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The effect of cadmium exposure on carbohydrates of the plasma membrane and the phosphorylation of proteins of epididimary spermatozoa of the Wistar rat
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In mammals, reproduction requires the recognition between two cells that are highly differentiated, the spermatozoon (which is asymmetric and mobile) and the oocyte (which is large and immobile), to originate a new individual. For this the following is required: 1) the species-specific union of the spermatozoon with the zona pellucida of the oocyte, 2) the acrosomal reaction by the spermatozoon, 3) the penetration of the extracellular layer of the oocyte by the spermatozoon, 4) the union of the sperm membrane with that of the oocyte, and 5) the fusion between gametes (fertilization) (Wassarman et al., 2001). For these events to take place, a solid structure is required in both the sperm and the oocyte, until the fusion between the gametes occurs.
Regarding the male gamete, it has been widely described that its continuous and efficient production depends on the proliferation of spermatogonia and its consequent differentiation into anatomically complete spermatozoa (Hess and Renato de Franca, 2008), for which it is necessary that the neuroendocrine hypothalamic–pituitary–gonadal axis functions coordinately, because spermatogenesis is a highly complex process, in which an orderly series of divisions is carried out –mitosis first, meiosis second– which in combination with spermiogenesis forms anatomically complete spermatozoa, from spermatogonia, with the participation of the appropriate microenvironment that is regulated by the Sertoli and Leydig cells, which, together with paracrine factors, such as cytosines and growth agents, regulate the mechanisms of cell division and renewal, and of interleukin 1, which is capable of modulating the functions of Sertoli cells, resulting in the adequate production and development of male gametes in the seminiferous tubules (Jenardhanan et al., 2016; Neto et al., 2016).
However, after its formation in the seminiferous tubules and subsequent release during spermiation in the light of the tubule, mammals spermatozoa are directed towards the rete testis, for which they will require a series of post-testicular processes, so that they are functional inside the oviduct and are able to execute an efficient fertilization; that is, they need to pass through a wide range of microenvironments made up of different fluids, whose origin and composition will be provided by organs such as the epididymis, the prostate, the seminal vesicle, and the oviduct. These fluids will exert in the spermatozoa an important succession of changes of maturation that transform the immobile cells, which also are incapable of fertilizing, into vigorously active ones with the capacity to unite and fuse with an oocyte, and at the right moment they will grant spermatozoa an induction that culminates in the acquisition of fertilizing capacity (Sostaric et al., 2008).
2. Epididymis: structure and function
The epididymis has been described as an organ composed of a single long and tightly-coiled tube. Its length varies according to each species, 1 m in the mouse, 3 m in the rat, from 5 to 7 m in the human, and from 70 to 80 m in the horse (Sostaric et al., 2008; Arrotéia et al., 2012; Robaire and Hinton, 2015). During embryonic development, the epididymis of mammals, like other components of the urogenital system, develops from the mesonephros (Sostaric et al., 2008; Robaire and Hinton, 2015), a tissue that originates in inferior animals a primitive kidney. This organ is located in the upper part of each testicle and it communicates with the rete testis by means of the efferent channels, connecting by these routes with the testicle; likewise, it is delimited with the beginning of the vas deferens (Arrotéia et al., 2012).
When analyzed anatomically, the epididymis is generally divided into four regions: the first is the initial segment, followed by the head (caput or cephalic region), the body (corpus or middle region), and the tail (cauda or caudal region) (Robaire and Hinton, 2015) (Figure 1); along all of them, the lumen of the epididymis is formed by a pseudostratified epithelium, integrated by the presence of different types of cells: a) main cells, b) basal cells, c) apical cells, d) narrow cells, e) clear cells, and f) halo cells. Some of them, such as the main and basal cells, can be found throughout the tubule, while the apical, narrow, clear, and halo cells (intraepithelial lymphocytes) are only present in some segments (Arrotéia et al., 2012; Robaire and Hinton, 2015), with which they establish different microenvironments. Together, all the epithelial cells that cover the epididymal duct form a luminal fluid environment, through the active secretion and absorption of macromolecules (proteins, glycoproteins) that are synthesized by the expression of different genes, as well as by small molecules (sugars, electrolytes), which, together with the contents derived from the testicles, provide a particular environment in which the spermatozoa that travel through the canal are modified when they come into direct contact with the luminal microenvironment, as a requirement of the maturation process (Tulsiani, 2006; Robaire and Hinton, 2015), together with the fact that they are responsible for eliminating cytoplasmic residues and protecting spermatozoa, thanks to the formation of an anatomical and immunological barrier during their passing through the entire epididymal tubule, as when they are stored (Hernández-Rodríguez et al., 2016).
Figure 1. Schematic organization of the rat epididymis. The four regions of the epididymis: head, body, and tail, as well as the initial segment, are represented. The lines indicate the place where the different regions are divided (Hernández- Rodríguez et al., 2016).
After the production of spermatozoa through the epithelium of the seminiferous tubules, they should move to the region of the head and then to the body of the epididymis to develop maturation before being collected and stored in the region of the tail (Robaire and Hinton, 2015). It is important that in this area a specific luminal environment allows the spermatozoa to be collected for even several weeks, because a metabolic quiescence that prevents spermatozoa from being activated before ejaculation is maintained (Sostaric et al., 2008). Sperm maturation gives the sperm its fertilizing capacity (Dacheux et al., 2009), which means that they acquire the potential to move progressively and the ability to recognize and enter the zona pellucida to fertilize an oocyte (Cornwall et al., 2007; Robaire and Hinton, 2015). Although the length of the epididymal tubule varies according to different mammals species, several studies indicate that the average transit of spermatozoa through the epididymis is highly similar and that it ranges between 9 and 11 days (Sostaric et al., 2008; Robaire and Hinton, 2015), so that their functional maturity in this organ can require up to 12 days in the human, 13 days in the ram, 10 to 11 days in the pig, and in the rat it can take from 8 to 11 days (Robaire and Hinton, 2015). Much of this variability is due to the time required to reach the caudal region, since in most species the transit through the head and body takes approximately one to three days each, and the movement of the spermatozoa in both regions is mainly achieved by the continuous peristaltic contractions of the smooth muscles in the wall of both regions of the epididymis, while in the cauda the smooth muscles are generally inactive, until being stimulated to contraction.
3. Changes in spermatozoa during epididymal maturation
Several studies indicate that for the mammals spermatozoon to develop the ability to move through the oviduct (acquisition of progressive mobility) and subsequently recognize and penetrate the zona pellucida, to finally fertilize and impregnate an oocyte (Tulsiani, 2006; Dacheux and Dacheux, 2014), changes need to occur in the composition of carbohydrates and proteins of the plasma membrane to modify the physiological properties of spermatozoa as they transit and interact with various molecules that produce epididymal cells (Tulsiani, 2006 Sostaric et al., 2008; Caballero et al., 2011; Perobelli et al., 2013; Robaire and Hinton, 2015).
Spermatozoa exit the testicle with the complete anatomical structure that is known (Figure 2), which is composed of a head that contains the nucleus with the DNA very compacted between protamines and whose anterior end is covered with the acrosome (a cap-shaped structure containing hydrolytic enzymes, whose participation is relevant during the process of fertilization), and a flagellum, necessary for motility, which morphologically is divided into four regions: a) the head with its connecting piece, which joins the head with the flagellum; b) the middle part, also called the neck, where the mitochondria are located in an optimal helical arrangement to generate the energy that the axoneme uses (composed of nine pairs of microtubules arranged radially around the central filaments) extending throughout the following region; c) the main part, which covers most of the flagellum and which is used for propulsion, by waves of efficient movement, to ensure remote transport, and finally; d) the terminal part of the flagellum, composed of a fibrous sheath (Cooper and Yeung, 2006, Arenas-Ríos et al., 2010). However, the spermatozoa still lack the fertilizing potential, for this reason they require post-testicular maturation, a complex and sequential process in which the totality of modifications that are experienced is still unknown; yet, it is known that in this transformation, the epididymis plays the main role (Nixon et al., 2014), favoring the occurrence of a series of changes that promote transformations at the morphological, physiological, and biochemical levels, the result of which is a spermatozoon with a high motility potential and with the full capacity for fertilization (Palomo-Peiró, 1995, Rodríguez-Tobón, 2016).
Figure 2. Anatomical structure of a rat’s spermatozoon.
3.1 Morphological changes
For some mammalian species, one of the important aspects in epididymal maturation is the migration and subsequent loss of the cytoplasmic droplet (CD). This can remain in the spermatozoon after the release of the germinal epithelium in spermiation, since generally the majority of the cytoplasm of the spermatid during its elongation is phagocytosed as a “residual body” by the Sertoli cell (Cooper, 2011). In a morphological study of ejaculated spermatozoa from humans, with various alterations, it was shown that there are two types of cytoplasm in sperm cells. One represents the remnant cytoplasm, which can be located attached to any part of the spermatozoon flagellum, and the other that is the “true” CD, which is more stable in the region of the spermatozoon neck. In the spermatozoa of the mouse epididymis, CD is located in the middle part of the flagellum (Cooper, 2005), so the difference in morphology and the position of the CDs between human and mouse spermatozoa reflect differences between species (Xu et al., 2003). However, in spermatozoa of the albino rat it has been reported that CD is lost during spermiation (O'Donnell et al., 2011). However, it is possible that CD is still observed in regions of the epididymis as a consequence of some alteration that occurred during spermiation, so it is required that the cells of the epididymal tubule phagocytize CD. The percentage of spermatozoa with CD decreases markedly, as they move from the region of the head to the tail of the epididymis (Cooper and Yeung, 2006; Arrotéia et al., 2012; Robaire and Hinton, 2015). Similarly, the epididymis propitiates that the unions between the protamines, which are nucleoproteins among which the DNA or the male genome is modified by an increase in the amount of disulfide bridges (Cooper and Yeung, 2006), are modified. The genetic information that the spermatozoon carries is inside a highly compacted and stable nucleus. Therefore, in turn, there is a gradual decrease in the size of the spermatozoon head, after being in the region of the head, and transported to the tail of the epididymis. Likewise, it has been described that during epididymal maturation there is a decrease in the size of the acrosome, which covers the spermatozoon nucleus, after the reorganization of various enzymes and other proteins that it contains (Cooper and Yeung, 2006; Arrotéia et al., 2012; Robaire and Hinton, 2015), to provide a better hydrodynamic displacement. Other modifications that occur during maturation are at the level of the mitochondria, fibers and microtubular components of the middle and main parts, with the purpose of giving rise to the efficiency in the progressive motility. All these changes in the spermatozoon prevent the presence of germ cells with an abnormal, incomplete or damaged genetic load in the ejaculate, and avoid the presence of infertile cells that could limit reproductive success (Cooper and Yeung, 2006; Arrotéia et al., 2012; Robaire and Hinton, 2015; Rodríguez-Tobón, 2016).
3.2 Physiological changes
The optimal functionality of spermatozoa depends on the processes that occur inside and outside of these cells. The formation of diverse microenvironments along the epididymis is very important, due to different molecules secreted towards the lumen of the tubule, and also, due to the changes between the cellular associations that are observed along the epididymal epithelium, which allows modification in the characteristics and composition of the surface of the plasma membrane of the spermatozoa to occur. This leads to the spermatozoa being able to prolong their survival within the female genital tract by maintaining the acrosome and plasma membranes as stable structures, when the glycosylation events occur in the components of the spermatozoon surface, and until the modifications that lead to the acrosome reaction occur. The appropriate combination of physiological changes, and their link with morphological and biochemical changes, favors that in spermatozoa the processes linked with the fertilizing capacity can occur, such as: a) the acquisition of a marked increase in the vigor of the motility, after the passage of the circular movement to rectilinear, b) the ability to experience the training process and c) the ability to interact with the zona pellucida and finally fertilize the oocyte (Hernández-Rodríguez et al., 2016).
3.3 Biochemical changes
All the components that conform the spermatozoon are surrounded by a plasma membrane that is highly complex and highly specialized (Srivastava and Olson, 1991; Dacheux and Dacheux, 2014). This is a vital component of high importance for the early events that occur during fertilization. However, a spermatozoon is a cell that loses the ability to synthesize proteins or some other type of molecule of biological importance in the last steps of spermatogenesis (Dacheux and Dacheux, 2014). It is for this reason, that the biochemical changes that occur in male gametes, throughout the journey through the epididymis, will depend on post-translational modifications (which occur in the residues of the amino acids that constitute proteins). They can be of several types: a) addition or b) removal of functional groups, or c) formation of links with other proteins.
Among the modifications of great importance that occur in the plasma membrane of spermatozoa, several changes are described that allow a better and specific spatial distribution in the components of the glycocalyx, which takes its name after the presence of abundant oligosaccharides in its composition, which have been linked to a large amount of proteins (Rodríguez-Tobón, 2016) and lipids, forming chains of glycoproteins and glycolipids, respectively. Both chains form the cell envelope that is in contact with the extracellular medium. In particular, modifications occur in glycosylation that allow the integration or separation of one or more glycan chains to a main chain by means of covalent bonds.
The biochemical changes that occur in the carbohydrate portions of the components of the plasma membrane of spermatozoa during maturation in the epididymis have been evaluated for some time, for it is known that the content of glycoconjugates in spermatozoa is a good indicator of the maturation in the epididymis (Tajiri et al., 2012). Because glycoconjugates are a highly complex form that participates in cell-to-cell recognition, in the union of the spermatozoon to the zona pellucida in the acrosomal reaction and in the spermatozoon-oocyte fusion (Cardona-Maya and Cadavid, 2005), it requires the use of lectins to evaluate the presence of these carbohydrates on the surface of the membrane of the spermatozoon (Srivastava and Olson, 1991).
Lectins are proteins of vegetable and animal origin with a high specificity of binding to the residues of the carbohydrates. In glycoconjugates (Hernández-Cruz et al., 2005) they work as a tool for the analysis of the variations in the distribution and density of the exposure of saccharides when the spermatozoon passes through the epididymis. Even though certain details of these changes have not been fully clarified, there is evidence of the participation of two sets of enzymes: a) glycosyltransferases, a group of synthetic enzymes that add sugar residues to a donor sugar (nucleotide sugar), and b) glycohydrolases, hydrolytic enzymes that separate residues of a sugar from existing glycoconjugates. Both groups are present in high concentrations in the luminal fluid of the epididymis that surrounds the spermatozoa, which favors modifications in the glycoconjugates of the spermatic surface (Tulsiani, 2006).
Among the sugars identified in the sperm membrane and whose presence is modified by the transport of these cells through the tubule of the epididymis, the following are found: a) sialic acid, which has been related to the total negative charge of the surface of the spermatozoon and which increases during the epididymal transit; in the rat, several researchers have reported that in the head of the sperm higher values of sialic acid are joined to the membrane and as they progress through the epididymal tubule, the unions in the tail of the spermatozoon decrease to lower values, so it is possible that the binding of sialic acid to the membrane may be relevant and linked to sperm maturation in that species (Hall and Killian, 1987) and as a preparation for the subsequent fusion of the spermatozoon with the oocyte; b) N-acetylglucosamine: the adequate presence and distribution of this carbohydrate is of great importance in the interaction process between gametes, in addition, in spermatozoa of some mammalian species, it has been described that the presence of N-acetylglucosamine is necessary to carry out the acrosomal reaction and eventually fertilization (Cardona-Maya and Cadavid, 2005; Tulsiani and Abou-Haila, 2012); the presence of both carbohydrates can be evaluated by the affinity they have with the Triticum vulgare agglutinin (WGA) lectin, which detects the residues found in glycans; c) mannose is a carbohydrate that permanently forms the surface of the membrane of the spermatozoon (Aliabadi et al., 2013), with its respective changes along the epididymis; it is important for its role in the recognition of the spermatozoon and the zona pellucida and for taking part in the fertilization process (Jiménez et al., 2006; Aliabadi et al., 2013); it has been reported that the Canavalia ensiformis agglutinin (Concanavalin A, Con A) lectin binds to mannose residues, which makes it possible to identify its presence on the spermatic surface; d) fucose is a carbohydrate that is related to the species-specific binding between gametes, by its adhesion with proteins from the zona pellucida (Tulsiani and Abou-Haila, 2012); the presence of fucose bound to sperm proteins and in the seminal plasma grants the ability of gametes to form a spermatozoa reserve in the oviduct after adding proteins to the epithelial cells that recognize fucose (Ignotz et al., 2001). Particularly, it was reported in the study by Hall and Killian (1987) that in the albino rat spermatozoa transiting the epididymis, the presence of plasma membrane fucose decreases three times in the region of the tail, as opposed to spermatozoa from the region of the head.
The changes that occur during the process of sperm maturation in the presence of membrane carbohydrates include other molecules, such as D-galactose, which decrease as the cell progresses along the epididymis, while the levels of N-acetylgalactosamine, on the contrary, are higher in the membrane of the spermatozoa of the tail than in those of the head of the epididymis. Other carbohydrate residues, such as -D-glucose and mannose, are present in a greater proportion in the membranes of the spermatozoa of the head and tail, but in very low amounts in the cells of the epididymal body. In relation to the content of mannose in the plasma membrane, the changes registered in the epididymis can be a good indicator of sperm maturation (Belmonte et al., 2002).
On the other hand, protein phosphorylation in tyrosine residues is another of the important intracellular changes in the spermatozoon in the acquisition of the fertilizing capacity during maturation (Filtz et al., 2014), because it is considered to be the essential molecular basis for the coordinated development of the progressive movement by the flagellum and with the subsequent training (hyperactivation) and acrosome reaction of the spermatozoon (Moseley et al., 2005; Mor et al., 2007) in the oviduct of the female. It has been found that the state of protein phosphorylation is regulated by a dependent pathway of cyclic adenosine monophosphate (cAMP) and by the enzymatic activity of two types of proteins: a) kinases and b) phosphatases, the former adding a phosphate group and the latter removing it. Because the spermatozoon is unable to synthesize new proteins, it can be argued that the dependence on protein phosphorylation is greater than that of other cell types and it is associated with the process of epididymal maturation by phosphorylation and dephosphorylation events mainly in proteins of the flagellum, participating in the acquisition of motility and in the ability to fertilize the oocyte, functioning as an on/off switch. In rats, proteins of a molecular weight between 50-64 and 91-127 kDa that are phosphorylated have been identified, located at the level of the entire acrosomal domain of the spermatozoon, and similarly at the level of the middle piece (Rodríguez-Tobón, 2016). In the research by Lewis and Aitken (2001), the impact of epididymal sperm maturation on the patterns of tyrosine phosphorylation in spermatozoa of the head and tail region of the epididymis in the Wistar rat was analyzed by immunohistochemistry. The results were: a) in the spermatozoa at the head region of the epididymis, the pattern of protein phosphorylation in tyrosine residues that occurred most frequently was the one observed in the head of the spermatozoon, whereas b) in the spermatozoa at the tail of the epididymis, the pattern of outstanding phosphorylation was the one that was noticed in the posterior part of the spermatozoon; that is to say, that the immunolocalization that predominated in the majority was concentrated in the part of the flagellum. The multiple changes that the sperm suffer when crossing the epididymal regions are fundamental (Tulsiani, 2003; Seligman et al., 2004) and depend on the specialization of the microenvironment in each section of the tubular lumen, through the synthesis and secretion of proteins and molecules that interact with the spermatozoa, and by the elimination of substances by the cells that make up the epididymal epithelium, whose functionality depends on the action of androgens (Robaire and Hamzeh, 2011; Robaire and Hinton, 2015).
4. Regulation of the function of the epididymis by androgens
The steroid hormones fulfill diverse functions along the embryonic development and in stages subsequent to birth (Blok et al., 1992), particularly in males, the androgens have effects that play an essential role in the masculinization of the fetus, in the formation of internal and external reproductive organs (Arrotéia et al., 2012) and in the development of male secondary sexual characteristics. Testosterone (T), the main androgen that circulates in the bloodstream, is synthesized and then secreted by Leydig cells located in the interstitial space of the testicles. This hormone can reach the cells of the epididymis through two different routes: a) through the efferent channels after leaving the rete testis, where it is mostly added to the androgen-binding protein (ABP), and b) through blood circulation, in which the free T can enter the cells by passive diffusion. The greater part that reaches the cells of the epididymal tubule is transformed by the reduction that causes the steroid enzyme 5-reductase to its most potent metabolite, 5-dihydrotestosterone (DHT) (Robaire and Hamzeh, 2011; Robaire and Hinton, 2015). The DHT is the steroid that has been determined as having the greatest power of action in maintaining the functions of the epididymis, which is achieved through its binding to the androgen receptor (AR) (Arrotéia et al., 2012). This is a member of the superfamily of nuclear receptors located on the X chromosome, whose gene encodes a multidomain receptor that is constituted by four functional domains: 1) an NH2- terminal domain (NTD), responsible for transcriptional activation, qualified as the more variable among nuclear receptors in both length and sequence; 2) a DNA binding domain (DBD), located in the central part of the AR molecule, which is the most conserved region within the family of nuclear receptors and the one that binds to DNA; 3) a ligand binding domain (LBD), responsible for the dimerization and activation of transcription, this domain regulates the interaction between the AR and the heat shock proteins and interacts with the NTD domain of the receptor to stabilize the androgen binding (Heinlein and Chang, 2002) and 4), a non-conserved and flexible hinge region, responsible for binding to the LBD and DBD domains and for regulating DNA binding, nuclear translocation and transactivation of the AR (Haelens et al., 2007).
In the target tissues, the regulation of the androgenic effects of AR occurs because it acts as a receptor-ligand transcription factor, because once in the cytosol the AR has been linked to T or DHT, it stops being inactivated by the separation of thermal shock proteins and it is transported to the nucleus of the cell, where in the form of a homodimer it subsequently binds to the hormone response elements, called specific androgen response elements (AREs). In the epididymis of the rat, the AR is immunolocalized in a specific manner, with greater intensity in the region of the head and body, and less intensity in the tail, suggesting that its presence and expression have specific functions in the epididymis (Robaire and Hamzeh, 2011; Robaire and Hinton, 2015) to control their proper performance, so the participation and regulation of androgens in the epididymis is of vital importance in relation to a good maturation and storage of spermatozoa.
Although the function of the epididymis depends mostly on the effects of androgens, it is known that they are not the only steroids that have action within their various regions, since T is also aromatized to 17-estradiol (E ), by means of the aromatase enzyme P450 within the epididymal epithelial cells, in which the presence of the alpha and beta forms of the estrogen receptor (REand RE) has been identified, both in the head and in other regions of the epididymis, and whose expression it seems to be of a specific species. The role of estrogen has been related to the reabsorption of the luminal fluid that occurs in the efferent ducts and in the initial segment of the epididymis; likewise, estrogens regulate the transport of fluid through the duct and they are responsible for the increase in the concentration of spermatozoa when they reach the head of the epididymis. Although the epididymis epithelium is described as androgen-dependent, estrogens also assist in the differentiation and maintenance of epithelial morphology (Arrotéia et al., 2012), which indicates that E fulfills a role in the functions the epididymis performs in the spermatozoa, that is, that the physiology of the epididymis is regulated by androgens and estrogens from the production and secretion of T, which is necessary to control the morphophysiology of epithelial cells and the expression and secretion of proteins, in addition to preventing cell death, which ensures the maturation and storage of spermatozoa (O'Hara et al., 2011; Kerkhofs et al., 2012), and the increase in male fertility.
However, there is a wide variety of compounds in the environment that directly affect the synthesis and concentration of T in various species, which leads to alterations in the functions regulated by steroid hormones, within which, the ones that are involved with the performance of the epididymis in the maturation of male gametes by means of epithelial cells can be affected, because the expression and secretion of proteins related to maturation and storage prior to sperm ejaculation can be modified, so that this may be one of the factors that increases male fertility problems.
5. Factors that affect male fertility
During the last years, the focus of various studies has been on the reduction of male reproductive quality, because of the aspects of health that have been evaluated and that are linked to male reproduction of negative consequences, for example, the repercussion caused by exposure to chemical agents found in the environment (Povey and Stocks, 2010). So it has been detected in humans and in experimental animals (mostly rodents) that pollutants released through various routes into the environment can alter the proper functioning of the endocrine system and cause a negative effect that modifies the production of spermatozoa, as well as damaging the functioning of the organs that integrate the reproductive system involved in the acquisition of the fertilizing potential of the spermatozoa, such as the prostate, the seminal vesicles and the epididymis, which can affect in this way the reproductive functionality of males (Anway et al., 2005; Wong and Cheng, 2011).
In general, in recent years the cases of infertility have increased around the world, which is a serious problem in reproductive health for young couples. Specifically, in Mexico, according to the most recent data from the National Institute of Statistics and Geography (INEGI), the number of couples with fertility problems corresponds to an approximate of 1.5 million, so one couple out of six, in their reproductive age, are having troubles to conceive (INEGI, 2016), that is, they face some type of infertility.
The World Health Organization (WHO) has clinically defined the concept of infertility as the impossibility of achieving pregnancy after one year of regular sexual intercourse without the use of any contraceptive method (WHO, 2010). According to this organization, approximately 15% of couples have fertility problems, so they require the assistance of medical specialists, which implies a great economic and emotional cost. Of the total fertility difficulties, it is known that 30% of them respond directly to difficulties in the reproductive physiology of women, another 30% is related in a concrete way to alterations that cause inconveniences in men, while the remaining 40% corresponds to a great variety of causes, which, when analyzed, concern both men and women, which is why it has been estimated that men are related to 50% of the reasons that cause infertility (Cui et al., 2016).
In males, the causes of infertility can be derived from a great diversity of conditions and most of them coincide with affectations that diminish significantly the quantity and the quality of the seminal parameters, in terms of concentration, motility and vitality of spermatozoa (Palma and Vinay, 2014). Although in a majority the problems that cause infertility in men are related to pathologies that can be treated when identified, as in the cases of varicocele and hypogonadotropic hypogonadism, in others, such as in testicular atrophy and in congenital malformations of the genitals, a conclusive diagnosis is possible, although a specific treatment cannot be offered. Despite the advances in clinical research that try to answer the reasons why some men fail to achieve reproductive success, about 34-40% of male fertility problems are still unknown, which is why they have been grouped as idiopathic infertility, whose etiology is ignored (Povey and Stocks, 2010; Arrotéia et al., 2012).
Among the pollutants dispersed in the environment that affect male fertility, heavy metals disturb reproductive physiology in different ways. Some of these are lead (Pb), mercury (Hg) and cadmium (Cd) (Wijesekara et al., 2015). These chemical elements are found in our environment constantly, mainly as a result of various anthropogenic activities, since Cd is commonly used to protect metals, such as iron and steel, against corrosion, salts, such as sulphite and Cd selenite are components of paints for the coloring of ceramic and plastics, likewise, Cd is used in the manufacture of nickel-cadmium hydroxide batteries for the automotive industry, in addition to being present in phosphorus fertilizers and pesticides used in agriculture (Cichy et al., 2014) and even the Cd compounds are poured into the water of rivers and lakes, so it can reach foods through irrigation and bioaccumulation, given that it is not expelled from organisms (Tbeileh et al., 2007; Saeed, 2013), which is why high concentrations are reported in fish and other sea foods for human consumption. Cigarette smoke, for both active and passive smokers, is another important cause of exposure to Cd (Ashraf, 2012); the general population comes into contact with it through different routes, including water, food and air, and in various industrial working areas (Siu et al., 2009), which is why it has been an issue for legislation in Mexico, by means of NOM- 009-SSA1-1993, and in other countries such as the United States, through the national toxicology program of the Agency for Toxic Substances and Disease Registry (ATSDR, 2012), which establishes that the concentrations of Cd to which the human being can be exposed to are from 0.25 to 0.50 ppm (corresponding to 0.25 and 0.5 mg of Cd per liter of water); however, in polluted areas, the concentrations of this metal are higher, so it requires a continuous analysis of the effect it can produce on the health and physiology of individuals.
6. Alterations in male fertility due to the effect of cadmium
Cadmium, like other heavy metals, does not have a physiological function in organisms, however, it can be incorporated into the body through some absorption pathways, because it is similar to essential elements such as calcium and zinc, in whose physiological processes can intervene and affect the regulation of the metabolism (Staessen et al., 1991; Yang and Shu, 2015). Once bio-accumulated within the organism, the Cd is eliminated with great difficulty by means of urine, because it binds closely to metallothioneins that are completely reabsorbed in the renal tubules. Since Cd does not have any participation in beneficial results in the body, it bioaccumulates from 20 to 40 years within the organism (ATSDR, 2012; Ronchetti et al., 2013). For this reason, it has been reported that Cd poisoning causes serious damage at the cellular level in vital organs such as the brain, the liver, the kidney and the bones, in addition to being classified as carcinogenic (ATSDR, 2012).
Postnatal exposure to Cd carries serious consequences in male sexual development, because the hypothalamic-pituitary-testis neuroendocrine axis is the target of the toxic effect, which gives rise to undesirable sequelae at the level of its three components. Particularly, in testes of animals exposed to this metal before puberty, a reduction in the development of the seminiferous epithelium and the concentration of T has been observed due to the fact that inside the testis the Cd decreases the activity of the steroidogenic enzymes: 3-hydroxysteroid dehydrogenase (3-HSD) and 17-hydroxysteroid dehydrogenase (17-HSD) (Ji et al., 2010; Lafuente, 2013), which are key in the transformation of cholesterol for the biosynthesis of T, together with that Cd also significantly reduces the number of Leydig cells (Ji et al., 2010), in which androgen synthesis occurs. Cd intoxication and dysregulation in the synthesis and concentration of T alter spermatogenesis (Järup and Akesson, 2009; Lafuente, 2013) and cause a decrease in motivation and sexual performance, in comparison with healthy individuals (Arteaga-Silva et al., 2015). At the same time, the Cd affects the androgen-dependent organs, such as the epididymis, reducing its size and weight.
However, little is known about the alterations produced by Cd in the different changes on spermatozoa as part of its maturation process to acquire its fertilizing potential. Thus, the damage of this metal in some biochemical and/or morphological process can lead to a physiological deficiency in spermatozoa that does not allow the reproductive success of the male. Most investigations in this regard have been made in samples of ejaculates from humans and rodents, these report a decrease in concentration, motility and sperm vitality (Järup, 2003; Haouem et al., 2008; ATSDR, 2012).
It has been determined that in patients with infertility, the presence of Cd in the blood has an inverse correlation with the concentration of spermatozoa in the ejaculate, that is, it reduces the number of spermatozoa as it increases the bioaccumulation of Cd, and the same occurs with the motility parameters, because the percentages are reduced (Benoff et al., 2009). Thus, the bioaccumulation in the male reproductive system and its presence in semen intervenes in the reproduction of males and favors the increase in male infertility, by reducing the sperm quality (Pant et al., 2003). In addition, it increases the percentage of morphological abnormalities in spermatozoa, which participate in the execution of the progressive movement in the recognition and in the subsequent fertilization of the oocyte. It has also been proved that Cd can cause the acrosomal reaction prematurely (Oliveira et al., 2009). Cd generates pro-oxidant mechanisms that stimulate the unbalance in the formation of reactive oxygen species (ROS), which produce an increase in the lipoperoxidation of the membranes. In spermatozoa of males exposed to Cd, this has been proven and correlated with azoospermia, because Cd has an adverse effect during spermatogenesis (Akinloye et al., 2006). At the level of the nucleus and the genetic information that is protected in the spermatozoon, it can cause diverse DNA damage (Xu et al., 2003; Taha et al., 2013), because there is an increase in the fragmentation and decompaction of this molecule, and therefore, part of the information stored in the nucleic acids can be lost. Because of this, it has been estimated necessary to continue investigating in greater detail the effect of Cd on the epididymis, since it may be that exposure per se is an alteration in the performance of the same, or that it is a consequence of the reduction in the concentration of T that affects the expression and secretion of proteins related to the maturation and storage of spermatozoa, which occurs as part of the functions performed by the epithelial cells that surround them.
7. The effect of Cadmium on the epididymis and the spermatozoa during maturation
Studies that report the effect of Cd on the epididymal function focus on sperm quality parameters in samples of ejaculates, both human and rodent. These studies show that because of Cd, there is a low concentration of spermatozoa and of the percentage of the living (Järup, 2003; Haouem et al., 2008; ATSDR, 2012). It is also known that Cd causes a high number of morphological abnormalities throughout the spermatozoon that affect progressive movement and that later limit the recognition and fertilization of the oocyte, in addition to the fact that Cd causes DNA damage in spermatozoa (Taha et al., 2013; Xu et al., 2013), through ROS, which can modify the structure and integrity of the membranes, this has been related to azoospermia in infertile men (Akinloye et al., 2006). Another negative effect on DNA is the decompaction of the sperm chromatin by the loss of the junctions in the disulfide bonds (S-S) between the cysteine residues that bind the protamines and together they stabilize the chromatin during the final stages of sperm maturation (Quintero-Vásquez et al., 2015). These alterations have also been associated with infertility. However, in studies with sperm exposure to CdCl2 in vitro, it does not have an impact on the integrity of sperm chromatin, but on the fragmentation of sperm DNA (Méndez et al., 2011).
In a study carried out by Ribeiro (2013) in Wistar rats to see the effect of Cd on the epididymal function, after administration in a single dose at different concentrations of CdCl2 (1.1, 1.4 and 1.8 mg/kg) it was observed that the greater the quantity, the less a presence of spermatozoa was found in the lumen of the tubule of the region of the initial segment, the head and the tail of the epididymis. In this same study, the diameter of the epithelium and the lumen of the tubule were evaluated, with an increase in the epithelial thickness of the epididymal head of the animals treated with 1.1 and 1.4 mg/kg of Cd, so it was suggested that this effect is due to an inability in the reabsorption of luminal fluid. Regarding the light of the tubule, in the tail region of the epididymis of animals treated with 1.8 mg/kg of Cd a greater proportion in the diameter of the light and a decrease in the number of spermatozoa was noticed; similar data reported that exposure to Cd decreases sperm concentration in the three regions of the epididymis and that the same occurs when examining motility (Benoff et al., 2009). It has been documented that these results may be due to the fact that Cd has a negative effect on the regulation and maintenance of the blood-epididymal barrier (Dubé and Cyr, 2012), which causes changes in the histology of the epididymis, including the thickness of the epithelium, necrosis of the epithelial cells, vasoconstriction and interstitial edema together with the infiltration of mononuclear cells, which leads to the alteration of the sperm maturation process, which can interfere and cause infertility (Adamkovicova et al., 2014).
Although the aforementioned studies analyze the effect of Cd on sperm quality parameters and some histological characteristics, it is necessary to consider the biochemical changes characteristic of epididymal maturation, such as the glycosylation of the sperm membrane that involves sugars as N-acetylglucosamine, sialic acid, mannose and fucose, as well as the action of Cd in the processes related to the acquisition of sperm motility, as is the case of the phosphorylation of tyrosine residues in sperm proteins.
7.1 The effect of Cadmium on sperm membrane glycosylation
The development of the ability of the spermatozoa to cross the cervical mucous membranes, approach the oocyte and fertilize it depends on the adequate and orderly realization of the glycosylation events that occur at the level of the plasma membrane during epididymal maturation. However, there are no research studies that broadly analyze the impact that Cd can have on these biochemical processes, although in a general way, it is possible to point out that they are affected as described below.
As previously mentioned, N-acetylglucosamine is a carbohydrate present in the plasma membrane of the spermatozoon that changes along the epididymal tubule, so it has been suggested that these modifications are related to sperm maturation (Hall and Killian, 1987). Tulsiani (2006) investigated that the union of N-acetylglucosamine to residues of glycoconjugates is carried out through the activity of the enzyme N-acetylglucosaminyltransferase, although there is not a well differentiated pattern of its activity; the description indicates that in the distal region of the head of the epididymis towards the proximal of the tail (near to the body of the epididymis), this activity of this enzyme is greater on spermatozoa (Tulsiani, 2006). On the effect of Cd on the activity of glycosylation enzymes such as N-acetylglucosaminidase, spermatozoa in carp ejaculate have been evaluated in vitro and no significant differences have been found between the different concentrations of Cd (1, 10 and 100 mg/L) and at different times of incubation (0, 4 and 24 hours), with respect to the spermatozoa of the control group (Sarosiek et al., 2009). However, in the analysis of our team we have observed that the in vivo exposure to Cd decreases the presence of N-acetylglucosamine in the spermatozoa of the three regions of the epididymis of Wistar rats exposed to this toxic, unlike the spermatozoa of each of the same regions of the epididymis of our control group; this could be visualized by means of the specific binding that this carbohydrate has with the lectin of Triticum vulgare or wheat germ agglutinin (WGA), a protein that can likewise recognize residues of sialic acid, a carbohydrate whose presence in the tissues of the carp (kidney, liver, muscle and gills) decreases as a result of exposure to Cd (Aktaç et al., 2010). However, until now it had not been described exactly if the Cd could affect the presence of sialic acid in the sperm that travel along the epididymis, which is why in our work group we analyzed the presence and modifications of this carbohydrate at the level of the plasma membrane as it progressed along the epididymis, in order to decipher how the epididymal maturation could be damaged in spermatozoa of Wistar rats exposed to a dose of 0.25 mg/kg of CdCl2. Our results suggest that there was always a low presence of sialic acid in the spermatozoa of the three regions of the epididymis, with respect to the spermatozoa corresponding to each study region of the control group. However, it is still necessary to confirm if this is due to a direct effect of Cd on the binding sites between sialic acid and its glycoconjugates or if Cd may be affecting per se the activity of the enzyme sialyltransferase, which is normally high in the region of the head of the epididymis and decreases significantly towards the tail area (Tulsiani, 2006).
Likewise, another cause for which Cd can alter the presence of sialic acid in spermatozoa is that its toxic effect on the epididymal epithelium is capable of damaging the secretion of this carbohydrate, since it has been documented that under normal conditions it is an event that must occur in the epididymis (Toshimori et al., 1988).
Exposure to Cd on the presence of other carbohydrates in the plasma membrane of the spermatozoon when transiting through the epididymis is an unknown process, as in the case of the addition of mannose and fucose in the membrane.
Mannose has been described as a carbohydrate located in the membrane of the spermatozoon (Jiménez et al., 2006; Aliabadi et al., 2013), with active function in the acrosomal reaction (Wu and Sampson, 2013) and with great relevance in the signaling of later processes, such as in the fertilization of the oocyte (Belmonte et al., 2002). For this reason, in the investigation we conducted, its presence was evaluated by its binding to the lectin of Canavalia ensiformis agglutinin (Concanavalin A, Con A) in spermatozoa of the three regions of Wistar rats epididymis with CdCl treatment. Low percentages of mannose were found in the area of sperm glycocalyx versus a high percentage of spermatozoa without the presence of this carbohydrate in the same region in the rats administered with Cd, results that were significantly opposite to those obtained in the spermatozoa of rats used as a control group. Thus, we consider that these variations in the presence of mannose in spermatozoa are caused by Cd and can affect both the maturation process and training and fertilization, so that the Cd could affect the potential fertilizer of the spermatozoa.
Another interest in our work group was to investigate if Cd generated any alteration in the fucose in the spermatozoa of Wistar rats exposed to this heavy metal, because to date there has been no description about it. However, it is important to mention that any type of modification in the presence and in the order in which fucose is integrated into the sperm glycocalyx, can cause male fertility problems (Tecle and Gagneux, 2015). From the analysis that was carried out with the lectin Ulex europaeus agglutinin (UEA), which is a protein related to the fucose residues, we obtained that in the spermatozoa of rats administered with CdCl we had an opposite pattern to those of the group control, that is, a low percentage was detected in the presence of this carbohydrate in spermatozoa of the head of the epididymis and was increasing towards the tail region, while in the spermatozoa of the head of the epididymis of control rats a large amount of percentage of fucose, which subsequently decreased toward the tail of the epididymis. It is worth mentioning that Tulsiani (2006) indicates that the fucose integration towards the plasma membrane of the sperm is carried out by the enzyme fucosyltransferase, which has a high activity in the head and is reduced towards the tail of the epididymis, so that the Cd could affect the synthesis and function of this enzyme, which is regulated from the epididymal epithelium and therefore be one of the causes in the differences observed in spermatozoa of the group treated with Cd.
8. The effect of Cadmium on the phosphorylation of protein tyrosine residues during sperm maturation
The phosphorylation of tyrosines has been considered as one of the most important intracellular changes that occur in the spermatozoon during epididymal maturation, because it has been taken as the essential molecular basis for the coordinated development of progressive movement, training and acrosomal reaction of the spermatozoon in the oviduct of the female, so that the alterations generated in this process by the exposure to Cd may be the main cause in male infertility.
One of the modifications caused by Cd during phosphorylation events is that it affects the activity of tyrosine kinases (enzymes with the ability to transfer a phosphate group to a tyrosine residue of a protein), or to the binding sites of substrate proteins, which is why this circumstance has been taken as an explanation to the studies that indicate that alterations in sperm motility occur in both animals and humans with Cd exposure (Da Costa et al., 2016). On the other hand, failures in sperm motility as a consequence of Cd may be due to energetic wear and a decrease in the concentration of ATP/AMP, since the phosphorylation state is regulated by a cyclic adenosine monophosphate-dependent pathway (CAMP), which influences the activity of protein kinases and phosphatases.
According to the in vitro study of Wang et al. (2016), in which they observed the effect of Cd (using different concentrations: 0.1, 0.5, 1, 5, 10 and 50 μM, in a basal medium) on the protein phosphorylation in spermatozoa of the tail of the epididymis of rat, the percentage of phosphorylated proteins in spermatozoa with exposure to CdCl increased compared with the control group, which was not administered with Cd. However, they point out that the increase of phosphorylated proteins in tyrosine residues is not a fact that could favor the development of sperm motility in all species, because when they analyzed the sperm motility parameters, except for the immobile, the others were canceled. Particularly, in the research that was carried out in our team, it was observed that a concentration of 0.25 mg/Kg of CdCl2 intraperitoneally also causes an increase of proteins that are phosphorylated, since there was a progressive increase of this process in sperm from the region of the head to the body of the epididymis, and later it was higher in those of the tail region of subjects treated with Cd, which was different from that found in spermatozoa of control subjects, whose protein phosphorylation pattern was similar between the areas of the head and the body of the epididymis, and decreased markedly in the region of the tail.
The information gathered in the various studies consulted for this chapter indicates that the spermatozoon’s acquisition of fertilizing capacity depends on the correct functionality of the epididymis and the different changes it causes on the spermatozoon, in time and space, throughout all of its parts. However, the alterations on the epithelium and the functions of the epididymis produced by Cd modify the changes required for maturation, in such a way that the addition of carbohydrates of the plasma membrane and the phosphorylation of proteins in tyrosine residues of the spermatozoa are affected on the three regions of the epididymis. Thus, exposure to Cd can disturb epididymal sperm maturation and be one of the causes of the various faults in the inhibition of motility and thus lead to increased problems when fertilizing the oocyte, increasing the current cases of male infertility.
17β-hydroxysteroid dehydrogenase (17β-HSD): It is an enzyme with the ability to convert estrone to estradiol.
3β-hydroxysteroid dehydrogenase (3β-HSD): It is an enzyme with the ability to synthesize pregnenolone to progesterone (17-hydroxypregnenolone to 17-hydroxyprogesterone) and dehydroepiandrosterone to androstenedione in the adrenal gland.
5α-dihydrotestosterone (DHT): It is the active metabolite of testosterone with high biological power, because DHT has an affinity 3 times higher than testosterone and 15-30 times higher than adrenal androgens with the androgen receptor. It is synthesized by the enzyme 5α-reductase that reduces the 4,5-link of testosterone. In men, approximately 5% of testosterone is reduced to DHT. In embryonic development, DHT is essential for the formation of the male external genitalia, and in the adult it acts as the main androgen in the prostate and hair follicles.
Acrosome: A cap-shaped structure that covers the nucleus with DNA, it is located in the anterior part of the spermatozoon head and contains hydrolytic enzymes whose participation is relevant during the process of fertilization.
DNA: Deoxyribonucleic acid, it is a complex chain of polymers that store all the genetic information of a living being.
Bioaccumulation: It is the process of accumulation of toxic chemical substances in different tissues of living organisms, whose concentrations are higher than those existing in the environment or in food, after entering through the respiratory, digestive and/or skin.
Cadmium (Cd): It is a chemical element whose atomic number is 48 and its chemical symbol is Cd. It is found in group 12 of the periodic table of the elements. It is considered as a heavy metal because it has a density equal to or greater than 5 g/ml, since its atomic weight is 112.40 g/mol. It is a soft, silver-white metal that generally does not occur in the environment in pure form, but forms compounds with other elements such as oxygen (O) (cadmium oxide, CdO), chlorine (Cl) (cadmium chloride, CdCl ) or sulfur (S) (sulfate and cadmium sulfide, CdSO and CdS).
Disulfide bridge (S-S): It is a strong covalent bond between thiol groups (-SH) of two cysteines. This type of link is of great relevance in the structure, folding and function of proteins.
Epididymis: It is an organ divided into head, body and tail, is composed of a single narrow long coiled tubule, whose length depends on the type of species. It is located on the upper part of the testicle and connects the efferent ducts with the vas deferens.
Estradiol (E2 or 17β-estradiol): It is a steroid sex hormone that is derived from cholesterol. To be formed, the aromatase enzyme takes testosterone as a substrate and it aromatizes it. In women it is produced by the granular cells of the ovaries.
Fertility potential: The capacity that spermatozoa acquire so that they can move progressively, recognize and bind to the zona pellucida to fertilize an oocyte.
Flagellum: It is a movable appendix with a whip that appears in a great diversity of unicellular organisms and in some cells of multicellular organisms. For the spermatozoon it is a necessary structure for mobility.
Gametes: From the Greek upameetḗ “wife” or μέgamétēs “husband”, they are haploid sex cells (n), originated by mitosis and meiosis from the germ cells. They are named according to the sex of the carrier, oocyte for the female and spermatozoon for the male. After their fusion, a zygote cell with two sets of chromosomes or diploid (2n) is originated.
Glycocalyx: A structure composed of abundant oligosaccharides linked to a large amount of proteins.
Glycohydrolases: A group of hydrolytic enzymes that separate residues of a sugar from existing glycoconjugates.
Glycosylation: Modifications that allow the integration or separation of one or more chains of glycans to a main chain by means of covalent bonds.
Glycosyltransferases: Group of synthetic enzymes that add sugar residues to a donor sugar (nucleotide sugar).
Idiopathic infertility: It is the disability with unknown etiology to achieve a pregnancy in a period no longer than a year.
Infertility: Impossibility of achieving a pregnancy after one year of regular sexual intercourse without the use of any contraceptive method.
In vivo: From the Latin “inside the living”, is a term that is used for techniques that are performed within a living organism. Tests performed on animals and clinical trials are considered as live forms of research.
Lectin: It is a protein of vegetable or animal origin with the ability to bind with high specificity to the residues of a specific sugar.
Oocyte: From the Latin ovŭlum, diminutive of ovum “egg”, it is a haploid sex cell carrying the female genetic material, which is large and immobile.
Phosphorylation: The addition of a phosphate group to any other molecule, for example to the amino acid of a protein. This process is the basic mechanism of energy transport from the place where it is produced to where it is required. Phosphorylation is one of the major processes that regulates the activity of proteins, particularly enzymes.
Plasma membrane: It is composed of a lipid bilayer that encompasses and shapes the cell. It is formed by phospholipids, glycolipids and proteins. It maintains the balance between the interior (intracellular medium) and the exterior (extracellular medium).
Adamkovicova, M., Toman, R., Cabaj, M., Massanyi, P., Martiniakova, M., Omelka, R., Krajcovicova, V. & Duranova, H. (2014). Effects of subchronic exposure to cadmium and diazinon on testis and epididymis in rats. The Scientific World Journal, 2014, 1-9.
Agency for Toxic Substances and Disease Registry (ATSDR). (2012). Toxicological profile for cadmium. Atlanta: Division of Toxicology and Human Health Sciences, Environmental Toxicology Branch.