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Innervation and serotoninergic receptors of the testis interact with local action of interleukin-1beta on steroidogenesis
Autonomic Neuroscience: Basic and Clinical 131 (2007) 21 – 27 www.elsevier.com/locate/autneu
Innervation and serotoninergic receptors of the testis interact with local action of interleukin-1beta on steroidogenesis Ida Gerendai a,⁎, Péter Banczerowski a,b , Valér Csernus c , Béla Halász a a
c
Neuroendocrine Research Laboratory, Hungarian Academy of Sciences and Semmelweis University, Department of Human Morphology and Developmental Biology, H-1094 Budapest, Tűzoltó u. 58., Hungary b National Institute of Neurosurgery, H-1145 Budapest, Amerikai út 51., Hungary Neurohumoral Regulation Research Group, Hungarian Academy of Sciences and Pécs University, Department of Human Anatomy, H-7643 Pécs, Szigeti út 12., Hungary Received 9 March 2006; received in revised form 22 May 2006; accepted 2 June 2006
Abstract Testosterone secretion by Leydig cells is affected by interleukin-1β (IL-1β). The aim of the present study was to investigate whether partial denervation of the testis or local administration of a serotonin (5-HT) receptor antagonist could alter the changes in testicular steoidogenesis induced by IL-1β. Intratesticular administration of IL-1β was combined with vasectomy or local injection of ketanserin (5-HT type 2 receptor antagonist) in immature hemicastrated rats and the effect of the interventions on testicular steroidogenesis was studied. One day after treatment with local injection of IL-1β induced a significant rise in testosterone secretion that could be prevented by vasectomy (that also means transection of the inferior spermatic nerve). In a model in which neither IL-1β nor ketanserin interfered with steroidogenesis, administration of the receptor antagonist just prior to IL-1β treatment significantly reduced testosterone secretion. Data indicate interaction between testicular nerves and IL-1β action and interaction between testicular 5-HT2 receptors and local effect of IL-1β on testosterone secretion. © 2006 Elsevier B.V. All rights reserved. Keywords: IL-1β; Testicular steroidogenesis; Vasectomy; Ketanserin
1. Introduction Cytokines are regulatory proteins involved in haemopoiesis, inflammation and immune responses. Several cytokines including the proinflammatory cytokine family, the interleukin-1 (IL-1) system have been implicated as local regulators of different organs, including the testis (Schlatt et al., 1997; Hedger and Meinhardt, 2003; Svechnikov et al., 2004). The family of IL-1 consists of two agonists, isotypes IL-1α and IL-1β encoded by separate genes, and the naturally occurring IL-1 receptor antagonist (Eisenberg et al., 1991; Dinarello, 1994). Two IL-1 receptors have been characterized: IL-1-type I and IL-1-type II receptors (Sims and Dower, 1994) that bind both IL-1α and IL-1β and the ⁎ Corresponding author. Tel.: +36 1 215 6920x3616; fax: +36 1 215 3064. E-mail address: [email protected] (I. Gerendai). 1566-0702/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2006.06.002
antagonist (Dinarello, 1996). In the rat testis IL-1α is constitutively produced and secreted by Sertoli cells (Syed et al., 1988; Gerard et al., 1991, 1992; Cudicini et al., 1997; Jonsson et al., 1999; Wahab-Wahlgren et al., 2000) and acts as a paracrine regulator in the control of a wide range of testicular functions including differentiation of the organ, steroidogenesis, and spermatogenesis (for review see Hedger and Meinhardt, 2003; Svechnikov et al., 2004). By contrast to IL-1α, IL-1β does not seem to be constitutively synthesized in the intact testis (Jonsson et al., 1999; Sultana et al., 2000; Wahab-Wahlgren et al., 2000), but in response to preinflammatory stimuli. Both in immature and adult testis, Leydig cells and testicular macrophages are able to express mRNA for IL-1β (Wang et al., 1991; Hales et al., 1992; Lin et al., 1993; Xiong and Hales, 1994; Cudicini et al., 1997; Gow et al., 2001) and secrete the protein (Jonsson et al., 2001). IL-1β has been reported to affect Sertoli cell
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functions (Petersen et al., 2002) and steroidogenesis. Testosterone secretion by Leydig cells has been demonstrated to be either inhibited (Lin et al., 1991; Hales, 1992; Khan et al., 1992; Mauduit et al., 1992) or stimulated (Verhoeven et al., 1988;Warren et al., 1990) by IL-1β. Recent in vitro (Svechnikov et al., 2001) and in vivo (Gerendai et al., 2005) studies indicate that the effect of IL1β on testicular steroidogenesis is age-dependent. In addition, the results of the in vivo experiments indicate that in immature rats the alterations in testosterone secretion following testicular administration of IL-1β depend also on the time elapsed between treatment and sacrifice and on the fact whether animals were hemicastrated or had two testes in situ (Gerendai et al., 2005). Besides the great number of biologically active substances produced by different cell types of the testis and exerting local modulatory actions, nerves supplying the organ are also known to be involved in the fine-tuning control of testicular functions (Setchell and Brooks, 1988; Gerendai and Halász, 1997). The nerves to and from the testis include the superior testicular nerve that runs along the testicular artery and the inferior testicular nerve which accompanies the ductus deferens (Kuntz and Morris, 1946). Catecholamines, noradrenaline and adrenaline released from sympathetic nerve endings activate catecholaminergic receptors located on Sertoli cells, Leydig cells and myoid cells of the testis and steroid production (Heindel et al., 1981; Moger et al., 1982; Anakwe and Moger, 1986; Poyet and Labrie, 1987; Mayerhofer et al., 1992, 1993; Setchell et al., 1994; Mayerhofer, 1996). Our previous studies indicated that the surgical or neurochemical partial denervation of the testis can alter local modulatory action of opioid peptides (Gerendai et al., 1989; Gerendai et al., 1992) and oxytocin (Gerendai et al., 1996) on steroidogenesis. Furthermore, Frungieri et al. (2002) have reported interaction between catecholamine actions on testosterone secretion and testicular serotonin type 2 (5-HT2) receptors. The aim of the present study was to investigate whether partial denervation of the testis or local administration of the serotonin receptor (5-HT2) antagonist could alter the changes in testicular steroidogenesis induced by IL-1β. Local treatment of the testis with the cytokine was combined with unilateral partial denervation of the organ by means of vasectomy. Our previous data have indicated that different types of denervation of the testis (vasectomy, pharmacological destruction of serotoninergic neural elements) could alter local peptide effects in different ways (Gerendai et al., 1996). In order to avoid that an overall effect due to total denervation could obscure the effect of one of the components of the innervation, partial denervation of the organ was performed. Vasectomy (interruption of the inferior spermatic nerve) is a useful method of partial testicular denervation, especially in immature animals, where no excretion is present towards the ductus deferens. Furthermore, recent morphological data show that the majority of efferent innervation of the testis originates in the pelvic
ganglia and sympathetic chain and reaches the organ with the deferential artery (Rauchenwald et al., 1995). The functional significance of the inferior spermatic nerve is indicated by studies which demonstrate the major contribution of the inferior spermatic nerve in the hemicastration-induced testosterone response (Frankel and Mock, 1982; Frankel and Chapman, 1984; Frankel and Wright, 1982; Zhu et al., 1995) and in control of steroidogenesis mediated by acetylcholinergic fibers (Zhu et al., 2002). In another experiment we combined cytokine treatment with injection of ketanserin (a highly selective 5-HT2 antagonist) to reveal a possible interaction between the effect of interleukin and serotonin. Intratesticular injection of different substances is a widely used method and the only approach to study effects of locally produced substances in vivo. Bergh and Söder (1990) published a detailed study concerning the effect of intratesticular injection of different substances (IL-1α, IL1β, histamine, serotonin, bradykinin) on vascular permeability and testicular histology. Our experiments were performed in immature hemicastrated rats as such animals were used in the majority of previous studies demonstrating the effect of local treatment of the testis with IL-1β (Gerendai et al., 2005) and the interactions between locally produced regulatory peptides and testicular nerves. 2. Materials and methods 2.1. Animals Pregnant CFY (originally Sprague-Dawley) rats were housed in a light- (lights on for 12 h) and temperature (22 ± 2 °C)-controlled room with free access to rat chow and tap water. After delivery, ten newborn male rats were placed with each lactating female. 2.2. Experiments, treatments Experiments were carried out in immature (21-day-old) animals. In each experiment IL-1β or physiological saline was injected into the right testis and immediately after treatment the other gonad was removed. Animals were subjected to two different types of interventions just prior to the injection of the cytokine and hemicastration. In experiment I unilateral vasectomy, in experiment II testicular administration of ketanserine were performed. The interventions were made under ether anaesthesia. Animals were sacrificed 1 day or 6 days after the interventions. The experimental procedures were approved by the Local Animal Care and Use Committee in accordance with NIH Guidelines for the Care and Use of Laboratory Animals. 2.3. Experiment I (vasectomy, intratesticular injection of IL1β, and hemicastration) Twenty-one-day old rats were divided into four experimental groups: (1) sham controls: intratesticular injection of
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2 μl physiological saline into the right testis and removal of the left testis; (2) local injection with 0.1 μg/2 μl/testis of IL1β on the right side and hemicastration; (3) right-sided vasectomy (cutting the ductus deferens and accompanying vessels and nerves between two ligations), right-sided intratesticular injection of 2 μl physiological saline, and hemicastration, and (4) first right-sided vasectomy, then local treatment of the right testis with 0.1 μg/2 μl/testis of IL1β, and hemicastration. Animals were sacrificed 1 day after surgery and treatment.
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bound and free steroids were separated by dextran-coated charcoal. The radioactivity was measured in a two phase liquid scintillation system. The sensitivity limit of the assay is 3 fmol/tube. The inter- and intraassay coefficients for variation were 9.80 and 5.98%, respectively. Due to the high cross reaction of the testosterone antiserum with DHT and the fact that in prepubertal testis the primary androgen produced by Leydig cells is not testosterone but androstendione, DHT, and 5-androstenediol (Wiebe, 1976), the steroid measured is a mixture of testosterone and 5-DHT, but for the sake of simplicity, it is referred as testosterone.
2.4. Experiment II (local treatment of the right testis with ketanserin and/or IL-1β, and hemicastration) In 21-day-old animals 5 μg/5 μl ketanserin or the same volume of physiological saline was injected into the right testis followed by treatment of the same gland with 0.1 μg/ 2 μl IL-1β or 2 μl of physiological saline. Immediately after treatment the left testis was removed. Animals were sacrificed 6 days later. The doses of the cytokine (Gerendai et al., 2005), ketanserin (Csaba et al., 1998) as well as the survival time applied were chosen on the basis of previous results or of preliminary studies (Gerendai et al., 1992, 1996). 2.5. Testicular incubation When animals were killed, testes were immediately removed, decapsulated and incubated in medium 199 containing 25 mM Hepes for 3 h, in a metabolic shaker at 35 °C. The volume of medium was 1 ml. After the incubation media were transferred to tubes and stored at − 20 °C until assayed for testosterone. The results are expressed as testosterone produced per testis (nmol/l/testis) during the incubation period. 2.6. Assay Testosterone concentrations of serum or tissue culture medium (Medium) were determined by RIA as previously described in details (Csernus, 1982). Briefly, 20 μl serum was extracted with 2 ml ether. The dried extract was dissolved in 500 μl assay buffer (ASB, 0.5 M PBS with 1 g/l gelatin, pH = 7.4). For standard, Calbiochem Testosterone for HPLC standard was used in a 9-step series ranging from 3.5 to 1000 fmol/tube. From 20 μl Medium samples, direct determination was made. In this case, to each standard tube 20 μl unused Medium was added. The RIA tubes contained the samples (in duplicates) or standards, 7 nl/tube antibody (CV-RT 17, 1:100 000 final dilution) and 12 000 cpm 3Hlabelled testosterone (100 fmol, The Radiochemical Center, Amersham) in a total volume of 0.7 ml ASB. The cross reactions of the antibody: 5α-DHT 45%, 5β-DHT 9%, and androstendione 2%. With other 27 natural and synthetic steroids examined, the antibody showed less than 0.05% cross reaction. After an overnight incubation at 4 °C the
Fig. 1. Effect of unilateral intratesticular injection of interleukin-1beta (IL1beta) and/or hemivasectomy on serum testosterone concentration, basal testosterone secretion in vitro and testicular weight in hemicastrated rats. Treatment, vasectomy and hemicastration were performed in 21-day-old rats, which were killed 1 day after treatment and/or surgery. Values are mean ± SEM. Figures below the horizontal axis indicate the number of animals. phys. s.: physiological saline; T: testosterone; UORCHX: unilateral orchidectomy; vas: vasectomy.
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LH and FSH levels from rat sera were determined by RIA utilizing National Hormone and Pituitary Program kits. For reference, rLH-RP-3 and rFSH-RP-2 preparations were used. The inter- and intraassay coefficients of variation were 7–9 and 4–6%, respectively. 2.7. Statistical analysis Results were analyzed by ANOVA followed by Student– Newman–Keuls multiple comparison methods. When only two means were compared, Student's t test was performed. Results were considered significant if p < 0.05.
3. Results 3.1. Experiment I In accordance with our previous data (Gerendai et al., 2005), in 21-day-old animals one day after unilateral intratesticular injection of IL-1β (0.1 μg/testis) and removal of the contralateral gonad, a significant rise both in basal testosterone secretion in vitro and serum testosterone concentration could be observed (p < 0.05). Unilateral vasectomy, by itself, did not interfere with the parameters studied. When vasectomy was performed just prior to local administration of the cytokine, IL-1β failed to enhance significantly steroidogenesis and serum testosterone concentration (Fig. 1, middle and upper panel). The weight of the remaining testis of different experimental groups did not differ significantly (Fig. 1, lower panel). 3.2. Experiment II In 21-day-old hemicastrated rats, injection of 0.1 μg/testis of IL-1β into the right testis did not alter the parameters studied 6 days after treatment. This is in accordance with our previous observations (Gerendai et al., 2005). Similarly, local treatment with ketanserin was also without effect. By contrast, administration of ketanserin just prior to IL-1β injection, resulted in a significant decrease both in steroidogenesis (p < 0.01) and serum testosterone level (p < 0.05) as compared to any other groups (Fig. 2, middle and upper panel). Administration of IL-1β did not interfere with testicular weight but ketanserin combined either with the cytokine or the vehicle resulted in a slight but significant (p < 0.05) decrease in the weight of the remaining testis as compared to physiological saline-treated control (Fig. 2, lower panel). No change in serum LH and FSH concentration could be observed in any experiment. 4. Discussion
Fig. 2. Effect of unilateral intratesticular injection of ketanserin (ketanser) and/or interleukin-1beta (IL-1beta) on serum testosterone concentration, basal testosterone secretion in vitro and testicular weight in hemicastrated rats. Treatments and hemicastration were performed in 21-day-old rats. Animals were sacrificed 6 days posttreatment. Values are mean ± SEM. Figures below the horizontal axis indicate the number of animals. phys. s.: physiological saline; T: testosterone; UORCHX: unilateral orchidectomy.
The results of the present study demonstrate for the first time (i) interaction between testicular innervation and IL-1βinduced changes in steroidogenesis, (ii) interaction between testicular 5-HT2 receptors and local effect of IL-1β on testosterone secretion. It has previously been reported that in immature rats vasectomy suppresses basal testosterone secretion in vitro (Gerendai et al., 1986a). The mechanism of action of vasectomy on steroidogenesis is not established. The lack of changes in the secretion of LH, FSH and other pituitary hormones (Rosemberg et al., 1974; Singhal et al., 1977) suggests a pituitary-independent, neurally mediated mechanism. This view is supported by the observations that in hypophysectomized animals hemivasectomy blocks the hemicastration-induced increase in testosterone secretion (Frankel et al., 1984) and that in rats with two testes
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hemivasectomy results in changes of both gonads (Gerendai et al., 1986a). Further studies have indicated that in immature, hemicastrated rats hemivasectomy performed just prior to testicular administration of oxytocin modified the peptide-induced changes in steroidogenesis (Gerendai et al., 1996). Similarly, in immature, vasectomized animals intratesticular treatment of naloxone or enkephalinamide failed to alter steroidogenesis (Gerendai et al., 1992) that is present in rats with intact nerves to and from the testis (Gerendai et al., 1986b, 1990). These findings are in agreement with the results of the present study, which indicate that local testicular action of IL-1β on steroidogenesis is altered by vasectomy, i.e., interruption of the inferior spermatic nerve that is composed of both sympathetic and parasympathetic elements. On the basis of these observations an interaction between testicular innervation and local action of the cytokine can be supposed. Interestingly enough in our second experiment IL-1β or the 5-HT2 receptor blocker ketanserin alone failed to affect steroidogenesis, combined treatment resulted in a significant reduction in steroidogenesis. The mechanism of this action is not known. It can be supposed that under conditions, such as 6 days posttreatment when IL-1β alone is not able to suppress steroidogenesis, the action of ketanserin is needed to obtain inhibitory action of the cytokine. Testicular 5-HT originates from local synthesis of interstitial cells, such as Leydig cells and mast cells (Gaytan et al., 1989; Tinajero et al., 1993) and from nerve endings (Campos et al., 1990) and serotonin receptors of the 5-HT2 subtype are present in rat and hamster Leydig cells (Tinajero et al., 1992; Frungieri et al., 2002). In vitro studies and in vivo experiments in adults indicate that 5-HT acting on 5-HT2 receptors suppresses steroidogenesis (Ellis et al., 1972; Kalla, 1979; Tinajero et al., 1992; Csaba et al., 1998). The action of serotonin on steroidogenesis, however, seems to be age-dependent: in immature rats intratesticular administration of 5-HT stimulated, while ketanserin or 5,7-dihydroxytryptamine (5,7DHT) suppressed it (Csaba et al., 1998). Testicular 5-HT has been reported to stimulate the corticotropin-releasing hormone secretion in rat Leydig cells, where it exerts an inhibitory action on gonadotropin-induced cyclic adenosine monophosphate generation and steroidogenesis (Dufau et al., 1993). Further studies in hamster Leydig cells have indicated interactions between 5-HT2 receptor system and adrenergic receptors (Frungieri et al., 2002). Taking into account the fact that ketanserin is a competitive antagonist of not only 5HT2, but also of 1-adrenoreceptors (Marwood, 1994; Israilova et al., 2002), and the observation which suggests that in the golden hamster testicular action of 1- and βadrenergics is exerted through 5-HT2 receptors (Frungieri et al., 2002), only additional investigations could clarify regulatory mechanisms that might involve cooperation of IL-1β with 5-HT2 and also with 1-adrenergic receptors. The involvement of testicular serotoninergic innervation in the control of steroidogenesis has also been observed. Intratesticular administration of the neurotoxin 5,7-dihydroxytryp-
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tamine, that selectively destroys serotoninergic neuronal elements (Gerson and Baldessarini, 1975) altered steroidogenesis (Csaba et al., 1998) and prevented the rise in steroid secretion induced by local administration of oxytocin (Gerendai et al., 1996). Further studies have to be performed to clarify the mechanism of interaction between testicular effect of IL-1β and testicular 5-HT2 receptors. The involvement of cerebral serotonin and norepinephrine and the role of the sympathetic system including also its peripheral components in the IL-1β-induced inflammatory responses, such as pyrogenic reaction, reduced food intake, stimulation of the hypothalamo–pituitary–adrenal axis, changes in behavior are well documented (Saigusa, 1989; Niijima et al., 1991; Ichijo et al., 1994; Kannan et al., 1996; Murakami et al., 1996; Ohashi and Saigusa, 1997; Saindon et al., 2001; Heneka et al., 2002, 2003; Wang et al., 2002; Madrigal et al., 2005; MohanKumar and MohanKumar, 2005). The demonstration of a direct neural pathway between the testis and the brain (Gerendai et al., 2000) and the observation that intracerebroventricular administration of IL-1β blunts the hCG-induced testosterone secretion through a catecholamine-mediated mechanism without alteration in gonadotrope hormone secretion (Selvage et al., 2004) strongly suggest a multifactorial mechanism which modulates the local action of the cytokine on testicular functions. Further studies are needed to provide explanation concerning the mechanism of action and interaction between testicular innervation, serotoninergic receptors and local interleukin effect. Acknowledgements This work was supported by grants from the National Research Fund (OTKA T-046624 to I.G.), and the Hungarian Academy of Sciences. References Anakwe, O.O., Moger, W.H., 1986. Catecholamine stimulation of androgen production by rat Leydig cells. Interaction with luteinizing hormone and luteinizing hormone-releasing hormone. Biol. Reprod. 35, 806–814. Bergh, A., Söder, O., 1990. Interleukin-1beta but not interleukin-1alpha, induces acute inflammation-like changes in the testicular microcirculation of adult rats. J. Reprod. Immunol. 17, 155–165. Campos, M.B., Vitale, M.L., Calandra, R.S., Chiocchio, S.R., 1990. Serotoninergic innervation of the rat testis. J. Reprod. Fertil. 88, 475–479. Csaba, Zs., Csernus, V., Gerendai, I., 1998. Intratesticular serotonin affects steroidogenesis in the rat testis. J. Neuroendocrinol. 10, 371–376. Csernus, V., 1982. Antibodies of high affinity and specifity for radioimmunological determination of progesterone, testosterone and estradiol-17-beta. In: Görög, S. (Ed.), Advances in Steroid Analysis. Akadémiai Kiadó, Budapest, pp. 171–177. Cudicini, C., Lejeune, H., Gomez, E., Bosmans, E., Ballet, F., Saez, J., Jegou, B., 1997. Human Leydig cells and Sertoli cells are producers of interleukins-1 and -6. J. Clin. Endocrinol. Metab. 82, 1426–1433. Dinarello, C.A., 1994. The biological properties of interleukin-1. Eur. Cytokine Netw. 5, 517–531. Dinarello, C.A., 1996. Biologic basis for interleukin-1 in disease. Blood 87, 2095–2147.
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Innervation and serotoninergic receptors of the testis interact with local action of interleukin-1beta on steroidogenesis Ida Gerendai a,⁎, Péter Banczerowski a,b , Valér Csernus c , Béla Halász a a
c
Neuroendocrine Research Laboratory, Hungarian Academy of Sciences and Semmelweis University, Department of Human Morphology and Developmental Biology, H-1094 Budapest, Tűzoltó u. 58., Hungary b National Institute of Neurosurgery, H-1145 Budapest, Amerikai út 51., Hungary Neurohumoral Regulation Research Group, Hungarian Academy of Sciences and Pécs University, Department of Human Anatomy, H-7643 Pécs, Szigeti út 12., Hungary Received 9 March 2006; received in revised form 22 May 2006; accepted 2 June 2006
Abstract Testosterone secretion by Leydig cells is affected by interleukin-1β (IL-1β). The aim of the present study was to investigate whether partial denervation of the testis or local administration of a serotonin (5-HT) receptor antagonist could alter the changes in testicular steoidogenesis induced by IL-1β. Intratesticular administration of IL-1β was combined with vasectomy or local injection of ketanserin (5-HT type 2 receptor antagonist) in immature hemicastrated rats and the effect of the interventions on testicular steroidogenesis was studied. One day after treatment with local injection of IL-1β induced a significant rise in testosterone secretion that could be prevented by vasectomy (that also means transection of the inferior spermatic nerve). In a model in which neither IL-1β nor ketanserin interfered with steroidogenesis, administration of the receptor antagonist just prior to IL-1β treatment significantly reduced testosterone secretion. Data indicate interaction between testicular nerves and IL-1β action and interaction between testicular 5-HT2 receptors and local effect of IL-1β on testosterone secretion. © 2006 Elsevier B.V. All rights reserved. Keywords: IL-1β; Testicular steroidogenesis; Vasectomy; Ketanserin
1. Introduction Cytokines are regulatory proteins involved in haemopoiesis, inflammation and immune responses. Several cytokines including the proinflammatory cytokine family, the interleukin-1 (IL-1) system have been implicated as local regulators of different organs, including the testis (Schlatt et al., 1997; Hedger and Meinhardt, 2003; Svechnikov et al., 2004). The family of IL-1 consists of two agonists, isotypes IL-1α and IL-1β encoded by separate genes, and the naturally occurring IL-1 receptor antagonist (Eisenberg et al., 1991; Dinarello, 1994). Two IL-1 receptors have been characterized: IL-1-type I and IL-1-type II receptors (Sims and Dower, 1994) that bind both IL-1α and IL-1β and the ⁎ Corresponding author. Tel.: +36 1 215 6920x3616; fax: +36 1 215 3064. E-mail address: [email protected] (I. Gerendai). 1566-0702/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2006.06.002
antagonist (Dinarello, 1996). In the rat testis IL-1α is constitutively produced and secreted by Sertoli cells (Syed et al., 1988; Gerard et al., 1991, 1992; Cudicini et al., 1997; Jonsson et al., 1999; Wahab-Wahlgren et al., 2000) and acts as a paracrine regulator in the control of a wide range of testicular functions including differentiation of the organ, steroidogenesis, and spermatogenesis (for review see Hedger and Meinhardt, 2003; Svechnikov et al., 2004). By contrast to IL-1α, IL-1β does not seem to be constitutively synthesized in the intact testis (Jonsson et al., 1999; Sultana et al., 2000; Wahab-Wahlgren et al., 2000), but in response to preinflammatory stimuli. Both in immature and adult testis, Leydig cells and testicular macrophages are able to express mRNA for IL-1β (Wang et al., 1991; Hales et al., 1992; Lin et al., 1993; Xiong and Hales, 1994; Cudicini et al., 1997; Gow et al., 2001) and secrete the protein (Jonsson et al., 2001). IL-1β has been reported to affect Sertoli cell
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functions (Petersen et al., 2002) and steroidogenesis. Testosterone secretion by Leydig cells has been demonstrated to be either inhibited (Lin et al., 1991; Hales, 1992; Khan et al., 1992; Mauduit et al., 1992) or stimulated (Verhoeven et al., 1988;Warren et al., 1990) by IL-1β. Recent in vitro (Svechnikov et al., 2001) and in vivo (Gerendai et al., 2005) studies indicate that the effect of IL1β on testicular steroidogenesis is age-dependent. In addition, the results of the in vivo experiments indicate that in immature rats the alterations in testosterone secretion following testicular administration of IL-1β depend also on the time elapsed between treatment and sacrifice and on the fact whether animals were hemicastrated or had two testes in situ (Gerendai et al., 2005). Besides the great number of biologically active substances produced by different cell types of the testis and exerting local modulatory actions, nerves supplying the organ are also known to be involved in the fine-tuning control of testicular functions (Setchell and Brooks, 1988; Gerendai and Halász, 1997). The nerves to and from the testis include the superior testicular nerve that runs along the testicular artery and the inferior testicular nerve which accompanies the ductus deferens (Kuntz and Morris, 1946). Catecholamines, noradrenaline and adrenaline released from sympathetic nerve endings activate catecholaminergic receptors located on Sertoli cells, Leydig cells and myoid cells of the testis and steroid production (Heindel et al., 1981; Moger et al., 1982; Anakwe and Moger, 1986; Poyet and Labrie, 1987; Mayerhofer et al., 1992, 1993; Setchell et al., 1994; Mayerhofer, 1996). Our previous studies indicated that the surgical or neurochemical partial denervation of the testis can alter local modulatory action of opioid peptides (Gerendai et al., 1989; Gerendai et al., 1992) and oxytocin (Gerendai et al., 1996) on steroidogenesis. Furthermore, Frungieri et al. (2002) have reported interaction between catecholamine actions on testosterone secretion and testicular serotonin type 2 (5-HT2) receptors. The aim of the present study was to investigate whether partial denervation of the testis or local administration of the serotonin receptor (5-HT2) antagonist could alter the changes in testicular steroidogenesis induced by IL-1β. Local treatment of the testis with the cytokine was combined with unilateral partial denervation of the organ by means of vasectomy. Our previous data have indicated that different types of denervation of the testis (vasectomy, pharmacological destruction of serotoninergic neural elements) could alter local peptide effects in different ways (Gerendai et al., 1996). In order to avoid that an overall effect due to total denervation could obscure the effect of one of the components of the innervation, partial denervation of the organ was performed. Vasectomy (interruption of the inferior spermatic nerve) is a useful method of partial testicular denervation, especially in immature animals, where no excretion is present towards the ductus deferens. Furthermore, recent morphological data show that the majority of efferent innervation of the testis originates in the pelvic
ganglia and sympathetic chain and reaches the organ with the deferential artery (Rauchenwald et al., 1995). The functional significance of the inferior spermatic nerve is indicated by studies which demonstrate the major contribution of the inferior spermatic nerve in the hemicastration-induced testosterone response (Frankel and Mock, 1982; Frankel and Chapman, 1984; Frankel and Wright, 1982; Zhu et al., 1995) and in control of steroidogenesis mediated by acetylcholinergic fibers (Zhu et al., 2002). In another experiment we combined cytokine treatment with injection of ketanserin (a highly selective 5-HT2 antagonist) to reveal a possible interaction between the effect of interleukin and serotonin. Intratesticular injection of different substances is a widely used method and the only approach to study effects of locally produced substances in vivo. Bergh and Söder (1990) published a detailed study concerning the effect of intratesticular injection of different substances (IL-1α, IL1β, histamine, serotonin, bradykinin) on vascular permeability and testicular histology. Our experiments were performed in immature hemicastrated rats as such animals were used in the majority of previous studies demonstrating the effect of local treatment of the testis with IL-1β (Gerendai et al., 2005) and the interactions between locally produced regulatory peptides and testicular nerves. 2. Materials and methods 2.1. Animals Pregnant CFY (originally Sprague-Dawley) rats were housed in a light- (lights on for 12 h) and temperature (22 ± 2 °C)-controlled room with free access to rat chow and tap water. After delivery, ten newborn male rats were placed with each lactating female. 2.2. Experiments, treatments Experiments were carried out in immature (21-day-old) animals. In each experiment IL-1β or physiological saline was injected into the right testis and immediately after treatment the other gonad was removed. Animals were subjected to two different types of interventions just prior to the injection of the cytokine and hemicastration. In experiment I unilateral vasectomy, in experiment II testicular administration of ketanserine were performed. The interventions were made under ether anaesthesia. Animals were sacrificed 1 day or 6 days after the interventions. The experimental procedures were approved by the Local Animal Care and Use Committee in accordance with NIH Guidelines for the Care and Use of Laboratory Animals. 2.3. Experiment I (vasectomy, intratesticular injection of IL1β, and hemicastration) Twenty-one-day old rats were divided into four experimental groups: (1) sham controls: intratesticular injection of
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2 μl physiological saline into the right testis and removal of the left testis; (2) local injection with 0.1 μg/2 μl/testis of IL1β on the right side and hemicastration; (3) right-sided vasectomy (cutting the ductus deferens and accompanying vessels and nerves between two ligations), right-sided intratesticular injection of 2 μl physiological saline, and hemicastration, and (4) first right-sided vasectomy, then local treatment of the right testis with 0.1 μg/2 μl/testis of IL1β, and hemicastration. Animals were sacrificed 1 day after surgery and treatment.
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bound and free steroids were separated by dextran-coated charcoal. The radioactivity was measured in a two phase liquid scintillation system. The sensitivity limit of the assay is 3 fmol/tube. The inter- and intraassay coefficients for variation were 9.80 and 5.98%, respectively. Due to the high cross reaction of the testosterone antiserum with DHT and the fact that in prepubertal testis the primary androgen produced by Leydig cells is not testosterone but androstendione, DHT, and 5-androstenediol (Wiebe, 1976), the steroid measured is a mixture of testosterone and 5-DHT, but for the sake of simplicity, it is referred as testosterone.
2.4. Experiment II (local treatment of the right testis with ketanserin and/or IL-1β, and hemicastration) In 21-day-old animals 5 μg/5 μl ketanserin or the same volume of physiological saline was injected into the right testis followed by treatment of the same gland with 0.1 μg/ 2 μl IL-1β or 2 μl of physiological saline. Immediately after treatment the left testis was removed. Animals were sacrificed 6 days later. The doses of the cytokine (Gerendai et al., 2005), ketanserin (Csaba et al., 1998) as well as the survival time applied were chosen on the basis of previous results or of preliminary studies (Gerendai et al., 1992, 1996). 2.5. Testicular incubation When animals were killed, testes were immediately removed, decapsulated and incubated in medium 199 containing 25 mM Hepes for 3 h, in a metabolic shaker at 35 °C. The volume of medium was 1 ml. After the incubation media were transferred to tubes and stored at − 20 °C until assayed for testosterone. The results are expressed as testosterone produced per testis (nmol/l/testis) during the incubation period. 2.6. Assay Testosterone concentrations of serum or tissue culture medium (Medium) were determined by RIA as previously described in details (Csernus, 1982). Briefly, 20 μl serum was extracted with 2 ml ether. The dried extract was dissolved in 500 μl assay buffer (ASB, 0.5 M PBS with 1 g/l gelatin, pH = 7.4). For standard, Calbiochem Testosterone for HPLC standard was used in a 9-step series ranging from 3.5 to 1000 fmol/tube. From 20 μl Medium samples, direct determination was made. In this case, to each standard tube 20 μl unused Medium was added. The RIA tubes contained the samples (in duplicates) or standards, 7 nl/tube antibody (CV-RT 17, 1:100 000 final dilution) and 12 000 cpm 3Hlabelled testosterone (100 fmol, The Radiochemical Center, Amersham) in a total volume of 0.7 ml ASB. The cross reactions of the antibody: 5α-DHT 45%, 5β-DHT 9%, and androstendione 2%. With other 27 natural and synthetic steroids examined, the antibody showed less than 0.05% cross reaction. After an overnight incubation at 4 °C the
Fig. 1. Effect of unilateral intratesticular injection of interleukin-1beta (IL1beta) and/or hemivasectomy on serum testosterone concentration, basal testosterone secretion in vitro and testicular weight in hemicastrated rats. Treatment, vasectomy and hemicastration were performed in 21-day-old rats, which were killed 1 day after treatment and/or surgery. Values are mean ± SEM. Figures below the horizontal axis indicate the number of animals. phys. s.: physiological saline; T: testosterone; UORCHX: unilateral orchidectomy; vas: vasectomy.
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LH and FSH levels from rat sera were determined by RIA utilizing National Hormone and Pituitary Program kits. For reference, rLH-RP-3 and rFSH-RP-2 preparations were used. The inter- and intraassay coefficients of variation were 7–9 and 4–6%, respectively. 2.7. Statistical analysis Results were analyzed by ANOVA followed by Student– Newman–Keuls multiple comparison methods. When only two means were compared, Student's t test was performed. Results were considered significant if p < 0.05.
3. Results 3.1. Experiment I In accordance with our previous data (Gerendai et al., 2005), in 21-day-old animals one day after unilateral intratesticular injection of IL-1β (0.1 μg/testis) and removal of the contralateral gonad, a significant rise both in basal testosterone secretion in vitro and serum testosterone concentration could be observed (p < 0.05). Unilateral vasectomy, by itself, did not interfere with the parameters studied. When vasectomy was performed just prior to local administration of the cytokine, IL-1β failed to enhance significantly steroidogenesis and serum testosterone concentration (Fig. 1, middle and upper panel). The weight of the remaining testis of different experimental groups did not differ significantly (Fig. 1, lower panel). 3.2. Experiment II In 21-day-old hemicastrated rats, injection of 0.1 μg/testis of IL-1β into the right testis did not alter the parameters studied 6 days after treatment. This is in accordance with our previous observations (Gerendai et al., 2005). Similarly, local treatment with ketanserin was also without effect. By contrast, administration of ketanserin just prior to IL-1β injection, resulted in a significant decrease both in steroidogenesis (p < 0.01) and serum testosterone level (p < 0.05) as compared to any other groups (Fig. 2, middle and upper panel). Administration of IL-1β did not interfere with testicular weight but ketanserin combined either with the cytokine or the vehicle resulted in a slight but significant (p < 0.05) decrease in the weight of the remaining testis as compared to physiological saline-treated control (Fig. 2, lower panel). No change in serum LH and FSH concentration could be observed in any experiment. 4. Discussion
Fig. 2. Effect of unilateral intratesticular injection of ketanserin (ketanser) and/or interleukin-1beta (IL-1beta) on serum testosterone concentration, basal testosterone secretion in vitro and testicular weight in hemicastrated rats. Treatments and hemicastration were performed in 21-day-old rats. Animals were sacrificed 6 days posttreatment. Values are mean ± SEM. Figures below the horizontal axis indicate the number of animals. phys. s.: physiological saline; T: testosterone; UORCHX: unilateral orchidectomy.
The results of the present study demonstrate for the first time (i) interaction between testicular innervation and IL-1βinduced changes in steroidogenesis, (ii) interaction between testicular 5-HT2 receptors and local effect of IL-1β on testosterone secretion. It has previously been reported that in immature rats vasectomy suppresses basal testosterone secretion in vitro (Gerendai et al., 1986a). The mechanism of action of vasectomy on steroidogenesis is not established. The lack of changes in the secretion of LH, FSH and other pituitary hormones (Rosemberg et al., 1974; Singhal et al., 1977) suggests a pituitary-independent, neurally mediated mechanism. This view is supported by the observations that in hypophysectomized animals hemivasectomy blocks the hemicastration-induced increase in testosterone secretion (Frankel et al., 1984) and that in rats with two testes
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hemivasectomy results in changes of both gonads (Gerendai et al., 1986a). Further studies have indicated that in immature, hemicastrated rats hemivasectomy performed just prior to testicular administration of oxytocin modified the peptide-induced changes in steroidogenesis (Gerendai et al., 1996). Similarly, in immature, vasectomized animals intratesticular treatment of naloxone or enkephalinamide failed to alter steroidogenesis (Gerendai et al., 1992) that is present in rats with intact nerves to and from the testis (Gerendai et al., 1986b, 1990). These findings are in agreement with the results of the present study, which indicate that local testicular action of IL-1β on steroidogenesis is altered by vasectomy, i.e., interruption of the inferior spermatic nerve that is composed of both sympathetic and parasympathetic elements. On the basis of these observations an interaction between testicular innervation and local action of the cytokine can be supposed. Interestingly enough in our second experiment IL-1β or the 5-HT2 receptor blocker ketanserin alone failed to affect steroidogenesis, combined treatment resulted in a significant reduction in steroidogenesis. The mechanism of this action is not known. It can be supposed that under conditions, such as 6 days posttreatment when IL-1β alone is not able to suppress steroidogenesis, the action of ketanserin is needed to obtain inhibitory action of the cytokine. Testicular 5-HT originates from local synthesis of interstitial cells, such as Leydig cells and mast cells (Gaytan et al., 1989; Tinajero et al., 1993) and from nerve endings (Campos et al., 1990) and serotonin receptors of the 5-HT2 subtype are present in rat and hamster Leydig cells (Tinajero et al., 1992; Frungieri et al., 2002). In vitro studies and in vivo experiments in adults indicate that 5-HT acting on 5-HT2 receptors suppresses steroidogenesis (Ellis et al., 1972; Kalla, 1979; Tinajero et al., 1992; Csaba et al., 1998). The action of serotonin on steroidogenesis, however, seems to be age-dependent: in immature rats intratesticular administration of 5-HT stimulated, while ketanserin or 5,7-dihydroxytryptamine (5,7DHT) suppressed it (Csaba et al., 1998). Testicular 5-HT has been reported to stimulate the corticotropin-releasing hormone secretion in rat Leydig cells, where it exerts an inhibitory action on gonadotropin-induced cyclic adenosine monophosphate generation and steroidogenesis (Dufau et al., 1993). Further studies in hamster Leydig cells have indicated interactions between 5-HT2 receptor system and adrenergic receptors (Frungieri et al., 2002). Taking into account the fact that ketanserin is a competitive antagonist of not only 5HT2, but also of 1-adrenoreceptors (Marwood, 1994; Israilova et al., 2002), and the observation which suggests that in the golden hamster testicular action of 1- and βadrenergics is exerted through 5-HT2 receptors (Frungieri et al., 2002), only additional investigations could clarify regulatory mechanisms that might involve cooperation of IL-1β with 5-HT2 and also with 1-adrenergic receptors. The involvement of testicular serotoninergic innervation in the control of steroidogenesis has also been observed. Intratesticular administration of the neurotoxin 5,7-dihydroxytryp-
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tamine, that selectively destroys serotoninergic neuronal elements (Gerson and Baldessarini, 1975) altered steroidogenesis (Csaba et al., 1998) and prevented the rise in steroid secretion induced by local administration of oxytocin (Gerendai et al., 1996). Further studies have to be performed to clarify the mechanism of interaction between testicular effect of IL-1β and testicular 5-HT2 receptors. The involvement of cerebral serotonin and norepinephrine and the role of the sympathetic system including also its peripheral components in the IL-1β-induced inflammatory responses, such as pyrogenic reaction, reduced food intake, stimulation of the hypothalamo–pituitary–adrenal axis, changes in behavior are well documented (Saigusa, 1989; Niijima et al., 1991; Ichijo et al., 1994; Kannan et al., 1996; Murakami et al., 1996; Ohashi and Saigusa, 1997; Saindon et al., 2001; Heneka et al., 2002, 2003; Wang et al., 2002; Madrigal et al., 2005; MohanKumar and MohanKumar, 2005). The demonstration of a direct neural pathway between the testis and the brain (Gerendai et al., 2000) and the observation that intracerebroventricular administration of IL-1β blunts the hCG-induced testosterone secretion through a catecholamine-mediated mechanism without alteration in gonadotrope hormone secretion (Selvage et al., 2004) strongly suggest a multifactorial mechanism which modulates the local action of the cytokine on testicular functions. Further studies are needed to provide explanation concerning the mechanism of action and interaction between testicular innervation, serotoninergic receptors and local interleukin effect. Acknowledgements This work was supported by grants from the National Research Fund (OTKA T-046624 to I.G.), and the Hungarian Academy of Sciences. References Anakwe, O.O., Moger, W.H., 1986. Catecholamine stimulation of androgen production by rat Leydig cells. Interaction with luteinizing hormone and luteinizing hormone-releasing hormone. Biol. Reprod. 35, 806–814. Bergh, A., Söder, O., 1990. Interleukin-1beta but not interleukin-1alpha, induces acute inflammation-like changes in the testicular microcirculation of adult rats. J. Reprod. Immunol. 17, 155–165. Campos, M.B., Vitale, M.L., Calandra, R.S., Chiocchio, S.R., 1990. Serotoninergic innervation of the rat testis. J. Reprod. Fertil. 88, 475–479. Csaba, Zs., Csernus, V., Gerendai, I., 1998. Intratesticular serotonin affects steroidogenesis in the rat testis. J. Neuroendocrinol. 10, 371–376. Csernus, V., 1982. Antibodies of high affinity and specifity for radioimmunological determination of progesterone, testosterone and estradiol-17-beta. In: Görög, S. (Ed.), Advances in Steroid Analysis. Akadémiai Kiadó, Budapest, pp. 171–177. Cudicini, C., Lejeune, H., Gomez, E., Bosmans, E., Ballet, F., Saez, J., Jegou, B., 1997. Human Leydig cells and Sertoli cells are producers of interleukins-1 and -6. J. Clin. Endocrinol. Metab. 82, 1426–1433. Dinarello, C.A., 1994. The biological properties of interleukin-1. Eur. Cytokine Netw. 5, 517–531. Dinarello, C.A., 1996. Biologic basis for interleukin-1 in disease. Blood 87, 2095–2147.
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