Introduction
Melatonin, an indolamine secreted by the pineal gland during the hours of darkness, plays a central role in a variety of important physiological processes, including reproduction (Morgan et al., Reference Morgan, Barrett, Howell and Helliwell1994). In particular, melatonin influences the timing of mammalian circadian rhythms and regulates the reproductive changes that occur in response to hours of daylight in seasonally breeding mammals (for reviews, Reiter, Reference Reiter1991a–Reference Reiterc; Bartness et al., Reference Bartness, Powers, Hastings, Bittman and Goldman1993).
It is well known that melatonin elicits its effects via specific receptors which are coupled to G protein and are characterized by a seven transmembrane-spanning domain (Dubocovich et al., Reference Dubocovich, Masana, Iacob and Sauri1997). To date, three high affinity melatonin receptor subtypes have been identified by molecular cloning studies: MT1 (previously termed mel 1a) and MT2 (mel 1b) melatonin receptors (Reppert et al., Reference Reppert, Godson, Mahle, Weaver, Slaugenhaupt and Gusella1995, Reference Reppert, Weaver, Ebisawa, Mahle and Kolakowski1996), while an additional subtype, mel 1c receptor, that is not expressed in mammals, has been found in Xenopus, chickens and zebrafish (Wiechmann et al., Reference Wiechmann, Campbell and Defoe1999). In addition, there is evidence for a nanomolar melatonin binding site in the brain and kidneys of hamsters (Duncan et al., Reference Duncan, Takahashi and Dubocovich1988; Pickering & Niles, Reference Pickering and Niles1990; Paul et al., Reference Paul, Lahaye, Delagrange, Nicolas, Canet and Boutin1999); the quinine reductase enzymes called MT3.
Melatonin receptors have been detected in several areas of central nervous system, including suprachiasmatic nuclei, hippocampus, cerebellar cortex, prefrontal cortex, basal ganglia, substantia nigra, ventral tegmental area, nucleus accumbens and retinal horizontal, amacrine and ganglion cells (Pandi-Perumal et al., Reference Pandi-Perumal, Trakht and Srinivasan2008).
Apart from the well characterized role of melatonin in the hypothalamus and pituitary, melatonin receptors have been also identified in several peripheral tissues (Pandi-Perumal et al., Reference Pandi-Perumal, Trakht and Srinivasan2008). Among these, an action at testicular level has recently been suggested. In fact, it has been demonstrated that melatonin inhibits testosterone production by Leydig cells of mammals (Olivares et al., Reference Olivares, Valladares, Bustos-Obregón and Núñez1989; Niedziela et al., Reference Niedziela, Lerchl and Nieschlag1995; Valenti et al., Reference Valenti, Guido, Giusti and Giordano1995, Reference Valenti, Thellung, Florio, Giusti, Schettini and Giordano1999; Kus et al., Reference Kuş, Akpolat, Ozen, Songur, Kavakli and Sarsilmaz2002) both in in vivo and in in vitro experiments. Moreover, melatonin binding sites in the gonads of several vertebrate species have been demonstrated (Vera et al., Reference Vera, Tijmes and Valladares1997; Shiu et al., Reference Shiu, Li, Siu, Xi, Fong and Pang2000; Clemens et al., Reference Clemens, Jarzynka and Witt-Enderby2001; Kus et al., Reference Kuş, Akpolat, Ozen, Songur, Kavakli and Sarsilmaz2002) and melatonin receptors coupled via a pertussis toxin-sensitive G-protein are present in adult rat Leydig cells (Valenti et al., Reference Valenti, Giusti, Guido and Giordano1997).
In order to acquire more information concerning the molecular evidence on melatonin receptor(s) in the rat, we investigated the expression of melatonergic receptors in testes and in different tissues. Here we report the cloning of MT1 and, for the first time, MT2 melatonin receptors, other than a melatonin-related orphan H9 receptor (also known as GPR50) (Gubitz & Reppert, Reference Gubitz and Reppert1999) from adult rat testes. Finally we analysed the expression of MT1 and MT2 mRNAs during development.
Materials and Methods
Animals and tissue collection
Sprague–Dawley rats (Rattus norvegicus) were housed under definite conditions (12D:12L) and they were fed standard food and provided with water ad libitum.
Animals at several stages of development, 1-day-, 3-day-, 1-week- and 2-week-old (infantile); 3-week- and 1-month-old (prepubertal); and 3-month-, 6-month-old (adult; three animal/each time) were killed by decapitation under ketamine anesthesia (100 mg/kg i.p.) in accordance with local and national guidelines covering experimental animals. Testes were dissected from rats at each stages, while from adult animals were dissected muscle, adrenal glands, spleen, exorbital glands, brain, lung and testes.
Specimens were collected at noon, according to Sallinen et al. (Reference Sallinen, Saarela, Ilves, Vakkuri and Leppäluoto2005), which showed no significant difference in the expression of MT1 and MT2 receptor mRNAs between midnight and noon. All tissues were quickly frozen by immersion in liquid nitrogen and stored at –80 °C until RNA extraction. In addition, some pieces of these tissues were fixed in Bouin's fluid for histological analysis.
RNA and first strand cDNA synthesis
Total RNA was isolated from different rat tissues according to Chomczynski & Sacchi (Reference Chomczynski and Sacchi1987). The tissues were homogenized in 10 ml lysis buffer (guanidine thiocyanate 4 M, sodium citrate 25 mM pH 7.0, 0.5% sarcosyl and β2-mercaptoethanol 0.1 M) followed by extraction with phenol acid:chloroform:isoamyl alcohol (50:49:1) and by precipitation with isopropanol. The resulting RNA pellet was dissolved in water.
Three microgrammes of total RNA were reverse-transcribed into cDNA using 40 ng random hexameric primers and 100 U Superscript III RT enzyme (Invitrogen) according to manufacturer's instruction.
PCR cloning and sequencing
Based on the published sequence of Rattus norvegicus melatonin receptors (MT1–MT2; EMBL data bank accession number AF130341–XM001074702) and melatonin-related receptor (H9; EMBL data bank accession number U52218) mRNAs, primers were designed to amplify cDNA in different tissues of Rattus norvegicus. For MT1 the primers were as follows: MT1 forward = 5′-CTACATTTGCCACAGTCTC-3′; RMEL1 reverse = 5′-CATATCCTTAAGTAGCAGAAAG-3′. For MT2 the primers were as follows: MT2 forward = 5′-CATGCTCCCCTCTACATCAG-3′ MT2 reverse = 5′-CAGGCGTAGCTTTCTCTCAG-3′. For H9 the primers were as follows: H9 forward = 5′-TCCAGTACAATGCGGATCTTC-3′; H9 reverse = 5′-CAGCAAACTGGTTGTCAGGAT-3′. PCR reactions (25 μl volume) were performed in the presence of 3 μl of cDNA, 1.5 U Taq polymerase (Invitrogen) and its buffer 1×, MgCl2 1.5 mM, dNTP 0.2 mM and 5 pmol of each primer. PCR amplification was carried out for 43 cycles (MT1–MT2) or 30 cycles (H9) with denaturing at 94 °C for 30 s, annealing at 56 °C (MT1) or 58 °C (MT2–H9) for 45 s and extension at 72 °C for 45 s, followed by a final extension at 72 °C for 7 min. Amplification products were electrophoresed on 1.2% agarose. The expected DNA fragments (253 bp for MT1, 267 bp for MT2 and 301 bp for H9) were purified by QIAGEN gel extraction kit (QIAGEN) and cloned into the pGemT easy vector according to the manufacturer's instruction. Sequence determination was performed on both strands with the dideoxynucleotide chain termination method (Sanger et al., Reference Sanger, Donelson, Coulson, Kössel and Fischer1974). The deduced aminoacid sequences were compared with the EMBL Genbank database.
PCR and quantification
The expression of MT1 mRNA in Rattus norvegicus testes at different stages of development, as well as MT1, MT2 and H9 mRNAs in several tissues, were evaluated using a semiquantitative RT-PCR, as described above. An appropriate region (347-bp fragment) of Rattus norvegicus GAPDH transcript (EMBL databank accession number NM_017008), was amplified with specific oligonucleotide primers (GAPDH forward: 5′-GCATCCTGCACCACCAACT-3′, GAPDH reverse: 5′-GCCTGCTTCACCACCTTCTT-3′) and used as control. GAPDH amplification was carried out for 30 cycles with denaturing at 94 °C for 30 s, annealing at 58 °C for 45 s and extension at 72 °C for 45 s, followed by a final extension at 72 °C for 7 min. The expected RT-PCR products were separated by agarose gel (1.2%) electrophoresis and the quantization was performed using GELDOC 1.00-UV fluorescent gel documentation system (BioRAD).
Statistical analysis
Data obtained from three separate sets of RT-PCR experiments were analysed using one-way analysis of variance (ANOVA). If the effects were significant, the Duncan's test was used for post-ANOVA multiple comparisons (p < 0.05). All data are presented as the mean ± standard error mean (SEM).
Results
Expression of MT1, MT2 and H9 mRNA in several rat tissues
Total RNA from several rat tissues (muscle, adrenal glands, spleen, exorbital glands, brain, lung and testes) was subjected to RT-PCR assay using specific primers for the membrane melatonin receptors (MT1 and MT2) and melatonin related receptor (H9).
Agarose gel electrophoresis of RT-PCR products using oligonucleotides for MT1 showed a single cDNA band of the expected size (253 bp) in all the analysed tissues (Fig. 1A). Differently, using oligonucleotides for MT2, a single cDNA band of the expected size (267 bp) was detected in muscle, brain, spleen, exorbital glands and testes, while no specific band was obtained in adrenal glands and lung (Fig. 1C).
Agarose gel electrophoresis of representative RT-PCR products. (A) MT1 mRNA expression in several rat tissues; (C) MT2 mRNA expression in several rat tissues; (E) H9 mRNA expression in several rat tissues. AG = adrenal gland; EX = exorbital gland. (B, D, F) GAPDH expression on the same samples used as control. C = control PCR; M = molecular marker.
Analysis of RT-PCR products using primers for H9, detected a specific band in brain, testes, adrenal glands and lung. This product had the predicted size (301 bp). No specific bands were obtained in the other tissues (Fig. 1E).
For each analysis, the amount of cDNA in each line was controlled using specific primers for the GAPDH (Fig. 1B, D, F).
In Table 1 is reported the different expression of MT1, MT2 and H9 transcripts in the several tissues analysed. All the transcripts are widely distributed in the whole brain as well as in the majority of peripheral rat tissues.
Performance of MT1, MT2 and H9 transcripts in several rat tissues.
Expression of melatonin receptors (MT1, MT2 and H9) in different tissues.
An in situ hybridization analysis was carried out to localize MT1, MT2 and H9 transcripts at testicular level; due to the low expression of the three transcripts in the testes, no signals have been detected (data not shown).
Expression of MT1 and MT2 mRNA in rat testes during development
In order to evaluate a possible role of melatonin in rat testes during post natal development, the expression of melatonin receptor (MT1 and MT2) transcripts was analysed by RT-PCR. The assessment demonstrated the persistent expression of both the mRNAs in rat testes at all the examined time points, from 1 day to 6 months of age (Figs. 2A, 3A). In detail, MT1 and MT2 transcripts were expressed at lower levels in testes of rats from 1 day to 1 week of age, than they lightly increased at 2 weeks of age and remained permanently expressed throughout development until 6 months. The amount of cDNA in each line was controlled using specific primers for GAPDH (Figs. 2B, 3B). In Figures 2C and 3C the relative ratios of the densitometry of Rattus norvegicus MT1 and MT2 cDNA respectively versus GAPDH cDNA are reported (p < 0.05).
Agarose gel electrophoresis of representative RT-PCR products. (A) MT1 mRNA expression in rat testicular samples from 1-day-old, 3-day-old (d), 1-week-old, 2-week-old, 3-week-old (w), 1-month-old, 3-month-old, 6-month-old (m) old rats. (B) GAPDH expression on the same samples used as control. C = control PCR; M = molecular marker. (C) Relative densitometry of the MT1 and GAPDH band measured with Gel Doc. a vs b = p < 0.05.
Agarose gel electrophoresis of representative RT-PCR products. (A) MT2 mRNA expression in rat testicular samples from 1-day-old, 3-day-old (d), 1-week-old, 2-week-old, 3-week-old (w), 1-month-old, 3-month-old, 6-month-old (m) old rats. (B) GAPDH expression on the same samples used as control. C = control PCR; M = molecular marker. (C) Relative densitometry of the MT2 and GAPDH band measured with Gel Doc. a vs b = p < 0.05.
Discussion
The involvement of melatonin in the reproductive process is well documented (Reiter, Reference Reiter1980, Reference Reiter1981; Arendt, Reference Arendt1986; Hazlerigg et al., Reference Hazlerigg, Morgan and Messager2001; Bittman et al., Reference Bittman, Kaynard, Olster, Robinson, Yellon and Karsch1985; Cassone et al., Reference Cassone, Warren, Brooks and Lu1993). Melatonin provokes reproductive alterations in response to changes in hours of daylight in seasonally breeding mammals (Reiter, Reference Reiter1991a–Reference Reiterc; Bartness et al., Reference Bartness, Powers, Hastings, Bittman and Goldman1993). It has been shown that melatonin can exert its regulatory role at various levels of the hypothalamic–pituitary–gonadal axis (Stankov & Reiter, Reference Stankov and Reiter1990; Aleandri et al., Reference Aleandri, Spina and Morini1996; Vanecek & Watanabe, Reference Vanecek and Watanabe1998). The presence of melatonin receptors in the brain's suprachiasmatic nuclei and in the pituitary pars tuberalis indicates the hypothalamic–pituitary axis as the main site through which melatonin may modulate reproductive functions in vertebrates (Anton-Tay et al., Reference Anton-Tay, Chou, Anton and Wurtman1968; Weaver, Reference Weaver1999). However, it has been determined that pineal-derived circulating melatonin is taken up by peripheral tissues including the testes (Wurtman & Axelrod, Reference Wurtman and Axelrod1966; Cardinali et al., Reference Cardinali, Vacas and Boyer1979; Reiter, Reference Reiter1981, Reference Reiter1991b). In addition, the ability of the testes to locally synthesize melatonin in a non-photoperiodic mammal (rat) and a bird (quail) has also been demonstrated (Tijmes et al., Reference Tijmes, Pedraza and Valladares1996; Kato et al., Reference Kato, Fu, Kotera, Sugahara and Kubo1999; Fu et al., Reference Fu, Kato, Kotera, Noguchi, Sugahara and Kubo2001; Stefulj et al., Reference Stefulj, Hörtner and Ghosh2001).
Many authors have demonstrated the existence of a mutual relationship between the pineal gland and the testes (for review see Reiter, Reference Reiter1980, Reference Reiter1981). In vitro experiments in hamster (Niedziela et al., Reference Niedziela, Lerchl and Nieschlag1995) and rat (Valenti et al., Reference Valenti, Guido, Giusti and Giordano1995) indicated that melatonin inhibits testosterone secretion by Leydig cells, while Wu et al. (Reference Wu, Chiao, Hsiao, Chen and Yen2001) observed that melatonin suppresses steroidogenesis through specific binding sites in hCG/cAMP analogue-treated MA-10 mouse Leydig cells.
Moreover, the characterization of melatonin receptors in testes has been mainly assayed by radioreceptor assay using 2-[125I]-iodomelatonin-binding analysis (Vera et al., Reference Vera, Tijmes and Valladares1997; Shiu et al., Reference Shiu, Li, Siu, Xi, Fong and Pang2000; Clemens et al., Reference Clemens, Jarzynka and Witt-Enderby2001); recently Frungieri et al. (Reference Frungieri, Mayerhofer, Zitta, Pignataro, Calandra and Gonzalez-Calvar2005), using RT-PCR and western blot techniques, have demonstrated the presence of MT1 but not MT2 sites in hamster testes. In addition, Reppert et al. (Reference Reppert, Weaver, Ebisawa, Mahle and Kolakowski1996) have cloned an orphan G protein-coupled receptor, designed H9 and also known as GPR50 (Gubitz & Reppert, Reference Gubitz and Reppert1999), from a human pituitary cDNA library. The H9 cDNA encodes a protein that is 45% identical at the amino acid level to human MT1 and MT2 melatonin receptors. It has also been reported an extensive and detailed analysis of melatonin-related receptor mRNA expression in the mouse central nervous system and various peripheral tissues including testes; the results suggested for this type of receptor a conserved function in neuroendocrine regulation and a potential role in coordinating physiological response in the CNS and peripheral tissues (Drew et al., Reference Drew, Barrett and Mercer2001). In this respect, it is also to be highlighted that rat testis development is regulated by maternal melatonin as well as by the beginning of the own melatonin rhythm of the offspring (Diaz et al., Reference Diaz, Diaz, Colmenero, Arce, Esquifino and Marìn1999).
The present study provides evidence for the existence of both MT1 and MT2 melatonin receptor transcripts other than the H9 melatonin-related receptor transcript, in adult rat testes and during development.
As shown in Table 1, our results indicate that MT1, MT2 and H9 transcripts are widely distributed in the whole brain as well as in peripheral rat tissues (muscle, adrenal glands, spleen, exorbital glands, lung and testes). Interestingly, MT1 is mostly expressed in the majority of peripheral tissues while MT2 is expressed in the above mentioned tissues with the exception of the adrenal glands and lung (Fig. 1). MT1, MT2 and H9 receptors are coexpressed in the brain, which represents the main target of melatonin action and, surprisingly, in the testes of the rat (Table 1).
The in situ hybridization performed in rat testes failed to show the localization of MT1, MT2 and H9 mRNAs probably because of the low expression of their mRNAs in the majority of the tissues examined (data not shown).
Lastly, RT-PCR analysis showed that the level of MT1 and MT2 transcripts increased in the testes of rats at 2 weeks of age and remained permanently expressed until six months. These results, in accordance with those previously reported by Diaz et al. (Reference Diaz, Diaz, Colmenero, Arce, Esquifino and Marìn1999), support the idea that melatonin exerts a role during rat development; in particular, the increasing levels of receptors mRNAs at the onset of puberty suggest that the expression of these receptors in the pups is mainly associated to their own melatonin rhythm, fully established in this period.
Until now, the only molecular evidence of melatonin receptor subtypes in vertebrate testes is MT1 described by Frungieri et al. (Reference Frungieri, Mayerhofer, Zitta, Pignataro, Calandra and Gonzalez-Calvar2005) in hamsters. The presence of MT1, MT2 and H9 transcripts in the rat testes strongly supports a role of the indolamine in the male gonads.
It is worth remembering that the physiological role of melatonin in the testes is still not fully understood. Probably the indoleamine represents an as-yet-unrecognized local inhibitory control of the testicular function. An inhibitory role of melatonin in vertebrate testicular activity is supported by many studies. Ultrastructural changes in Leydig cells have been demonstrated in rat after pinealectomy (Kus et al., Reference Kus, Sarsilmaz, Ogetürk, Yilmaz, Keleştimur and Oner2000) and in mice after melatonin treatment for 22 consecutive days (Redins et al., Reference Redins, Redins and Novaes2002). A prolonged exposure to indolamine reduces the number and affinity of melatonin receptor binding sites on rat Leydig cell membranes and causes hypersensitization to LH challenge, resulting in higher cAMP and testosterone secretion (Valenti et al., Reference Valenti, Fazzuoli, Giordano and Giusti2001). In addition, our recent studies, using the testes of the frog Rana esculenta as a model, support the hypothesis of an inhibitory role exerted by melatonin in vertebrate testes. In fact, we highlighted that melatonin inhibits both the cellular proliferation of primary spermatogonia (d'Istria et al., Reference d'Istria, Palmiero, Serino, Izzo and Minucci2003) and the proliferation and/or differentiation of mast cells (Izzo et al., Reference Izzo, d'Istria, Serino and Minucci2004) induced in vivo and in vitro by 17β-estradiol. In addition, our observations indicate that melatonin might act on Leydig cells, as after in vivo or in vitro melatonin treatments many Leydig cells display degenerative morphological changes (d'Istria et al., Reference d'Istria, Serino, Izzo, Ferrara, De Rienzo and Minucci2004).
This study provides molecular evidence of the presence of both MT1 and, for the first time, MT2 melatonin receptors other than of H9 melatonin-related receptor in adult rat testes and during development. These data strongly support the hypothesis that melatonin acts directly in male vertebrate gonads. However, further studies are required to assess the biological relevance of melatonin in testicular regulation. Consequently, a suitable model to verify the role of indolamine in vertebrate testicular activity may be provided by rat testes.
Acknowledgements
We thank Dr Janet Gates for revision of the manuscript and Mrs Antonella Pennino for technical assistance. This work was supported by grants from Ricerca di Ateneo ‘Seconda Università di Napoli’ and from Regione Campania ‘L.R. n.5, 2006’.
