Results

Specific oligonucleotides derived from rat, mice, and Siberian hamster sequences allowed amplification of DNA products with the predicted size, in both the PT and the SCN. Sequence analysis revealed that the 977 bp Syrian hamster mt1 cDNA sequence (after excluding both 3' and 5' ends corresponding to the universal primer sequences) presents 86.5%, 88.8% and 94.7% of sequence identity with the rat, mouse and Siberian hamster mt1 sequences, respectively (7). Furthermore, the Syrian hamster deduced amino acid sequence presents a 96.9 and 92.3% similarity with the Siberian hamster and mouse deduced amino acid sequences, respectively. According to Siberian hamster and mouse amino acid sequences, this Syrian hamster amino acid sequence would include the seven presumed transmembrane domains characterizing G protein-coupled melatonin receptors, and a portion of both the first extracellular and the last intracel-lular domains (Figure 1).

Quantitative autoradiography revealed that in the PT, the melatonin receptor density declined from PN 0 to PN 8. Indeed, at PN 8, binding was only 40% of the value measured at birth (Figure 2 and Table 1). Afterwards, PT melatonin receptor density slightly went up until it got stabilized around 25 fmol/mg of protein (Figure 3A and Table 1). Similarly, the mt1 mRNA expression decreased from PN 0 to PN 8 (Figure 2 and Table 1), and slightly went up from PN 8 to PN 60 (Figure 3A and Table 1). Regression analysis revealed that mt1 mRNA expression and melatonin binding values were highly correlated (r = 0.98, P < 0.003, between PN 0 and PN 60).

Figure 1. Alignment of the deduced amino acid sequences of the mt1 melatonin receptor subtype in Syrian hamster, Siberian hamster, mouse and rat. The shaded residues are those who differ from the consensus. Analysis were performed using Lasergene Softwares (Dnastar USA). The seven presumed transmembrane domains (I-VII) are highlighted on the deduced amino acid sequence by solid bars. The sequence has been deposited in GenBank under the AF061158 accession number.

Figure 1. Alignment of the deduced amino acid sequences of the mt1 melatonin receptor subtype in Syrian hamster, Siberian hamster, mouse and rat. The shaded residues are those who differ from the consensus. Analysis were performed using Lasergene Softwares (Dnastar USA). The seven presumed transmembrane domains (I-VII) are highlighted on the deduced amino acid sequence by solid bars. The sequence has been deposited in GenBank under the AF061158 accession number.

Table 1. Post natal development of both the specific 2-iodo-melatonin binding (in fmol/mg protein) and the specific hybridization of an antisense mt1 cRNA riboprobe (in DPM) in both the PT (A) and the SCN (B) between PN 0 and PN 60 (adulthood). Each value is the mean ±

SEM of 5 animals

Table 1. Post natal development of both the specific 2-iodo-melatonin binding (in fmol/mg protein) and the specific hybridization of an antisense mt1 cRNA riboprobe (in DPM) in both the PT (A) and the SCN (B) between PN 0 and PN 60 (adulthood). Each value is the mean ±

SEM of 5 animals

Age

PN 0

PN 8

PN 21

PN 30

PN 60

PT 2-iodo-melatonin specific

binding (fmol/mg prot)

50.1 ± 1.1

18.5 ± 0.7++

19.3 ± 0.8**'+

24.4 ± 1.4**

26.1 ± 1.3**

PT mt1 mRNA

expression (DPM)

130 ± 6

52 ± 3++

63 ± 4**'+

59 ± 2**'+

79 ± 4**

SCN 2-iodo-melatonin specific

binding (fmol/mg prot)

12.1 ± 0.3

4.5 ± 0.1++

1.7 ± 0.3**

1.1± 0.1**

0.9 ± 0.1**'

SCN mt1 mRNA

expression (DPM)

52 ± 3

30 ± 2**

30 ± 2**

30 ± 2**

22 ± 1.2**

**: P <0.001 when compared with values obtained at PN 0;++: P < 0.001 when compared with values obtained at PN 0 andPN 60; +: P <0.01 when compared with values obtained at PN 60; *:P < 0.05 when compared with values obtained at PN 21.

**: P <0.001 when compared with values obtained at PN 0;++: P < 0.001 when compared with values obtained at PN 0 andPN 60; +: P <0.01 when compared with values obtained at PN 60; *:P < 0.05 when compared with values obtained at PN 21.

In the SCN of the same animals, the melatonin receptor density was 12.1 ± 0.3 fmol/mg protein at PN 0, and then dramatically decreased until a basal value was reached between PN 30 and PN 60 (Figs. 2 and 3B, Table 1). The mti mRNA expression decreased rapidly from PN 0 to PN 8 (Fig. 2 and Table 1), plateaued between PN 8 and PN 30, and afterwards declined slightly until adult age (Fig. 3B and Table 1). In contrast with PT data, after PN 8, the dramatic decrease in SCN binding capacities did not correlate to the mt1 mRNA expression variations (r = 0.45, p < 0.55, between PN 8 and PN 60).

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