Results

3.1. Isolation of Two Putative Melatonin Receptor Fragments

The PCR amplification of pike genomic DNA using degenerate primers allowed to obtain fragments of the appropriate length (504 bp), which were cloned and sequenced. Of 20 clones analyzed, 2 putative melatonin receptor fragments—P4 and P8—were identified which are 70% identical (Figure 2). Among all the cloned melatonin receptors, P4 is most similar to the zebrafish Mel1a melatonin receptor (Z1.4), with which it shares 84% amino acid identity. P8 displays similarities with the zebrafish Melib melatonin receptor (Z2.6), with which it shares 79% amino acid identity.

3.2. Expression of P4 and P8 mRNA

P4 and P8 mRNA distribution was examined by northern blot analysis in several brain areas, including the pituitary as well as in the liver and retina. Hybridization with the P8 probe revealed the presence of one transcript (approximately 5.4kb) highly expressed in the optic tectum (Figure 3). No hybridization signal was apparent in other brain areas, pituitary or liver, either with the P8 or with the P4 probe (not shown).

The expression of the P4 and P8 genes was also examined using a RT-PCR assay (Figure 4). No amplification was detected in the controls, thereby indicating that genomic DNA was not amplified. The P4 labeled mRNA was expressed in several brain areas and the retina. Actin served as a control to verify the amount of template for each sample. Although no precise quantification could be done, our results suggest high levels of expression in the optic tectum and diencephalon. Noticeable expression was also found in the olfactory bulbs, telencephalon and retina whereas threshold levels were detected in the pituitary and the cerebellum. The P8 transcript was detected in the same tissues but the relative pattern of expression was apparently different. A high expression was found in the optic tectum and telencephalon, and a much lower expression was apparent in the pituitary, olfactory bulbs, diencephalon and cerebellum. A very low hybridization signal was obtained in the retina. No expression was detected in the ovaries, liver and intestine with either probe.

3.3. Binding of 125I-Melatonin in Pituitary Sections and Membranes

In vitro radioautography on pituitary sections indicated that the labeling did not cover the whole surface of the gland (Figure 5). Rather, it appeared as a moon quarter in the antero-ventral part. Alternative sections incubated in the presence of an excess (1 ^M) of melatonin displayed absolutely no binding.

To study the binding of 125I-melatonin on pituitary membranes we used the parameters set up for the binding on membrane preparations from the brain (11). Under these conditions, 125I-melatonin bound in a saturable fashion to pituitary membranes (Figure 6). Scatchard re-plot of the data indicated that Bmax was 2.8 fmol/mg prot. and Kd was 556 pM. Nonspecific binding increased linearly with increasing 125I-melatonin concentrations.

4. DISCUSSION

This study reports the cloning of two receptor fragments (P4 and P8) from the pike that contain structural motifs typical of the G protein-coupled melatonin receptor family. The comparison of their deduced amino-acid sequences with the sequences from the zebrafish melatonin receptors (29) supports the view that the P4 and P8 fragments correspond, respectively, to the MeL and MeU receptor subtypes. Northern blot analysis indicated that expression of the MeU was high in the optic tectum. No

TMS-IV

Pike P4

Zebrafish 21.4 MEL1A Chicken MEL1A Human MEL1A Consensus

CHSMKYNKLF SDRNTICYVS LVWVLTILAI APNWFAESLQ YDPRVYSCTF CHSLKYDKLF SNKNTVCYVI LVWALTVLAI VPNWFVESLQ YDPRVFSCTF CHSLKYDKLY SDKNSLCYVG LIWVLTWAI VPNLFVGSLQ YDPRIYSCTF CHSLKYDKLY SSKNSLCYVL LIWLLTLAAV LPNLRAGTLQ YDPRIYSCTF

LQ YDPR scir

1 QO

XHS-V

Pike P4 AQSVSSLYTI TWWHFILP ISIVTYCYLR IWILVIHVRR RVKPDTRTKL Zebrafish Z1.4 MEL1A AQSVSSLYTI MVWVHFIVP IGIVTYCYLR IWILVIQVPR RVKPDSRPKI

Chicken MEL1A AQSVSSAYTI AWFFHFILP IAIVTYCYLR IWILVIQVRR RVKPDNNPRL

Human MEL1A AQSVSSAYTI AWVFHFLVP MIIVIFCYLR IWILVLQVRQ RVKPDRKPKL

Consensus

AQSVSS YTI W HT P IV CYLR IWILV V RVKPD

7Kfl

tmc-VT

TMS-

Pike P4

Zebrafish Z1.4 MEL1A Chicken MEL1A Human MEL1A Consensus

KPHDLRNFLT MFWFVLFAV CWAPLNFIGL AVAINPR.LG LNIPEWLFTA KPHDFRNFLT MFWFVLFAV CWAPLNFIGL AVAIHPR.LG QSIPEWLFTA KPHDFRNFVT MFWFVLFAV CWAPLNFIGL AVAVDPETII PRIPEWLFVS KPQDFRNFVT MFWFVLFAI CWAPLNFIGL AVASDPASMV PRIPEWLFVA

KP D RNT I MFWFVLFA CWAPLNFIGL AVA P

IPEWLF

Pike P4

Zebrafish Z1.4 MEL1A Chicken MEL1A Human MEL1A Consensus

VII SYF SYF SYY SYY SY

TMS-IV

Pike P8

Zebrafish Z2.6 MEL1B Chicken MEL1B Human MEL1B Consensus

CHSFSYDKFY SYRNTLLLVA LIWLLTILAI IPNFFVGSLQ YDPRVYSRTF CHSFAYGRLC SFRNTLLLVA LIWALTVLAI LPNFFVGSLS YDPRVYSCTF CHSFAYDKVY SCWNTMLYVS LIWVLTVIAT VPNFFVGSLK YDPRIYSCTF CHSMAYHRIY RRWHTPLHIC LIWLLTWAL LPNFFVGSLE YDPRIYSCTF

LIW LT A PNFFVGSL YD PR YS TF

TMS-V

Pike P8

Zebrafish Z2.6 MEL1B Chicken MEL1B Human MEL1B Consensus

AQAVSTSYTI TVWIHFIVP IAWTFCYLR IWILVIQVRR KVKSEVRPRL TQTASSSYTV VWWHFLVP IAWTFCYLR IWVLVIQVRR KVKSEERSRV VQTASSYYTI AVWIHFIVP ITWSFCYLR IWVLVLQVRR RVKSETKPRL IQTASTQYTA AWVIHFLLP IAWSFCYLR IWVLVLQARR KAKPESRLCL Q S YT VW HF P I W FCYLR IW LV Q RR K Z

Pike P8

Zebrafish Z2.6 MEL1B Chicken MEL1B Human MEL1B Consensus

vT MÏWEVÎ.PAÏ ôtfGftiMPïéL AVAI'DPERVA PRIPE'

KPSDMRNFVT

RPSDLRNFVT MFWFVLFAI CWAPLNLIGL WAINPEVMA PRVPEWLFW KPSDFRNFLT MFWFVIFAF CWAPLNFIGL AVAINPSEMA PKVPEWLFII KPSDLRSFLT MFWFVI FAI CWAPLNCIGL AVAINPQEMA PQIPEGLFVT PSD R F T MFWFV FA CW PLN ISL VAI P A P PE LF

Pike P8

Zebrafish Z2.6 MEL1B Chicken MEL1B Human MEL1B Consensus

VII SYF SYF SYF SYL SY

Figure 2. Deduced amino acid sequences of pike P4 and P8 fragments and their comparison with the Zebrafish, Chicken and Human melatonin receptors. Genbank accession numbers are U31823 (Zebrafish Z1.4), U31824 (Zebrafish Z2.6), U31820 (Chicken Mel,,), U30609 (Chicken MeU), U14108 (Human Mel,.) and U25341 (Human Mel1b). TMS: transmembrane domain.

Pituitary Optic Tectum

Figure 3. Expression of P8 putative (Mel1b) melatonin receptor mRNA assessed by northern blot analysis. Each lane contained 2.5^g of poly(A)+RNA. The lower portion of the blot depicts the hybridization 7 i>Q — pattern obtained with an actin probe to verify equal j __loading of lanes. The blot was exposed to X-ray film for one week at -80°C.

hybridization signal was detected in other brain areas with either probe, suggesting that the levels of expression were low. However, RT-PCR analysis, using specific primers, indicated that all the brain areas expressed the Mel1a and Mel1b subtypes. Radioautographic studies also report a widespread distribution of the 125I-melatonin binding sites in the brain of the goldfish (21), the trout (3), the salmon (8) and the pike (not shown). Our data agree with previous investigations which showed that 125I-melatonin could bind to membrane preparations from pike brain (11). Binding was saturable reversible and sensitive to GTP, suggesting the presence of G-protein coupled melatonin receptors. In addition, displacement experiments suggested the presence of two components with affinities in the femtomolar and nanomolar range of concentrations respectively (11).

The interesting finding of this study was that the P8 probe and, to a lesser extent, the P4 probe allowed to evidence some expression in the pituitary. To determine whether this expression was correlated with the presence of melatonin binding sites, we investigated the binding of 125I-melatonin to sections and membranes. Indeed, 125I-melatonin bound in a saturable manner to pike pituitary membranes. The dissociation constant was within the range of that found with the brain membranes (11). In contrast, the number of binding sites was 10- to 15-fold lower in the pituitary than in the brain. This resulted in a low signal-to-noise ratio in the pituitary. Tissue sections from the pituitary exhibited a specific binding, but localized only in the antero-ventral part of the gland. Our results contrast with previous investigations indicating that no melatonin binding sites are present in the pituitary of nonmammalian vertebrates

Figure 4. Tissue-specific distribution of P4 and P8 melatonin receptor mRNA assessed by RT-PCR. Sample (3 ^g) of each tissue total RNA was subjected to reverse transcriptase using Dynabeads oligo(dT)25 followed by PCR using two additional oligonucleotides (4S2-4A2 or 8S2-8A2) as described in Materials and Methods and in Fig. 1. One percent of the amplified cDNA products were Southern-blotted and probed with a 32P-labeled specific melatonin receptor subtype fragment internal to the PCR fragment (+). For each tissue, a control group, in which reverse transcriptase was omitted, was processed in parallel (-). Actin served as a control to verify the amount of template for each sample.

Figure 4. Tissue-specific distribution of P4 and P8 melatonin receptor mRNA assessed by RT-PCR. Sample (3 ^g) of each tissue total RNA was subjected to reverse transcriptase using Dynabeads oligo(dT)25 followed by PCR using two additional oligonucleotides (4S2-4A2 or 8S2-8A2) as described in Materials and Methods and in Fig. 1. One percent of the amplified cDNA products were Southern-blotted and probed with a 32P-labeled specific melatonin receptor subtype fragment internal to the PCR fragment (+). For each tissue, a control group, in which reverse transcriptase was omitted, was processed in parallel (-). Actin served as a control to verify the amount of template for each sample.

Figure 5. 2-[125I]iodomelatonin binding sites in the pituitaries revealed by in vitro radioautography. Images were produced on Xray films by 20^m coronal tissue sections through pituitaries incubated with l00pM of 2-[125I]iodomelatonin. Sections incubated in the presence of an excess (1 ^M) of melatonin were devoid of any labeling (not shown).

including fish (refs in 2,11). The difficulty to evidence melatonin binding sites in the pituitary might result from a number of factors: species investigated, low levels of expression, age of the animals, possible nycthemeral and/or circannual rhythms of expression.

Altogether, our data speak in favor of the presence of melatonin receptors in the pituitary of the pike. Preliminary investigations indicate that melatonin modulates cyclic AMP levels in this organ, suggesting that these are functional receptors. A comparison of the radioautography sections with histological sections from freshly fixed pike pituitaries suggests that the binding is associated to an area containing gonadotrophs as well as prolactin and growth hormone cells.

In conclusion, the present paper is the first to report the presence of melatonin receptors in the pituitary of a non-mammalian vertebrate. This opens new lines of

0 100 200 300 400 500 600 700 800 2-[l25I]iodomelatonin (pM)

Bound (fmol/mg protein)

Bound (fmol/mg protein)

Figure 6. Equilibrium saturation binding of 2-[125I]iodomelatonin to pike brain membranes. Membranes were prepared and binding performed as described in Materials and Methods, in the presence of increasing concentration of 2-[125I]iodomelatonin. In the top graph, the specific binding (solid circles) is defined as total binding minus non-specific binding (open circles), determined in the presence of 50^M of melatonin. Scatchard plot of the experimental data with the best least-squares regression line is shown in the bottom graph. Each experiment used pooled homogenates from 60 pituitaries. Means ± SEM (n = 3). One representative experiment out of 2.

investigation related to the melatonin-mediated photoperiodic control of neuroendocrine functions in fish. Future investigations will aim to identify which cell type(s) and which hormonal output(s) is (are) modulated by melatonin in the pike pituitary. Interestingly enough, physiological concentrations of melatonin modulated 1) GtH II release from cultured pituitary fragments of the Atlantic croaker (15), and 2) gonadotropin release by neonatal rat gonadotrophs challenged with LH-RH (31). The fish pituitary offers interesting perspectives to study molecular and cellular events related to the transduction of the melatonin signal in ectotherms.

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