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Population Council, Center for Biomedical Research, New York, New York 10021
Correspondence: Address all correspondence and requests for reprints to: Dolores D. Mruk, Ph.D., or C. Yan Cheng, Ph.D., Population Council, Center for Biomedical Research, 1230 York Avenue, New York, New York 10021. E-mail: D-Mruk{at}popcbr.rockefeller.edu or Y-Cheng{at}popcbr.rockefeller.edu
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Abstract |
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 Top Abstract I. IntroductionII. Sertoli CellsIII. Sertoli-Sertoli and Sertoli...IV. Mechanism of Germ...V. Concluding RemarksReferences |
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I. Introduction |
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TopAbstractI. IntroductionII. Sertoli CellsIII. Sertoli-Sertoli and Sertoli...IV. Mechanism of Germ...V. Concluding RemarksReferences |
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On this note, we attempt to provide a balanced but informative treatment of Sertoli and germ cells in the testis, in particular the functions of Sertoli cells, and the interactions taking place that confer germ cell movement (e.g., cell junction restructuring) during spermatogenesis. Although brief accounts are given in many subject areas, readers are encouraged to refer to the excellent review articles cited throughout the text and to references cited therein, in particular the role of Sertoli and germ cell proteins in cell-cell interactions and the regulation of spermatogenesis (for reviews, see Refs.4 and 8, 9, 10, 11, 12). We focus our discussion largely on Sertoli-Sertoli and Sertoli-germ cell interactions at the level of cell junctions rather than the paracrine and/or autocrine factors used by these cells for communication because this latter subject area has recently been reviewed (for reviews, see Refs.8 , 9 , and 11). Furthermore, recent advances in the field have clearly shown that most, if not all, of the junction types found in the testis are nonstatic structural entities. In other words, they are dynamic structures that serve as platforms for signal transduction events that regulate the opening and closing of these junctions (see Section III). As such, we first briefly introduce the different types of junctions found in the testis (Table 1
). A detailed discussion on the structure and functions of Sertoli cells is found in Section II, which is followed by in-depth discussions on the different types of junctions, their constituent proteins, and regulation. These are followed by a description of a current biochemical model that regulates the movement of germ cells not only across the blood-testis barrier but also the remaining seminiferous epithelium during spermatogenesis.
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). As such, the term adherens junction refers only to one of the four types of anchoring junctions. In addition, the ectoplasmic specialization and tubulobulbar complex are two of the best-studied adherens junction types in the testis (Table 1). Although their presence in the testis has been demonstrated, they remain to be poorly characterized (Table 1) (for reviews, see Refs.15, 16, 17, 18). For example, it is known that germ cells, such as pachytene spermatocytes and round spermatids, must remain attached to Sertoli cells throughout the 14 stages of the epithelial cycle in the rat, yet the molecules that confer this adhesiveness are not all known (for reviews, see Refs.15 and 18).
In the following sections, we will limit our discussion to the biology of Sertoli-Sertoli and Sertoli-germ cell interactions at the level of cell junctions, emphasizing recent advances in the field. We will also highlight specific areas that deserve additional investigation. We begin by summarizing the three junction types found in the testis and the proteins that constitute these junctions, which will be discussed in greater detail (see Section III) after a discussion on the structure and function of Sertoli cells. This is done because until recently much of the information on junction dynamics in the testis was derived from studies using Sertoli cells (for reviews, see Refs.4 , 6 , 19 , and 20). Figure 1 is a schematic drawing of the seminiferous epithelium in the rat testis illustrating the relative locations of the different types of junctions found between Sertoli cells, as well as between Sertoli and germ cells. Figure 2 is an electron micrograph showing the intimacy between the seminiferous epithelium and the basement membrane (21). As shown in Fig. 1, Sertoli cell tight junctions that create the blood-testis barrier also anatomically divide the seminiferous epithelium into basal and adluminal compartments (for review, see Ref.3). Although it is obvious that the current biochemical architecture of tight and adherens junctions in the testis will be rapidly updated, it is our intention to summarize recent advances in this rapidly evolving field so that this review can serve as a guide for future studies. Every effort was made to credit original investigators with their findings, but because of the large number of references, we often chose to cite reviews rather than original articles. As such, we apologize to investigators in the field for possible oversights on our part.
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0.5–1.2 x 106 cells/cm2) on Matrigel-coated bicameral units or dishes (22, 23, 24, 25, 26, 27, 28, 29, 30). This model has been extensively characterized by different laboratories (30, 31, 32, 33, 34, 35, 36, 37). Studies on the regulation of blood-testis barrier dynamics are based on in vivo models of blood-testis barrier damage induced by cadmium chloride, glycerol, or occludin peptide (38, 39, 40, 41, 42, 43). These have also been previously characterized (for review, see Ref.44). For studies relating to the regulation of adherens junction dynamics, such as the ectoplasmic specialization (5, 45, 46, 47, 48, 49), an in vitro Sertoli-germ cell coculture system was used (5, 50, 51, 52). Unfortunately, no in vivo models to study adherens junction dynamics in the testis were available until recently (45, 49, 53, 54); these now include the use of 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364; for review, see Ref.4) and androgen/estrogen implants to suppress the endogenous testosterone level (for review, see Ref.55). Both induce the loss of cell adhesion between Sertoli and germ cells, in particular spermatids. Detailed discussions on these in vitro and in vivo models are found in Sections III and IV. |
II. Sertoli Cells |
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TopAbstractI. IntroductionII. Sertoli CellsIII. Sertoli-Sertoli and Sertoli...IV. Mechanism of Germ...V. Concluding RemarksReferences |
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), the somatic constituents that extend from the base to the apex of the seminiferous epithelium, interact directly with developing germ cells throughout spermatogenesis. To this end, several studies have shown that these cells have an indispensable role in the development and movement of germ cells (for reviews, see Refs.61, 62, 63, 64, 65, 66, 67, 68, 69). The following discussion on Sertoli cell structure and function will provide the foundation for the discussion of Sertoli-germ cell interactions.
A. Sertoli cell structure
Since their discovery in the late 1800s, Sertoli cells have been extremely difficult to study morphologically because they have a constantly changing, three-dimensional relationship with developing germ cells throughout the 14 stages of the epithelial cycle (for reviews, see Refs.6 , 67 , and 70), yet Sertoli cells continue to be one of the most-studied cell types in the seminiferous tubule. They are irregularly shaped, columnar cells that extend from the basal to the adluminal compartment and occupy a volume of approximately 17–19% in the seminiferous epithelium of adult rats (71, 72) (Fig. 2). Sertoli cells are in direct contact with the basement membrane, a modified form of the extracellular matrix in the testis (21) (Figs. 1 and 2). They have an enormous surface area (Fig. 2), which allows them to sustain a vast number of developing germ cells at a Sertoli:germ cell ratio of approximately 1:50 in the adult rat testis (71, 73). This attribute, in itself, is crucial not only to spermatogenesis, but also to germ cell movement.
From a morphological standpoint, Sertoli cells at any one time can be classified into one of two categories: 1) type A, or 2) type B (for reviews, see Refs.18 , 67 , and 70). In type A Sertoli cells, mature spermatids ready for release into the tubule lumen are lodged deep within cytoplasmic crypts, whereas in type B Sertoli cells these cytoplasmic crypts are either barely visible or completely absent. Therefore, during the 14 stages of the epithelial cycle, type A Sertoli cells must transform themselves into type B Sertoli cells to adapt to the cellular changes that take place during germ cell development and movement. These observations have been supported by eloquent morphological studies in the rat, which have demonstrated that there are drastic changes in cell shape in stages II, VII, VIII, IX–XI, and XIII–XIV, illustrating that Sertoli cells function together with germ cells in forming the epithelial cycle (for reviews, see Refs.3 , 74 , and 75).
B. Sertoli cell functions
To date, several Sertoli cell functions, most of which are directly related to germ cell development and movement, have been described. These include: 1) providing structural support; 2) creating an impermeable and immunological barrier; 3) participating in germ cell movement and spermiation; 4) nourishing germ cells via their secretory products, and several others (for reviews, see Refs.62 , 66 , 69 , and 74), all of which are discussed in the following sections. It must be noted, however, that several of these functions were determined by studies using Sertoli cells cultured in vitro, and the physiological relevance of some of these functions in Sertoli cells in vivo remains to be determined.
1. Structural support.
Sertoli cells provide support for developing germ cells within the seminiferous epithelium by participating directly in the deposition of extracellular matrix components and permitting the formation of specialized cell junctions. Another striking feature of Sertoli cells is their well-developed cytoskeleton, which is responsible for the collective organization of the seminiferous epithelium (for reviews, see Refs.3 , 7 , 15 , 16 , 18 , and 70). Morphological studies have shown that the Sertoli cell cytoskeleton: 1) maintains shape; 2) positions and transports organelles within the cell; 3) forms and stabilizes the cell membrane at sites of cell-cell and cell-extracellular matrix contact; 4) positions, anchors, and aids in the movement of developing germ cells; and 5) participates in the release of mature spermatids from the seminiferous epithelium at spermiation. It consists of three major components, namely actin, intermediate filaments, and microtubules, with each component having a unique pattern of distribution that changes in association with the 14 stages of the epithelial cycle (for reviews, see Refs.7 , 67 , and 76). Collectively, these components play a crucial role in facilitating germ cell movement.
a. Actin.
Actin filaments are composed of actin monomers of molecular mass (Mr) 42 kDa, each of which polymerizes into a linear chain with a helical twist approximately 8-nm wide (for reviews, see Refs.77 and 78) and exists in six different isoforms in mammalian tissues (79, 80). In the Sertoli cell, actin plays an extremely important role in maintaining structure and conferring cell contractility, but its presence at the cell periphery at sites of cell contact also suggests that it participates in motility-related processes. There are two pools of actin, namely monomeric G-actin and polymeric F-actin, that coexist in equilibrium in all eukaryotic cells, including Sertoli cells. The predominance of one pool affects the overall status of the cytoskeleton by causing polymerization or depolymerization of actin filaments. This, in turn, affects cell shape and movement (for reviews, see Refs.81 and 82). Figure 3A depicts a typical Sertoli cell cultured in vitro. Notice the F-actin filaments (green) that were visualized by using Oregon Green 488 phalloidin. Also shown in this figure is immunoreactive vinculin (red), an adaptor protein (Section III.A.5.b), and the Sertoli cell nucleus, which was stained with DAPI (blue). Figure 3B shows two typical Sertoli cells cultured in vitro for approximately 7 d and immunostained for zyxin (green), another adaptor protein found at the focal contact and leading edge of cytoplasmic processes. Recently, zyxin was demonstrated to associate with actin in Sertoli cells by immunoprecipitation (83). Actin filaments are also major components of ectoplasmic specializations and tubulobulbar complexes, modified anchoring junction types present between Sertoli cells, as well as between Sertoli and germ cells, illustrating that both of these junctions participate in the movement of developing germ cells (for reviews, see Refs.7 and 67). It is also of interest that mouse haploid germ cells express two unique actins designated T-actin 1 and T-actin 2, which share 40% homology with other actins (84). T-actin 1 is confined to the spermatid cytoplasm, whereas T-actin 2 is restricted to the sperm head and tail (84), suggesting that these germ cell-specific actins are important to the morphogenesis of germ cells during spermiogenesis.
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) because they confer desmosome-like junctions between Sertoli cells and between Sertoli cells-spermatocytes/round spermatids (for review, see Ref.87). Although the exact role of intermediate filaments in Sertoli cells is not entirely known, their distribution within these cells suggests that they participate in maintaining the integrity of the seminiferous epithelium (88, 89, 90).
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, ß, 
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, and 
(104), but the major building blocks of the microtubule cytoskeleton are - and ß-tubulins (105). In general, microtubules appear to have a much clearer role in Sertoli cell function (for reviews, see Refs.7 and 106, 107, 108). They are known to: 1) maintain a columnar shape; 2) position and transport intracellular organelles; 3) translocate spermatids within the seminiferous epithelium; and 4) adjust the contour of the Sertoli cell membrane to adapt to the irregularly shaped spermatid heads that are lodged within crypts during spermatogenesis (for reviews, see Refs.6 and 7). It is not surprising, therefore, that the most striking changes in microtubule organization occur when mature spermatids associate with Sertoli cells (109). Microtubules are also found at the site of the ectoplasmic specialization (110, 111). Although the presence of actin, intermediate filaments, and microtubules in the testis has been implicated in the events of germ cell movement, the mechanisms underlying the regulation of the Sertoli cell cytoskeleton during different stages of the epithelial cycle and germ cell movement remain to be investigated.
2. Create an impermeable barrier.
The blood-testis barrier (also known as the seminiferous epithelial barrier), a modified occluding junction, is formed by approximately 15–18 d of age in the rat as constituted by Sertoli cell tight junctions located in the basal third of the seminiferous epithelium (Refs.2 and 112, 113, 114, 115 ; for reviews, see Refs.116, 117, 118) (Fig. 1). The blood-testis barrier compartmentalizes the epithelium into two (112, 119), rather than three, compartments as previously postulated (2, 114). One is a basal compartment in which spermatogonia, preleptotene, and leptotene spermatocytes reside. The other is an adluminal compartment in which meiotic spermatocytes and spermatids in various stages of spermatogenesis and spermiogenesis exist.
a. Concept of the blood-testis barrier.
The concept of the blood-testis barrier was first developed in the 1970s based on two important findings (for reviews, see Refs.112 , 120 , and 121). First, there were drastic differences in the composition of fluids and proteins obtained from the rete testis and seminiferous tubule lumen, and the testicular lymph and blood plasma. For example, it was demonstrated that fluid collected from the rete testis was higher in potassium and chloride than that collected from the blood plasma (Ref.122 ; for review, see Ref.123). In addition, the total protein content of the rete testis fluid was only approximately 1/20 to 1/50 that of the plasma (124). Second, there were variations in the rate in which radioactive tracers or dyes could pass from the blood plasma into testicular fluids (114, 125, 126, 127, 128). Although these results suggested that a barrier existed, concrete evidence, not to mention the physiological significance of such a barrier, was for the most part lacking until the 1980s when cell biology and morphology studies confirmed earlier observations.
b. Functions of the blood-testis barrier.
Three main functions are ascribed to the blood-testis barrier. The blood-testis barrier: 1) creates a specialized environment; 2) regulates the passage of molecules; and 3) serves as an immunological barrier. First, the blood-testis barrier contributes directly to the complex structural organization of the testis by creating a specialized environment necessary for germ cell development and movement. As such, Sertoli cells are obliged to synthesize, secrete, and efficiently deliver products that are essential for the growth and differentiation of developing germ cells (Refs.129 and 130 ; for reviews, see Refs.131, 132, 133, 134, 135, 136). For instance, the synthesis of testicular transferrin, which was first reported by Thorbecke et al. (137) and later by Skinner and Griswold (138), is the classic example of this Sertoli cell characteristic (for reviews, see Refs.136 and 139). Because germ cells residing in the adluminal compartment do not have access to serum iron, a transport mechanism orchestrated by Sertoli cells must exist. Nevertheless, recent in vitro studies have demonstrated that germ cells can indeed stimulate Sertoli cell transferrin mRNA and protein levels, suggesting that germ cells govern their own iron requirements via their interactions with Sertoli cells (140, 141, 142). One must be cautioned, however, that some of the earlier reported effects of germ cells on Sertoli cell function may indeed be artifacts because residual trypsin, which was used to isolate germ cells from the testis, can mimic some of the reported effects on Sertoli cells (143).
Tight junctions that constitute the blood-testis barrier also regulate the movement of products, such as nutrients and wastes, both into and out of the seminiferous epithelium (for review, see Ref.144). Although studies that have specifically focused on this tight junction attribute in the testis are by far limited, the regulated movement of solutes across tight junctions continues to be well studied in other organs, such as the kidney and intestine in normal (e.g., food absorption in the small intestine) and pathological (e.g., migration of neutrophils across the tight junction barrier during inflammation) responses. In the testis, the best example of regulated movement of products lies in the biology of FSH, whose major site of action is on Sertoli cells. However, FSH cannot enter the seminiferous tubule fluid readily (for reviews, see Refs.145, 146, 147). Therefore, it is not unexpected that receptors for FSH are found in the basal compartment residing on the Sertoli cell membrane (146, 148, 149, 150, 151, 152).
The third main function of the blood-testis barrier is to create an immunological barrier, ensuring that the organism’s immune system does not recognize unique antigens present on the cell surfaces of haploid germ cells. This would result in the organism being immunized against its own spermatozoa. In addition, recent studies have shown that Sertoli and germ cells maintain a unique antiviral defense system via the production of interferons (IFNs), ILs, and cytokines (153, 154). The blood-testis barrier also prevents the entrance of Igs and lymphocytes into the adluminal compartment (for review, see Ref.155).
c. Regulation of the blood-testis barrier.
The blood-testis barrier is one of the tightest barriers in the mammalian body comparable to the blood-brain barrier, and tight junction barriers in the endothelium of blood vessels and the epidermis. However, the blood-testis barrier is different in that it must open periodically to accommodate the passage of germ cells during spermatogenesis. For instance, developing preleptotene/leptotene spermatocytes must traverse the blood-testis barrier, which is known to take place in late stage VIII–early IX of the epithelial cycle (2). This process requires that Sertoli-Sertoli and Sertoli-germ cell junctions be disassembled and reassembled. This also illustrates the unusual dynamic nature of the blood-testis barrier. However, the mechanism(s) by which germ cell movement takes place remains to be elucidated. Additionally, the factor(s) that regulates the formation and maintenance of the blood-testis barrier is currently not known. For instance, gonadotropins have been implicated, but this is largely a speculation that has remained relatively unexplored. Until recently, the lack of information on the biology of the blood-testis barrier has largely stemmed from the lack of suitable in vitro and in vivo models that could be used to study Sertoli cell tight junction dynamics. These will be discussed below.
i. An in vitro model.
Sertoli cells cultured in vitro on Matrigel-coated dishes or bicameral units at a cell density ranging between 0.5 and 1.2 x 106 cells/cm2 are known to establish a functional tight junction barrier (24, 31, 32, 35, 36, 37). The assembly and maintenance of the tight junction barrier was assessed by transepithelial electrical resistance (TER); polarized secretion of Sertoli cell proteins; restricted diffusion of [3H]inulin, [125I]BSA, or fluorescein isothiocyanate (FITC)-dextran (Mr, 4.4 and 35.6 kDa); or maintenance of nonequilibrium of media across the Sertoli cell epithelium in bicameral units (24, 27, 31, 32, 35, 36, 37, 48). Using this system, several laboratories, including ours, have shown that Sertoli cell tight junction barrier function is regulated by an array of molecules, including intracellular calcium, cAMP, cytokines, protease inhibitors, protein kinases and phosphatases, and others (for reviews, see Refs.4 and 44). However, this in vitro system is not flawless. For instance, the Sertoli cell tight junction that constitutes the blood-testis barrier is one of the tightest barriers in the mammalian body (Section II.B.2). However, the tightness of the Sertoli cell tight junction barrier in vitro is only approximately 100 ohm·cm2 (22, 24, 27, 28, 29, 34, 57) vs. 1000–2000 ohm·cm2 in Madin-Darby canine kidney (MDCK) cells and keratinocytes (156, 157, 158) when barrier function was assessed by TER measurement. These results seemingly suggest that Sertoli cell tight junctions in vitro are only moderately tight when compared with other cell types. Recent studies have shown that the inclusion of androgens (27, 28), cAMP (34), and protease inhibitors (29) into Sertoli cell cultures during tight junction assembly can increase the tightness of the tight junction permeability barrier in vitro, illustrating that many biological factors present in vivo contribute to the tightness of this barrier in the testis. In short, these additional data demonstrate that this in vitro model is useful for studying tight junction dynamics.
Indeed, this in vitro model was used to verify some of the in vivo observations regarding Sertoli cell tight junction barrier function. For instance, recent studies have shown that extracellular matrix components, such as laminin and heparan sulfate, can promote the formation of the blood-testis barrier in vivo (Ref.159 ; for review, see Ref.160), whereas another report illustrated that laminin can also regulate the Sertoli cell tight junction barrier in vitro (161). To determine whether a relationship between extracellular matrix proteins and the assembly of the Sertoli cell tight junction permeability barrier does indeed exist (162, 163), this in vitro model was also used. Interestingly, the inclusion of an anticollagen 3(IV) antibody into Sertoli cells cultured at high density (1.2 x 106 cells/cm2) on Matrigel-coated bicameral units was able to perturb the assembly of the tight junction barrier, indicating that this protein is essential to tight junction functionality (163). This result also brings on a rare significance all on its own, because tight junctions in the testis are located in the basal third of the seminiferous epithelium closest to the basement membrane (21) [comprised of laminin, type I and IV collagen, heparan sulfate, and entactin (164)]. This is unlike other epithelia where tight junctions are located apically, farthest away from the extracellular matrix. What is not known, however, is whether this anticollagen-induced effect on Sertoli cell tight junction assembly is mediated via integrins or other yet-to-be identified molecules that constitute the focal adhesion or hemidesmosome. It has also been suggested that peritubular myoid and endothelial cells contribute, at least in part, to the functionality of the blood-testis barrier (97, 165), but the significance of this is presently unknown because there are no in vitro or in vivo studies examining the effects of these two cell types on the Sertoli cell tight junction barrier.
Other studies have demonstrated that the formation of the blood-testis barrier is not dependent upon germ cells because the disappearance of these cells from the seminiferous epithelium in vivo did not prevent the formation of the barrier (166, 167). Additional studies have demonstrated that the formation of the Sertoli cell tight junction permeability barrier is dependent on temperature and hormones (27, 33, 34, 168). For instance, the addition of testosterone (0.001–10 µM) into Sertoli cells cultured on bicameral units in vitro was able to enhance TER across the Sertoli cell epithelium (33, 34). This is not entirely unexpected because the testis is an androgen-dependent organ. On the other hand, treatment of rats with estradiol valerate did not affect the passive diffusion of [3H]inulin into the tubule lumen, although testicular weight and the serum testosterone level were shown to be altered (169), illustrating that estrogens do not regulate Sertoli cell tight junction barrier function.
There is also limited evidence to suggest that protease inhibitors can affect Sertoli cell tight junctions. A recent report by Okanlawon and Dym (29) demonstrated that chloroquine (an antimalarial drug and a protease inhibitor that has the property of increasing the pH within lysosomes and endocytic vesicles) was able to dose-dependently increase TER, making the Sertoli cell tight junction barrier tighter, when compared with Sertoli cells cultured in the absence of chloroquine. Unfortunately, the effects of protease inhibitors on Sertoli cell tight junction dynamics remain limited to this single report, and there is not a single study in which the effects of proteases are examined. In studies in which other cell types and cell lines such as MDCK cells were used, proteases were found to induce the aberrant formation of tight junctions. For example, the application of trypsin, a serine protease, together with calcium to the basolateral surface of confluent MDCK monolayers resulted in the formation of basally situated tight junction strands, which was accompanied by a transient increase in TER (170). These results seemingly suggest that this increase in tight junction barrier function is a normal physiological response of an epithelium to a potential cellular hazard. What is not known is whether the cellular distributions of occludin and claudin were altered in response to trypsin treatment. The participation of protease inhibitors was also studied in the human adenocarcinoma cell line, Caco-2, when these cells were cultured in the presence of nontoxic concentrations of either leupeptin or antipain (Ref.171 ; for review, see Ref.172). It was shown that these protease inhibitors could effectively block tight junction barrier assembly in vitro. These results, taken collectively, demonstrate the involvement of proteases and protease inhibitors in tight junction dynamics, yet similar studies in the testis await to be performed.
ii. In vivo models.
Once formed, the blood-testis barrier cannot be easily disrupted, such as by heat or irradiation (166, 173). However, ip administration of cadmium salts (e.g., cadmium chloride) can cause the blood-testis barrier to break down, resulting in complete degeneration of the seminiferous epithelium (28, 37, 40, 41, 174). This in vivo model of blood-testis barrier disruption is irreversible, and an explanation that would help to understand the destructive effects of cadmium chloride in the testis is still lacking. In studies using cell lines, the effects of cadmium chloride were shown to be concentration- and time-dependent, decreasing the tight junction barrier by TER, increasing the permeability of mannitol, and altering the localizations of occludin, zonula occludens (ZO)-1, and E-cadherin (175). In another recent in vitro study from our laboratory, Sertoli cells were cultured in the presence of cadmium chloride (noncytotoxic concentrations), which induced a disruption of the tight junction permeability barrier as assessed by TER. As anticipated, the tight junction barrier failed to reseal upon removal of cadmium chloride (27). However, the addition of testosterone (1 x 10–9 M) allowed reassembly of the Sertoli cell tight junction permeability barrier after cadmium chloride was removed from cultures, and a 200-fold increase in testosterone even protected Sertoli cells from cadmium chloride-induced damage (27). This suggests that this androgen can protect Sertoli cell tight junctions from being damaged.
Another in vivo model of blood-testis barrier destruction is the glycerol model (Refs.176, 177, 178, 179, 180 ; for review, see Ref.181). Similar to cadmium chloride, intratesticular injection of glycerol irreversibly disrupts the blood-testis barrier and, consequently so, spermatogenesis. However, glycerol does not affect Leydig cell steroidogenesis or serum FSH and LH levels (176). In a recently published article by Wiebe et al. (42), the effects of glycerol on the testis were expanded when the localizations of cytoskeletal and tight junctional proteins such as F-actin, tubulin, and occludin were examined by immunofluorescence. It was shown that glycerol, in addition to upsetting the localization of occludin, was capable of perturbing microfilament and microtubule organization in the testis. Unfortunately, comparable studies on the effects of glycerol on Sertoli cells in vitro are lacking. Moreover, results obtained from the study of these two models suggest that a primary disruption of the blood-testis barrier can also lead to a loss of adherens junction function since the treatment of rats with cadmium chloride or glycerol results in the loss of germ cells from the seminiferous epithelium, causing aspermatogenesis and infertility (for review, see Ref.44).
A third in vivo model to study blood-testis barrier dynamics is based on a recent study from this laboratory. When a single dose of a synthetic occludin peptide (NH2-GSQIYTICSQFYTPGGTGLYVD-COOH, corresponding to amino acid residues 209–230 in the second extracellular loop of rat occludin) (Fig. 4) was administered intratesticularly at 1.5–10 mg/testis, it caused reversible germ cell loss from the seminiferous epithelium (43). Morphological analysis of treated testes revealed that more advanced germ cells, such as elongate spermatids, began to deplete from the epithelium 1–2 wk after administration of the peptide (43). By 4 wk post treatment, there was a massive depletion of germ cells from the seminiferous epithelium in virtually all of the tubules examined. This event also associated with a disruption of the integrity of the blood-testis barrier when barrier function was monitored by micropuncture technique, detecting leakage of [125I]BSA from the systemic circulation into the lumens of the rete testis and seminiferous tubules (43). Figure 5 shows recent results using immunofluorescent microscopy to monitor the integrity of the blood-testis barrier by localizing occludin to this site. Damage of blood-testis barrier function was associated with an extensive disruption (Fig. 5, A—E) and loss of occludin immunostaining intensity (Fig. 5F) at the site of the blood-testis barrier. In addition, tubules in occludin peptide-treated testes shrunk significantly, with the tubule diameter reducing by as much as 20–30% when compared with control testes receiving either vehicle or a myotubularin synthetic peptide (43). Thereafter, germ cells began to repopulate the epithelium. By 7–8 wk, some spermatocytes were clearly visible in all of the tubules examined. By 10–16 wk (depending on the dose of occludin peptide used), the morphology of the seminiferous epithelium was indistinguishable from that of control rats, showing full recovery from occludin peptide-induced damage. The fact that testes recovered almost completely illustrates that spermatogonia were not destroyed. Occludin peptide-induced germ cell loss was also accompanied by a reversible disruption of the blood-testis barrier when assessed by micropuncture technique that quantified [125I]BSA in rete testis and seminiferous tubule fluids after administration of [125I]BSA through the jugular vein (43). Moreover, recovery of the blood-testis barrier coincided with the recovery of the seminiferous epithelium when examined histologically (43). It is likely that this peptide exerts its effects by interfering with the homotypic interactions that exist between two occludin molecules in apposing Sertoli cell membranes, thereby disrupting blood-testis barrier function. Once the peptide is metabolized and/or cleared, the blood-testis barrier reseals. Thus, these results illustrate that occludin peptide-induced reversible damage of the blood-testis barrier can be a useful in vivo model to study the dynamics of the blood-testis barrier if adequately characterized.
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) (for reviews, see Refs.182, 183, 184). In Sertoli cells cultured in vitro, for instance, the mRNA level of TGF-ß3 was found to decrease approximately 2-fold during the assembly of the tight junction barrier from d 1–3, which coincided with a transient but significant increase in the expression of occludin and ZO-1 (22). However, during the maintenance phase of the tight junction permeability barrier from d 5–8, the level of TGF-ß3 increased. The reason for this increase is not immediately known, but it is likely that TGF-ß3 is not needed for the maintenance of the Sertoli cell tight junction barrier.
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). These observations were verified when an in vivo model of blood-testis barrier disruption induced by cadmium chloride was used in which 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole (SB202109, a specific p38 MAPK inhibitor) could effectively block the cadmium chloride-mediated loss of occludin from the site of the blood-testis barrier (38). This illustrates that the dynamics of the blood-testis barrier are regulated, at least in part, by TGF-ß3 via the p38 MAPK pathway.
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can also perturb Sertoli cell tight junction barrier function by affecting the production of occludin, collagen, matrix metalloprotease-9 (MMP-9), and tissue inhibitor of metalloproteases-1 (TIMP-1) (163). It was postulated that TNF- induces the production of Sertoli cell collagen 3(IV), MMP-9, and TIMP-1, as well as promoting the activation of pro-MMP-9. Because activated MMP-9 cleaves the collagen network in the basement membrane, the Sertoli cell tight junction barrier is perturbed. This creates a negative feedback loop that causes TNF- to induce the synthesis of collagen and TIMP-1, which are needed to reassemble the disrupted collagen network and tight junction barrier (163). TNF- may also limit the activity of MMP-9. As such, this cytokine likely regulates collagen, MMP-9, and TIMP-1 which collectively maintain the integrity of the blood-testis barrier in vivo (163).
Many other cytokines and growth factors have also been investigated with regard to their effects on tight junction barrier assembly and disassembly. However, these studies were all performed in nongonadal cell types and cell lines. For instance, cytokines such as TGF-, epidermal growth factor (EGF), and IL-1 have all been documented to affect tight junction barrier function in different epithelial cell types and cell lines in vitro (Table 3). Such a disturbance may be mediated via: 1) a redistribution and/or shut-down in the production of tight junction proteins, or 2) remodeling of the actin cytoskeleton (for reviews, see Refs.182 and 184). If adequately expanded, these studies should shed light on the regulation of tight junction dynamics in the testis.
Interestingly, a recent study has shown that nitric oxide (NO) also plays an important role in the regulation of Sertoli cell tight junction dynamics via its effects on soluble guanylate cyclase and protein kinase G (PKG). For instance, significant decreases in the expression of inducible (iNOS) and endothelial NO synthase (eNOS) were detected during the assembly of the Sertoli cell tight junction permeability barrier in vitro, seemingly suggesting that NO can down-regulate tight junction barrier function (30). Indeed, the inclusion of zinc II protoporphyrin-IX (ZnPP, a specific inhibitor of NOS that can also inhibit soluble guanylate cyclase) into Sertoli cell cultures facilitated tight junction barrier assembly, whereas the presence of 8-bromo-cyclic GMP (cGMP) perturbed tight junction barrier function (30). Furthermore, C29H25N3O5 (KT-5823, a specific inhibitor of PKG) could also facilitate Sertoli cell tight junction barrier assembly (30). Lastly, iNOS and eNOS were both shown to physically interact with occludin, but not with N-cadherin and nectin-3 as demonstrated by coimmunoprecipitation (30). Collectively, these data have demonstrated that NO regulates Sertoli cell tight junction dynamics via the NOS/soluble guanylate cyclase/PKG signaling pathway. Needless to say, these data also illustrate that blood-testis barrier dynamics are under the regulation of multiple variables, some operating independently and others cooperatively.
e. Additional comments.
After nearly three decades of study by reproductive biologists, a much finer physiological distinction has arisen between the blood-testis barrier (e.g., occluding zonule) and Sertoli cell tight junction (e.g., occluding macule) based on the fact that the assembly of the blood-testis barrier and Sertoli cell tight junction are not synchronous events in vivo (213, 214, 215). The occluding zonule, which separates the seminiferous epithelium into basal and adluminal compartments, is the basis of the blood-testis barrier. The fibrils that compose the occluding zonule, or blood-testis barrier, are continuous, encircling each Sertoli cell and forming an impermeable barrier. In the rat, the blood-testis barrier forms by approximately 15–18 d of age (for reviews, see Refs.16 and 116). The occluding macule, on the other hand, is constituted by a focal seal through which interstitial fluids or tracers can readily pass. Morphological studies have shown that the fibrils that constitute the occluding macule in the testis are discontinuous and situated immediately above and below the occluding zonule (213, 214, 215). It must be noted that in other epithelial cell types and cell lines, such as MDCK cells, the occluding zonule is synonymous with the tight junction.
3. Assisting in germ cell translocation.
Sertoli cells are directly involved in the movement of developing germ cells because these cells lack an architecture characteristic of migrating cells, such as fibroblasts (for review, see Ref.3). A possible mechanism underlying germ cell movement may involve members of the GTPase superfamily, as shown in a series of recent studies from our laboratory (45, 50). Moreover, other studies have shown that the translocation of elongating/elongate spermatids across the seminiferous epithelium is largely conferred by the ectoplasmic specialization (Section III.A.3) (for reviews, see Refs.20 , 61 , and 65). It has been shown that microtubules, together with several motor proteins such as dynein (216, 217) and myosin VIIa (218), ATPases, and GTPases (for review, see Ref.219) present at the site of the ectoplasmic specialization assist in the migration of elongating/elongate spermatids across the seminiferous epithelium. This is analogous to a train (e.g., elongating/elongate spermatid) on a railroad track (e.g., microtubule) whose motion is driven by an engine (e.g., dynein, myosin VIIa). Because this hypothesis has been recently reviewed (for reviews, see Refs.20 and 216), it will not be discussed any further. However, many questions still remain unanswered. For instance, if the movement of germ cells must coincide with their developmental status, then what signal(s) dictates the timely movement of spermatids across the epithelium during the epithelial cycle? What is the mechanism(s) that regulates the restructuring of the ectoplasmic specialization? Do germ cells signal to Sertoli cells when they need to be lifted upward? To answer these questions, the biochemical architecture of the ectoplasmic specialization must first be completely elucidated.
Sertoli cells also assist in the translocation of early meiotic spermatocytes from the basal to the adluminal compartment during the epithelial cycle in the seminiferous epithelium. This process, which likely requires the disassembly of Sertoli-Sertoli and Sertoli-germ cell adherens junctions, is a highly selective process. For instance, type A spermatogonia cannot be lifted but must remain attached to the basement membrane, suggesting that an intriguing mechanism must exist to determine which specific cell types can be lifted from the basement membrane to the basal compartment.
4. Spermiation.
Sertoli cells are indispensable to the release of mature spermatids from the seminiferous epithelium. This process involves a cascade of events, some of which include: 1) encapsulation of spermatid heads by Sertoli cell cytoplasmic processes; 2) expulsion of spermatids from Sertoli cell crypts; and 3) release of spermatid heads (for reviews, see Refs.3 and 6).
5. Phagocytosis.
After the release of mature spermatids from Sertoli cell cytoplasmic crypts, Sertoli cells phagocytose the residual bodies that were released from spermatids and any germ cells that may have degenerated during spermatogenesis (220, 221, 222, 223). In this respect, Sertoli cells function as macrophages by maintaining the integrity of the seminiferous epithelium.
6. Pinocytosis and receptor-mediated endocytosis.
In addition to phagocytosis, studies have shown that Sertoli cells undergo active pinocytosis and receptor-mediated endocytosis in the adluminal and basal compartments of the epithelium, respectively (222, 223, 224).
7. Secretory function.
Sertoli cells are secretory cells. This is backed by the observation that approximately 15% of the total proteins produced by Sertoli cells are glycoproteins that are destined to be secreted (for reviews, see Refs.9 , 35 , 62 , 63 , 132 , 133 , 225 , and 226). As part of an attempt by many investigators to understand the many functions of Sertoli cells, in particular the relationship between these cells and germ cells, numerous research articles were published that identified and characterized an array of proteins; these include 1) proteases, 2) protease inhibitors, 3) hormones, 4) energy substrates, 5) growth factors, 6) paracrine factors, and 7) extracellular matrix components, as Sertoli cell secretory products (for reviews, see Refs.9 , 11 , 62 , 63 , 132 , 133 , 134 , 225 , and 227, 228, 229, 230). Brief descriptions on some of these proteins as they relate to germ cell movement are found below.
a. Proteases and protease inhibitors.
Sertoli cells synthesize and secrete proteases and protease inhibitors, which participate in nearly all cellular processes involving tissue maintenance, repair, growth, and development. Proteases and their inhibitors have also been implicated in the events of germ cell movement (for reviews, see Refs.225 , 231 , and 232). For example, it was postulated that cathepsin L plays a role in spermiation because its expression was highest in stage VII before germ cell release when compared with other stages of the epithelial cycle (47, 233, 234). However, subsequent studies have shown that the mRNA levels of cathepsins D (an aspartic protease) and S (a cysteine protease) peaked in stages VII–IX and VII–VIII, respectively (47). Thus, if cathepsin L is indeed required to cleave Sertoli cell-elongate spermatid junctions at spermiation, then what are the roles of cathepsins D and S? Needless to say, each protease or protease inhibitor likely works with other molecules so that a unique physiological effect relating to spermatogenesis and germ cell movement can be executed. Parvinen’s group also showed enhanced secretion of plasminogen activator (PA, a serine protease) in stages VII–VIII of the epithelial cycle (235), suggesting that this protease may also be required to cleave Sertoli-germ cell junctions to permit for the release of elongate spermatids into the tubule lumen. In yet another study from our laboratory, it was demonstrated that there was a transient but significant increase in overall serine protease activity during the adhesion of germ cells to Sertoli cells in vitro, but preceding the formation of Sertoli-germ cell junctions (5). Taken collectively, these results suggest that proteases need to be activated before junction disassembly (47, 233, 234) and assembly (5).
Studies in which changes in protease inhibitors correlated with junction assembly and/or disassembly in the testis include cystatin C (236) and 2-macroglobulin [2-MG (237)]. In both cases, the localizations of these protease inhibitors were reduced in stage VII when proteases such as urokinase-type PA (u-PA) and cathepsin L were highest (Refs.5 and 233 ; for review, see Ref.225), illustrating the existence of a ying-yang relationship between proteases and protease inhibitors. Furthermore, it was shown that 2-MG associated tightly with elongating spermatids in stages I–VI of the epithelial cycle, apparently protecting the epithelium from unwanted proteolysis (237). These results also illustrate the significance of protease inhibitors in germ cell movement in the seminiferous epithelium.
b. Growth, autocrine, and paracrine factors.
Growth, autocrine, and paracrine factors are secretory molecules that bind to cell surface receptors to induce a cascade of signal transduction events that affect cell growth, differentiation, and function (for reviews, see Refs.13 , 14 , 238 , and 239). In the testis, these factors play a crucial role in the maintenance of spermatogenesis and germ cell movement (for reviews, see Refs.240, 241, 242, 243, 244, 245, 246, 247). For instance, TGF-, a member of the EGF family (for reviews, see Refs.248, 249, 250) and putative Sertoli, germ, and peritubular myoid cell product (251), stimulates the proliferation of prepubertal Sertoli cells (252). On the other hand, several different TGF-ß isoforms are known to exist (for reviews, see Refs.253, 254, 255, 256, 257), and the presence of TGF-ß1, -ß2, and -ß3 (23, 258, 259) and their receptors (260, 261, 262) have all been demonstrated in the testis. Interestingly, the TGF-ßIII receptor, also known as betaglycan (for reviews, see Refs.253 and 255), was recently shown by Vale and colleagues to bind inhibin (Refs.263 and 264 ; for review, see Ref.265). Moreover, it appears that TGF- and -ß elicit opposite effects on cell division and differentiation in several different cell types, such as granulosa (266) and Leydig (267, 268) cells, suggesting that these cytokines may work synergistically but have opposite effects in regulating specific cellular events.
c. Extracellular matrix components.
It has been known for several decades that the extracellular matrix plays an extremely important role in cellular movement, such as in the invasion and metastasis of malignant cells. However, its participation in germ cell movement is less clear. In the testis, the extracellular matrix at the basal lamina surrounding seminiferous tubules constitutes the basement membrane, which ultimately associates with Sertoli cells and developing germ cells (Fig. 1). It is largely composed of laminin, type IV collagen, heparan sulfate proteoglycan, and entactin (for review, see Ref.21). Sertoli cells are known to secrete components of the extracellular matrix, such as collagen (269, 270, 271, 272) and laminin (270, 272), that contribute to the structural integrity of the seminiferous epithelium, whereas peritubular myoid cells produce fibronectin (269). Indeed, a previous study has shown that laminin is crucial in maintaining the integrity of the Sertoli cell tight junction and blood-testis barriers (161). Recently, collagen was also shown to play an important role in regulating the Sertoli cell tight junction barrier in vitro, possibly via an interaction with TNF-, MMP-9, and TIMP-1 (163). Indeed, it has been shown that TNF- can stimulate Sertoli cell collagen 3(IV) production by as much as 12-fold vs. controls without TNF- (163) and that germ cells and testicular macrophages are the major source of TNF- in the testis (273). Collectively, these data illustrate that Sertoli, germ, and peritubular myoid cells contribute to the making and maintenance of the extracellular matrix, which in turn affects junction dynamics.
8. Provision of nutrients.
Sertoli cell products, many of which are important for germ cell survival, need to be synthesized and efficiently delivered in sufficient quantities to spermatocytes and spermatids residing behind the blood-testis barrier. These products include amino acids, carbohydrates, lipids, vitamins, and metal ions. This transfer of nutritive products from Sertoli to germ cells is largely made possible because an intricate relationship exists between these two cell types (for review, see Ref.62). This is exemplified best when one isolates and then attempts to culture germ cells for an extended period of time because these cells are incapable of survival past 24 h (for review, see Ref.274). The finding that stem cell factor, a putative Sertoli cell product, can prevent germ cell apoptosis in vitro (275), suggests that germ cell survival is largely dependent on Sertoli cells. Indeed, earlier studies have shown that germ cells can survive up to approximately 8 d in vitro when cocultured with Sertoli cells (276). Other factors that can enhance the viability of germ cells both in vivo and in vitro include hormones [e.g., testosterone (277), dihydrotestosterone, and estradiol (278)] and cytokines [e.g., TNF- (279)].
a. Sertoli cell metabolism.
The metabolism of Sertoli cells continues to be poorly understood because many of the investigations that were conducted thus far are in vitro studies, and they may not reflect the actual in vivo condition (for reviews, see Refs.62 , 227 , and 228). For instance, the hormonal environment of Sertoli cells cultured in vitro is drastically different from that of in vivo because these cells are cultured in serum-free, chemically defined media. If serum is added into Sertoli cell cultures, results obtained from these experiments become misleading because the presence of the blood-testis barrier in vivo restricts access of serum proteins into the adluminal compartment. As such, the interpretation of these results in the context of the testis in vivo is often difficult, if not impossible. Nevertheless, morphological studies of Sertoli cells have shown that these cells have a large surface area, which remains in close contact with developing germ cells in all stages of the epithelial cycle. This is of great importance to the biology of the testis when one considers that a single Sertoli cell can sustain approximately 50 developing germ cells (71, 73). As such, the net output of energy is enormous, which is important not only to spermatogenesis, but also to germ cell development and movement (for review, see Ref.133). This is best exemplified in the observation that Sertoli cells can efficiently metabolize glucose into lactate (the preferred energy source of germ cells) and pyruvate (280, 281), which is likely channeled via gap junctions to germ cells to maintain their survival.
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III. Sertoli-Sertoli and Sertoli-Germ Cell Interactions |
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TopAbstractI. IntroductionII. Sertoli CellsIII. Sertoli-Sertoli and Sertoli...IV. Mechanism of Germ...V. Concluding RemarksReferences |
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. In this section, these junctions and their constituent proteins as they relate to junction dynamics and germ cell movement are discussed in greater detail.
A. Cell junctions present in the testis
1. Occluding junctions.
Tight junctions are the only known example of occluding junctions (for reviews, see Refs.282, 283, 284, 285) (Figs. 1 and 6 and Table 1). In the testis, the tight junction is an area of close contact between plasma membranes of neighboring cells composed of fibrils, usually 50–100 in number, that completely encircle the basal domain of each cell. This divides the Sertoli cell, forming a basal and an adluminal compartment. Although the biochemical composition of these strands is not entirely known, it is believed that each of these fibrils represents a row of tightly arranged transmembrane proteins, such as occludin and claudin, that seal the paracellular space (for reviews, see Refs.282, 283, 284, 285). Unlike tight junctions found in other epithelia, in the testis these junctions have a unique location in that they are closest to the basement membrane, which is a modified form of the extracellular matrix (for reviews, see Refs.13 , 14 , and 16). In addition, the blood-testis barrier as constituted by Sertoli cell tight junctions is not assembled until 15–18 d of age in the rat testis, at which time a specialized microenvironment is created. This microenvironment is essential for germ cell development and movement.
a. Functions of occluding junctions.
Tight junctions (for reviews, see Refs.13 and 14) have two main functions; they 1) create a barrier, and 2) form a boundary. First, tight junctions create a semipermeable barrier, which restricts the passage of molecules. This is known as the barrier function of tight junctions. However, the passage of molecules depends largely on their molecular weight and chemical nature (for reviews, see Refs.144 and 286, 287, 288). In fact, tight junction permeabilities are known to vary among tissues so as to adjust to the physiological needs and functions of different organs (for reviews, see Refs.118 , 184 , and 289, 290, 291). Studies have also shown that tight junction permeability can be regulated by different factors and physiological conditions. For instance, sodium orthovanadate, a protein tyrosine phosphatase inhibitor, was able to perturb tight junction permeability in MDCK cells when the diffusion of inulin across the epithelium was examined (292), suggesting that an increase in protein tyrosine phosphorylation can perturb tight junction barrier function. These results were corroborated and expanded in a recent report from our laboratory which illustrated that inhibitors of protein tyrosine and serine/threonine phosphatases could perturb the assembly and maintenance of the Ser
