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BOR - Papers in Press, published online ahead of print October 1, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.021329
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BIOLOGY OF REPRODUCTION 70, 267–276 (2004)
DOI: 10.1095/biolreprod.103.021329
© 2004 by the Society for the Study of Reproduction, Inc.

Minireview

Nitric Oxide/Nitric Oxide Synthase, Spermatogenesis, and Tight Junction Dynamics1

Nikki P.Y. Lee, and C. Yan Cheng2

Population Council, 1230 York Avenue, New York, New York 10021


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 CONSTITUENT PROTEINS OF THE...
 CURRENT STATUS OF RESEARCH...
 DIVERGENT EFFECTS OF NO/NOS...
 CONCLUDING REMARKS AND FUTURE...
 REFERENCES
 
During spermatogenesis, preleptotene and leptotene spermatocytes, residing in the basal compartment of the seminiferous epithelium, must traverse the blood-testis barrier (BTB) to gain entry to the adluminal compartment for further development at late stage VIII and early stage IX of the epithelial cycle. As such, the timely opening and closing of the BTB is crucial to spermatogenesis. A compromise in this process can lead to infertility. Moreover, the BTB is unique in its relative localization in the seminiferous epithelium compared to the tight junctions (TJs) found in other epithelia. Sertoli cell TJs are situated near the basal lamina in the testis, closest to the basement membrane (a modified form of extracellular matrix [ECM]), unlike TJs found in other epithelia, which are found nearest the apical portion of an epithelium, farthest away from ECM. Needless to say, BTB function in the testis is maintained by intricate regulatory mechanisms. In addition to hormones and cytokines, nitric oxide (NO) was recently shown to be a putative TJ regulator in the testis. Perhaps equally important, TJ dynamics in the testis were shown to be regulated, at least in part, by occludin, a TJ-integral membrane protein, via the NO/soluble guanylate cyclase/cGMP/protein kinase G signaling pathway. This minireview summarizes recent advances in the field regarding the role of NO in testicular function, with special emphasis regarding its role in TJ dynamics and the likely implications of these studies for male contraceptive development.

cyclic adenosine monophosphate, cyclic guanosine monophosphate, nitric oxide, signal transduction, spermatogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
CONSTITUENT PROTEINS OF THE...
 CURRENT STATUS OF RESEARCH...
 DIVERGENT EFFECTS OF NO/NOS...
 CONCLUDING REMARKS AND FUTURE...
 REFERENCES
 
In the mammalian testis, the blood-testis barrier (BTB) is located near the basal lamina, which effectively divides the epithelium into basal and adluminal compartments (Fig. 1). During late stage VIII through early stage IX of the epithelial cycle, preleptotene and leptotene spermatocytes residing in the basal compartment adjacent to the basement membrane must traverse the BTB to gain entry to the adluminal compartment [1] for further development while differentiating into haploid spermatids. Without this timely movement of developing preleptotene and leptotene spermatocytes across the BTB, spermatogenesis is halted, leading to infertility. However, the mechanism(s) that governs BTB dynamics has remained largely obscure until recently.



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  		FIG. 1. A schematic drawing that illustrates the current molecular architecture of the three TJ-integral membrane proteins and their associated peripheral proteins at the site of the BTB as well as the three possible signaling pathways that regulate the opening and closing of the Sertoli cell tight junction. Two other TJ-integral membrane proteins, namely CAR (coxsackie virus and adenovirus receptor) and CRBI (Crumbs homolog 1), found in other epithelia are not shown here since their presence in the testis remains to be identified (for a review see [9]). Sertoli cell TJs that constitute the BTB physically divide the seminiferous epithelium into adluminal and basal compartments (for reviews, see [2, 4]). Underneath the basal compartment is the tunica propria, which is composed of a noncellular zone and a cellular zone. The noncellular zone is constituted by the basement membrane (in the testis, the basement membrane is a modified form of extracellular matrix composed largely of type IV collagen, laminin, heparan sulfate proteoglycan, and entactin; for a review, see [81]) adjacent to the seminiferous epithelium; behind this is a layer of type I collagen fibrils. The cellular zone is constituted by a layer of myoid cells; behind this myoid cell layer lies the lymphatic endothelium (for reviews, see [3, 81]). Three classes of TJ-integral membrane proteins, namely occludins, claudins, and JAMs, are found in the testis (for a review, see [2]). These proteins in turn interact with adaptors, such as ZO-1, ZO-2, afadin, cingulin, and membrane-associated guanylate kinase with inverted orientation (MAGI), tethering actin filament to the TJ-integral membrane proteins and recruiting proteins to the BTB site (for a review, see [2]). Other regulatory proteins, such as NOS, p38 mitogen-activated protein kinase (MAPK), JNK, ILK, GSK, and p130cas, have recently been identified at the site of TJs in the testis that regulate the opening and closing of the Sertoli cell TJ barrier based on in vitro and/or in vivo studies [5–7 16]. The three signaling pathways that are known to regulate Sertoli cell TJ dynamics are as follows: First, NO stimulates sGC to synthesize cGMP, leading to TJ disruption [16]. The cGMP can also activate PKG, which in turn can affect TJ dynamics via its effects on occludin [16], reducing the level of occludin at the site of Sertoli cell TJ, thereby opening up the TJ barrier (see pathway I). Second, TNF{alpha} activates the ILK/GSK/p130cas/JNK signaling pathway, which in turn affects the level of occludin [7] (see pathway II). Moreover, TNF{alpha} can also regulate TJ dynamics via its effects on the production of tissue inhibitor of metalloproteases (TIMP)-1, collagen, and matrix metalloprotease (MMP)-9, which in turn affects homeostasis of the basement membrane and the Sertoli cell TJ integrity [7]. Third, TGFß3 activates MAP/ERK (extracellular signal-regulated kinase) kinase kinase 2 (MEKK2) and p38 MAPK to perturb Sertoli cell TJs via its effects on the level of occludin and ZO-1 at the site of the BTB [5, 6] (see pathway III). (This figure was prepared based on [2, 5–7, 16])

The BTB creates a unique microenvironment for germ cell development; it immunologically segregates most of the germ cell antigens, except those residing on spermatogonia and preleptotene/leptotene spermatocytes, from the systemic circulation; and maintains cell polarity (for reviews, see [2–4]). The significance of the BTB to spermatogenesis has been known for decades, but how the barrier function is regulated has remained obscure until recently. Studies have shown that BTB dynamics in vitro and/or in vivo are regulated by cytokines, such as transforming growth factor (TGF) ß3 and tumor necrosis factor (TNF) {alpha}, via two defined signaling pathways, such as the TGFß3/MEKKs (MAP [mitogen-activated protein]/ERK kinase kinases)/p38 MAP kinase [5, 6] and the TNF{alpha}/integrin-linked kinase (ILK)/glycogen synthase kinase (GSK)/p130cas/c-Jun N-terminal kinase (JNK) MAP kinase [7] signaling pathways. Because the role of cytokines in BTB dynamics has recently been reviewed [8, 9], this is not discussed herein. However, emerging evidence has clearly illustrated the pivotal role of nitric oxide/nitric oxide synthase (NO/NOS) in epithelial barrier function in many organs, including the testis, and in spermatogenesis. Therefore, it is our goal to summarize recent advances in this field and to discuss the possible relevance of these findings regarding male contraceptive development.


   CONSTITUENT PROTEINS OF THE BTB
TOP
 ABSTRACT
 INTRODUCTION
 CONSTITUENT PROTEINS OF THE...
CURRENT STATUS OF RESEARCH...
 DIVERGENT EFFECTS OF NO/NOS...
 CONCLUDING REMARKS AND FUTURE...
 REFERENCES
 
Recent studies have shown that tight junctions (TJs) at the site of the BTB are constituted by three classes of TJ-integral membrane proteins, namely occludins, claudins, and junctional adhesion molecules (JAMs) (for reviews, see [2, 10, 11]) (Table 1 and Fig. 1). In turn, these transmembrane proteins structurally associate with different adaptors (Table 1), forming a functional TJ complex at the site of the BTB (for reviews, see [2, 8, 12]) (Fig. 1). For instance, the C-terminus of occludin, claudin, or JAM interacts with zonula occludens (ZO)-1 (for reviews, see [12, 13]), which in turn tethers to the underlying actin filaments either directly or via binding to afadin [14] and cingulin [15]. Equally important, these TJ proteins can structurally interact with other regulatory proteins, such as NOS [16], implicating these TJ constituent proteins in the induction of downstream signaling events, which in turn can regulate the opening and closing of TJs (for reviews, see [17, 18]).


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  		TABLE 1. TJ proteins and their interacting partners in the seminiferous epithelium of the testis.a

Several recent reviews have discussed the structural and functional features of these TJ-integral membrane proteins and their peripheral binding partners as well as recent advances in the field that investigate the regulation of TJ dynamics by cytokines using both in vitro and in vivo models (for reviews, see [2, 8, 9]). As such, details of this subject area are not elaborated herein. In brief, the functionality of these TJ-integral membrane proteins is regulated by and large via protein phosphorylation involving putative protein kinases and phosphatases (for a review, see [2]). For instance, protein kinase (PK) C was shown to phosphorylate JAM [19] and occludin [20] at Ser284 and Ser338, respectively, which in turn can affect the cellular distribution of these proteins at the TJ site, regulating TJ dynamics. However, what triggers these kinases and phosphatases to regulate TJ dynamics remains obscure. Interestingly, recent studies have shown that growth factors, such as TGFß3 [6, 21] and TNF{alpha} [7], as well as other regulators, such as cAMP [22, 23] and NO/NOS [16], play a crucial role in the regulation of TJ dynamics in the testis (Fig. 1), and the downstream regulators of these signaling pathways are indeed putative protein kinases. Other recent studies have shown that NOS is indeed a crucial permeability regulator in multiple epithelia (for a review, see [24]). Perhaps most important of all, a recent report has shown that the use of an inhibitor against NOS, zinc(II) protoporphyrin IX (ZnPP), can also affect Sertoli cell TJ dynamics by exerting its effects on occludin, possibly via the NOS/soluble guanylate cyclase (sGC)/cGMP signaling pathway [16]. Obviously, preleptotene and leptotene spermatocytes that must traverse the BTB likely are the cell types responsible for induction of the signaling function to open the TJ barrier. Nonetheless, the details of this mechanistic event are entirely unknown.


   CURRENT STATUS OF RESEARCH ON NO/NOS IN THE TESTIS
TOP
 ABSTRACT
 INTRODUCTION
 CONSTITUENT PROTEINS OF THE...
 CURRENT STATUS OF RESEARCH...
DIVERGENT EFFECTS OF NO/NOS...
 CONCLUDING REMARKS AND FUTURE...
 REFERENCES
 
Introduction

A homodimer, NOS is composed of two identical monomers (~130–160 kDa each) (for reviews, see [25, 26]). The NOS converts L-arginine to NO and L-citrulline in the presence of cosubstrates and cofactors (for reviews, see [26, 27]) (Fig. 2). Three NOS isoforms, namely neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS), have been found in mammalian cells to date (for a review, see [28]). The physiological roles of NO/NOS are diverse because of the versatile NO-mediated downstream signaling pathways (for reviews, see [29, 30]) (Fig. 3). For instance, NO is implicated as a crucial regulator in inflammation (for a review, see [31]). Equally important, NOS is an important physiological regulator of the endocrine system (for a review, see [32]). The complexity of NO-mediated downstream signaling pathways is further complicated by the diversified biological effects elicited by different concentrations of NO.



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  		FIG. 2. A flowchart that depicts the NO synthesis pathway. The NOS synthesizes NO and L-citrulline, the by-product of the enzymatic reaction, from L-arginine. This enzymatic conversion requires the presence of cosubstrates, namely O2 and nicotinamide adenine dinucleotide phosphate (NADPH), and cofactors, namely tetrahydrobiopterin (BH4), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), heme, Ca2+, and Zn2+. (This figure was prepared based on [25–28])



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  		FIG. 3. A flowchart that depicts the different signaling pathways mediated by NO/NOS that are known or are being implicated in the regulation of Sertoli cell TJ dynamics. The level and activity of NOS can be stimulated by several different regulators, such as growth factors, ions, cytokines, and hormones, triggering the NO-mediated downstream signaling pathways (for reviews, see [29, 33, 34]). A high concentration of NO (>1 µM) leads to DNA deamination, nitrotyrosine formation, and DNA oxidation. On the other hand, a low concentration of NO (<1 µM) can stimulate other cellular downstream signaling pathways. In addition, NO can activate sGC and adenylate cyclase (AC) to synthesize cGMP and cAMP, respectively. The cAMP activates PKA. Likewise, the cGMP has more diverse effects by activating phosphodiesterase (PDE), cyclic nucleotide-gated channel (CNG), and PKG. Both cAMP and cGMP per se are known regulators of Sertoli cell TJ dynamics [16, 22, 23]. The PDE can negatively control the levels of cAMP and cGMP. In addition, NO can stimulate mitogen-activated protein (MAP) kinases, such as p38, c-Jun N-terminal kinase (JNK), and MAP/ERK kinase 1 and 2 (MEK1/2). In the testis, it has been shown that cAMP/PKA and cGMP/PKG are putative regulators of TJ dynamics [16, 22, 23]. DHT, 5{alpha}-Dihydrotestosterone; EGF, epidermal growth factor; LPS, lipopolysaccharides; VEGF, vascular epithelial growth factor; T, testosterone; GFs, growth factors

High-concentration effects When the cellular concentration of NO is higher than 1 µM, the predominant NO-mediated effects include DNA deamination, oxidation, or nitration via interaction of NO with either oxygen or superoxide radicals. Thus, the effects of NO are mostly detrimental at high concentrations (for reviews, see [29, 33]) (Fig. 3).

Low-concentration effects Unlike the indirect effects of NO at high concentration (i.e., >1 µM), the actions of NO are direct without interacting with oxygen or superoxide radicals when its concentration is less than 1 µM (for reviews, see [29, 33]). For instance, NO directly interacts with sGC to induce synthesis of cGMP, which further activates cGMP-regulated phosphodiesterase (PDE), protein kinase G (PKG), and cyclic nucleotide-gated channels (for reviews, see [34, 35]) (Fig. 3). In addition, the MAP kinase signaling pathways can also be induced by NO (for reviews, see [36, 37]) (Fig. 3). Collectively, low concentrations of NO can regulate different physiological activities via different signaling pathways.

Sperm Function

In the testis, NOS has been shown to regulate an array of functions, including sperm motility and maturation, as well as germ cell apoptosis in the testis [38–40]. Remarkably, the first implication of NO in sperm motility stems from localization studies, which demonstrate the presence of all three types of NOS (eNOS, iNOS, and nNOS) in spermatozoa [40–44]. These results seemingly suggest the crucial role of NO/NOS in the normal functioning of spermatozoa. Indeed, abnormal sperm motility was shown to be associated with aberrant eNOS expression patterns [40]. Also, treatment of spermatozoa with NOS inhibitors led to reduced motility [44]. In addition, NO and sGC, the downstream effector of NO, are also involved in sperm capacitation and acrosome reaction (for reviews, see [45, 46]).

Germ Cell Apoptosis and Leydig Cell Function

Interestingly, eNOS [38, 39, 47] and iNOS [48], but not nNOS, are known to be involved in germ cell apoptosis. For instance, eNOS localizes with apoptotic germ cells undergoing DNA fragmentation but not with normal germ cells, suggesting its role in germ cell degeneration [38, 39, 47]. In addition, iNOS is a positive regulator of germ cell apoptosis [48]. Because iNOS-/- mice have significantly heavier testes, resulting from an increase in germ cell number (mostly in pachytene spermatocytes and round spermatids), it has been suggested that iNOS is crucial to germ cell apoptosis by maintaining germ cell number in the epithelium [48]. In addition, iNOS-mediated NO production partly facilitates {alpha}-fodrin proteolysis, promoting germ cell necrosis [49]. Whether iNOS and eNOS use the same apoptotic pathway(s) to regulate germ cell number in the seminiferous epithelium is not known, but clearly, both iNOS and eNOS are positive regulators of germ cell apoptosis.

Based on these findings, NOS homologous mutants are anticipated to be infertile. Surprisingly, however, NOS-/- mice, including nNOS-/- [50], iNOS-/- [51], and eNOS-/- [52], are all fertile, age normally, and die naturally, illustrating that other molecules, such as reactive oxygen species, superoxide dismutase and catalases, can work synergistically to supersede NOS function in regulating spermatogenesis. However, NOS-/- mice display other unusual physiological behaviors and phenotypes. For instance, eNOS-/- mice developed hypertension [52], and iNOS knockout mice had defects in cholesterol homeostasis and atherogenesis [53]. Likewise, nNOS-/- mice had stomach and pyloric stenosis enlargement [50]. Despite these findings, these data fail to negate the significance of NO/NOS in testicular function. Rather, these findings suggest that the function of NO is so crucial to spermatogenesis that a knockout of the NOS gene can lead to an enhanced production of other reactive oxygen species regulators that compensate for the loss in NOS (for a review, see [54]). For instance, recent studies have delineated the roles of NO/NOS in the regulation of Sertoli cell TJ dynamics [16] (see below). Also, NO can inhibit Leydig cell steroidogenesis via its effects on the cholesterol side-chain cleavage enzyme, cytochrome P450scc, without affecting cGMP and cAMP levels [55].

Localization of NOS in the Testis

All three NOS isoforms are found in the testis, displaying distinctive yet overlapping cellular distribution patterns (Table 2). The nNOS, iNOS, and eNOS are found in both Sertoli and germ cells in the seminiferous epithelium [16, 38, 39, 47, 48, 56–59]. They are also found in Leydig cells [38, 39, 47, 57, 59–63] (Table 2) as well as in myoid cells, endothelial cells, myofibroblasts, and spermatozoa [38–41, 47, 56, 62, 64] (Table 2). Remarkably, a testis-specific, truncated form of nNOS (TnNOS) has recently been shown to localize exclusively to Leydig cells but not to Sertoli and germ cells [65, 66], implicating a potential role in steroidogenesis. However, it is not known whether TnNOS-/- mice are infertile. Interestingly, eNOS, but not iNOS and nNOS, was specifically associated with degenerating germ cells [38, 39, 47, 57], implicating a role in germ cell apoptosis. Furthermore, it is not apparent if any of these NOSs are stage-specific proteins in the seminiferous epithelium throughout the epithelial cycles.


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  		TABLE 2. Localization of NOS in the testisa

Regulation of NOS in the Testis

Because NOS is found in all cell types in the testis, this seemingly suggests that NO/NOS is needed for spermatogenesis. Unfortunately, until recently, it has been unknown how NOS is regulated. Hormones and cytokines are the two classes of molecules that are known to regulate NOS in the testis (Table 3). Because of the inducible nature of iNOS, it is not surprising that most of these earlier studies were limited to iNOS (Table 3). For instance, interleukin-1ß has a stimulatory effect on iNOS and NO production by Leydig cells [63]. On the other hand, the level of iNOS in Sertoli cells is induced by TNF{alpha} [16]. Furthermore, the effect of a single hormone or cytokine can be further potentiated in the presence of another cytokine, hormone, or biological factor, such as lipopolysaccharide [67, 68]. The levels of iNOS in Leydig and Sertoli cells can be induced by factors released from round spermatids [59], implicating a regulatory role of germ cells on Sertoli and Leydig cell NOS function. Most of the results shown in Table 3 regarding the effects of cytokines and/or hormones on the expression of different NOSs are consistent between different laboratories. For some, however, the discrepancy likely results from different experimental conditions, such as the age and/or species of animals used for the isolation of Sertoli and/or Leydig cells, different incubation times, and different potencies of the recombinant proteins.


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  		TABLE 3. Effects of cytokines and hormones on NOS expression in rat testicular cellsa

NOS-Interacting Proteins in the Testis

A regulatory molecule largely exerts its effects via protein-protein interactions within a cell, and NOS is no exception. Unlike other proteins, few NOS-interacting partners are known in the testis, yet several reports have identified NOS-interacting partners in other epithelia (for reviews, see [69, 70]). For instance, eNOS was shown to associate with caveolin-1 [71], suggesting its localization to the site of caveolaes in the endothelial cell membrane. Moreover, eNOS had been shown to interact with dynamin-2, a GTP-binding protein [72].

Table 4 summarizes the results of recent studies that identify the binding partners of different NOSs in the testis. Both eNOS and iNOS were shown recently to associate structurally with occludin [16]. Perhaps equally important, eNOS and iNOS were shown to bind actin, {alpha}-tubulin, and vimentin, the three structural cytoskeletal elements in the seminiferous epithelium [16]. These results provide compelling proof that eNOS and iNOS are constituent proteins of TJs and, possibly, cell-cell actin-based adherens junctions (AJs) and desmosome-like junctions that regulate junction-restructuring events in the seminiferous epithelium. On the other hand, eNOS was found to colocalize with sGC and cGMP in the lamina propria of the seminiferous tubules by immunohistochemistry [56]. Also, iNOS and sGC were shown to colocalize to the same site in Sertoli cells in the seminiferous epithelium [56]. Furthermore, nNOS colocalizes with cGMP and sGC in Leydig cells [61].


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  		TABLE 4. Putative interacting partners of NOS in the testisa

Collectively, these results strongly implicate that the NOS/sGC/cGMP is a putative signaling pathway used by the testis to regulate spermatogenesis. More important, using different inhibitors, the NOS/NO/sGC/cGMP/PKG signaling pathway has been identified as being crucial in regulating TJ dynamics (see below) [16]. For instance, ZnPP, an inhibitor of NOS and sGC, can promote the assembly and maintenance of the Sertoli cell TJ barrier, similar to the effect of KT-5823 (C29H25N3O5), a specific inhibitor of PKG [16], suggesting that a lowering of the intracellular cGMP level favors the assembly of the Sertoli cell TJ barrier. Indeed, inclusion of 8-bromo-cGMP at 1 mM can perturb the Sertoli cell TJ-barrier function. Interestingly TNF{alpha}, TGFß2, and TGFß3, known to perturb Sertoli cell TJ-barrier function in vitro [5, 7, 21], can induce Sertoli cell NOS production and/or expression as well [16], suggesting that cytokines can also regulate TJ-barrier function via their effects on NO production.


   DIVERGENT EFFECTS OF NO/NOS IN REGULATION OF PERMEABILITY BARRIER AND JUNCTION DYNAMICS
TOP
 ABSTRACT
 INTRODUCTION
 CONSTITUENT PROTEINS OF THE...
 CURRENT STATUS OF RESEARCH...
 DIVERGENT EFFECTS OF NO/NOS...
CONCLUDING REMARKS AND FUTURE...
 REFERENCES
 
It is increasingly clear that NO is a crucial regulator of TJ dynamics in the BTB of the testis and in the blood-brain and the blood-retina barriers. However, it is apparent that the effects of NO on the permeability barrier are inconsistent between different laboratories (for reviews, see [24, 73]) (Table 5). For instance, 3-morpholino-sydnonimine, an NO donor, was shown to facilitate TJ assembly in rat retinal pigment epithelial cell monolayers [74]. In contrast, no alteration in the basal permeability of the blood-brain barrier was detected in another study using an NOS inhibitor [75]. These intriguing results seemingly suggest that the effects of NO/NOS on the permeability barrier depend largely on the cell type and the system being used for the investigation. Another possibility is the biphasic effects of different concentrations of cAMP and cGMP, the two downstream signaling molecules of NO, on TJ-barrier functions. For instance, cAMP analogs can either facilitate or perturb Sertoli cell TJ at 4–20 or 100–500 µM, respectively [16, 22, 23]. In addition, a recent report has illustrated that the testis uses the NOS/NO/sGC/cGMP/PKG pathway to regulate Sertoli cell TJ dynamics [16]. Nonetheless, these results clearly demonstrate the divergent effects of NO/NOS and its downstream effectors in the regulation of permeability barrier function (Table 5).


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  		TABLE 5. Effects of NO on the permeability barrier function of Sertoli cells and other epithelial cells in vitro and the putative downstream structural targets.a

Table 5 summarizes several recent reports using either inhibitors or stimulators of NOS or scavengers or donors of NO to investigate their effects on the TJ-barrier function of epithelial cells in vitro including the Sertoli cell. Interestingly, it is apparent that these chemical entities exert their effects via changes in the levels and/or distribution of proteins at the site of the TJ of the corresponding cell epithelium, including occludin, ZO-1, and the underlying actin network (Table 5).

At present, the downstream target protein(s) of NO at the site of BTB are not entirely known. However, recent studies have suggested that occludin and actin are the likely putative downstream targets of NO in the testis (Table 5). For instance, the presence of ZnPP, an inhibitor of NOS and sGC, not only could facilitate assembly of the Sertoli cell TJ barrier but also stimulate occludin production by Sertoli cells [16]. In addition, eNOS is a putative regulator of platelet endothelial cell adhesion molecule (PECAM)-1, colocalizing with PECAM-1 to the same cell-cell contact sites in the microvascular endothelial bEnd.3 cell line [76]. Besides, NO has been shown to regulate actin dynamics by modulating the level of cGMP, a downstream signaling molecule of NO/NOS [77, 78], which in turn regulates the homeostasis of the intracellular G-actin:F-actin ratio, affecting actin dynamics by increasing the overall intracellular G-actin level and depolymerizing the actin network. Furthermore, vimentin (an intermediate filament protein) colocalizes with and is a putative substrate of PKG (a downstream molecule of NOS) in neutrophils [79]. Collectively, these results seemingly suggest that several structural proteins at the site of cell junctions, such as occludin, actin, and vimentin, are the putative downstream targets of NO/NOS.


   CONCLUDING REMARKS AND FUTURE PERSPECTIVES
TOP
 ABSTRACT
 INTRODUCTION
 CONSTITUENT PROTEINS OF THE...
 CURRENT STATUS OF RESEARCH...
 DIVERGENT EFFECTS OF NO/NOS...
 CONCLUDING REMARKS AND FUTURE...
REFERENCES
 
The testis is one of the most dynamic organs in the mammalian body and produces millions of germ cells each day in a way that is homologous to the bone marrow, where blood cells, such as erythrocytes, are actively produced. It has been estimated that as many as 150 x 106 spermatozoa are produced each day from a healthy human male [80]. Unlike other epithelia, TJs in the testis are unique in their location and function and must open periodically to permit germ cell movement, illustrating that multiple signaling pathways likely are being used to ensure successful and continual production of spermatozoa. For instance, unlike other epithelia, TJs in the seminiferous epithelium are closest to the basement membrane (a modified form of extracellular matrix [81]) and are present side-by-side with AJs and desmosomes. In all other epithelia and endothelia, TJs are farthest away from extracellular matrix, being located at the apical portion of a cell epithelium. Underneath these TJ structures are AJs, followed by desmosomes (for a review, see [2]); these structures are referred to as junctional complexes.

In this review, we have summarized recent studies in the field investigating how NO/NOS regulates TJ-barrier function. For instance, Sertoli cell TJ dynamics have been shown to be regulated via the NOS/NO/sGC/cGMP/PKG signaling pathway, which in turn affects the level of occludin at the site of TJs. Many of the recent studies in the literature used cells isolated from retina, intestine, kidney, or testis (e.g., Sertoli cells), as reviewed herein; the results of these in vitro studies will be helpful in future studies when they are translated to the in vivo level. Obviously, by identifying the pathways that regulate TJ dynamics in the testis, such as the NOS/NO/sGC/cGMP/PKG pathway, this information will have a significant impact on male contraceptive development. For instance, if the BTB can be temporarily shut down using specific inhibitors, such as ZnPP and KT-5823, the known NOS inhibitor and PKG inhibitor, respectively, that were shown to facilitate the Sertoli cell TJ-barrier function in vitro [16], making the TJ barrier tighter. This will deny access of preleptotene and leptotene spermatocytes across the BTB, and spermatogenesis will be disrupted, resulting in male infertility.

Needless to say, new chemical entities will need to be synthesized to eliminate possible cytotoxicity, which can be assayed and screened rapidly using the in vitro Sertoli cell culture system to monitor their efficacy in regulating Sertoli cell TJ-barrier function before they are used for in vivo studies (for reviews, see [2, 8]). If such effects can be exerted locally, at the site of cell junctions in the seminiferous epithelium, by manipulating the intracellular NO concentration of Sertoli cells, then the hypothalamus-pituitary-testicular axis is not compromised, and side effects, if any, are minimal. Perhaps this can be achieved by conjugating an inhibitor of a crucial signaling molecule of the NOS/NO pathway, such as PKG, to a modified FSH mutant protein having the ability to bind to its receptor without the hormonal activity. As such, its disruptive effect on NO production can be limited to Sertoli cells at the site of the BTB. Obviously, this is a significant area of research that deserves much attention by investigators in male contraceptive development.


   FOOTNOTES
 
1 Supported in part by grants from the CONRAD Program (CICCR CIG-96-05B, CIG-01-72 to C.Y.C.), National Institutes of Health (NICHD, UOI HD45908 to C.Y.C.; U54 HD29990, Project 3 to C.Y.C.; U54 HD13541-20S to C.Y.C.) and the Noopolis Foundation. N.P.Y.L. was supported in part by a Hong Kong University Research Scholarship Award. Back

2 Correspondence: C. Yan Cheng, Population Council, Center for Biomedical Research, 1230 York Avenue, New York, NY 10021. FAX: 212 327 8733; y-cheng{at}popcbr.rockefeller.edu Back

Received: 24 July 2003.

First decision: 11 August 2003.

Accepted: 23 September 2003.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 CONSTITUENT PROTEINS OF THE...
 CURRENT STATUS OF RESEARCH...
 DIVERGENT EFFECTS OF NO/NOS...
 CONCLUDING REMARKS AND FUTURE...
 REFERENCES
 

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