The role of reduced glutathione (GSH) in the process of spermatic capacitation

1. Introduction

The last piece of sexual reproduction is the fusion between the male and female gametes that generates a new individual, involving processes orchestrated by the neuroendocrine system and, particularly in the case of the spermatozoon, the process to be fully fit for fertilization, which ends in the female reproductive tract (O’Donnell et al., 2006).

The production of male gametes takes place inside the testicle, through a process known as spermatogenesis. The gametes then enter the epididymis to complete their maturation through structural and functional changes, so that they can complete the capacitation and acrosome reaction (AR) in the female reproductive tract and conclude with fertilization (O’Donnell et al., 2006; Cunningham and Klein, 2003).

After the spermatozoa have traveled through the caput and the corpus of the epididymis, they are stored in the cauda, before being ejaculated. It has been reported that the transit of sperm by the pig epididymis takes about 9 days (Shalender Bhasin, 2006; Franca and Cardoso, 1998). Ejaculation is generated by the central nervous system, triggering the muscular fibers that surround the epididymis to expel the spermatozoa through the vas deferens, into the urethra and, along with the secretions of the accessory sex glands, forming the semen (Shalender Bhasin, 2006).

Spermatozoa must go through capacitation and AR before fertilizing the ovum. In humans, this process seems to be regulated, in part, by oxidation-reduction reactions (De Lamirande and Gagnon, 1998; De Lamirande and Gagnon, 2003) involving reactive oxygen species (ROS). Spermatozoa, like other cells under aerobic conditions, are capable of producing ROS, which can be harmful to themselves, causing damage to the membrane and DNA, affecting sperm motility and viability (Claude Gagnon and Lamirande, 2006; Medeiros, 2008). However, there is evidence indicating that the generation of ROS (Figure 1) is involved in the acquisition of the spermatozoa fertilizing capacity (De Lamirande and Gagnon, 1998; De Lamirande and Gagnon, 2003). It has been reported that the generation of controlled amounts of ROS, such as the superoxide anion (O2-) and its dismutation product, hydrogen peroxide (H2O2), are involved in the regulation and in the physiological functions of spermatozoa (De Lamirande and Gagnon, 1998; De Lamirande and Gagnon, 2003).

Figure 1. Outline of the Redox cycling in the ROS regulation (modified from Aitken and Roman, 2008).

The first studies did not reveal a positive interaction between ROS and spermatozoa (Aitken et al., 2016). MacLeod in 1943 (MacLeod, 1943) reported, on human sperm, loss of motility due to high oxygen concentrations, directly involving H2O2 in this alteration; Tosic and Walton, in 1946 (Tosic and Walton, 1946), studying bull spermatozoa, found that H2O2 altered the spermatic function –mainly cellular respiration. Later, in 1947 Evans and collaborators (Evans, 1947) reported that the main function of sea urchin spermatozoa can be affected by oxidative stress; similarly, in 1993, Aitken (Aitken et al., 1993) and his group reported that the catalase enzyme, and not the superoxide dismutase, was capable of inhibiting the deleterious effect of ROS, particularly H2O2. Nowadays, studies continue to show that oxidative stress can affect physiological processes in spermatozoa, such as inhibiting capacitation and motility (Morielli and O'Flaherty, 2015).

The mentioned damages were shown in ejaculated spermatozoa, however, there are also studies in which damage produced by ROS is reported in epididymal sperm maturation. In 2017 Liu and O’Flaherty (Liu and O’Flaherty, 2017) found, in the cauda of the rat epididymis, spermatozoa with low motility and oxidized DNA, when there was oxidant stress generated by a hydroperoxide (tert-Butyl hydroperoxide [t-BHP]). Accordingly, they also reported an increase in another antioxidant enzyme, peroxiredoxin (PRDX) in the cauda of the epididymis, which proves that the epididymis defends spermatozoa against oxidative stress (Liu and O’Flaherty, 2017). Regarding this, it has been described that human spermatozoa, like that of pigs, have characteristically high levels of polyunsaturated fatty acids (PUFA), these insaturations make them very vulnerable to free radicals, generating lipid peroxides and aldehydes, having as a direct consequence the inhibition of spermatozoa motility, thus compromising the fertilizing capacity of the spermatozoon (Jones et al., 1979; Roca et al., 2005; Malo et al., 2010). However, ROS participate in spermatozoa physiology, particularly during sperm capacitation, where they have been associated with increased phosphorylation of tyrosine residues, directly stimulating the tyrosine kinase enzymes (TK) and soluble adenylate cyclase (ACs), and inhibiting tyrosine phosphatase (TP) (Ford, 2004).

2. Spermatic capacitation

Spermatic capacitation is essential to ensure the oocyte fertilization; it is an extremely complex process that occurs during the transit of the spermatozoa through the female reproductive tract (Figure 2) (O'Flaherty, 2015; López Úbeda and Matas Parra, 2015). During this process, the spermatozoon undergoes biochemical and membrane changes in its energy metabolism, as well as in its enzymatic activity (De Lamirande and Gagnon, 1998); changes that do not occur adequately without prior adequate spermatogenesis (in the testicle) and epididymal sperm maturation (Cervantes et al., 2008).

Figure 2. A) After ejaculation, a heterogeneous spermatozoa population reaches the female reproductive tract. B) Only a few spermatozoa can reach the oviduct. C) Only properly capacitated spermatozoa can fertilize the oocyte. Dead spermatozoon (gray), damaged (red), normal (blue), hyperactivated (green-blue) and capacitated (green) (López Úbeda and Matas Parra, 2015).

Among the changes undergone by the plasma membrane, the reordering of proteins and lipids in the plasma membrane stands out, among which the outflow of cholesterol is one of the most important, modulated by the secretions of the female reproductive tract that have a high albumin concentration, which absorbs cholesterol, giving fluidity to the membrane, activating the bicarbonate and calcium channels (Vadnais and Althouse, 2011). The increase in the concentration of these ions in the sperm cytoplasm stimulates adenylate cyclase, which begins to produce cAMP from ATP. This production activates a cAMP-dependent protein kinase (protein kinase A) (Cross, 2004); the plasma membrane and the outer acrosomal membrane acquire the ability to fuse with one another as their fluidity and permeability are modified (Gadella, 2008). The final result of this remodeling allows the spermatozoa to bind to the ovum zona pellucida and to carry out the acrosomal reaction (Rosado, 1988; Gadella, 2008).

2.1 Spermatic hyperactivation

Hyperactivation is a physiological change of the capacitated spermatozoa. That is, its flagellar movement changes from a progressive motility to a greater flagellum movement and flexion, a great amplitude in the lateral displacement of the head, as well as an irregular and tortuous trajectory (Claude Gagnon and Lamirande, 2006). Theres is hyperactivation because the sperm must be strong enough to penetrate the layer of granulosa cells and the zona pellucida of the oocyte, as it must also be able to move in the fluid of the female reproductive tract, until its membrane touches the ovum (Tulsiani, 2006). In order for this to happen, spermatozoa maintain precise ionic gradients through their plasma membrane, which are regulated by Na+/K+ and Ca2+ dependent ATPases; this is how alteration in the ion concentration activates acrosome enzymes as well as enzymes related to changes in motility (Gadella, 2008).

The induction of hyperactivation requires intracellular Ca2+ and an increase of cAMP and intracellular pH. The hyperactivation of the spermatozoon is due to the fact that Ca2+ binds to fixative proteins in the external arm of dynein, which is found in the internal part of the flagellum (Inaba, 2003). Dynein transforms the chemical energy from the ATP hydrolysis, generating the mechanical energy necessary for motility (Gagnon and De Lamirande, 2006). Ca2+ can come from several places, such as intracellular reserves, mediated by inositol 1,4,5-trisphosphate (IP3) receptors or the mitochondria, and extracellularly by Na+/H+ and Na+/Ca2+ transport systems (Gadella, 2008). The removal of cholesterol from the spermatozoon plasma membrane can also directly activate ion channels, increasing the concentration of intracellular Ca+2 (Darszon et al., 2011).

There are also reports about the possible participation of a sodium and bicarbonate cotransporter (NBC, an acronym for Na+/bicarbonate cotransporter) possibly activated by extracellular Ca+2, which favors the entry of Na+ and HCO3-, where the latter is essential for the activation of the ACs enzyme, which is soluble in the cytosol, thus increasing the production of cAMP (Darszon et al., 2011). Likewise, a sodium-proton exchanger channel (sNHE, an acronym for Na+/hydrogen exchanger) has been reported, which works by exchanging Na+ and H+ and, along with a specific proton channel (Hv) regulated by Zn+2, elevating the intracellular pH (from 6.0 to 7.5, relevant for the activation of ion channels [Ca+2, Cl- and K+]), favoring hyperactivation. At the same time, the increase in cAMP levels activates PKA, stimulating TK (protein tyrosine kinase), and so this triggers tyrosine phosphorylation, a process associated with spermatic capacitation (Darszon et al., 2011).

3. Glutathione

The antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) are responsible for protecting the sperm against oxidative stress. Particularly GPx is responsible for metabolizing hydrogen peroxide (H2O2) produced endogenously, using reduced glutathione (GSH) as a substrate, which is oxidized to glutathione disulfide (GSSG) and can return to its reduced state by the action of the glutathione reductase enzyme (GR) to regain antioxidant activity, a process known as the glutathione redox cycle (Martínez-Sámano, 2011).

Now, from the chemical point of view, GSH is a low molecular mass compound, with a sulfhydryl group (-SH), formed (Figure 3) by the amino acids: glutamic acid, glycine and cysteine (Glu-Gly-Cys), also referred to as L-γ-glutamyl-cysteinyl-glycine, its molecular formula is C10H17N3O6 S and its molecular weight is 307.33 g/mol. The oxidized glutathione is L-γ- glutamyl-L-cysteinyl-glycine disulfide (GSSG), and its molecular formula is C20H32N6O12S2. (Sarrasague et al., 2006).

Figure 3. Representation of A) reduced glutathione (GSH), and B) glutathione disulfide (oxidized, GSSG) (Martínez-Sámano, 2011).

The concentration of intracellular glutathione is among the range of 1 to 11 mM in somatic mammalian cells (García-Giménez et al., 2013; Lu, 2013), while more specialized studies have reported a GSH concentration in pig spermatozoa of 3.84 ± 0.21 nM/108, and of 4.47 ± 0.46 nM/108 in human sperm (Gadea et al., 2004; Gadea et al., 2011). Correspondingly, it has been reported that the cytosolic concentration of GSH in somatic mammalian cells represents between 70% and 85% while the nuclear and mitochondrial concentration represent between 15% and 30%; a small percentage in endoplasmic reticulum has been similarly reported (García-Giménez et al., 2013; Lu, 2013). In the same way, GSH has important functions in xenobiotics detoxification, and it serves as well for the storage and transport of cysteine (Martínez-Sámano, 2011). On the other hand, GSH is essential for cell proliferation and has an important role in the inhibition of apoptosis, since its decrease allows the activation of caspases and the progression of apoptosis. A very important GSH function is to maintain the oxide-reduction potential of the cell, since it keeps the thiol groups of the proteins in a reduced state and thus allows the generation of several cascades of intracellular signaling; PKC and PKA are an example of this (Martínez-Sámano, 2011).

GSH is synthesized in the cells cytoplasm by the consecutive action of two enzymes: γ-glutamyl-cysteine (γ-GluCys) synthetase (also known as glutamate cysteine ligase, GCL), which uses glutamate and cysteine as a substrate to form the dipeptide γ-glutamylcysteine –this enzyme is considered the limiting step in the subsequent glutathione synthesis, glycine, in a reaction catalyzed by glutathione synthetase, forms GSH. ATP is an energy donor for both reactions. The intracellular concentration of GSH is regulated by the inhibition of GCL by the final product, GSH. Thus, there is a cellular balance between synthesis and consumption (Figure 4) (Sarrasague et al., 2006; Martínez-Sámano, 2011; Lu, 2013).

Figure 4. Representation of the glutathione synthesis (modified from Martínez-Sámano, 2011; Lu, 2013).

3.1 Glutathione metabolism

During the ROS detoxification, GSH is involved in two types of reactions: the non-enzymatic interaction with radicals such as O2-, nitric oxide and OH•; another reaction in which it participates is when it provides an electron for the peroxides reduction in the GPx catalyzed reaction (Fernández-Checa, 2008). The final product of GSH oxidation is GSSG, regenerated by GR, which transfers electrons from NADPH to GSSG, reducing itself to NADP+. During the reactions catalyzed by GPx and GR, GSH is not degraded, but recycled and thus can be reused (Martínez-Sámano, 2011; Dringen et al., 2000).

In contrast, during the generation of S-glutathione conjugates by glutathione S-transferases (GST) or by GSH exiting the cells, the internal level of GSH decreases, so the GSH has to be replaced by de novo synthesis (Dringen et al., 2000). Therefore, extracellular GSH and S-glutathione conjugates are substrates for the enzyme γ-glutamyl transpeptidase (γ-GT), which catalyzes the transfer of γ-glutamyl from GSH (or the S-glutathione conjugates) to an acceptor molecule and, therefore, generates the dipeptide cysteinylglycine (or the S-cysteinylglycine conjugate) and conjugated γ-glutamyl. The dipeptide cysteinylglycine can be hydrolyzed by cysteine and glycine ectopeptidases, amino acids that can subsequently be transported through the cell by specific transporters and participate in the de novo synthesis of GSH (Dringen et al., 2000).

When the spermatic cells are released from the testicle, their nucleus is extremely packed to perform transcriptional activity, however, they present changes during their passage through the epididymis (epididymal spermatozoa maturation), through the female reproductive tract (capacitation) and in the moment before fertilization (acrosome reaction) (Halliwell, 2006). These changes could be achieved by modifications in the existing proteins that, in turn, could be modulated by signals originating from the different spermatic environments, in this way the spermatozoa physiology will depend on networks of complex intracellular and extracellular signals (Halliwell, 2006).

Among the most widely known extracellular signals are the participation of cytokines, hormones and neurotransmitters that bind to specific receptors on the cell surface, which, when linked to said receptors, can generate several types of intracellular signals, such as changes in the ion concentration, activation of G proteins, such as cGMP, and activation of PKA and PKC kinases. These changes, consecutively, will cause the release of second messengers: cAMP, Ca2+ and phospholipid metabolites and protein phosphorylation; essential processes for cellular functions –the acquisition of fertilizing capacity, in the case of spermatozoa (Cisneros-Mejorado et al., 2014; Darszon et al., 2011).

Since ROS intervene in the spermatic function during the spermatozoa transit from the testicle to the oocyte, and because they are produced by the same spermatozoa, antioxidants, among which is GSH/GPX, can delay or inhibit oxidation, therefore, they could function as capacitation regulators (Rodwell, 2007).

It has been observed that ROS participate in tyrosine phosphorylation, directly influencing the intracellular levels of cAMP, thanks to the stimulation of adenylate cyclase (ACs) and,  consequently, protein phosphorylation (Rivlin et al., 2004). In addition, H2O2 can increase tyrosine phosphorylation through the inhibition of tyrosine phosphatase (TP) activity (Hecht and Zick, 1992).

4. Practical applications of GSH

GSH has been commonly used to improve the spermatic quality of several species, especially in frozen spermatozoa, using varying concentrations of GSH (Gadea et al., 2005). However, the positive effect of ROS has been determined by adding molecules that counteract their production or effect, as some antioxidants (Gadea et al., 2005). An example of this is reported by Gadea and collaborators in 2005 (Gadea et al., 2005), who found that GSH can have a counterproductive effect on spermatic capacitation when added at a concentration of 5mM on pig spermatozoa, since the viable capacitated sperm decrease, with respect to the control, and at 1mM; in contrast, the sperm that penetrate the oocyte are increased, at 1mM of GSH the motility is better than at 5 and 10mM (Gadea et al., 2005; Zhang et al., 2016), on the other hand, in humans and in pigs the basal concentration of GSH decreases in frozen spermatozoa as compared to fresh spermatozoa (Gadea et al., 2004; Gadea et al., 2011).

Recent studies have shown that not only pig spermatozoa suffer damage when frozen and unfrozen; in 2016, Lucio et al. reported a decrease in total progressive motility and mitochondrial activity in unfrozen dog spermatozoa using the CASA study (computer assisted sperm analysis) –the spermatozoa were treated with 20 mM GSH, comparing it with the control without GSH (Lucio et al., 2016).

Regarding this, it is evident that the GSH concentration is essential to maintain a redox balance in spermatozoa, not only in domestic species such as the pig or the dog; in 2017 Angrimani et al. reported a decrease in mitochondrial activity in Leopardus tigrinus spermatozoa depending on the concentration of GSH, this phenomenon could be the result of ROS inhibition due to the high concentrations of this antioxidant, thus blocking the physiological activity of ROS in spermatozoa (Angrimani et al., 2017).

An example of the GSH versatility is its use in different techniques, not only for frozen-unfrozen spermatozoa or evaluation of its activity in processes such as sperm capacitation, in 2017 Zambrano et al. reported the use of GSH at a concentration of 15 mmol/L combined with ICSI (intracytoplasmic sperm injection) pretreatment means, finding an improvement in chromatin decondensation of bull spermatozoa, compared to treatments without GSH (Zambrano et al., 2017).

The antioxidant, enzymatic and non-enzymatic spermatozoon system is very important for its maturation, first in the testicle, then in the epididymis and finally in the reproductive tract of the female, therefore, the GSH could be participating in the regulation of these processes.

5. Conclusions

The process of spermatic capacitation is fundamental for the spermatozoon to fulfill its main function, fertilizing the ovum; however, the process is regulated by several factors, where the participation of reactive oxygen species stands out. The oxidation suffered by the spermatozoon must be regulated so that it is not counterproductive in its physiology, this regulation is given, among other factors, by the GSH, since in conjunction with the enzyme antioxidant system, it provides the appropriate redox environment for the spermatozoon to acquire its fertilizing capacity.