxidant might promote oxysterol formation in the mitochondrial membranes, leading to activation of the intrinsic apoptotic cascade, oxidative DNA damage, motility loss, and ultimately, cell death. Thus, peroxynitrite generation may drive spermatozoa along a capacitation highway that should ultimately culminate in capacitation and fertilization of the oocyte. However, if the latter does not occur, the continued generation of ROS will eventually lead these cells into the cul de sac of apoptosis. Although the net result of entering this pathway will be an apoptotic death for a vast majority of spermatozoa, such a regulated demise may well be necessary to promote the silent phagocytosis of these foreign cells by the female immune system [36]. The idea that capacitation and apoptosis are part of a redox-regulated continuum is summarized in Figure 1. Such suggestions may be highly speculative, but then good papers invariably act as stimuli for the hypothesis generation-refutation-refinement cycle that drives scientific research. Proposed capacitation-apoptosis highway. During capacitation, the generation of ROS, particularly peroxynitrite (ONOO−), leads to oxysterol production, which facilitates the removal of cholesterol from the plasmalemma and a consequential increase in membrane fluidity as well as other changes, including tyrosine phosphatase inhibition and increased cAMP production. All of these changes promote acquisition of a capacitated state. If fertilization does not occur, the continued generation of oxysterols in the mitochondrial membranes leads to the induction of apoptosis characterized by enhanced mitochondrial superoxide generation, lipid peroxidation, cytochrome c (CYC) release, caspase activation, oxidative DNA damage, and death. AIFM, apoptosis-inducing factor, mitochondrion-associated; BAD, BAX, and BAK1, proapoptotic members of the Bcl-2 family; DIABLO(SMAC), diablo homolog-(Drosophila); ENDOG, endonuclease G; H2O2, hydrogen peroxide; NO, nitric oxide; OH•, hydroxyl radical.

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