The Blood-Testis and Blood-Epididymis Barriers Are More than Just Their Tight Junctions

Abstract

The terms blood-testis barrier (BTB) or blood-epididymis barrier (BEB), are often described as Sertoli cell-Sertoli cell tight junctions (TJs) or TJs between the epithelial cells in the epididymis, respectively. However, in reality, the BTB and BEB are much more complex than just the TJ. The focus of this minireview is to remind readers that the complete BTB and BEB are comprised of three components: anatomical, physiological, and immunological. The TJs form the anatomical (physical) barrier that restricts passage of molecules and cells from entering or exiting the lumen. The physiological barrier is comprised of transporters that regulate movement of substances in or out of the lumen, thus creating a microenvironment, which is critical for the proper development and maturation of germ cells. The immunological barrier limits access by the immune system and sequesters the majority of the autoantigenic germ cells. Combined with the overall immune-privilege of the testis, this suppresses detrimental immune responses against the autoantigenic germ cells. These three components on their own do not create a complete functional barrier; instead, it is the interaction between all three components that create a barrier of maximal competence.

The fully functional blood-testis and -epididymis barriers are comprised of anatomical, physiological, and immunological barriers that act together to create barriers of maximal competence.

INTRODUCTION

When one thinks about either the blood-testis barrier (BTB) or the blood-epididymis barrier (BEB), an image of their tight junctions probably immediately comes to mind. In this Minireview, we clarify that the barriers in both the testis and epididymis are more complex and are not comprised only of epithelial cell tight junctions (the physical or anatomical barrier), but also include physiological and immunological barriers. It is really the complex interaction between all three of these barriers that constitute the functional BTB and BEB.

Like other blood-tissue barriers, the BTB and BEB are not impenetrable walls, but are rather restrictive in nature; that is, they restrict or control the entry of molecules or even cells present in circulating blood/interstitium from entering a tissue or compartment. For the testis and epididymis, this barrier is between the blood vessels and the luminal compartment of the seminiferous tubules or the epididymal lumen, respectively. In contrast to the brain, where the blood-brain barrier is located at the endothelial tight junctions lining the blood vessels [1, 2], in the testis and epididymis, the primary physical component of the BTB and BEB is the tight junctions formed between adjacent epithelial cells lining the seminiferous tubules (Sertoli cells) [3–5] or epididymal duct [6–8]. While this physical barrier (i.e., tight junctions between epithelial cells) is very important for the functions of the BTB and BEB, the common misconception is that this physical barrier is the sole component of the BTB and BEB. However, the fully functional BTB and BEB are much more complex, and consist of anatomical/physical (tight junctions, basolateral and apical membranes), physiological (transporters, channels at the basolateral and apical membranes, and the paracellular route) and immunological (inside and outside the tubule/duct) components, which are all necessary to make up the functioning barrier in the testis and epididymis. It is how these three barriers interact that is the key.

The primary function of the BTB and BEB is to create the appropriate microenvironment to control germ cell development (mitosis, meiosis, and differentiation) and maturation, to sequester autoantigenic germ cells to prevent harmful autoimmune responses, and to shield germ cells from cytotoxic molecules [9]. Therefore, besides a physical barrier, it is also necessary to have a physiological component to control entry and exit of molecules, and an immunological component to protect the germ cells. This review is not meant to be a comprehensive description of all the individual components of each part of the BTB and BEB, which are reviewed individually in detail elsewhere [10–15], but instead to emphasize the point that all three components are important for complete BTB and BEB function. Here, we describe the three components of the BTB and BEB, with specific emphasis on the importance of the physiological and immunological components.

BASIC ANATOMY OF THE TESTIS AND EPIDIDYMIS

The testis consists of two compartments: the highly convoluted seminiferous tubules, and the interstitial space [16]. The interstitium includes Leydig cells, blood vessels, macrophages, lymphocytes, connective tissue, and lymphatic vessels [17]. The Leydig cells within the interstitial space produce testosterone, which is important to maintain spermatogenesis [18, 19] and the male secondary sex characteristics [20]. The seminiferous epithelium is the site of spermatogenesis and includes the Sertoli cells and germ cells [16]. This seminiferous epithelium is surrounded by one or more layers (depending on the species) of peritubular myoid cells [21]. The Sertoli cells extend from the basement membrane to the lumen surrounding the germ cells, and are responsible for providing structural and functional support to the developing germ cells [22]. Adjacent Sertoli cells are connected by tight junctions, which are located in the basal third of the seminiferous epithelium [3, 4, 23, 24]. The junctions separate the epithelium into an adluminal and basal compartment, thereby separating the germ cells located in the adluminal compartment from the blood supply [3, 4, 23–27]. This allows the Sertoli cells to control the microenvironment within the seminiferous tubules.

The epididymis is a highly convoluted duct/tube and, for most species, can be divided into distinct anatomical regions: initial segment, caput, corpus, and cauda [28]. The interstitial component that surrounds the duct contains blood vessels, connective tissue septa that divide the duct into smaller segments, lymphatic vessels, macrophages, and lymphocytes [29]. The epididymal epithelium comprises several cell types, including principal, basal, narrow, apical, halo, and clear cells surrounded by multiple layers of peritubular myoid cells [25, 30]. From a functional perspective, the epididymis plays several important roles including sperm maturation, sperm transport, sperm storage, and sperm protection [29–32]. Similar to the BTB, the BEB is important in regulating the microenvironment within the duct to allow for sperm maturation and to sequester autoantigenic spermatozoa from the immune system.

In the next three sections, the three principal components of the BTB and BEB are discussed and include: anatomical (physical), physiological (permeability), and immunological barriers.

ANATOMICAL (PHYSICAL) BARRIER

One function of the BTB/BEB, which is carried out by the anatomical barrier, is to restrict entry of molecules into the adluminal compartment of the testis or the lumen of the epididymis. In the testis and the epididymis the major anatomical component of the BTB and the BEB is the tight junction complexes formed between Sertoli cells (Sertoli cell-Sertoli cell tight junctions) [33] or epithelial cells of the epididymis, respectively (Fig. 1, A and B). For the testis, substances within the circulation flow past the endothelial cells lining the blood vessels through the interstitial space to the myoid cells and Sertoli cells before reaching the lumen of the seminiferous tubules [21]. While there are several similarities between the endothelial cells of the testis and brain, in contrast to the brain, where the anatomical blood-brain barrier is located at the endothelial cell junctions [1, 2], in the testis, several studies provide evidence for a high degree of permeability of the testicular blood vessels [25, 34–36]. In rodents, but not larger mammals, the peritubular myoid cells create a semipermeable barrier excluding the entry of large molecules, such as carbon particles and limiting the entrance of smaller molecules (e.g., lanthanum) in ∼85–90% of tubules [3, 4, 37]. In all species, tight junctions formed between adjacent Sertoli cells [38], almost completely prevent entrance of molecules (e.g., lanthanum) from entering the lumen of the seminiferous tubules [3, 4, 21, 23, 24, 39]. These Sertoli cell-Sertoli cell tight junctions (zonula occludens) are similar to other tight junctions in that they are composed of occludins, claudins, and junctional adhesion molecules [10, 40–42]. However, they are quite unique compared to other epithelial tight junctions in that they are located basally rather than apically [21].

FIG. 1.

The anatomical and physiological barrier. Anatomical barrier. Substances within the circulation enter the interstitial space, but most of them (dots) are prevented from entering the adluminal compartment (thin line) of the testis (A) and the lumen of the epididymis (B) by the anatomical component (tight junctions) of the BTB/BEB. In the testis (A), the adluminal compartment (thin line) of the seminiferous epithelium is separated from the basal compartment (thick line) by the tight junctions. The basal membrane, along with the apical membrane and tight junctions, is what forms the Sertoli cell barrier, which is the complete anatomical barrier. In the epididymis (B), the lumen is separated from the epididymal epithelium by tight junctions between adjacent epithelial cells. Physiological barrier. At the adluminal (thin line) and basal (thick line) membranes are the sites of specific transporters that control the movement of molecules (dots and stars) between the compartments, creating unique microenvironments for germ cell development and maturation in the testis (A). In the epididymis (B) these transporters also create a unique microenvironment along the length of the epididymal duct through variation in the gradient of molecules being exported (dots and stars in the lumen). AM, apical membrane; B, blood vessel; BM, basal membrane; E, epididymal epithelial cell; GC, germ cell; L, Leydig cell; M, myoid cell; S, Sertoli cell; TJ, tight junction.

In an attempt to more precisely describe the BTB, Russell and Peterson [33], used the term the “Sertoli cell barrier.” This term more adequately defined the barrier location at the seminiferous epithelium instead of, as the name BTB implies, at the blood vessels. This description also took into account that the barrier does not have direction, and was a permeability barrier, which we have identified as being the “physiological” barrier. They recognized that it was both the occludin junctions, together with the body of the Sertoli cell, that ultimately formed the Sertoli cell (SC) barrier and acted to restrict the passage of molecules into and out of the seminiferous tubule lumen. Furthermore, they specifically referred to the tight junctions of the BTB as the Sertoli cell-Sertoli cell junctions, which we believe is a more accurate description of the anatomical component of the BTB.

Similarly to the BTB, the anatomical site of the BEB has been considered to be the tight junctional complex between the epididymal epithelial cells. Friend and Gilula [43] wrote, “Among the various epithelial cell contacts examined, the zonula occludens of the epididymis is the most highly developed.” This has been confirmed by several freeze-fracture and ultrastructural studies that show an extensive tight junction meshwork [44–46]. Also similar to the junctions between Sertoli cells, the junctional complexes between epididymal epithelial cells contain many proteins, including claudins, connexins, and catenins [47–50]. Lanthanum and horseradish peroxidase have been used to probe the integrity of the anatomical BEB, and similarly to the BTB, these tracers do not penetrate the tight junctions between the epididymal epithelial cells [6, 46].

Therefore, the components that make up the anatomical barrier in both the testis and epididymis are the basolateral membrane, the apical membrane, and the tight junctional complex between the cells (Fig. 1, A and B). Each will contribute in their unique way to restrict the passage of molecules from entering and exiting the lumen and allowing each tissue to form a very unique luminal fluid milieu. The permeability properties of the basolateral and apical membranes are being considered here as the physiological barrier, but we recognize that their physical structure alone will restrict many substances from entering and exiting the seminiferous tubular and epididymal lumen.

PHYSIOLOGICAL (PERMEABILITY) BARRIER

While the anatomical barrier restricts passage of molecules into the lumen, specific transporters located along the basolateral and apical membranes of the Sertoli cells and epididymal epithelial cells regulate the movement of molecules in or out of the lumen [11]. This physiological barrier is required to create the appropriate microenvironment within the seminiferous epithelium and lumen for development (meiosis and differentiation) and maturation of the germ cells. The whole idea of a physiological barrier within the testis probably originated with the paper by Setchell et al. in 1969 [51], although it was well known before that time that vascular perfused dyes were excluded from the seminiferous tubular lumen. The experiments were performed to examine the permeability of the testis to molecules of differing molecular weight. Radioactive substances were injected into blood, rete testis fluid was collected, and the amount of radioactivity collected was compared to that injected into blood. From this study, the authors stated, “It is suggested that this permeability barrier will regulate the access to the seminiferous epithelium of some constituents of blood plasma, isolate the germinal cells immunologically and help to maintain the concentration differences between rete testis and lymph or blood plasma” [51]. The following year, Dym and Fawcett [3] published their paper showing that lanthanum nitrate failed to penetrate the tight junctions between the Sertoli cells, thereby linking the permeability barrier described by Setchell et al. [51] with an anatomical barrier [3, 51]. Unfortunately, although the tight junctions do, indeed, prevent many molecules from entering the lumen of the seminiferous tubule, the contribution of the basolateral and apical membranes with specific transporters has not been recognized as a major contributor to the restrictive movement of substances from blood to lumen. Therefore, the physiological barrier reflects primarily the permeability of the basolateral and apical membranes to molecules (Fig. 1, A and B). This permeability depends upon the presence and specificity of a number of transporters/carriers. Molecules are needed for proper cellular function (basolateral membrane permeability) and for developing germ cells and maturing spermatozoa (apical membrane permeability) in the testis and epididymis, respectively. Note that the flow of molecules is not one way, from blood to cell to lumen, but also from lumen to cell to blood, making the permeability properties of the apical and basolateral membranes quite complex and not trivial.

One minor criticism from these earlier BTB physiological experiments was that the measurements were of the permeability of the seminiferous tubule and rete testis rather than the seminiferous tubule alone. To this end, micropuncture experiments, in which fluid was removed directly from the seminiferous tubule, confirmed that, indeed, there was a permeability barrier across the seminiferous tubule [5, 52, 53]. Similar experiments were performed demonstrating the permeability characteristics of the epididymal epithelium [54–57]. Further studies showed clearly that many ions, organic solutes, and peptides are transported into and out of the testis [21, 54, 58–64] and the epididymal epithelium via specific transporters [65–73]. These carriers were also quite specific for their substrates, only allowing the required molecules to enter the Sertoli and epididymal cell, and then presumably, for some molecules, to cross the apical membrane and enter into the lumen (Fig. 1, A and B).

Another key point that should be made is that the physiological barrier in both the testis and epididymis is highly dynamic; that is, it is constantly changing. It is well known that the Sertoli cell itself is highly dynamic [22, 74], changing its three-dimensional structure during the course of spermatogenesis and spermiogenesis. As the Sertoli cell changes, the physiological barrier also changes to meet the needs (e.g., nutritional) of the germ cells and the Sertoli cells. However, although the tight junctions are also dynamic in the sense that they undergo remodeling (opening and closing), it would appear that they remain static regarding their function; that is, no matter at which stage of the cycle, the integrity of the junctions remain intact. Likewise, for the physiological barrier in the epididymis; the tight junctions remain intact, yet the physiological barrier changes from one region to the next. This is reflected in the ever-changing luminal fluid composition along the duct (Fig. 1B); one can imagine that, even from cell to cell from initial segment to cauda, the physiological barrier changes! It is extraordinary to realize that all these events are highly coordinated, and the mechanism(s) by which this coordination is achieved is not known.

IMMUNOLOGICAL BARRIER

Immunological Barrier in Testis

The term immune-privilege is often used synonymously with BTB. In fact, it is often stated that immune-privilege in the testis is because of the BTB. However, immune-privilege in the testis is more complex, and the whole testis, not just the adluminal compartment, is immune-privileged. The immunoprotective ability of the space outside of the BTB was demonstrated by several studies in which allogeneic or xenogeneic tissues, such as fragments of skin, pancreas, or parathyroid, transplanted into the testis interstitium survived for an extended duration of time [75–77]. For instance, transplantation of allogeneic or xenogeneic pancreatic islets within the interstitial space of the testis not only resulted in prolonged graft survival compared to non-immune-privileged sites, kidney, and liver, but also normalized blood glucose levels in diabetic rodents and Rhesus monkeys [77, 78].

If the whole testis is immunologically privileged, what role, if any, does the BTB play in testicular immune-privilege and creating an immunological barrier to protect autoantigenic germ cells? It has been proposed that the BTB acts as an immunological barrier by sequestering germ cell antigens and preventing entry of leukocytes and antibodies into the adluminal or luminal compartments of the testis. This is partially true in that antibodies and immune cell entry is hindered by the SC barrier under normal conditions (Fig. 2A) [79–83]. However, in the testis, although most of the autoantigenic germ cells are sequestered behind the SC barrier, preleptotene spermatocytes and spermatogonia are located within the basal compartment of the seminiferous epithelium, and express antigens that can evoke an immune response (Fig. 2A) [84]. Moreover, there is recent evidence that germ cell-specific antigens expressed only by germ cells in the adluminal compartment, such as LDHc4 (testis-specific isoform of lactate dehydrogenase) [85], can be transferred across the SC barrier and detected in immune complexes within the interstitium of the testis (Fig. 2A) [86]. It is thought that this allows for the induction of immunologic tolerance to these germ cell autoantigens [86]. Therefore, as stated by Tung, “The idea of complete sequestration of testis autoantigens as the pre-eminent basis of immunologic unresponsiveness no longer holds” [87]. Instead, testicular immune-privilege also involves local production of anti-inflammatory cytokines and immunosuppressive factors by cells within the testis, such as Sertoli cells, Leydig cells, testicular macrophages, and regulatory T cells, which control the immune response [12, 13]. The importance of Sertoli cells in creating an immune-privileged environment has been demonstrated by transplantation experiments where Sertoli cells placed in ectopic sites were able to survive and protect cografted allogeneic and xenogeneic cells [88]. This protection is likely due to the expression of immunoregulatory factors, as the protected cotransplanted cells were located in the vicinity of, but not surrounded by, the Sertoli cells at the graft site [88–90].

FIG. 2.

The immunological barrier. The immunological component prevents entry of immunoglobulins (Y) and immune cells (macrophages and T cells). In the testis (A), autoantigenic germ cells reside in the basal and adluminal compartments. Germ cell autoantigens are also present within Sertoli cells and are transported by Sertoli cells to the interstitial compartment, where they are located outside the seminiferous tubules (dots). In addition, secreted immunomodulatory factors within the interstitial space control immune responses. Restriction of movement of antibodies and immune cells across the Sertoli cell barrier, together with the controlled presentation of antigens, and secretion of immunomodulatory factors creates a unique immune-privileged site in the testis. In the epididymis (B), tight junctions of the epididymal epithelium form the major component of the immunological barrier. Autoantigenic germ cells are present in the lumen, immunoglobulins can cross the epithelial cells, and immune cells have also been observed within the epithelium adjacent to epithelial cells. B, blood vessel; E, epididymal epithelial cell; GC, germ cell; L, Leydig cell; M, myoid cell; MΦ, macrophage; S, Sertoli cell; T, T cell; TJ, tight junction.

The involvement of immunomodulatory factors and other immunoregulatory mechanisms in forming a functional immunological barrier is further supported by experiments where the permeability of the SC barrier was altered in mice by knockout of the tight junction protein, claudin 11, or treatment with a mutant occludin peptide [91, 92]. Even though the barrier was permeable to bovine serum albumin or inulin, an autoimmune reaction was not generated, as testicular autoantibodies were not detected in serum or within the adluminal compartment, and no CD4 T cell infiltrate was detected in the testis [91, 92], indicating that there is more to testicular immune-privilege than just the anatomical barrier. However, since the germ cells were lost after disruption of the barrier in these mice, possibly before an immune response could be generated, further studies need to be designed to specifically address the importance of the tight junctions before concluding that the SC barrier does not play a role in immune-privilege. In contrast to immune protection in the testis, even when the BTB is intact, autoimmune orchitis can develop, leading to destruction of the developing germ cells. In addition, survival of the tissue transplanted into the interstitium and cografted with Sertoli cells is highly variable and can be rejected, suggesting that a delicate balance of immune regulation exists in the testis [75–78, 89].

Overall, the immunological component of the BTB is an important part of testicular immune-privilege. By preventing antibodies and immune cells from entering into the tubule, sequestering the majority of the autoantigens and allowing for controlled antigen exposure/presentation, this could decrease the immune response to the germ cells (Fig. 2A). Combined with the other immunomodulatory properties of the testis, this leads to creation of an effective immune-privileged environment throughout the testis that, under normal circumstances, protects the autoantigenic germ cells from immunological destruction.

Immunological Barrier in Epididymis

The function of the immunological barrier in the epididymis is to protect the sperm from attack by the immune system. Primarily, this function is carried out by the BEB, which sequesters sperm antigens and restricts entry of immunoglobulins and immune cells (Fig. 2B). The BEB prevents sperm antigens from escaping the duct and contacting immune cells, thus preventing induction of an autoimmune reaction [14]. Under normal conditions, monocyte-macrophages, CD4⁺ T cells, and CD8⁺ T cells, which are located throughout the epididymal epithelium and interstitial space (Fig. 2B) [81, 93], are not detected within the epididymal lumen [81]. Typically, immunoglobulins do not cross the BEB [81, 94, 95], and, although less than 2% of serum IgG levels are detected within the lumen of the cauda epididymis under normal circumstances [96], this can be accounted for by leakiness of the rete testis [96].

While several immunoregulatory factors are expressed in the epididymis [15], there is little direct evidence to support that the whole epididymis is immune-privileged. Only one study has examined the survival of allografts after transplantation into the interstitial space of the epididymis [97]. In this study, allogeneic parathyroid tissue was transplanted into the epididymis, and a slight prolonged survival (21 ± 6.22 days) of the grafts was observed only when the spleen was removed before transplantation and the lymphatic drainage bypassed the regional lymph nodes. In the case of intact spleen, grafts were rejected, with mean graft survival of 7.57 ± 0.9 or 12 ± 2.68 days if the lymphatics drained directly to the regional lymph nodes or bypassed the regional lymph nodes, respectively. This graft survival time was much shorter than the survival of allografts transplanted into the testis [75–78, 88], indicating that immune protection in the interstitium of the epididymis is limited.

In response to injection of spermatozoa or testicular germ cells into the epididymal interstitial space, spermatic granulomas were formed [98]. Moreover, intravenous injection of Bordetella pertussigens induced an inflammatory reaction and infiltration of leukocytes within the epididymal interstitium [99]. Despite the increase in inflammatory cells within the interstitium, the epididymal tubules were not damaged, indicating that the tight junctions provided a sufficient immunological barrier [99]. In contrast, when similar studies were performed (injection of spermatozoa, testicular germ cells, or Bordetella pertussigens) in the testis, no infiltrate was detected and spermatic granulomas were not formed in the interstitial space [98, 99].

Together, this supports the importance of the tight junctions in the epididymis in forming an effective immunological barrier, and indicates that, while the immunological barrier in the testis includes the interstitial space, this may not be the case for the epididymis.

CONCLUSION

The BTB and BEB consist of three components: anatomical, physiological, and immunological. Each of these components, not just the Sertoli cell-Sertoli cell junctions or epithelial tight junctions of the epididymis, contributes to form the BTB and BEB of maximum competence. Together, they create a unique, anatomical, physiological, and immunological microenvironment, which is responsible for proper development of the germ cells into fully functional sperm.