The role of epididymosomes in the transfer of proteins during the epididimary maturation

1. Introduction

In vertebrates such as mammals, spermatozoa produced in the seminiferous tubules within the male gonads is not able to fertilize the ovule. They must undergo a series of morphological and biochemical modifications as they transit throughout the epididymal tubule in order to train to carry out the acrosome reaction, the recognition and fertilization of the female gamete within the female genital tract (Cervantes et al., 2008; Cooper, 2011; Rodríguez-Tobón et al., 2015a). The acrosome reaction is a unique event of acrosome exocytosis, essential for the spermatozoon to fertilize the homologous ovule (Beltran et al., 2016). This series of modifications in spermatozoa during transit through the epididymis is known as epididymal sperm maturation, and is caused jointly by the epithelial tissue and the microenvironment generated by the same tubule cells (Caballero et al., 2010; Rodríguez-Tobón, 2011).

2. The epididymal tubule

The epididymis is a single tightly-coiled tube attached to the cranial part of the testicle. Its length varies from 3 to 80 m depending on the species. It is responsible for the transport, maturation and storage of spermatozoa (Robaire et al., 2006, Sullivan et al., 2007). In mammals, 3 main anatomical regions (head, body and tail) have been recognized (Figure 1), in addition to the initial segment that connects with the efferent tubules (Arenas-Ríos et al., 2005, Robaire et al., 2006). Several cell types have been described in the epididymal duct responsible for the synthesis, secretion and absorption of substances that will come into direct contact with spermatozoa (Serre and Robaire, 1998). The apical cells have been described in the initial part and in the intermediate zone of the epididymis of the adult rat and occasionally in other epididymal segments in elderly rats (Adamali and Hermo, 1996). These cells resemble the main cells, but differ in that these cells have a characteristic spherical nucleus located in the apical part and they have no contact with the basement membrane (Adamali and Hermo, 1996, Robaire et al., 2006). Because apical cells also participate in processes of secretion and endocytosis, they have been considered to modify the luminal environment of the epididymal tubule, for they are involved in the degradation and eventual internalization of proteins (Adamali and Hermo, 1996; Andonian and Hermo, 1999).

Figure 1. The epididymis main regions: head, body and tail.

Narrow cells, as well as apical cells, only appear in the epithelium of the initial segment and in the intermediate zone of the rat and mouse’s epididymis. They are characterized by numerous cup-shaped apical vesicles that are involved in endocytosis, in addition to secreting H+ ions towards the lumen of the epididymal tubule. These cells are morphologically differentiated from the main and the apical cells for their different proteins, and also because they can be precursors of clear cells (Serre and Robaire, 1998, Robaire et al., 2006).
Clear cells are not present in the initial segment of the rat’s epididymis (Moore and Bedford, 1979), but in many species, including humans, they can be found in all three regions of the epididymis (Robaire et al., 2006). They are characterized by performing a much greater endocytic function than the corresponding one performed by the adjacent main cells, considered to be specialized in phagocytosis, especially in the tail of the epididymis. In addition, they participate along with the narrow cells in the acidification of the luminal fluid (Oko et al., 1993, Andonian and Hermo, 1999, Robaire et al., 2006).
Halo cells are small cells with a narrow cytoplasmic border, located throughout the epididymis. These cells are normally found at the base of the epithelium and contain a nucleus conformed by a variable number of dense granules. They have been described as lymphocytes or monocytes, only they are not typical migratory lymphocytes. Among the main differences are that their granules contain multivesicular bodies and a large endoplasmic reticulum.
Anatomically, each epididymal region can be divided into intra- regional segments, forming lobes of coiled epididymal tubules, separated by connective tissue septa, which provide support and stability to the organ itself. In addition, the epithelial cells of each region of the epididymis express different genes and synthesize various proteins that are released into the lumen, with other substances, establishing the microenvironment. When spermatozoa transit through the epididymis, they are in direct contact with that specific luminal microenvironment (Turner et al., 2003; Robaire et al., 2006; Aitken et al., 2007), as in the case of the head and the body, where sperm maturation occurs in the majority of mammal species. However, in the case of the Corynorhinus mexicanus bat, sperm maturation (Cervantes et al., 2008; Rodríguez-Tobón et al., 2015a; Rodríguez-Tobón et al., 2015b) and storage (Turner et al., 2003; Robaire et al., 2006) have been reported to take place in the tail.

3. Epididymal sperm maturation

The heterogeneous microtopography of the sperm membrane is partly the key to understanding the complex functions of the spermatozoon during its post-testicular existence (Eddy et al., 1985, Schroter et al., 1999, Eddy, 2006). The ionic composition, the organic molecules and the different proteins that have contact with spermatozoa during their transit through the epididymis have been observed to cause the continuous modifications that allow the increase in the negative charge of the plasma membrane, the loss of cytoplasmic droplet, the change in the phospholipid/cholesterol coefficient, the restructuring or remodeling of the acrosome shape, the decrease in the diameter of the mitochondria, the increase in progressive movement and the addition, elimination and modification of proteins (Lewis and Aitken, 2001, Ecroyd et al., 2004, Naz and Rajesh, 2004, Sidhu et al., 2004, Sullivan et al., 2007, Fabrega et al., 2011). Together, these modifications experienced by the spermatozoon are known as epididymal sperm maturation.

4. Apocrine secretions in the epididymis

The epithelial cells of the epididymis show regional-specific differences in their function and protein secretion patterns (Girouard et al., 2009). One of the characteristics of the main and narrow cells of many mammalian species is the formation of bubbles in the apical part, which interact with spermatozoa transiting through the epididymal tubule, involved in the transfer of proteins from the epithelial tissue to the sperm membrane (Saez et al., 2003).

Figure 2. Scheme of apocrine secretion in the main cells of the epididymal tubule.

Prostasomes are vesicles formed in the epithelial tissue of the male’s duct and secreted in seminal plasma; these are found in several species of mammals including rabbits, primates, sheep, dogs, cats, cattle, horses, rats and humans (Saez et al., 2003; Rejraji et al., 2006; Girouard et al., 2009). Based on the classic process of exocrine secretion, several studies have mentioned that the epithelial cells of the epididymal tubule are involved in the processes of apocrine secretion (aposomes) (Aumuller et al., 1999). However, because they are inside the epididymal tubule, they have been popularly named as “epididymosomes”. These vesicles present an electrodense ultrastructure similar to exosomes. Their diameter varies in size ranging from ~ 10 nm to 500 nm (Griffiths et al., 2008).
Yanagimachi et al. (1985) were the first to describe the interaction of membrane vesicles found in the epididymal lumen with spermatozoa. They considered that the sperm membrane is susceptible to numerous changes during maturation. Thus, they assumed that vesicles would be involved in the transport of different molecules such as cholesterol, enzymes and/ or glycoproteins from the epididymal tissue to the sperm membrane as spermatozoa descends from the head to the tail of the epididymis.

It has been known for some decades that the epididymal epithelium secretes the proteins that interact with the spermatozoon during its transit through the different regions of the epididymis to generate a fully functional gamete (Robaire et al., 2006). It is now accepted that epididymosomes are involved in the transfer of proteins to the spermatozoon related to motility, interaction with the zona pellucida and fertility potential (Sullivan and Saez, 2013). In addition, proteins such as Rab (small G proteins) and SNARE (N-ethylmaleimide sensitive factor binding protein receptor) have been recognized, which are related to the transit and fusion of vesicles (Sullivan and Saez, 2013). Epididymosome lipids composition varies along the epididymal tubule. In bulls and murids, the phospholipid/cholesterol ratio increases 1.5 times from the head to the tail of the epididymis. In the particular case of the mouse, epididymosomes are enriched in sphingomyelin and polyunsaturated fatty acids (mainly arachidonic acid) (Rejraji et al., 2006).

It has been suggested that, as in the sperm membrane, epididymosomes also have regions rich in sphingomyelin and cholesterol, known as “lipid rafts” or detergent-resistant membrane regions (DRMs) (Girouard et al., 2009). In bulls epididymosomes, the bovine epididymal protein (P25b) is associated with DRMs and transferred to the spermatozoon DRMs so that they could be targets of proteins secreted by the epididymis to a specific region of the sperm membrane (Sullivan and Saez, 2013) However, there are other proteins such as the macrophage migration factor (MIF) and aldose reductase that are not associated with DRMs (Sullivan and Saez, 2013).

5. Epididymosomes and their participation in epididymal maturation

In mammals, a mature spermatozoon has different functional domains in the plasma membrane that may be regulating the interactions between epididymosomes and spermatozoa. In the head is the function of recognition of the zona pellucida; for the penetration of the ovule, the middle piece provides the energy for movement and the tail aids in flagellar motility (Cooper, 2011). Therefore, the proteins acquired by the spermatozoon during maturation are expected to have a strategic and functional location in the different parts of its structure.

These microdomains that specialize in cell signaling and vesicle trafficking events are rich in glycosylphosphatidylinositol (GPI) and transmembrane signaling molecules such as protein tyrosine kinases (PTK). When transferring proteins from epididymosomes to spermatozoa, some can be established as membrane proteins (Kirchhoff and Hale, 1996) or absorbed into the internal structures of the same cell (Frenette et al., 2005).

Many of the molecular modifications that occur during sperm maturation involve structural proteins, receptors on the surface of the membrane, ion channels, hormones, cytokines and water, as well as proteins from the extracellular matrix. The activity and availability of many of the proteins is regulated by proteases, however, the proteases involved in the process are unknown (Netzel-Arnett et al., 2009).

The secreted proteins in the cephalic and corporal region of the epididymis are involved in the acquisition of a notable motility and the ability to bind the zona pellucida, as well as in the fusion with the plasma membrane of the oocyte (Turner et al., 2003). These proteins are also related to protection against oxidative stress and elimination of defective spermatozoa (Sullivan et al., 2005; Rejraji et al., 2006; Sullivan et al., 2007). Proteins secreted in the epididymis caudal region are necessary to maintain viability and fertility potential of stored spermatozoa (Saez et al., 2003).

In species such as bull, pig, sheep, rodents and horses, the incorporation of proteins into spermatozoa from the epididymal epithelial tissue occurs by regions (Belleannee et al., 2011, Baker et al., 2012), some are directed to the part of the membrane that covers the acrosome and others to the middle part of the flagellum (Griffiths et al., 2008; Martin-DeLeon, 2015). Some  molecules  in  the  acrosome  are  modified  during  sperm maturation, mainly those that contain carbohydrate chains such as D-galactosidase and N-acetyl-D-galactosamine, which molecular size decreases during glycosylation and/or proteolysis due to the substitution of several glycoconjugates (Toshimori, 1998). This molecular reduction has been determined in rabbit (Mukerji and Meizel, 1979), human (Baba et al., 1989), guinea pig (Anakwe et al., 1991), rat (Nagdas et al., 1992), and pig (Baba et al., 1994). In guinea pig and chimpanzee, certain glycoconjugates added to spermatozoa during spermiogenesis to the acrosome membrane and the entire plasma membrane are subsequently redistributed to the posterior region of the head of the spermatozoon, reducing its size during maturation, and finally migrating to the inner membrane of the acrosome during the acrosome reaction (Myles et al., 1984; Cowan et al., 1986; Phelps et al., 1990; Overstreet et al., 1995).

Recent studies show that the composition of epididymosomes is quite varied in terms of proteins, some of which are more common and others more specific, which indicates that their expression is restricted from one population of vesicles to another, depending on the region of the epididymis where they occur (Sullivan and Saez, 2013). Different epididymal populations have been reported in the epididymis of different species of mammals, some differentiate according to their ultrastructure and enzymatic composition and others to their density, lipids composition and proteins (Fornes et al., 1995, Frenette et al., 2010). Thus, each population of epididymosomes is capable of transferring a particular group of proteins to the spermatozoon during maturation and interacting differently with it.

The proteins secreted by the epididymal tissue, some bound to GPI, are secreted directly to the epididymal lumen and transferred to the spermatozoon by means of apolipoprotein J or clusterin, which is known to carry lipids in a great variety of biofluids, like the one that is found in the epididymal tubule. Clusterin is found abundantly in the lumen of the epididymis, and it is related to facilitating the sperm-binding of proteins bound to GPI, as well as its removal during the modifications undergone by the membrane (Martin-DeLeon, 2015). The main proteins transported by this route are: the murine sperm cell adhesion molecule (SPAM1), the pathogenesis related protein (GLIPR1L1), which is involved in the binding between the spermatozoon and the zona pellucida of the oocyte; as well as with some members of the families of hyaluronidases (HYAL3, HYAL5 and HYAL2), membrane-bound serine proteases (PRSS21), P34H (human), P26h (hamster) and P25b (bull) (Legare et al., 1999; Frenette and Sullivan, 2001; Zhang et al., 2004; Martin-DeLeon, 2015).

Epididymosomes, like clusterin, transport proteins bound to GPI, so it is considered that the delivery of proteins, whether vesicular or not, is mediated by hydrophobic interactions (Sullivan and Saez, 2013). The proteins associated with the epididymosomes identified so far are: CD52 (formerly HE5), a protein that forms part of the surface of lymphocytes found in humans, primates and rats; the murine sperm cell adhesion molecule (SPAM1)/PH-20, whose homologue has been reported in humans, primates, bulls and rats; the P26h protein, secreted in the epididymis and localized in hamster spermatozoa (related to the binding of the spermatozoon to the zona pellucida) and its orthologs P34H (human), P25m (mouse), P25b (bull) and P31m (primate); in addition to Glutathione peroxidase type 5 (GPX5), associated to the protection against oxidative stress and DNA integrity; Methylmalonate-semialdehyde dehydrogenase (MMSDH); the macrophage migration factor (MIF), which is associated to the spermatozoon dense fibers, may be related to the elimination of Zn2+ that affects free sulfhydryl groups in the sperm flagellum (Sullivan et al., 2005; Caballero et al. al., 2010; Sullivan and Saez, 2013; Martin-DeLeon, 2015); the proteins aldose reductase and sorbitol dehydrogenase, which can modulate the motility during the transit of the spermatozoa through the epididymis; Liprin α3, related to the acrosome reaction; Protein kinase cSrc related to the motility of sperm (Krapf et al., 2012, Sullivan and Saez, 2013) and finally; Ubiquitin and Sperm-Associated Protein 1 (ELSPBP1), both related to the elimination of defective spermatozoa, where ubiquitin carries out the enzymatic degradation of proteins by proteasome.

Finally, the family of proteins that contain a disintegrin and metalloprotease domain (ADAMs) is a group of membrane-anchored proteins whose members are widely distributed in different species and present in a wide variety of tissues (Cho, 2012). ADAM7 is one of the proteins that has been located at the level of the sperm plasma membrane, transported by epididymosomes (Oh et al., 2009). Due to its characteristics as a membrane protein, it has generated the idea that there is fusion between spermatozoa and epididymosomes. However, within the literature reported, there is controversy regarding whether these vesicles fuse or not to the sperm membrane (Martin-DeLeon, 2015).

Schwarz et al. (2013) showed that the epididymosomes obtained from the cephalic region of the bull epididymis have a greater fusogenic capacity with the sperm membrane obtained from this region, in comparison with the vesicles obtained from the caudal region. The fusion of spermatozoa with epididymosomes was analyzed evaluating the compensation of the auto off of the fluorescence of the R18. They determined that the fusion rate between epididymosomes and spermatozoa depends both on proton concentration and time, since the decrease in pH favors the fusion of membranes in 180 seconds. The melting rate gradually decreased during a period of 10 min (Schwarz et al., 2013). Additionally, the results were corroborated with fluorescence microscopy analysis at pH 5 and 6.5. After the in vitro fusion, the fluorescence signal was increased, being predominantly visible in the head, neck and middle part of the spermatozoon.

6. Perspectives

Recent studies seek to determine which proteins are part of the vesicles secreted by the epididymal epithelial tissue, which will later bind to the spermatozoa that transit through the epididymis from head to tail, in order to check which proteins are present in spermatozoa not found in the newly formed cells in the testicles. This has allowed many of these proteins to be considered potential targets to prevent the fertility of spermatozoa. However, the research for exclusive molecules of spermatozoa must continue to prevent affecting the functionality of any other types of cells.