Localization of TNFα and TNFα receptors in the testis
By enriching for specific germ cell populations using unit gravity sedimentation and analyzing for the secretion of bioactive TNFα and for TNFα mRNA De et al [
26] have shown that round spermatids secreted TNFα and both pachytene spermatocytes and round spermatids contained TNFα mRNA, though at two different transcript sizes [
26]. Results from in situ hybridization studies confirmed the presence of TNFα mRNA in the round spermatids and pachytene spermatocytes and detected mRNA in testicular interstitial macrophages as well [
26].
Interstitial testicular macrophages have also been described a possible a source of TNFα. Early studies by Hutson [
27] on collagenased dispersed rat testicular macrophages found TNFα activity in the conditioned media. However, later studies from the same investigators, found that no TNFα was detected in testicular interstitial fluid or in testicular macrophages isolated without collagenase [
28] suggesting that TNFα is not constitutively expressed in the normal testis. Treatment of non-collagenase isolated rat testicular macrophages with lipopolysaccharides (LPS) did induce TNFα secretion indicating that TNFα may be produced by testicular macrophages under certain conditions [
28].
To determine which cell type TNFα may be acting upon De et al [
26] performed Northern blot analysis for TNFR1 in isolated cell populations and found that Sertoli and Leydig cells contained the mRNA for TNFR1. Using cultured porcine Sertoli cells Mauduit and coworkers [
29] demonstrated that Sertoli cells bound radiolabeled TNFα via TNFR1 and that treatment with follicle stimulating hormone (FSH) upregulated TNFR1. TNFR2 or the p75 TNF receptor was not detected [
29].
Taken together results of the above studies suggest that TNFα is normally produced by germ cells, notably round spermatids, and that the receptors are on Sertoli and Leydig cells.
Role of TNFα in the seminiferous epithelium
Numerous functional roles for TNFα in the mammalian have been described by a number of laboratories. In germ cells TNFα has been reported to increase the expression of cytochrome P450 aromatase, an enzyme responsible for the synthesis of estrogen from androgens [
30]. The TNFα-mediated increase in cytochrome P450 aromatase was found to be predominantly in pachytene spermatocytes, while an inhibitory effect was observed in round spermatids [
30].
TNFα also acts in indirect ways to support spermatogenesis. As stated earlier, TNFα can stimulate the activation of the transcription factor NFκB and, indeed in, cultures of rat Sertoli cells TNFα stimulated nuclear NFκB binding [
31]. The intracellular androgen receptor (AR) in Sertoli cells binds and mediates testosterone signaling. Studies by Delfino et al [
32] found that TNFα stimulated binding of NFκB to the AR promoter and increased endogenous AR in primary cultures of Sertoli cells.
TNFα has also been shown to suppress Mullerian inhibiting substance (MIS). MIS is essential for normal sex differentiation and its expression is strictly regulated. Expression of MIS in Sertoli cells is high in the fetal to prepubertal period but then decreases after puberty. Studies by Hong et al [
33] have shown that TNFα is the molecule responsible for suppression of MIS. Treatment of testis organ cultures with TNFα caused a decrease in MIS expression and testis from TNFα knockout mice showed high and prolonged MIS expression. The suppressive ability of TNFα on MIS was found to be due to the activation of NFκB which subsequently associated with and repressed the transcription factor SF-1 [
33].
Transferrin is a major transporter of iron into cells [
34]. In the testis where the blood-testis barrier prevents macromolecules and several electrolytes from entering the adluminal compartment iron is transported into the compartment from the circulatory system by transferrin produced in Sertoli cells [
35]. Early studies showed that transferrin production is regulated by germ cells [
36]. More recently, TNFα was found to increase transferrin mRNA and protein levels in Sertoli cells in vitro [
37]. The effect of TNFα on Sertoli cell transferrin production was also found to be dependent on the stage of the cycle of the seminiferous epithelium with Sertoli cells from stages IX-XI and XIII most clearly responding [
38]. These results together with those indicating that TNFα is secreted by germ cells [
26] suggest that TNFα produced by germ cells acts in a paracrine manner to upregulate transferrin production by Sertoli cells and thus maintain spermatogenesis.
TNFα has also been shown to stimulate lactate dehydrogenase A expression in Sertoli cells [
39]. Postmeiotic germ cells utilize lactate derived from Sertoli cells rather than glucose as an energy substrate [
40]. Lactate dehydrogenase A is a key enzyme involved in lactate production. Treatment of Sertoli cells with TNFα caused a dose-dependent increase in lactate dehydrogenase A mRNA expression [
39]. In other studies it was also found that TNFα increased the activity of lactate dehydrogenase in cultured Sertoli cells [
41]. These results again suggest an important paracrine mode of action for TNFα on maintaining spermatogenesis. Monocarboxylate transporters (MCTs) are responsible for the transportation of lactate across the plasma membrane and recent studies have demonstrated the presence of MCT2 in differentiated murine germ cells and suggest it may play a role in lactate uptake [
42]. Interestingly, TNFα was found to inhibit MCT2 expression. These results led Boussouar et al [
42] to hypothesize that the inhibitory effect of TNFα on MCT2 in germ cells was related to the stimulatory effect of TNFα on Sertoli cell lactate production and would prevent an over accumulation of lactate in the germ cells.
Studies by Pentikainen et al [
43] have found that TNFα inhibited in a concentration dependent manner the germ cell apoptosis observed in segments of human seminiferous epithelium cultured under serum-free conditions. TNFα down-regulated Fas ligand (FasL) production by segments of seminiferous epithelium; thus, Pentikainen et al [
43] suggested that TNFα produced by germ cells works in a paracrine fashion to down-regulate FasL production by Sertoli cells and promote germ cell survival. TNFα has also been reported to increase both membrane-bound and soluble forms of Fas in cultured Sertoli cells [
44]. Sertoli cells treated with low concentrations of TNFα secreted the soluble form of Fas that was capable of inhibiting a basal level of apoptosis. At higher concentrations of TNFα, however, upregulation of membrane bound Fas was predominant and the germ cells were susceptible to FasL-mediated apoptosis. These results suggest that under physiologically low concentrations of TNFα the soluble form of Fas is produced by Sertoli cells and acts as a survival factor for germ cells; whereas, at high concentrations TNFα such as during inflammation TNFα induces membrane bound Fas which primes the Sertoli cells for FasL induced cell death [
44].
Another way in which TNFα exerts a paracrine effect on Sertoli cells is through the stimulation of insulin-like growth factor binding protein-3 (IGFBP-3) [
45]. By increasing IGFBP-3 levels in treated Sertoli cells TNFα was able to antagonize the action of IGF-1 on FSH binding to Sertoli cells [
45]. TNFα was able to increase IL-1α and IL-6 production from cultures of rat Sertoli cells [
46]. These data suggest that TNFα can alter the cytokine secretion profile of Sertoli cells that may affect Sertoli cell functions and ultimately spermatogenesis.
Studies investigating the signal transduction pathways stimulated by TNFα in Sertoli cells have found that treatment of murine Sertoli cells with TNFα resulted in the activation of JNK as well as p38 MAPK [
47]. By employing a specific p38 inhibitor it was demonstrated that activation of the p38 MAPK pathway led to the production of IL-6 by TNFα stimulated Sertoli cells and activation of the JNK pathway increased surface expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) [
47]. Since TNFα plays a role in increasing cell adhesion molecules, De Cesaris et al [
47] suggested that the effect of TNFα on Sertoli cells may be pathogenic in the outbreak of autoimmune disorders in the testis.
TNFα has also been shown to effect Sertoli cell tight junction assembly, collagen α 3(IV) synthesis, and enzymes and their inhibitors that effect the extracellular matrix (ECM) [
48]. By increasing the expression of matrix metalloprotease-9, TNFα has been found to perturb Sertoli cell tight junction formation; however, TNFα also increased the inhibitor of metalloprotease-1 and collagen α 3(IV) production suggesting a feedback mechanism to replenish the collagen network and reform tight junctions [
48]. These results have led Siu et al [
48] to suggest that TNFα influences the dynamics of the Sertoli cell tight junctions through effects on the ECM.
Glutathione S-transferase-α (GSTα) is an enzyme expressed by Sertoli cells involved primarily in detoxification. TNFα was found to decrease basal as well as hormone-stimulated GSTα in cultures Sertoli cells [
49]. Benbrahim-Tallaa et al [
49] suggested that increased levels of TNFα in the testis may alter the detoxification processes against genotoxic products during spermatogenesis.
TNFα in combination with other cytokines and lipopolysaccharides has been shown to stimulate inducible nitric oxide synthase (iNOS) and nitrite in Sertoli cells as well as seminiferous pertitubular cells [
50]. Similarly treating Sertoli cells with a combination of cytokines that included TNFα, IL-1β, and IL-6 caused a more than additive effect on transferrin and cGMP secrection [
51].
Based on the above studies TNFα has numerous roles in normal testicular functions (Table
1). At least one study suggests that the levels of TNFα produced in the testis may be an important factor. Low levels of TNFα may preferentially regulate normal testicular homeostasis; whereas, elevated levels influence or initiate pathological conditions in the testis.
Table 1
Summary of the Effects of TNFα on Sertoli, Leydig, and Peritubular Cells
increase cytochrome | decrease testosterone [ 52‐ 56] | |
| | |
increase NFκB activity [ 31] | | |
| | |
| | |
increase transferrrin [ 37, 38] | | |
| | |
| | |
| | |
| | |
| | |
| | |
increase ICAM and VCAM [ 47] | | |
| | |
increase TIMP-1 and collagen [ 48] | | |
| | |
Role of TNFα in testicular interstitial cells
Since testicular macrophages may be a source of TNFα and since testicular macrophages and Leydig cells are intimately associated through membrane digitations a role for TNFα in Leydig cell steroidogenesis has been investigated. Treatment of isolated murine Leydig cells with TNFα resulted in a significant decrease in basal testosterone secretion as well as cAMP-stimulated testosterone production [
52]. This TNFα inhibitory affect on the cAMP-stimulated testosterone production in Leydig cells was found to be due to a decrease in mRNA and protein levels of two cytochrome P450 enzymes important in testosterone biosynthesis, cholesterol side-chain cleavage enzyme and 17α-hydroxylase/C
17–20 lyase [
52]. Furthermore, Li et al [
53] have demonstrated that the inhibitory affect of TNFα on 17α-hydroxylase/C
17–20 lyase gene expression is mediated via protein kinase C. The inhibitory affect of TNFα on LH/hCG-induced testosterone secretion has also been reported in porcine Leydig cells [
54]. In these studies the mechanism of the inhibitory affect of TNFα was reported to be due to a decrease in steroidogenic acute regulatory protein mRNA and protein levels [
54]. Results from two independent studies have also shown that the TNFα inhibitory affects on Leydig cell testosterone secretion may involve a sphingomyelin/ceramide-dependent pathway [
55,
56]. Thus, results from numerous laboratories employing mouse, rat, or porcine Leydig cells have reported an inhibitory affect of TNFα on testosterone production; however, it remains unclear whether Leydig cells are normally exposed to TNFα under normal testicular homeostasis or only after testicular macrophages are activated.
Besides having an affect on Leydig cell testosterone production TNFα has also been reported to increase plasminogen activator inhibitor-1 (PAI-1) expression in rat testicular peritubular cells indicating that it may be involved in controlling protease activity [
57]. The authors suggest, however, that the biological effects of TNFα on PAI-1 may be secondary to due to epidermal growth factor receptor (EGFR) signaling since TNFα also increased EGFR mRNA and EGF binding in the peritubular cells [
57]. Table
1 summarizes the key roles of TNFα in the mammalian testis.