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Regular Article |
a Department of Biology, School of Education, Waseda University, Shinjuku-ku, Tokyo 169-8050, Japan
b Laboratory of Biological Chemistry, Showa University School of Pharmaceutical Sciences, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
c Department of Anatomy, Showa University School of Medicine, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
ABSTRACT
Silefrin is a sodefrin-like, female-attracting pheromone comprising 10 amino acids that was isolated from the abdominal gland of the sword-tailed newt, Cynops ensicauda. Hormonal effects on the silefrin precursor mRNA expression and silefrin content in the abdominal gland were investigated in the present study by using Northern blot analysis and radioimmunoassay, respectively. In the abdominal gland of newts treated with prolactin (PRL) plus testosterone propionate (TP), silefrin precursor mRNA expression was markedly enhanced as compared with that in the newts injected with saline, PRL, or TP. Values for radioimmunoassayable silefrin content in the abdominal gland paralleled those for the silefrin precursor mRNA levels. Moreover, silefrin precursor mRNA signals, as revealed by in situ hybridization, as well as stainability of immunoreactive silefrin were much more intense in the epithelial cells of the abdominal gland of the PRL-plus-TP-treated animals than in those of controls. We thus conclude that PRL and androgen are important factors for enhancing silefrin synthesis.
pheromones, prolactin, reproductive behavior, testosterone
INTRODUCTION
In the majority of urodeles, fertilization takes place internally by means of spermatophores transferred from the male to the female. Because they have no specific copulatory organs, the male is required to persuade the female to successfully transfer the spermatophores into her cloaca by courtship [1]. Although there are considerable variations in the courtship pattern among species and/or genera, the behavior in newt species belonging to the genera Cynops and Triturus is common in that the male does not capture or grasp the female during courtship, the contact between them being minimal. The male accesses the female's cloaca with his snout, and if he recognizes that the female is in a sexually developed state, the male blocks her path and vibrates his tail to direct water around the cloaca toward the female's snout. During this initial stage of courtship, the male projects numerous minute tubules that are connected to the abdominal gland of the cloaca [2, 3], suggesting that the male newt releases pheromonal substances to attract the female.
There is good reason to believe that the abdominal gland is one of the sources of female-attracting pheromones; males of Cynops pyrrhogaster and Triturus cristatus with their abdominal glands ablated are less able to attract females than intact ones [4, 5]. Electrophysiological study has also shown that the olfactory mucosa of the T. cristatus female is activated by an extract of the male's abdominal gland [6]. This prompted us to isolate the active substance from the abdominal gland of newts C. pyrrhogaster and C. ensicauda. As a result, we obtained female-attracting decapeptide pheromones, sodefrin and silefrin, from C. pyrrhogaster and C. ensicauda, respectively. Both of these pheromones comprise 10 amino acids, with two amino acid variations of each other at positions 3 and 8 [7, 8]. It has been confirmed that each pheromone is effective in attracting only conspecific females. In addition, cloning of cDNAs encoding these peptides revealed that both peptides were derived from their precursor proteins [9].
Prolactin (PRL) and sex steroids are known to play important roles in a series of reproductive events in urodeles [10]. Involvement of PRL and of androgen in the expression of courtship behavior has been demonstrated in T. cristatus [11, 12] and C. pyrrhogaster [13, 14]. In the Cynops male, development of the lateral gland (which secretes substances) that constitutes the spermatophore sac and of the abdominal gland (which secretes sodefrin) is dependent on PRL and testosterone [15]. In the Cynops female, responsiveness of the nasal sinus, a putative vomeronasal organ, to sodefrin is elevated markedly by the combination of PRL and estradiol [16]. Development of oviducts that secrete jelly substances that coat the eggs is also dependent on PRL and estradiol [17].
In this paper, we describe that PRL, in combination with androgen, enhances the synthesis of silefrin, judging from the elevation of both the silefrin precursor mRNA level as assessed by Northern blot analysis and in situ hybridization, and the immunoassayable silefrin content in the abdominal gland of C. ensicauda.
MATERIALS AND METHODS
Animals and Treatments
Adult male newts of C. ensicauda weighing about 6–7 g were purchased from a dealer, kept in a tank at 22°C under a 12L:12D illumination condition, and fed with Tubifex worms. After 1 wk of acclimation, hypophysectomy was performed under anesthesia with 0.1% m-aminobenzoic acid ethylester methanesulfonate (MS222; Sankyo Co., Tokyo, Japan). One day after the operation, hormone treatment was commenced. Ovine PRL (Sigma Chemical Co., St. Louis, MO) was dissolved in saline, and testosterone propionate (TP; Sigma) was dissolved in a minute volume of ethanol and then suspended in saline. Animals were divided into four groups, each consisting of eight animals, and were administered saline, 1 IU PRL, 5 µg TP, or a combination of 1 IU PRL and 5 µg TP every other day for 2 wk. The injection volume was 50 µl in each case. Doses for PRL and TP and duration of the hormone treatment were determined on the basis of our previous experiments [14, 15, 18]. On the day after the last injection, each bilobal abdominal gland was dissected out, weighed, and divided into two single lobes. One lobe was used for Northern blot analysis, and the other was subjected to RIA for silefrin content. For immunohistochemistry and in situ hybridization, abdominal glands were obtained from another batch of hypophysectomized animals injected with saline or with a combination of PRL and TP, as described earlier.
Northern Blot Analysis
Silefrin precursor mRNA was assessed by Northern blot analysis with silefrin precursor cDNA [9] as a probe. Total RNA (3 µg) extracted from newts injected with saline or PRL and/or TP was electrophoresed in denaturing agarose gels (2.2 M formaldehyde) and transferred to a nylon membrane. The RNA was fixed on the membrane by ultraviolet cross-linking. The membrane was prehybridized for 3 h at 65°C in hybridization solution consisting of 6x standard saline citrate (SSC; 0.15 M NaCl and 15 mM sodium citrate), 0.2% (w/v) BSA, 0.4% (w/v) Ficoll 400, 0.4% (w/v) polyvinyl-pyrrolidone, and 1% (w/v) SDS. Hybridizations with the radiolabeled cDNA were performed for 16 h at 65°C after adding the probe to the pre[chhybridization solution. The cDNA was labeled by the random-priming method with a Bcabest labeling kit (TaKaRa, Tokyo, Japan) according to the manufacturer's instructions. The filter was washed with 10x SSC containing 0.1% SDS for 30 min at 65°C and placed in contact for 30 min at room temperature with the imaging plate of a BAS-2000II (FujiFilm, Tokyo, Japan). Densitometry was performed on the Northern blot autoradiographs with a BAStation (FujiFilm). After removal of the label by boiling in 0.1x SSC/0.1% SDS for 10 min, rehybridization was achieved with a newt ß-actin cDNA. For normalization, the densitometry values for the silefrin precursor mRNA were divided by those for the ß-actin mRNA.
Radioimmunoassay of Silefrin
Silefrin was synthesized (American Peptide, Sunnyvale, CA) according to the amino acid sequence deduced from the nucleotide sequence of a cDNA previously cloned [9]. For radioiodination, silefrin with a tyrosine extension on its N terminus (Tyr-silefrin) was synthesized (American Peptide). For antibody production, a silefrin that had been extended on its C terminus with a cysteine residue (American Peptide) was coupled to keyhole limpet hemocyanin (Pierce, Rockford, IL) with m-maleimidobenzoyl-N-hydroxysuccinimide ester. An antiserum against silefrin was generated in a rabbit by means of the lymph node injection technique, as described previously [8, 18]. The radioiodination of Tyr-silefrin was carried out at room temperature according to the modified lactoperoxidase method described elsewhere [8, 16]. The specific radioactivity of the radioligand, calculated by the method of Greenwood et al. [19], was about 0.3 MBq/µg. The RIA was carried out according to the procedure described elsewhere [8]. Intra- and interassay coefficients of variation were 2.7 and 3.8%, respectively, and the sensitivity of this assay was 34.3 pg of silefrin standard per 100 µl assay buffer.
In Situ Hybridization
The tissue was fixed with 4% paraformaldehyde in PBS overnight. After cryoprotection with 20% sucrose and embedment in O.C.T. compound (Tissue-Tek, Miles Inc, IN), the fixed tissue was rapidly frozen with liquid nitrogen. Frozen sections were cut in 10-µm slices with a Leitz 1720 digital cryostat (Ernst Leitz, Midland, ON, Canada) and placed on 3-aminopropyltriethoxyxilane-coated slides. The sections were treated with 0.2 N HCl for 20 min to quench free aldehyde groups and then washed sequentially with PBS for 5 min and with 2x SSC and 0.1% sarkosyl for 10 min. Next, they were prehybridized for 3 h at room temperature in prehybridization solution consisting of 50% formamide, 0.1 M Tris, 4x SSC, 1x Denhardt solution, 1 mM EDTA, 2% sarkosyl, and 0.25 mg/ml salmon sperm DNA. Then, the sections were incubated overnight at room temperature with 1 x 106 dpm 35S-labeled probe or the labeled probe containing excessive nonlabeled probe for negative control in hybridization buffer containing 10% dextran sulfate and 1.4% ß-mercaptoethanol in prehybridization solution. The sections were washed in 2x SSC and 0.1% sarkosyl at room temperature for 30 min and in 0.1x SSC and 0.1% sarkosyl at 50°C for 80 min, dehydrated through a graded series of ethanol (70–100%), and then air dried. The sections were exposed to X-ray film (Hyperfilm ßmax; Amersham Pharmacia Biotech Inc., Piscataway, NJ) for 1 wk, dipped in nuclear track emulsion (NTB2; Eastman Kodak Co., Rochester, NY) at 42°C, air dried, and then exposed for 1 wk. They were then developed in Kodak D 19 developer (at 20°C for 2 min), fixed in Kodak Rapid Fixer, counterstained with hematoxylin and eosin, and then coverslipped before being analyzed with a microscope.
Immunohistochemistry
The fixation and embedment of the abdominal gland were performed as described above. Frozen sections were cut at a thickness of 10 µm with a Leitz 1720 digital cryostat (Ernst Leitz) and were placed on gelatin-coated slides. The sections were treated with 50 mM NH4Cl in PBS for 20 min to quench free aldehyde groups and were subsequently washed with PBS. The sections were then incubated first with 20% normal goat serum for 30 min and then with anti-silefrin diluted 1:1000 with 1% BSA-PBS or with antiserum (1:1000 in 1 ml) preadsorbed with silefrin (10 µg/ml). After the sections had been washed with PBS, they were incubated with rhodamine-labeled, affinity-purified goat antibody to rabbit IgG (Jackson Immunoresearch, West Grove, PA), diluted with PBS, and coverslipped using Perma Fluor (Lipshaw Immunon, Pittsburgh, PA).
Statistical Analysis
The significance of the results was assessed by analysis of variance and Duncan's multiple range test. A P value of less than 0.05 was considered significant.
RESULTS
Effects of PRL and/or TP on silefrin precursor mRNA expression in the abdominal gland of the hypophysectomized male C. ensicauda were investigated by Northern blot analysis. In the group treated with the combination of PRL and TP, the silefrin precursor mRNA expression was significantly higher than that in the control animals (Fig. 1). On the other hand, the mRNA levels in the group treated with either PRL or TP were not statistically different from those in the saline-injected control.
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Figure 2 shows the effect of PRL and/or TP on the silefrin content in the abdominal glands from hypophysectomized newts. The immunoreactive silefrin content in the TP-treated group was significantly higher than that in the controls. However, PRL alone did not noticeably increase the silefrin content. A combination of both hormones brought about a marked increase in the pheromone content.
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The effect of PRL and TP on the expression of silefrin precursor mRNA in the abdominal glands of male C. ensicauda was investigated by in situ hybridization. In the abdominal glands of PRL plus TP-treated newts, enlargement of the gland was noted, and intense mRNA signals were observed in the epithelial cells (Fig. 3a). As a negative control experiment, hybridization was performed with the probe added with an excess of nonlabeled silefrin precursor cDNA. No specific signals were seen in the epithelial cells in this case (Fig. 3b). In the abdominal glands of saline-injected newts, the mRNA signals were much weaker than those in the PRL plus TP-treated newts (Fig. 3c) and were rather close to those in the negative-control sections in terms of intensity (Fig. 3d).
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Using the antiserum against silefrin, we demonstrated immunoreactive silefrin in the epithelial cells of the abdominal gland. In the glands from PRL plus TP-treated newts, immunofluorescent silefrin was abundantly present in the epithelial cells (Fig. 4a). In the saline-injected group, the glands consisted of ducts with an extremely small lumen (Fig. 4b'), and the immunofluorescence in the epithelial cells was weak (Fig. 4b) as compared with that for the PRL-plus-TP-treated group. Antiserum preadsorbed with an excess of silefrin profoundly reduced the immunofluorescence seen in the saline-injected group (Fig. 4c).
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DISCUSSION
Prolactin and androgen are known to be involved in various phenomena related to urodele reproduction. Prolactin is believed to induce migration to water, where reproduction takes place [20]. Recently, endogenous PRL was demonstrated to be prerequisite for the aquatic life of C. pyrrhogaster [21]. Immunoneutralization of the circulating PRL by injecting antiserum against newt PRL to the aquatic newts resulted in the migration to the land from the water.
Sexually active Cynops and Triturus males vibrate their tails vigorously in front of a female partner during the early stage of courtship. Administration of PRL and androgen elicits this behavior in the sexually inactive male newts [12, 14]. Evidence for the involvement of endogenous PRL in courtship behavior was also provided by the finding that both the incidence and frequency of the behavior occurring spontaneously during the breeding season declined after the administration of antiserum against PRL [21].
Tail fin growth of several species of urodeles is also stimulated by PRL [17, 22, 23]. A broad tail with a well-developed fin is effective in the male for sending the courtship pheromonal substance by a water stream toward the female's snout. It is of interest to note that estradiol inhibits the PRL-induced tail fin growth [17].
Mauthner cells in urodeles are located at the level of the VIIIth cranial nerve root in the medulla oblongata. Electrophysiological, neuroanatomical, and behavioral studies revealed that Mauthner cells are involved in the tail movement [24, 25]. Prolactin plus testosterone induces enlargement of both the cell bodies and nuclei of Mauthner cells [26] and increases the number of synapses on somata of Mauthner cells [27]. Thus, PRL plus testosterone not only supports the pheromone production but also contributes to the distribution of the pheromone by enhancing the Mauthner cell function.
It is well known that testosterone secreted by the testis is often aromatized to estradiol. Therefore, it cannot be denied that some of the above-mentioned instances are mediated through estradiol. As regards the induction of courtship, however, testosterone seems to act directly to elicit the courtship behavior because dihydrotestosterone, but not estradiol, administered together with PRL enhances both the incidence and frequency of the behavior [14].
Elsewhere, we obtained a result that the combination of PRL and TP increased the immunoassayable sodefrin content in the abdominal gland of hypophysectomized and castrated C. pyrrhogaster [18]. In order to study the effects of PRL and/or testosterone on the abdominal gland function in C. ensicauda, we used hypophysectomized specimens with testes left intact instead of hypophysectomized and castrated ones. It has been confirmed that there is little difference in the histological feature in the abdominal gland between the hypophysectomized and castrated newts and the hypophysectomized ones [15]. Both exhibit similar degenerative changes in the glandular structure. By the present experiment, it was revealed that PRL and testosterone act synergistically on the abdominal gland of C. ensicauda to cause the enhancement of silefrin precursor mRNA expression and the increase of silefrin content as well as the structural development of the abdominal gland. This indicates that PRL plus testosterone enhances the syntheses of the pheromone.
When annual changes in the plasma levels of PRL and testosterone were studied in C. pyrrhogaster [28, 29], the levels of both hormones were found to be high during the spring breeding season. Likewise, a parallel elevation of PRL and testosterone levels in T. carnifex during the winter breeding season was reported by Zerani et al. [30]. The presence of receptors for androgen [31] and PRL [32] in the abdominal gland was demonstrated recently. In mammals, PRL-sex steroid hormone interaction has been studied at their receptor levels. It has been reported that in the rat prostate, PRL-receptor mRNA expression is enhanced by testosterone, estradiol, and PRL [33]. In the rat mammary gland, PRL-receptor mRNA is increased by estradiol and PRL [34]. In the rat corpus luteum, lactogenic hormones (PRL and placental lactogens) elevate the expression of estrogen receptors 
and ß [35]. The mechanisms of synergistic action of PRL and androgen on various organs in urodeles, however, have been scarcely understood. In this sense, the abdominal gland will be a good model for studying the PRL-androgen interaction at their receptor levels.
ACKNOWLEDGMENTS
We thank Professor K. Wakabayashi, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, for supplying the goat anti-rabbit IgG serum (HAC-RBA2–05GTP91).
FOOTNOTES
1 This work was supported in part by a grant-in-aid from the Ministry of Education, Science, and Culture of Japan and by research grants from Waseda University, Asahi Glass Research Foundation, and Uehara Memorial Life Science Foundation. 
2 Correspondence: Sakaé Kikuyama, Department of Biology, School of Education, Waseda University, Nishi-waseda 1-6-1, Shinjuku-ku, Tokyo 169-8050, Japan. FAX: 81 3 3207 9694; kikuyama{at}mn.waseda.ac.jp
Accepted: August 3, 2000.
Received: May 1, 2000.
REFERENCES
-dihydrotestosterone levels in the adult male newts, Cynops pyrrhogaster. Endocrinol Jpn 1983; 21:1–6. and ß in the rat corpus luteum of pregnancy: regulation by prolactin and placental lactogens. Endocrinology 1998; 139:2432–2442.
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