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Involvement of the neurosteroid 7α-hydroxypregnenolone in the courtship behavior of the male newt Cynops pyrrhogaster
Hormones and Behavior 62 (2012) 375–380
Contents lists available at SciVerse ScienceDirect
Hormones and Behavior journal homepage: www.elsevier.com/locate/yhbeh
Involvement of the neurosteroid 7α-hydroxypregnenolone in the courtship behavior of the male newt Cynops pyrrhogaster Fumiyo Toyoda a,⁎, Itaru Hasunuma b, Tomoaki Nakada c, Shogo Haraguchi d, Kazuyoshi Tsutsui d, Sakae Kikuyama b, d a
Physiology Department-I, Nara Medical University, Nara 634-8521, Japan Department of Biology, Faculty of Science, Toho University, Chiba 274-8510, Japan Department of Comparative and Behavior Medicine, Nippon Veterinary and Life Science University, Tokyo 180-8602, Japan d Department of Biology, Waseda University, and Center for Medical Life Science of Waseda University, Tokyo 162-8480, Japan b c
a r t i c l e
i n f o
Article history: Received 13 March 2012 Revised 26 June 2012 Accepted 3 July 2012 Available online 13 July 2012 Keywords: Newt Courtship Behavior Neurosteroid 7α-hydroxypregnenolone Dopamine
a b s t r a c t Reproductive behavior in amphibians, as in other vertebrate animals, is controlled by multiple hormones. A neurosteroid, 7α-hydroxypregnenolone, has recently been found to enhance locomotor activity in the male newt, Cynops pyrrhogaster. Here, we show that this neurosteroid is also involved in enhancing the expression of courtship behavior. Intracerebroventricular (ICV) injection of 7α-hydroxypregnenolone enhanced courtship behavior dose-dependently in the sexually undeveloped males that had been pretreated with prolactin and gonadotropin, which is known to bring the males to a sexually developed state. But, unlike the case in the locomotion activity, 7α-hydroxypregnenolone did not elicit the behavior in males receiving no prior injections of these hormones. ICV administration of ketoconazole, an inhibitor of the steroidogenic enzyme cytochrome P450s, suppressed the spontaneously occurring courtship behavior in sexually active males. Supplementation with 7α-hydroxypregnenolone reversed the effect of ketoconazole in these animals. It was also demonstrated that the effect of the neurosteroid on the courtship behavior was blocked by a dopamine D2-like, but not by a D1-like, receptor antagonist. These results indicate that endogenous 7α-hydroxypregnenolone enhances the expression of the male courtship behavior through a dopaminergic system mediated by a D2-like receptor in the brain. © 2012 Elsevier Inc. All rights reserved.
Introduction During the breeding season, the male red-bellied newt, Cynops pyrrhogaster, attracts a female partner using the female-attracting pheromone sodefrin (Kikuyama et al., 1995). This pheromone is secreted from the male's abdominal gland into the surrounding water and, through vigorous vibrations of the tail, the water is directed towards the female's snout. The male then creeps in front of the targeted female, who starts to follow the male, with her snout making contact with his tail. The male then discharges spermatophores, which are captured by the female's cloacal orifice and transported into the cloacal cavity (Kikuyama et al., 2003). Long-term research conducted by our group has demonstrated that multiple hormones, including androgen (Toyoda et al., 1993), prolactin (PRL) (Toyoda et al., 1993, 1996, 2005), gonadotropin (GTH) (Toyoda et al., 1993), and arginine vasotocin (AVT) (Toyoda et al., 2003), are involved either directly or indirectly in the expression of this behavior by the breeding red-bellied male newts. This is exemplified by the large increase ⁎ Corresponding author at: Physiology Department-I, Nara Medical University, Shijo-chou 840, Kashihara, Nara 634-8521, Japan. Fax: +81 744 29 0306. E-mail address: [email protected] (F. Toyoda). 0018-506X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2012.07.001
in plasma concentrations of PRL (Matsuda et al., 1990), GTH (Tanaka et al., 1980), and androgen (Tanaka and Takikawa, 1983) in male newts during the breeding season in comparison to the non-breeding season. Various regions of the vertebrate brain are able to synthesize bioactive steroids from cholesterol (for reviews, see Baulieu, 1997; Compagnone and Mellon, 2000; Do Rego et al., 2009; Mellon and Vaudry, 2001; Tsutsui et al., 1999, 2003). Studies in mammals and various birds have revealed that these ‘brain-born’ steroids, termed neurosteroids, regulate various aspects of behavior activities, including aggressiveness, anxiety, sexual behavior, and locomotion (Matsunaga et al., 2004; Meieran et al., 2004; Melchior and Ritzmann, 1994; Tsutsui et al., 2008). In amphibians, Matsunaga et al. (2004) reported that a neurosteroid, 7α-hydroxypregnenolone, is present in abundance in the brain of the red-bellied newt. The synthesis of this neurosteroid was observed to reach a maximum level in the male newt during the spring breeding period concomitant with increases in their locomotor activity. Moreover, intracerebroventricular (ICV) injection of 7α-hydroxypregnenolone to non-breeding males caused an acute elevation of locomotor activity, an action shown to be mediated by dopamine via a dopamine D2-like receptor.
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Material and methods
otherwise. New test animals were used for each test, and no animal was ever used repeatedly. The incidence and frequency of the targeted courtship behavior, i.e., vibration of the tail in front of the female partner, were monitored for 1 h using the method described by Toyoda et al. (1993). Incidence and frequency were expressed as the percentage of animals exhibiting the behavior and the mean number of times the behavior was recorded per test animal over the 1-h test period, respectively.
Animals
Statistical analyses
Adult male and female newts (C. pyrrhogaster) were captured in the field during the spring (April and May) and summer (July) and brought into the laboratory for use in our experiments. During the spring, we focused on capturing newts which were spontaneously exhibiting courtship behavior in the field. Those captured during the summer were not displaying courtship behavior. It has been shown that newts are not sexually active during the summer unless they receive an appropriate hormonal treatment (Toyoda et al., 1993). The groups of male and female newts were kept separately in tanks in the laboratory and fed daily with Tubifex worms. Prior to injection or sacrifice, the animals were anesthetized with 0.1% m-aminobenzoic acid ethylester methanesulfonate (Sigma Chemical Co., St. Louis, MO, USA). All experimental procedures were approved by the Animal Care and Use Committee of Nara Medical University.
The incidence of tail vibration behavior was statistically analyzed using Fisher's two-tailed exact tests (Siegel, 1956). Other experimental data were analyzed using the Kruskal–Wallis one-way analysis of variance (ANOVA) followed by the Mann–Whitney U test. A P value of b 0.05 was considered to be significant.
During the breeding season, the sexually developed male red-bellied newt moves around more actively than the female, searching for a sexually mature female. Once a potential mate has been located, the male starts to vibrate his tail vigorously. The aim of the study reported here was to ascertain whether 7α-hydroxypregnenolone is involved in the expression of this first stage of courtship behavior.
Reagents Human chorionic gonadotropin (HCG) and ovine PRL were purchased from Teikoku Hormone Mfg. Co. (Tokyo, Japan) and Sigma Chemical Co., respectively. These hormones were dissolved in isotonic saline before being injected into the experimental animals. 7α-hydroxypregnenolone was purchased from Steraloids (Newport, RI, USA), mammalian dopamine D1-like receptor antagonist (SCH23390) and mammalian dopamine D2-like receptor antagonist (sulpiride) were obtained from Tocris (Ellisville, MO, USA), and ketoconazole, an inhibitor of the cytochrome P450 steroidogenic enzymes, and testosterone propionate (TP) were purchased from Sigma Chemical Co. Prior to use, these reagents were dissolved in a vehicle, dimethyl sulfoxide (DMSO; Wako, Pure Chemical, Osaka, Japan). Injections ICV injections of 7α-hydroxypregnenolone, ketoconazole, SCH23390, sulpiride, and their vehicle (DMSO) were performed according to the method described by Toyoda et al. (2003). Briefly, a glass micropipette (tip diameter=50 μm) filled with 1 μl of the chosen solution and connected to a microsyringe was inserted, with the help of a micromanipulator to facilitate proper placement, into the third ventricle to a depth of approximately 1 mm through a small hole drilled (drill bit diameter= 0.5 mm) in the parietal bone posterior to the bregma. Ten seconds after the micropipette had been inserted, the contents of the micropipette were infused over a 5-second period, and 20 s later, the micropipette was removed. The hole was filled with acrylic resin (Shofu, Kyoto, Japan). Immediately after the injection, the animals were returned to water. The accuracy of the injection was confirmed in a preliminary experiment by visually inspecting the brains of the newts injected with 1 μl of 0.15% methylene blue dissolved in isotonic saline. Observation of male courtship behavior Each test male was paired with a sexually developed female captured in the field or with a “summer” female which had achieved sexual maturity following intraperitoneal (IP) injections of PRL (1 IU) and HCG (25 IU) on 7–10 successive days. The first behavioral test was conducted 2 h after the last injection of hormones and/or drugs, unless stated
Results Effect of 7α-hydroxypregnenolone on the expression of courtship behavior To determine the effect of 7α-hydroxypregnenolone on the expression of courtship behavior, we observed the tail vibration behavior of “summer” male newts which had received IP injections of PRL (1 IU) and HCG (25 IU) every other day for 1 week followed by ICV injections of various doses of 7α-hydroxypregnenolone. Control animals received the vehicle only. Primed male newts receiving ICV injections of 7α-hydroxypregnenolone showed an increased incidence and frequency of the tail vibration behavior that was dosedependent (Fig. 1A). The minimum effective amount was 10 ng. In “summer” males that had not been pretreated with PRL and HCG, the ICV injections of 7α-hydroxypregnenolone barely elicited the tail-vibrating behavior, even at a dose of 100 ng (Fig. 1B). Effect of ketoconazole on the expression of courtship behavior In order to determine whether endogenous 7α-hydroxypregnenolone is involved in eliciting the courtship behavior, we tested the effect of ketoconazole, an inhibitor of cytochrome P450s, on male newts captured in spring that were spontaneously exhibiting the courtship behavior in the field. Twenty-four hours after capture, “spring” male newts received an ICV injection of 2 μg of ketoconazole. Control animals received the vehicle only. Every 2 h until 8 h after injection, each male was paired with a test female captured similarly in the field, and courtship behavior was monitored. The suppressive effect of ketoconazole on the expression of the behavior became conspicuous 6 and 8 h after the treatment (Fig. 2A). In another set of experiment, effect of ICV injection of various doses of ketoconazole on the courtship behavior of “spring” males captured in the field was examined. Eight hours after the injection, the behavioral test of each male was performed by introducing a test female captured similarly in the field, and courtship behavior was monitored. ICV injection of ketoconazole decreased both the incidence and frequency of the tail-vibrating behavior. The minimum effective dose of ketoconazole was 2 μg (Fig. 2B). The ability of 7α-hydroxypregnenolone to restore the courtship behavior suppressed by ketoconazole was tested by injecting the male newts that showed suppressed tail-vibrating behavior due to a 2-μg ICV injection of ketoconazole with 100 ng 7α-hydroxypregnenolone at 6 h after the ICV injection of 2 μg ketoconazole. After an elapse of 2 h following ICV administration of the neurosteroid or its vehicle, each male was subjected to the behavioral test. As shown in Fig. 3, the group of newts that had shown suppressed tail-vibrating behavior following injection with 2 μg ketoconazole resumed the tail-vibrating behavior following the subsequent administration of 7α-hydroxypregnenolone. Injection of
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the vehicle only had no effect on the ketoconazole-suppressed tailvibration behavior. Involvement of the dopaminergic system in 7α-hydroxypregnenoloneinduced courtship behavior The effect of dopamine D1- and D2-like receptor antagonists on the expression of courtship behavior was studied to confirm that the action of 7α-hydroxypregnenolone is mediated through the dopaminergic system. Sexually active “spring” males were captured, kept in the laboratory for 24 h, and then injected ICV with the dopamine D1-like receptor antagonist SCH23390 or the D2-like receptor antagonist sulpiride, in a dose of 1 μg in vehicle. The behavioral test revealed that the dopamine D1-like receptor antagonist did not abolish spontaneous expression of the courtship behavior (Fig. 4A), whereas this behavior was suppressed by the D2-like receptor antagonist. The spontaneous behavior arrested by sulpiride was not restored by ICV injection of 100 ng 7α-hydroxypregnenolone when administered simultaneously with the inhibitor (Fig. 4B). Discussion Our study establishes that the neurosteroid 7α-hydroxypregnenolone, which was initially found to elevate the locomotor activity in the male red-bellied newt (Matsunaga et al., 2004), is also effective in enhancing the tail-vibrating behavior uniquely performed by the sexually active male red-bellied newt. The ICV administration of 7αhydroxypregnenolone dose-dependently increased the incidence and frequency of the tail-vibrating behavior in sexually inactive male newts that had been primed with PRL and GTH. In this experiment, care was taken in terms of intervals and duration of the hormonal treatment to ensure that these animals did not spontaneously achieve complete sexual maturity without the exogenously supplied neurosteroid. In contrast,
7α-hydroxypregnenolone was notably ineffective in inducing the behavior in the sexually inactive male newts that had not been pretreated with PRL and GTH, i.e. which had received saline injections. According to Matsunaga et al. (2004), 7α-hydroxypregnenolone elevates the level of locomotor activity in newts captured during the non-breeding period. The differential effectiveness of 7αhydroxypregnenolone for enhancing locomotion versus courtship behavior may come from the fact that the expression of courtship behavior is due to the involvement of multiple hormones (Iwata et al., 2000), whereas locomotor activity is likely to be enhanced by the neurosteroid alone. We have earlier studied the hormonal factors involved in eliciting courtship behavior in intact, hypophysectomized, and castrated newts, respectively, captured in the breeding or non-breeding season (see Kikuyama et al., 2003). PRL was found to promote the expression of the courtship behavior in the male newt, particularly in the presence of androgen (Toyoda et al., 1993). In subsequent studies involving the injection, either ICV or IP, of antiserum against newt PRL and antibody against newt PRL receptor into sexually active male newts, Toyoda et al. (1996, 2005) demonstrated that endogenous PRL acts centrally to elicit courtship behavior. Androgen is another important factor contributing to the expression of courtship behavior. In this context, GTH is regarded as an indirect factor that enhances androgen secretion (Toyoda et al., 1993). An anti-androgen, flutamide, administered by ICV injection to sexually active male newts was found to block the expression of courtship behavior more effectively than flutamide injected IP (unpublished data). This result suggests that androgen acts centrally to enhance the expression of courtship behavior. Once we found that exogenously administered 7α-hydroxypregnenolone was effective in enhancing courtship behavior, we examined whether endogenous 7α-hydroxypregnenolone is involved in the expression of this behavior in sexually active male newts. Ketoconazole,
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Ketoconazole (µg) Fig. 2. Effects of duration of the time after ICV injection of ketoconazole (A) and the dosage of ketoconazole (B) on the incidence and frequency of spontaneously occurring courtship behavior in the spring newt. In the experiment (A), each group consisting of 8 animals received ICV injection of ketoconazole or vehicle. Behavioral test was performed every 2 h until 8 h after the injection. Each point and vertical bar represent the mean and SEM, respectively. The value with an asterisk is significantly different from the respective control value at P b 0.05. In the experiment (B), each group consisted of 11 animals. Each column and vertical bar represent the mean and SEM, respectively. Means with different superscripts are significantly different at P b 0.05.
an inhibitor of cytochrome P450s, reduces the synthesis of 7αhydroxypregnenolone in the newt brain (Matsunaga et al., 2004). As anticipated, we found that ketoconazole suppresses the spontaneously occurring courtship behavior and that subsequent treatment with 7αhydroxypregnenolone reverses this effect. This result clearly indicates
that endogenous 7α-hydroxypregnenolone plays an important role in eliciting courtship behavior. Matsunaga et al. (2004) observed that 7α-hydroxypregnenolone increased the release of dopamine from the brain tissue in culture and that the dopamine D2-like receptor antagonist abolished neurosteroid-
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induced locomotor activity. Based on these results, these authors concluded that the stimulatory effect of 7α-hydroxypregnenolone on the locomotor activity of the newt is mediated by the dopaminergic system. In the present set of experiments, we showed that a D2-like receptor antagonist, sulpiride, but not a D1-like receptor antagonist, SCH2339, abolished the spontaneously occurring courtship behavior by acting centrally. Since even supplementation with an ample amount of 7αhydroxypregnenolone did not reverse the effect of this D2-like receptor antagonist, the neurosteroid is likely to act as an activator of dopaminergic system in this case as well. Considering that the neurosteroid synthesis is enhanced only in the breeding male (Matsunaga et al., 2004) but never in the female (Haraguchi et al., 2010), it may contribute to the rapid encounter of the male with a sexually mature female by increasing the locomotor activity and to the successful transfer of the sperm to the female partner by enhancing the courtship activity. Thus, it is considered that 7α-hydroxypregnenolone may widely activate the motility of the animal and that its action may not be specific for the tail vibration.
Our results suggest that 7α-hydroxypregnenolone can be added to the list of endogenous substances that are involved in the expression of courtship behavior. In terms of their interactions involved in eliciting this courtship behavior, it is of interest to note that PRL appears to act directly on neurosteroidogenic neurons in the preoptic nucleus to regulate the synthesis of 7α-hydroxypregnenolone in the male newt (Haraguchi et al., 2010). It was revealed that hypophysectomy decreases the synthesis of 7α-hydroxypregnenolone in the brain and that administration of PRL to the hypophysectomized newts causes a dose-dependent increase in 7α-hydroxypregnenolone synthesis. Moreover, the presence of the PRL receptor in the preoptic neurons expressing cytochrome P4507α (CYP7B), a steroidogenic enzyme that catalyzes the formation of 7α-hydroxypregnenolone was demonstrated. Therefore, it is likely that at least one of the roles of PRL in eliciting the male courtship behavior is to stimulate 7α-hydroxypregnenolone synthesis. This report is the first to demonstrate that a neurosteroid is involved in enhancing an amphibian sexual behavior. Future studies should be directed towards analyzing the precise roles of the hormones, such as PRL, AVT and androgen as well as of the
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neurosteroid 7α-hydroxypregnenolone and the site(s) of their respective action in eliciting the courtship behavior of the male redbellied newt. Acknowledgments This study was supported by research grants from the Ministry of Education Science and Culture of Japan to F.T. (22570075) and S.K. (21570071). References Baulieu, E.E., 1997. Neurosteroids: of the nervous system, by the nervous system, for the nervous system. Recent Prog. Horm. Res. 52, 1–32. Compagnone, N.A., Mellon, S.H., 2000. Neurosteroids: biosynthesis and function of these novel neuromodulators. Front. Neuroendocrinol. 21, 1–56. Do Rego, J.L., Seong, J.Y., Burel, D., Leprince, J., Luu-The, V., Tsutsui, K., Tonon, M.C., Pelletier, G., Vaudry, H., 2009. Neurosteroid biosynthesis: enzymatic pathways and neuroendocrine regulation by neurotransmitters and neuropeptides. Front. Neuroendocrinol. 30, 259–301. Haraguchi, S., Koyama, T., Hasunuma, I., Vaudry, H., Tsutsui, K., 2010. Prolactin increases the synthesis of 7α-hydroxypregnenolone, a key factor for induction of locomotor activity, in breeding male newts. Endocrinology 151, 2211–2222. Iwata, T., Toyoda, F., Yamamoto, K., Kikuyama, S., 2000. Hormonal control of urodele reproductive behavior. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 126, 221–229. Kikuyama, S., Toyoda, F., Ohmiya, Y., Matsuda, K., Tanaka, S., Hayashi, H., 1995. Sodefrin: a female-attracting peptide pheromone in newt cloacal glands. Science 267, 1643–1645. Kikuyama, S., Tanaka, S., Moore, F.L., 2003. Endocrinology of reproduction. In: Jamieson, B.G.M. (Ed.), Reproductive Biology and Phylogeny. : Reproductive Biology and Phylogeny of Urodela, vol. 1. Science Publishers, Enfield, pp. 275–321. Matsuda, K., Tanaka, S., Yamamoto, K., Kikuyama, S., 1990. Annual changes of plasma prolactin levels in the newt, Cynops pyrrhogaster. Zool. Sci. 7, 1143.
Matsunaga, M., Ukena, K., Baulieu, E.E., Tsutsui, K., 2004. 7α-Hydroxypregnenolone acts as a neuronal activator to stimulate locomotor activity of breeding newts by means of the dopaminergic system. Proc. Natl. Acad. Sci. U. S. A. 101, 17282–17287. Meieran, S.E., Reus, V.I., Webster, R., Shafton, R., Wolkowitz, O.M., 2004. Chronic pregnenolone effects in normal humans: attenuation of benzodiazepine-induced sedation. Psychoneuroendocrinology 29, 486–500. Melchior, C.L., Ritzmann, R.F., 1994. Pregnenolone and pregnenolone sulfate, alone and with ethanol, in mice on the plus-maze. Pharmacol. Biochem. Behav. 48, 893–897. Mellon, S.H., Vaudry, H., 2001. Biosynthesis of neurosteroids and regulation of their synthesis. Int. Rev. Neurobiol. 46, 33–78. Siegel, S., 1956. Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York. Tanaka, S., Takikawa, H., 1983. Seasonal changes in plasma testosterone and 5αdihydrotestosterone levels in the adult male newt, Cynops pyrrhogaster pyrrhogaster. Endocrinol. Jpn. 30, 1–6. Tanaka, S., Hanaoka, Y., Takikawa, H., 1980. Seasonal changes in gonadotropin activity of the pituitary gland in the adult male Japanese red-bellied newt, Cynops pyrrhogaster pyrrhogaster. Zool. Mag. 89, 187–191. Toyoda, F., Ito, M., Tanaka, S., Kikuyama, S., 1993. Hormonal induction of male courtship behavior in the Japanese newt, Cynops pyrrhogaster. Horm. Behav. 27, 511–522. Toyoda, F., Matsuda, K., Yamamoto, K., Kikuyama, S., 1996. Involvement of endogenous prolactin in the expression of courtship behavior in the newt, Cynops pyrrhogaster. Gen. Comp. Endocrinol. 102, 191–196. Toyoda, F., Yamamoto, K., Ito, Y., Tanaka, S., Yamashita, M., Kikuyama, S., 2003. Involvement of arginine vasotocin in reproductive events in the male newt, Cynops pyrrhogaster. Horm. Behav. 44, 346–353. Toyoda, F., Hasunuma, I., Yamamoto, K., Yamashita, M., Kikuyama, S., 2005. Prolactin acts centrally to enhance newt courtship behavior. Gen. Comp. Endocrinol. 141, 172–177. Tsutsui, K., Ukena, K., Takase, M., Kohchi, C., Lea, R.W., 1999. Neurosteroid biosynthesis in vertebrate brains. Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 124, 121–129. Tsutsui, K., Matsunaga, M., Ukena, K., 2003. Biosynthesis and biological actions of neurosteroids. Avian Poult. Biol. Rev. 14, 63–78. Tsutsui, K., Inoue, K., Miyabara, H., Suzuki, S., Ogura, Y., Haraguchi, S., 2008. 7αhydroxypregnenolone mediates melatonin action underlying diurnal locomotor rhythms. J. Neurosci. 28, 2158–2167.
Contents lists available at SciVerse ScienceDirect
Hormones and Behavior journal homepage: www.elsevier.com/locate/yhbeh
Involvement of the neurosteroid 7α-hydroxypregnenolone in the courtship behavior of the male newt Cynops pyrrhogaster Fumiyo Toyoda a,⁎, Itaru Hasunuma b, Tomoaki Nakada c, Shogo Haraguchi d, Kazuyoshi Tsutsui d, Sakae Kikuyama b, d a
Physiology Department-I, Nara Medical University, Nara 634-8521, Japan Department of Biology, Faculty of Science, Toho University, Chiba 274-8510, Japan Department of Comparative and Behavior Medicine, Nippon Veterinary and Life Science University, Tokyo 180-8602, Japan d Department of Biology, Waseda University, and Center for Medical Life Science of Waseda University, Tokyo 162-8480, Japan b c
a r t i c l e
i n f o
Article history: Received 13 March 2012 Revised 26 June 2012 Accepted 3 July 2012 Available online 13 July 2012 Keywords: Newt Courtship Behavior Neurosteroid 7α-hydroxypregnenolone Dopamine
a b s t r a c t Reproductive behavior in amphibians, as in other vertebrate animals, is controlled by multiple hormones. A neurosteroid, 7α-hydroxypregnenolone, has recently been found to enhance locomotor activity in the male newt, Cynops pyrrhogaster. Here, we show that this neurosteroid is also involved in enhancing the expression of courtship behavior. Intracerebroventricular (ICV) injection of 7α-hydroxypregnenolone enhanced courtship behavior dose-dependently in the sexually undeveloped males that had been pretreated with prolactin and gonadotropin, which is known to bring the males to a sexually developed state. But, unlike the case in the locomotion activity, 7α-hydroxypregnenolone did not elicit the behavior in males receiving no prior injections of these hormones. ICV administration of ketoconazole, an inhibitor of the steroidogenic enzyme cytochrome P450s, suppressed the spontaneously occurring courtship behavior in sexually active males. Supplementation with 7α-hydroxypregnenolone reversed the effect of ketoconazole in these animals. It was also demonstrated that the effect of the neurosteroid on the courtship behavior was blocked by a dopamine D2-like, but not by a D1-like, receptor antagonist. These results indicate that endogenous 7α-hydroxypregnenolone enhances the expression of the male courtship behavior through a dopaminergic system mediated by a D2-like receptor in the brain. © 2012 Elsevier Inc. All rights reserved.
Introduction During the breeding season, the male red-bellied newt, Cynops pyrrhogaster, attracts a female partner using the female-attracting pheromone sodefrin (Kikuyama et al., 1995). This pheromone is secreted from the male's abdominal gland into the surrounding water and, through vigorous vibrations of the tail, the water is directed towards the female's snout. The male then creeps in front of the targeted female, who starts to follow the male, with her snout making contact with his tail. The male then discharges spermatophores, which are captured by the female's cloacal orifice and transported into the cloacal cavity (Kikuyama et al., 2003). Long-term research conducted by our group has demonstrated that multiple hormones, including androgen (Toyoda et al., 1993), prolactin (PRL) (Toyoda et al., 1993, 1996, 2005), gonadotropin (GTH) (Toyoda et al., 1993), and arginine vasotocin (AVT) (Toyoda et al., 2003), are involved either directly or indirectly in the expression of this behavior by the breeding red-bellied male newts. This is exemplified by the large increase ⁎ Corresponding author at: Physiology Department-I, Nara Medical University, Shijo-chou 840, Kashihara, Nara 634-8521, Japan. Fax: +81 744 29 0306. E-mail address: [email protected] (F. Toyoda). 0018-506X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2012.07.001
in plasma concentrations of PRL (Matsuda et al., 1990), GTH (Tanaka et al., 1980), and androgen (Tanaka and Takikawa, 1983) in male newts during the breeding season in comparison to the non-breeding season. Various regions of the vertebrate brain are able to synthesize bioactive steroids from cholesterol (for reviews, see Baulieu, 1997; Compagnone and Mellon, 2000; Do Rego et al., 2009; Mellon and Vaudry, 2001; Tsutsui et al., 1999, 2003). Studies in mammals and various birds have revealed that these ‘brain-born’ steroids, termed neurosteroids, regulate various aspects of behavior activities, including aggressiveness, anxiety, sexual behavior, and locomotion (Matsunaga et al., 2004; Meieran et al., 2004; Melchior and Ritzmann, 1994; Tsutsui et al., 2008). In amphibians, Matsunaga et al. (2004) reported that a neurosteroid, 7α-hydroxypregnenolone, is present in abundance in the brain of the red-bellied newt. The synthesis of this neurosteroid was observed to reach a maximum level in the male newt during the spring breeding period concomitant with increases in their locomotor activity. Moreover, intracerebroventricular (ICV) injection of 7α-hydroxypregnenolone to non-breeding males caused an acute elevation of locomotor activity, an action shown to be mediated by dopamine via a dopamine D2-like receptor.
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Material and methods
otherwise. New test animals were used for each test, and no animal was ever used repeatedly. The incidence and frequency of the targeted courtship behavior, i.e., vibration of the tail in front of the female partner, were monitored for 1 h using the method described by Toyoda et al. (1993). Incidence and frequency were expressed as the percentage of animals exhibiting the behavior and the mean number of times the behavior was recorded per test animal over the 1-h test period, respectively.
Animals
Statistical analyses
Adult male and female newts (C. pyrrhogaster) were captured in the field during the spring (April and May) and summer (July) and brought into the laboratory for use in our experiments. During the spring, we focused on capturing newts which were spontaneously exhibiting courtship behavior in the field. Those captured during the summer were not displaying courtship behavior. It has been shown that newts are not sexually active during the summer unless they receive an appropriate hormonal treatment (Toyoda et al., 1993). The groups of male and female newts were kept separately in tanks in the laboratory and fed daily with Tubifex worms. Prior to injection or sacrifice, the animals were anesthetized with 0.1% m-aminobenzoic acid ethylester methanesulfonate (Sigma Chemical Co., St. Louis, MO, USA). All experimental procedures were approved by the Animal Care and Use Committee of Nara Medical University.
The incidence of tail vibration behavior was statistically analyzed using Fisher's two-tailed exact tests (Siegel, 1956). Other experimental data were analyzed using the Kruskal–Wallis one-way analysis of variance (ANOVA) followed by the Mann–Whitney U test. A P value of b 0.05 was considered to be significant.
During the breeding season, the sexually developed male red-bellied newt moves around more actively than the female, searching for a sexually mature female. Once a potential mate has been located, the male starts to vibrate his tail vigorously. The aim of the study reported here was to ascertain whether 7α-hydroxypregnenolone is involved in the expression of this first stage of courtship behavior.
Reagents Human chorionic gonadotropin (HCG) and ovine PRL were purchased from Teikoku Hormone Mfg. Co. (Tokyo, Japan) and Sigma Chemical Co., respectively. These hormones were dissolved in isotonic saline before being injected into the experimental animals. 7α-hydroxypregnenolone was purchased from Steraloids (Newport, RI, USA), mammalian dopamine D1-like receptor antagonist (SCH23390) and mammalian dopamine D2-like receptor antagonist (sulpiride) were obtained from Tocris (Ellisville, MO, USA), and ketoconazole, an inhibitor of the cytochrome P450 steroidogenic enzymes, and testosterone propionate (TP) were purchased from Sigma Chemical Co. Prior to use, these reagents were dissolved in a vehicle, dimethyl sulfoxide (DMSO; Wako, Pure Chemical, Osaka, Japan). Injections ICV injections of 7α-hydroxypregnenolone, ketoconazole, SCH23390, sulpiride, and their vehicle (DMSO) were performed according to the method described by Toyoda et al. (2003). Briefly, a glass micropipette (tip diameter=50 μm) filled with 1 μl of the chosen solution and connected to a microsyringe was inserted, with the help of a micromanipulator to facilitate proper placement, into the third ventricle to a depth of approximately 1 mm through a small hole drilled (drill bit diameter= 0.5 mm) in the parietal bone posterior to the bregma. Ten seconds after the micropipette had been inserted, the contents of the micropipette were infused over a 5-second period, and 20 s later, the micropipette was removed. The hole was filled with acrylic resin (Shofu, Kyoto, Japan). Immediately after the injection, the animals were returned to water. The accuracy of the injection was confirmed in a preliminary experiment by visually inspecting the brains of the newts injected with 1 μl of 0.15% methylene blue dissolved in isotonic saline. Observation of male courtship behavior Each test male was paired with a sexually developed female captured in the field or with a “summer” female which had achieved sexual maturity following intraperitoneal (IP) injections of PRL (1 IU) and HCG (25 IU) on 7–10 successive days. The first behavioral test was conducted 2 h after the last injection of hormones and/or drugs, unless stated
Results Effect of 7α-hydroxypregnenolone on the expression of courtship behavior To determine the effect of 7α-hydroxypregnenolone on the expression of courtship behavior, we observed the tail vibration behavior of “summer” male newts which had received IP injections of PRL (1 IU) and HCG (25 IU) every other day for 1 week followed by ICV injections of various doses of 7α-hydroxypregnenolone. Control animals received the vehicle only. Primed male newts receiving ICV injections of 7α-hydroxypregnenolone showed an increased incidence and frequency of the tail vibration behavior that was dosedependent (Fig. 1A). The minimum effective amount was 10 ng. In “summer” males that had not been pretreated with PRL and HCG, the ICV injections of 7α-hydroxypregnenolone barely elicited the tail-vibrating behavior, even at a dose of 100 ng (Fig. 1B). Effect of ketoconazole on the expression of courtship behavior In order to determine whether endogenous 7α-hydroxypregnenolone is involved in eliciting the courtship behavior, we tested the effect of ketoconazole, an inhibitor of cytochrome P450s, on male newts captured in spring that were spontaneously exhibiting the courtship behavior in the field. Twenty-four hours after capture, “spring” male newts received an ICV injection of 2 μg of ketoconazole. Control animals received the vehicle only. Every 2 h until 8 h after injection, each male was paired with a test female captured similarly in the field, and courtship behavior was monitored. The suppressive effect of ketoconazole on the expression of the behavior became conspicuous 6 and 8 h after the treatment (Fig. 2A). In another set of experiment, effect of ICV injection of various doses of ketoconazole on the courtship behavior of “spring” males captured in the field was examined. Eight hours after the injection, the behavioral test of each male was performed by introducing a test female captured similarly in the field, and courtship behavior was monitored. ICV injection of ketoconazole decreased both the incidence and frequency of the tail-vibrating behavior. The minimum effective dose of ketoconazole was 2 μg (Fig. 2B). The ability of 7α-hydroxypregnenolone to restore the courtship behavior suppressed by ketoconazole was tested by injecting the male newts that showed suppressed tail-vibrating behavior due to a 2-μg ICV injection of ketoconazole with 100 ng 7α-hydroxypregnenolone at 6 h after the ICV injection of 2 μg ketoconazole. After an elapse of 2 h following ICV administration of the neurosteroid or its vehicle, each male was subjected to the behavioral test. As shown in Fig. 3, the group of newts that had shown suppressed tail-vibrating behavior following injection with 2 μg ketoconazole resumed the tail-vibrating behavior following the subsequent administration of 7α-hydroxypregnenolone. Injection of
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the vehicle only had no effect on the ketoconazole-suppressed tailvibration behavior. Involvement of the dopaminergic system in 7α-hydroxypregnenoloneinduced courtship behavior The effect of dopamine D1- and D2-like receptor antagonists on the expression of courtship behavior was studied to confirm that the action of 7α-hydroxypregnenolone is mediated through the dopaminergic system. Sexually active “spring” males were captured, kept in the laboratory for 24 h, and then injected ICV with the dopamine D1-like receptor antagonist SCH23390 or the D2-like receptor antagonist sulpiride, in a dose of 1 μg in vehicle. The behavioral test revealed that the dopamine D1-like receptor antagonist did not abolish spontaneous expression of the courtship behavior (Fig. 4A), whereas this behavior was suppressed by the D2-like receptor antagonist. The spontaneous behavior arrested by sulpiride was not restored by ICV injection of 100 ng 7α-hydroxypregnenolone when administered simultaneously with the inhibitor (Fig. 4B). Discussion Our study establishes that the neurosteroid 7α-hydroxypregnenolone, which was initially found to elevate the locomotor activity in the male red-bellied newt (Matsunaga et al., 2004), is also effective in enhancing the tail-vibrating behavior uniquely performed by the sexually active male red-bellied newt. The ICV administration of 7αhydroxypregnenolone dose-dependently increased the incidence and frequency of the tail-vibrating behavior in sexually inactive male newts that had been primed with PRL and GTH. In this experiment, care was taken in terms of intervals and duration of the hormonal treatment to ensure that these animals did not spontaneously achieve complete sexual maturity without the exogenously supplied neurosteroid. In contrast,
7α-hydroxypregnenolone was notably ineffective in inducing the behavior in the sexually inactive male newts that had not been pretreated with PRL and GTH, i.e. which had received saline injections. According to Matsunaga et al. (2004), 7α-hydroxypregnenolone elevates the level of locomotor activity in newts captured during the non-breeding period. The differential effectiveness of 7αhydroxypregnenolone for enhancing locomotion versus courtship behavior may come from the fact that the expression of courtship behavior is due to the involvement of multiple hormones (Iwata et al., 2000), whereas locomotor activity is likely to be enhanced by the neurosteroid alone. We have earlier studied the hormonal factors involved in eliciting courtship behavior in intact, hypophysectomized, and castrated newts, respectively, captured in the breeding or non-breeding season (see Kikuyama et al., 2003). PRL was found to promote the expression of the courtship behavior in the male newt, particularly in the presence of androgen (Toyoda et al., 1993). In subsequent studies involving the injection, either ICV or IP, of antiserum against newt PRL and antibody against newt PRL receptor into sexually active male newts, Toyoda et al. (1996, 2005) demonstrated that endogenous PRL acts centrally to elicit courtship behavior. Androgen is another important factor contributing to the expression of courtship behavior. In this context, GTH is regarded as an indirect factor that enhances androgen secretion (Toyoda et al., 1993). An anti-androgen, flutamide, administered by ICV injection to sexually active male newts was found to block the expression of courtship behavior more effectively than flutamide injected IP (unpublished data). This result suggests that androgen acts centrally to enhance the expression of courtship behavior. Once we found that exogenously administered 7α-hydroxypregnenolone was effective in enhancing courtship behavior, we examined whether endogenous 7α-hydroxypregnenolone is involved in the expression of this behavior in sexually active male newts. Ketoconazole,
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Ketoconazole (µg) Fig. 2. Effects of duration of the time after ICV injection of ketoconazole (A) and the dosage of ketoconazole (B) on the incidence and frequency of spontaneously occurring courtship behavior in the spring newt. In the experiment (A), each group consisting of 8 animals received ICV injection of ketoconazole or vehicle. Behavioral test was performed every 2 h until 8 h after the injection. Each point and vertical bar represent the mean and SEM, respectively. The value with an asterisk is significantly different from the respective control value at P b 0.05. In the experiment (B), each group consisted of 11 animals. Each column and vertical bar represent the mean and SEM, respectively. Means with different superscripts are significantly different at P b 0.05.
an inhibitor of cytochrome P450s, reduces the synthesis of 7αhydroxypregnenolone in the newt brain (Matsunaga et al., 2004). As anticipated, we found that ketoconazole suppresses the spontaneously occurring courtship behavior and that subsequent treatment with 7αhydroxypregnenolone reverses this effect. This result clearly indicates
that endogenous 7α-hydroxypregnenolone plays an important role in eliciting courtship behavior. Matsunaga et al. (2004) observed that 7α-hydroxypregnenolone increased the release of dopamine from the brain tissue in culture and that the dopamine D2-like receptor antagonist abolished neurosteroid-
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induced locomotor activity. Based on these results, these authors concluded that the stimulatory effect of 7α-hydroxypregnenolone on the locomotor activity of the newt is mediated by the dopaminergic system. In the present set of experiments, we showed that a D2-like receptor antagonist, sulpiride, but not a D1-like receptor antagonist, SCH2339, abolished the spontaneously occurring courtship behavior by acting centrally. Since even supplementation with an ample amount of 7αhydroxypregnenolone did not reverse the effect of this D2-like receptor antagonist, the neurosteroid is likely to act as an activator of dopaminergic system in this case as well. Considering that the neurosteroid synthesis is enhanced only in the breeding male (Matsunaga et al., 2004) but never in the female (Haraguchi et al., 2010), it may contribute to the rapid encounter of the male with a sexually mature female by increasing the locomotor activity and to the successful transfer of the sperm to the female partner by enhancing the courtship activity. Thus, it is considered that 7α-hydroxypregnenolone may widely activate the motility of the animal and that its action may not be specific for the tail vibration.
Our results suggest that 7α-hydroxypregnenolone can be added to the list of endogenous substances that are involved in the expression of courtship behavior. In terms of their interactions involved in eliciting this courtship behavior, it is of interest to note that PRL appears to act directly on neurosteroidogenic neurons in the preoptic nucleus to regulate the synthesis of 7α-hydroxypregnenolone in the male newt (Haraguchi et al., 2010). It was revealed that hypophysectomy decreases the synthesis of 7α-hydroxypregnenolone in the brain and that administration of PRL to the hypophysectomized newts causes a dose-dependent increase in 7α-hydroxypregnenolone synthesis. Moreover, the presence of the PRL receptor in the preoptic neurons expressing cytochrome P4507α (CYP7B), a steroidogenic enzyme that catalyzes the formation of 7α-hydroxypregnenolone was demonstrated. Therefore, it is likely that at least one of the roles of PRL in eliciting the male courtship behavior is to stimulate 7α-hydroxypregnenolone synthesis. This report is the first to demonstrate that a neurosteroid is involved in enhancing an amphibian sexual behavior. Future studies should be directed towards analyzing the precise roles of the hormones, such as PRL, AVT and androgen as well as of the
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neurosteroid 7α-hydroxypregnenolone and the site(s) of their respective action in eliciting the courtship behavior of the male redbellied newt. Acknowledgments This study was supported by research grants from the Ministry of Education Science and Culture of Japan to F.T. (22570075) and S.K. (21570071). References Baulieu, E.E., 1997. Neurosteroids: of the nervous system, by the nervous system, for the nervous system. Recent Prog. Horm. Res. 52, 1–32. Compagnone, N.A., Mellon, S.H., 2000. Neurosteroids: biosynthesis and function of these novel neuromodulators. Front. Neuroendocrinol. 21, 1–56. Do Rego, J.L., Seong, J.Y., Burel, D., Leprince, J., Luu-The, V., Tsutsui, K., Tonon, M.C., Pelletier, G., Vaudry, H., 2009. Neurosteroid biosynthesis: enzymatic pathways and neuroendocrine regulation by neurotransmitters and neuropeptides. Front. Neuroendocrinol. 30, 259–301. Haraguchi, S., Koyama, T., Hasunuma, I., Vaudry, H., Tsutsui, K., 2010. Prolactin increases the synthesis of 7α-hydroxypregnenolone, a key factor for induction of locomotor activity, in breeding male newts. Endocrinology 151, 2211–2222. Iwata, T., Toyoda, F., Yamamoto, K., Kikuyama, S., 2000. Hormonal control of urodele reproductive behavior. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 126, 221–229. Kikuyama, S., Toyoda, F., Ohmiya, Y., Matsuda, K., Tanaka, S., Hayashi, H., 1995. Sodefrin: a female-attracting peptide pheromone in newt cloacal glands. Science 267, 1643–1645. Kikuyama, S., Tanaka, S., Moore, F.L., 2003. Endocrinology of reproduction. In: Jamieson, B.G.M. (Ed.), Reproductive Biology and Phylogeny. : Reproductive Biology and Phylogeny of Urodela, vol. 1. Science Publishers, Enfield, pp. 275–321. Matsuda, K., Tanaka, S., Yamamoto, K., Kikuyama, S., 1990. Annual changes of plasma prolactin levels in the newt, Cynops pyrrhogaster. Zool. Sci. 7, 1143.
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