Caspases in spermatozoa

1. Introduction

Caspases are proteins that cut other proteins into aspartic acid residues that execute a type of cell death called apoptosis. Apoptosis is a process that is orderly carried out without triggering an inflammatory response. In spermatozoa, this type of death is not defined, but the presence of such proteins in these cells has been related to various alterations such as freezing or to human pathologies.

2. What is apoptosis?

Apoptosis is a process of cell death that eliminates individual cells. It is carried out in an orderly and regulated manner, which is why it is often defined as “programmed cell death” (preparation of the machinery necessary to perform an activity at the desired time); however, this is not the only type of programmed cell death (Galluzzi et al., 2015; Kroemer et al., 2009; Parrish, Freel and Kornbluth, 2013). In somatic cells, apoptosis presents a series of morphological changes such as the loss of the integrity of the plasma membrane, condensation and chromatin fragmentation, as well as the packing of cell fragments into vesicles formed from plasma membrane (apoptotic bodies) (Earnshaw et al., 1999; McIlwain et al., 2013). In addition to the morphological changes, biochemical modifications can be recognized, such as changes in the Surface of the membrane that induce the externalization of phosphatidylserine (PS) (Elmore, 2007); the breaking of the DNA into regular fragments, normally multiples of 200 bp approx. (Elmore, 2007); as well as the cleavage between subsets of polypeptides and proteins (Earnshaw et al., 1999).

The cutting of proteins is carried out by a family of proteases called caspases (Earnshaw et al., 1999). This form of cell death can be activated in response to various types of cell stress, DNA damage, growth factor deprivation, endoplasmic reticulum stress and environmental signals, among other reasons. It can also be activated by other factors such as radiation, toxins, hypoxia, hyperthermia and free radicals (factors that cause stress within the cell) (Green and Llambi, 2015).

3. What are caspases?

Caspases are proteins that cut after aspartic acid residues (Cade and Clark, 2015, Grunewald et al., 2009), and this cut depends on a cysteine residue present in its catalytic site (McIlwain et al., 2013). They are proteins generated as proenzymes or zymogens (a protein that generates an active enzyme); when they are inactive, they are called procaspases. To activate them, it is necessary that a series of changes occur that allow them to mature and fulfill their function. Procaspases are structured by an N-terminal pro-domain (section of a protein that contributes to its three-dimensional conformation) followed by a long subunit and a short one (Figure 1). In some procaspases this subunit is separated by a small space eliminated from the zymogen during the maturation of the enzyme.

Figure 1. Apoptotic initiator and executioner caspases structure and general process of activation. Author: Blanca Patricia-López Trinidad.

All cleavage involved in the maturation of the procaspases is carried out in the carboxyl of the aspartate residues. Another structure that can be identified in these proteins is the pro-domain, within which two motifs can be identified (highly conserved amino acid sequence): the death effector domain (DED) and that it is apparently involved in the interaction with other proteins that signal death such as MORT1/FADD and TRADD; and the caspase recruiting domain (CARD), which is important for the interaction of mature caspases with other enzymes, besides their interaction with adapter and regulatory proteins (Earnshaw et al., 1999).

Caspases with short pro-domains, of around 23 amino acids (for example caspase 6 and 7), are apparently involved in the initiation of the apoptotic response, for which reason they are called initiators. Caspases with pro- domains of around 219 amino acids (for example caspase 10) are called executioners, these must be activated by the initiator caspases (Figure 1) (Earnshaw et al., 1999).

For a procaspase to be activated, it is necessary to go through a series of processes that will help it to mature; the first step is its dimerization with another procaspase, since this type of enzymes is activated through induced proximity, that is, when a protein called adapter interacts with the pro- domain of another protein of the same identity (Figure 1). This results in the formation of a tetramer composed of two equal units that join in pairs. This process continues with at least two more splits, which separate the long subunit from the small subunit. The new ends are folded over the dimers, promoting conformational changes to create two active sites in what will now be the mature caspase, followed by the removal of the pro-domains. However, it has been suggested that this elimination is not always necessary (Figure1) (Cade and Clark, 2015, Earnshaw et al., 1999, Green and Llambi, 2015).

3.1 Types of caspases

As mentioned above, caspases can be classified according to the domains they contain, initiators have a pro-domain, a long domain and a short domain, and executioner caspases, which only have the long and the short domain. Another way to classify them is according to their function as inflammatory or apoptotic caspases. The latter can be divided according to their structure and function as initiators or executioners (Figure 2).

Figure 2. Types of caspases. Author: Blanca Patricia López-Trinidad.

The apoptotic initiator caspases (AIC) are stable monomers and are activated by dimerization (Figure 1). After dimerization they acquire the capacity of self processing, which is why they can cleave the union between the pro-domain and the union between the long subunit and the short subunit. This same procedure is carried out by the executioners (with the difference that they do not have a pro-domain). Even after their dimerization, apoptotic executioner caspases (AEC) are not yet active, and they need to be cleaved by AIC to be active. As well as apoptotic caspases, inflammatory caspases are activated by dimerization (Figure 2) (Cade and Clark, 2015).

4. Caspases activation pathways

Caspase 8 is the most studied initiator caspase of the extrinsic apoptosis pathway, that is, the one that is activated by signs of death coming from the outside. One of the important extrinsic pathways is related to the activation of the tumor necrosis factor receptor (TNFR) superfamily, which has an extracellular domain rich in cysteine and a cytoplasmic domain of about 80 amino acids called death domain (DD) (Elmore, 2007). This receptor receives a death ligand (for example: FasL or TNF-alpha) inducing its oligomerization, favoring the recruitment of the death- inducing signaling complex (DISC). DISC is formed by proteins that contain and interact through the DD domain (Figure 3), such as FADD (Fas-associated protein with death domain) or TRADD (Tumor necrosis factor receptor type 1-associated death domain).

FADD or TRADD expose their DED (Death Effector Domain) domains which helps them interact with caspase 8 that also contains a DED domain, which causes the monomers of caspase 8 to come in proximity and dimerize. This encourages the activation and the necessary cleavages to occur so that the enzyme fully matures (see above, Figure 1). From this step, the pathway can stem into several alternatives; caspase 8 may activate the effector caspases 3 and 7 or it may cleave or cut Bid (pro-apoptotic protein that belongs to the BH3 family), which triggers mitochondrial death (Nair et al., 2014) (Figure 3A).

Figure 3A. Schematization of the general activation pathway of some caspases. Author: Blanca Patricia López-Trinidad.

The most studied caspase in cell death by the intrinsic pathway is caspase 9 since it will become part of the apoptosome. This is regulated both by external and internal factors, causing changes in the mitochondrial membrane, such as the formation of mitochondrial permeability transition pores (MPT), which causes the loss of membrane potential and the release of two main groups of pro-apoptotic proteins that are normally sequestered in the mitochondria and that escape to the cytosol (Elmore, 2007). The first group of pro-apoptotic proteins is formed by cytochrome c, Smac/DIABLO and HtrA2/Omi, which are proteins that activate the mitochondrial pathway dependent on caspase. Cytochrome c binds Apaf 1 and pro-caspase 9 to form the apoptosome, which is a heptamer of 7 subunits of each of the aforementioned proteins. This union will favor the activation of procaspase 9, turning it into active caspase 9 (Figure 3B). On the other hand, Smac/DIABLO and HtrA2/Omi induce apoptosis by inhibiting the inhibitor of apoptosis proteins (IAP) (Elmore, 2007, Green and Llambi, 2015, Kuhlbrandt, 2015, Schultz and Harrington, 2003).

Figure 3B. Schematization of the general activation pathway of some caspases. Author: Blanca Patricia López-Trinidad.

Caspase 1 is the most important in inflammatory processes; it is mainly associated with pyroptosis, a process of inflammatory cell death, accompanied by the release of cytosolic content, coinciding with apoptosis in the disintegration of DNA, but in this phenomenon the cleavage is visualized in the form of ladder. During this process, the caspase that fulfills the initiator and the executioner function is caspase 1 (Lin and Zhang, 2017). This caspase is involved in the processing of cytosines (small proteins secreted mainly by T helper cells by monocytes) (Zhang and An, 2007), which have a specific effect on the interaction and communication between cells. Particularly, caspase 1 can activate the precursor of interleukin 1 (Pro-IL-1, cytosine involved in inflammatory processes).

The above description will help to understand the functioning of the caspases that, until now, have been found in spermatozoa.

5. Caspases in spermatozoa

In general, the presence of caspases (1, 3, 9, 8) in ejaculated spermatozoa has always been associated with poor sperm quality and its presence can be related to cell death. Next, we will discuss the reports of the presence of caspases and the conditions under which they are presented.

5.1 Caspases associated with male infertility

In several studies, the relationship between the presence of active caspases and the low motility of spermatozoa has been analyzed. The first studies were carried out in 2002 by Weng and collaborators; they separated the spermatozoa of infertile patients, as well as of the healthy donors, using discontinuous percoll concentrations (45% and 90%), and marking the spermatozoa with fluorescent antibodies for active caspases. A greater amount of active caspase 3 was found in the low mobility portion (45% percoll), exclusively in the middle part of the spermatozoon, in both the healthy donors and the infertile patients. Therefore, they propose that caspase-dependent apoptosis can be triggered in the region where the mitochondria and the cytoplasmic drop of abnormal and immature sperm are located.

Together, they determined the general activity of the caspases and found that, in infertile patients, caspase activity is greater, even in the high motility fraction. Because the spermatozoon has little cytoplasm, the authors propose that the activity of caspase 3 in healthy patients is more efficient, so it requires low levels. In addition, in the high motility fraction they show a negative correlation between the motility and the activity of the caspases. In this same study, they quantify DNA fragmentation and observe a positive correlation between DNA fragmentation and the presence of caspase 3 in spermatozoa (Weng et al., 2002).

However, they were not the only ones that detected caspases related to problems in the sperm parameters, since Paasch and collaborators in 2003 determined caspases in spermatozoa by previously separating the sample between those that presented PS exposure and those that did not. In general, the donors had low levels of caspases compared to infertile patients. However, spermatozoa with externalization of PS showed high levels of caspases in both healthy donors and in infertile patients (Paasch et al., 2003). This is in coincidence with investigations in which it is argued that modifications of membrane functionality can also be linked to the activity of caspase 3, which cleaves protein kinases, thus modifying the bidirectional migration of phospholipids through the lipid bilayer, by the retention of the activity of the scramblase (Frasch et al., 2000). Paasch and colleagues also argue that the presence of caspases is a residual element of spermatogenesis, and that it is prone to be activated due to membrane damage. Furthermore, the presence of caspases indicates a defect or incomplete remodeling of the cytoplasm during spermatogenesis, since, they found staining of caspases (1 and 9) in the middle piece, specifically in the residual cytoplasm of patients, while in the donors it was located in the pos-acrosomal region.

Other studies have focused on determining the relation of caspases, as well as the release of cytochrome c, finding an increase in reactive oxygen species (ROS) and the concentration of cytochrome c in seminal plasma, in addition to finding signals of caspase 3 in the middle piece. Therefore, there is a possible relation between the increase in damage indicated by oxidative stress and CP-mediated apoptosis in male patients with a factor of infertility.

5.2 Activation of caspases by freezing

One of the techniques that support assisted animal reproduction is the freezing of gametes, which consists of subjecting the cells to low temperatures to decrease their metabolism and preserve them. However, it is essential to utilize cryoprotectants, which are compounds that dehydrate cells to prevent the formation of crystals inside and outside of them.

Among the most common cryoprotectants is glycerol. In 2005, Grunewald et al. tested two different concentrations of glycerol (7% and 14%) and observed the effect it had on the activation of caspases during the freezing process. They found that freezing causes an increase in all the caspases measured in this study (1, 3, 8 and 9), both in healthy donors and in infertile patients. Among the peculiarities that they report is that both caspase 8 and 9 increase at high concentrations of glycerol, and significantly, they also increase in the samples from infertile patients. Whereas the precursors of caspase 3 (32kDa) decrease when concentrations of antioxidants such as glutathione increase (Grunewald et al., 2005).

In 2004, Paasch had already reported the activation of caspases during freezing, mentioning that freezing leads to the activation of caspases 1, 8, 9 and 3, and that it is also related to the externalization of phosphatidylserine. To corroborate this last point, positive and negative cells were separated to phosphatidylserine, finding an increase of all the caspases in the portion of spermatozoa that presented the exposure to phosphatidylserine (Paasch et al., 2004).

Cryopreservation, as well as cryoprotectants, can trigger programmed cell death that is associated with the integrity of the membrane and the activation of caspases. This may occur because the cryopreservation of spermatozoa leads to several structural and functional alterations of the plasma membrane, which can lead to the loss of the stability of the lipid bilayer and to a sublethal cryodamage of spermatozoa. This lack of integrity in the membrane can initiate the caspase cascade. Cryoprotectants, such as glycerol itself, can contribute to the activation of caspases via the toxic effects on the mitochondria, since cytotoxic stress involves mitochondrial perturbations, followed by DNA fragmentation (Grunewald et al., 2005).

5.3 Activation of caspases by peroxide and progesterone in spermatozoa

It has been repeatedly shown that calcium plays an important role in the signaling of death in somatic cells and, consequently, in the activation of caspases. Progesterone can be a powerful mobilizer of calcium in spermatozoa (Demaurex and Distelhorst, 2003), allowing the exit of storage locations or generating a series of reactions that allow the entry of this ion. There are several intracellular components that are sensitive to calcium concentrations, such as the mitochondria, so that a concentration elevation can induce apoptosis, since it can promote the release of factors, such as cytochrome C, which also impair mitochondrial function.

In spermatozoa the effect of progesterone on the activation of caspases has been proved, finding that, from an exposure of 15 and up to 120 minutes with 20 μM of progesterone, there is an increase in the amount of active caspase 3, as well as, caspase 9. This effect was accentuated when the spermatozoa were subjected for 60 minutes with 10 μl of progesterone. These effects are diminished when spermatozoa are treated with DEVD-CMK or z-LEHD-FMK (caspase inhibitors 3 and 9) (Bejarano et al., 2008). Other studies have also reported an increase in active caspase 3 using the same progesterone concentration (Bejarano et al., 2008). The importance of the above lies in the fact that in somatic cells, calcium plays an important role in the signaling of death, however, in spermatozoa, a dual role can be attributed to this component, since progesterone is a steroid hormone that induces the entry of calcium into the spermatozoon and causes multiple physiological responses essential for fertilization to occur (Lishko et al., 2011), and it can also carry out processes such as the death of the same cell.

On the other hand, hydrogen peroxide (H2O2) is a ROS that, in addition to being able to mobilize calcium, can make modifications to other proteins, causing the activation of caspases and eventually, death (Bejarano et al., 2008). In spermatozoa, using 10 μM of H2O2 for 60 minutes, the highest caspase 3 activation is obtained (Bejarano et al., 2008, Demaurex and Distelhorst, 2003) and, ironically, high H2O2 concentrations do not produce a notable change in the activation of the caspases (Bejarano et al., 2008). It is important to consider that, while ROS can cause damage to the spermatozoon, they can also participate in important processes in sperm physiology, activating or inactivating proteins to carry out processes such as the activation of mobility by increasing the cyclic adenosine monophosphate (cAMP) and protein phosphorylation for the acquisition of its fertilizing capacity. Thus, among the negative and positive effects of ROS on spermatozoa, it seems that good functioning will depend on its proper regulation by antioxidants (Arenas-Ríos et al., 2014).

5.4 Other associations

The activation of caspases in spermatozoa has also been related to smokers, since the negative impact of cigarette smoke on male infertility has been demonstrated (Soares and Melo, 2008). In 2011 (El-Melegy and Ali, 2011), a study was carried out to analyze the percentage of active caspase 3 in spermatozoa from healthy donors and in smoker and non- smoker patients. The study showed that the infertile patients who were smokers presented a greater amount of this caspase, compared to the other groups. This condition was exacerbated significantly in those who were strong smokers, specifically that they had been smoking for 17 years and consuming at least 23 cigarettes a day (El-Melegy and Ali, 2011). This may be due to a deregulation in the hypothalamus- pituitary-testis axis or to mild hypoxia caused by the disruption of the testicular microcirculation, but a toxic effect of the chemical compounds of cigarettes on the testicular epithelium is more related, together with an adverse effect caused by ROS in spermatozoa, which could be the cause of the damage linked to the low concentration of antioxidants (El- Melegy and Ali, 2011).

Although in many studies the comparison between healthy and infertile men is made, infertile men do not have a pathology associated with their infertility such as varicocele. Varicocele is characterized by an abnormal and retrograde dilatation of the blood fluid in the testicular veins, this condition causes infertility (Tsili et al., 2017). In addition to causing infertility, it has been shown that this condition favors an increase in the amount of caspase 9 in spermatozoa. Oxidative stress is one of the probable causes of damage in these spermatozoa since it has been proposed that during the varicocele a stress condition is generated (Zalata et al., 2011).

Other studies relate caspases to the maturational status of spermatozoa. These investigations are based on the separation of spermatozoa through centrifugation in a concentration gradient, obtaining a fraction that were considered as mature spermatozoa (the spermatozoa found at the interface between the gradient of 47% and 90%) and immature (those found only in the 90% gradient). In both cases the number of caspases 9, 8 and 3 was determined, finding a greater amount in immature spermatozoa (Paasch et al., 2003). This may be due to incomplete maturation during transit through the epididymis (Paasch et al., 2003).

6. Conclusion

Despite several studies associate poor sperm quality with the presence of caspases (1, 3, 9, 8), these studies are inconclusive, since the signaling pathways in which they participate specifically in spermatozoa are unknown. In addition, it must be considered that what happens with these proteins during the spermatozoa transit through the epididymis is unknown. Thus, it is necessary to carry out more studies that help to clarify the role of caspases in sperm physiology during its formation and subsequent post-testicular maturation.

Abbreviations

AEC: Apoptotic executioner caspases.
AIC: Apoptotic initiator caspases.
Apaf 1: Apoptotic protease activating factor 1.
bp: Base pairs.
CARD: Caspase recruitment domain.
DD: Death domain.
DED: Death effector domain.
DIABLO: Direct IAP-binding protein with low pI.
DISC: Death-inducing signaling complex.
DNA: (deoxyribonucleic acid).
DNA: (deoxyribonucleic acid). Polynucleotide composed by the covalent binding among deoxyribonucleotide units; the carrier of genetic information.
FADD: Fas-associated protein with death domain.
FasL: Fas ligand. HtrA2/Omi: mitochondrially-located serine protease HtrA2, also known as Omi.
IAP: Inhibitor of apoptosis proteins.
Pro-IL-1 : Interleukin-1 beta precursor. PS: Phosphatidylserine.
ROS: Reactive oxygen species Smac: Second mitochondria-derived.