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.

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.

It appears that some controversy exists on the exact cell type in the mammalian testis that produces IL-1; however, the procedures used and the age of the animals may account for some of these differences. Early studies in the rat identified an IL-1-like factor in the testis that first appears during puberty, is stage dependent, and is correlated with spermatogonial DNA synthesis 58. Results of autoradiographic studies on membrane preparations from the mouse testis revealed that IL-1α displayed significantly higher binding then IL-1β and the highest binding density was observed in the interstitial areas of the testis 59. By employing RT-PCR and in situ hybridization techniques Jonsson et al 60 have demonstrated that the IL-1-like protein is IL-1α and not IL-1β or IL-6. Extracts of seminiferous tubule segments also demonstrated the presence of bioactive IL-1 but not IL-6. Results of the in situ hybridization studies revealed the IL-1α mRNA was localized to Sertoli cells in animals greater than 20 days of age. In younger rats no IL-1α transcripts were detected in the testis. The IL-1α mRNA was present in Sertoli cells at all stages of the seminiferous epithelium except stage VII which was negative 60. Interestingly the presence of IL-1α in Sertoli cells is dependent upon the presence of germ cells. In animals depleted of germ cells either by exposure to radiation prenatally or by treating adults with busulfan no IL-1α mRNA was detected in Sertoli cells 60. Studies by Cudicini et al 61 have demonstrated the presence of IL-1α in human Sertoli cells but also demonstrated the presence of IL-1α and IL-1β in isolated Leydig cells. Rozwadowska et al 62 also demonstrated IL-1α mRNA expression in seminiferous tubules and IL-1β mRNA expression in the interstitium of human testis. Further, Hales et al 63 demonstrated the presence of IL-1 mRNA in testicular macrophages. In contrast, Haugen et al 64 reported the presence of IL-1α protein and mRNA in isolated postmeiotic germ cells.

To further complicate matters regarding IL-1 in the testis, Gustafsson et al 65 reported that three isoforms of IL-1α are produced in the testis that may be posttranslational modifications of the IL-1α precursor and their differential effects are unknown. Also the IL-1 receptor antagonist, IL-1Ra, has been reported to be constitutively produced by isolated mouse Sertoli cells, but since the molecule was found intracellularly and no IL-1Ra bioactivity could be detected in the conditioned media 66 an inhibitory role may be unlikely.

Both IL-1Rs have been reported in the testes of mouse, rat, and human. IL-1RI and IL-1RII mRNA were observed in Sertoli, Leydig, and testicular macrophages from all three species whereas, only the rodent germ cells expressed IL-1RI and IL-1RII mRNA 67.

IL-1α has been reported to be a potent growth factor for immature Sertoli cells. Recent studies by Petersen et al 68 have not only identified the IL-1RI in cultures of Sertoli cells from 8 to 9 day rats but also demonstrate that IL-1α enhances Sertoli cell proliferation. Treatment of Sertoli cells with a combination of IL-1α and FSH showed a synergistic affect on proliferation 68. These results suggest that IL-1α may serve as a growth factor for Sertoli cells (Table 2).

The intimate association of Leydig cells and interstitial macrophages has prompted several studies investigating possible immuno-endocrine interactions. Numerous early studies have reported that IL-1 inhibits basal and LH/hCG-stimulated testosterone production by Leydig cells 697071. However, other studies have shown that IL-1β can stimulate testosterone levels in Leydig cells 7273. The apparent discrepancies in the results may come from differences in the experimental conditions or methodologies. In cultures of isolated rat Leydig cells from animals 10 to 20 days old IL-1β caused a dose-dependent increase in the incorporation of ³H-thymidine into DNA 74. IL-1α also had an affect on ³H-thymidine incorporation; however, it was much less potent than IL-1β. The effect of IL-1β was not observed in Leydig cell isolated from older animals suggesting that IL-1β may play a role in the proliferation of Leydig cells during prepubertal development 74.

Mice that lack a functional IL-1 signaling receptor (IL-1RI) exhibit normal fertility, have normal serum testosterone, and normal testicular levels of steroidogenic enzymes suggesting that IL-1 receptor signaling is not required in Leydig cell steroidogenesis 75. However, in studies where intratesticular macrophages have been depleted either via the injection of silica or by genetic mutation the development of normally functioning Leydig cells is impaired 76. These observations lead Hales 69 to suggest that during testicular development and under non-inflammatory conditions interstitial macrophages provide the appropriate microenvironment to support Leydig cells. Table 2 summarizes the key functional roles of IL-1α and IL-1β in the mammalian testis.

Recent studies from our laboratory have reported an increase in the proinflammatory cytokines TNFα and IL-1β after ischemia/reperfusion (IR) of the testis and suggest a role for the proinflammatory cytokines as early mediators of IR-injury in the testis 22. In the human, ischemia of the testis can result during a medical condition known as testicular torsion in which there is twisting of the spermatic cord. It is a medical emergency and surgical intervention is usually required for reperfusion of the testis; however, even after reperfusion testicular atrophy is common. Early studies from our laboratory have shown that IR of the rat testis results in a permanent loss of spermatogenesis despite the return of blood flow 77 and the continued presence of functional Leydig 78 and Sertoli cells 79. The IR-induced loss of spermatogenesis has been shown to be the result of germ cell-specific apoptosis 202180. Occurring contemporaneously with the IR-induced germ cell-specific apoptosis is an increase in neutrophils 2021 and reactive oxygen species 19 in the testis. Germ cell-specific apoptosis after IR of the testis is dependent upon the recruitment of neutrophils to the testis 5, and we have recently reported that an increase in proinflammatory cytokines after IR of the testis is correlated with activation of signaling pathways leading to neutrophil recruitment 22.

As early as 0.5 hr after IR of the murine testis an increase in the expression of both TNFα and IL-1β is observed 22 and this precedes the activation of JNK. JNK is a stress-related kinase that plays a critical role in the cellular response to many types of cellular stress. Two transcription factors downstream of JNK, ATF-2 and c-jun, are also activated, and immunolocalization of activated, phosphorylated, JNK (phospho-JNK) demonstrated immunoreactivity in intratesticular blood vessels 22. Corresponding with this was an increase in E-selectin mRNA and protein 4 hr after IR of the testis 22. Indeed, the E-selectin promoter is known to contain a positive regulatory domain II site which has been shown to bind a heterodimer of ATF-2 and c-jun 81. E-selectin is an endothelial cell adhesion molecule responsible for the tethering and slow rolling of neutrophils to endothelial cells 82. These results suggest that an increase in TNFα and/or IL-1β after IR of the testis stimulates the activation JNK signaling pathway leading to the expression of E-selectin in endothelial cells and ultimately neutrophil recruitment (Figure 1). In order to determine if TNFα, IL-1β, or both proinflammatory cytokines were eliciting this response, the cytokines were injected either alone or in combination to the testis and phosphorylation of JNK and neutrophil recruitment was examined. Results from these experiments revealed that injection of IL-1β mimicked the effects of IR on the testis, causing an activation of JNK and a recruitment of neutrophils to the testis 22. Thus, an increase in IL-1β after IR of the testis stimulates an intracellular signaling cascade in testicular endothelial cells resulting in neutrophil recruitment and eventually germ cell death. The cell type responsible for the increase in IL-1β production after IR of the testis is currently unknown. Even though TNFα did not have a role in neutrophil recruitment or JNK activation in the testis, preliminary experiments in our lab suggest that the increase in TNFα may lead to the activation of NFκB in Sertoli cells (Lysiak and Turner, unpublished) and may influence germ cell apoptosis by increasing FasL expression in Sertoli cells. Identification of the mechanisms involved in germ cell-specific apoptosis after IR may lead to the development of therapies aimed at interrupting specific pathways in order to prevent the IR-induced loss of spermatogenesis.

Autoimmune orchitis can occur spontaneously in dogs 83 and mink 84 and after vasectomy in rabbits 85 and guinea pigs 86. Mice that have been thymectomized within 4 days of birth also develop a similar autoimmune orichitis 87. In humans several idiopathic diseases of the testis have been described that are similar to autoimmune orchitis and are associated with male infertility 23. Animals injected with homologous testis antigen also develop autoimmune orchitis 88. This animal model system is known as experimental autoimmune orchitis (EAO) and is widely used to study this disease.

A role for proinflammatory cytokines in EAO has been described by Yule and Tung 24, who demonstrated that injection of a TNF neutralizing antibody abrogated the effects of EAO. By generating T-lymphocyte clones from mice with EAO and injecting them into normal mice Yule and Tung 24 demonstrated the T-lymphocyte clones induced the testicular autoimmune disease. The T-lymphocytes responsible for the transfer of EAO were found to be of the CD4⁺ subset and have a cytokine secretion profile of TNFα, IL-2, and IFNγ. To determine if TNFα had a role in the development of EAO orchitogenic T-lymphocytes were injected into recipient mice along with an anti-TNFα neutralizing antibody. Results from these experiments demonstrated that when TNFα was neutralized in vivo, orchitis elicited by an orchitogenic T-lymphocyte clone was significantly attenuated suggesting an important role for TNFα in EAO 24.

Recent studies by Suescun et al 25 provide further evidence for the involvement of TNFα in EAO. Rats with EAO exhibited a significant increase in the number of TNFα-positive testicular macrophages. Testicular macrophages isolated from animals with EAO and cultured secreted increased amounts of TNFα in the media compared to macrophage isolated from control animals 25. An increase in the number of TNFR1 positive germ cells during EAO was also noted suggesting a possible role for TNFα in germ cell apoptosis during EAO. Suescun et al 25 postulate that TNFα is probably involved in the progression of EAO because of its ability to increase endothelial cell permeability, to facilitate monocyte extravasation in the testis, and to activate T cells and macrophages through both autocrine and paracrine mechanisms. In biopsies of human testicular pathologies involving inflammation an increase in the number of macrophages and in TNFα expression by testicular macrophages has also been observed adding further evidence for the involvement of TNFα in testicular inflammation 89.