Neuroendocrinology of melatonin in reproduction: recent developments.

The circadian melatonin rhythm with high levels in the dark period is important for the synchronization of reproductive response to appropriate environmental conditions in animals. The target sites of melatonin action on reproductive functions remain to be clarified. Using autoradiography (ARG) and radioreceptor binding assays with 2[125I]iodomelatonin, a melatonin agonist, as the radioligand, studies on the sites of melatonin action have increased significantly in the last ten years. The recent cloning of melatonin receptor subtypes also allowed the characterization of receptor(s) to the molecular level. Earlier reports have documented that the hypothalamic-pituitary axis plays a vital role in the regulation of reproduction by melatonin. This is supported in part by the demonstration of melatonin receptors in the suprachiasmatic nuclei (SCN) in the brain and pars tuberalis (PT) in the pituitary. However, the nature of SCN and PT involvement in the reproductive action of melatonin remains unknown. In addition to the hypothalamus and pituitary, the two classical sites of melatonin action, other targets have been identified. The recent demonstration of 2[125I]iodomelatonin binding sites or melatonin receptors in the testis, epididymis, vas deferens, prostate, ovary and mammary gland suggest the concept of multiple sites of melatonin action on the reproductive system. The presence of melatonin receptors in the said tissues is consistent with earlier reports of direct melatonin actions on different levels of the reproductive system. This multiple levels of melatonin action, from the hypothalamus, pituitary, gonads to other reproductive tissues form a robust system of photoperiodic control in animal reproduction. This would guarantee successful gestation and delivery of the offspring at a time with optimum food availability and ultimately favourable for the survival of species. Molecular and cellular studies of melatonin signaling system(s), its regulation and effects on downstream functional events in the future may provide new insights and directions for the study of the physiology and pharmacology of fertility and contraception in animals and humans.

Melatonin receptors in the chicken kidney are up-regulated by pinealectomy and linked to adenylate cyclase.

The effect of pinealectomy on the characteristics of melatonin receptors in the chicken kidney was studied. One-day-old chicks were operated and kept under a 12 h/12 h light/dark photoperiod. Six weeks after operation, the animals were sacrificed at mid-light and mid-dark. Serum melatonin was determined by radioimmunoassay and kidney melatonin receptors were studied by radioreceptor assay using the melatonin agonist 2-[125I]iodomelatonin as the radioligand. Pinealectomy significantly reduced the mid-dark serum melatonin level and abolished the diurnal rhythm of 2-[125I]-iodomelatonin binding in the kidney. The density of 2-[125I]-Iodomelatonin binding sites in the kidney at mid-dark was increased significantly to a value comparable to the mid-light density after pineal ablation. Our results suggest that melatonin receptors in the chicken kidney are directly regulated by melatonin in the circulation. The coupling of kidney melatonin receptors to adenylate cyclase was investigated. The basal and forskolin-stimulated cAMP production in chicken kidney explants was studied following melatonin or melatonin plus pertussis toxin treatment. Levels of cAMP in chicken kidney explants were extracted and determined by radioimmunoassay. Melatonin had no effect on basal cAMP levels. However, melatonin significantly inhibited the forskolin-stimulated cAMP accumulation at a concentration of 10 pmol/l. Inhibitory effects of melatonin on the forskolin-stimulated cAMP increase in the chicken kidney were totally blocked by preincubating the kidney tissue with 1.0 micrograms/ml pertussis toxin. Our results suggest that kidney melatonin receptors may modulate the adenylate cyclase leading to biological responses in the renal system.

Melatonin implant decreases the density of 2[125I]iodomelatonin binding sites in the chicken spleen.

Changes in 2[125I]iodomelatonin binding to the chicken spleen following disruption of the normal melatonin rhythm by a slow-release melatonin implant were investigated. Melatonin capsules were implanted in 6-week-old male and female chickens reared under 12 h light/12 h darkness. The mid-light and mid-dark serum melatonin was measured throughout the period of implantation. Spleen weight and characteristics of splenic 2[125I]iodomelatonin binding sites were studied during the mid-light at 4 or 8 weeks after implantation. Melatonin implantation disrupted the normal diurnal rhythm of serum melatonin and elevated the mid-light level to the control mid-dark value until 5 weeks after implantation. The melatonin implant caused a significant decrease in the density of 2[125I]iodomelatonin binding sites at 4 weeks after implantation while there were no significant changes in the binding affinity. The reduction in 2[125I]iodomelatonin binding may be a result of down-regulation of the melatonin receptors by elevated serum melatonin. Alternatively, it may be due to an indirect suppression of 2[125I]iodomelatonin binding sites by the elevated sex hormones since melatonin implants increased the levels of testosterone in the male and estradiol in the female. Implantation of melatonin capsules significantly increased the body and spleen weights in the male chicken. These weight changes may be partly explained by the anabolic and erythropoietic action of the increased circulating testosterone.

Localization and characterization of [125I]iodomelatonin binding sites in duck gonads.

The characterization and localization of [125I]iodomelatonin binding sites in the gonads advances the understanding of possible regulatory sites of melatonin action. With the availability of [125I]iodomelatonin as a biologically active radioligand, our study utilized a combined approach of autoradiography for anatomical resolution together with an established radioligand binding assay to assess mid-light [125I]iodomelatonin binding in the testes and ovaries of ducks. In the autoradiography study, specific [125I]iodomelatonin binding was shown to be homogeneous throughout the testes, while in the ovaries, specific [125I]iodomelatonin binding appeared to be concentrated around the follicle. Radioligand binding assay results indicated a single class of binding sites with the maximum number of [125I]iodomelatonin binding sites measured at 1.91 +/- 0.70 fmol/mg protein in testicular membrane and 4.54 +/- 0.64 fmol/mg protein in ovarian membrane. [125I]Iodomelatonin binding affinity, characterized by equilibrium dissociation constants of 29 +/- 6 pmol/L in testicular membrane and 53 +/- 9 pmol/L in ovarian membrane, was in accordance with circulating melatonin levels, suggesting an appropriate concentration for eliciting a physiological response. [125I]Iodomelatonin binding in duck gonads satisfied all the criteria for a binding site, being rapid, stable, saturable, reversible, specific, and of high affinity, and may indicate a direct pineal-gonadal connection.

2-[125I]iodomelatonin binding sites in the testis and ovary: putative melatonin receptors in the gonads.

Through the synthesis and secretion of the hormone, melatonin, the pineal has been assigned the role of synchronizing a reproductive response to appropriate environmental conditions. Theoretical melatonin target sites may occur at several levels of the hypothalamic-pituitary-gonadal hierarchy, including a direct action on the gonads. The availability of a biologically active radioligand, 2-[125I]iodomelatonin, has provided the opportunity to examine the possible direct melatonin action on the gonads. 2-[125I]Iodomelatonin binding sites were identified in the testes and ovaries of chickens, ducks and quail but were not measurable in mammalian gonads, with the exception of tree shrew testes. The avian gonadal 2-[125I]iodomelatonin binding sites were stable, saturable, reversible, specific and of high affinity. 2-[125I]Iodomelatonin appeared to label a single class of binding sites as evidenced by the linearity of Rosenthal analysis of the specific binding data, the Hill coefficients close to unity and the monophasic competition curves. The high affinity on the gonadal 2-[125I]iodomelatonin binding sites, characterized by apparent equilibrium dissociation constants in the low picomolar range, was in accordance with circulating levels of melatonin suggesting that they may be physiologically relevant. Autoradiography indicated that these 2-[125I]iodomelatonin binding sites were widely distributed throughout the testes but localized in ovarian follicles in the birds studied. Specific inhibition of testicular 2-[125I]iodomelatonin binding by a guanine nucleotide analog has provided evidence that the 2-[125I]iodomelatonin binding sites in chicken testes may be coupled to a guanine nucleotide binding protein-effector system, thus promoting the idea that testicular 2-[125I]iodomelatonin binding sites may mediate a cascade of intracellular events. Although no circadian rhythm in the density or affinity of 2-[125I]iodomelatonin binding in chicken ovaries was found, there was a decrease in 2-[125I]iodomelatonin binding affinity at middark in chicken testes with no change in the number of testicular 2-[125I]iodomelatonin binding sites. The present evidence is in line with the hypothesis of a direct melatonin action on the gonads and further investigations on the above problem will be rewarding.

[125I]iodomelatonin binding sites in mammalian and avian kidneys.

[125I]Iodomelatonin binding sites have been identified and characterized in kidneys of birds and mammals. These binding sites in the kidneys of guinea pig, duck and chicken were found to be stable, reversible, saturable, specific and of high affinity. The binding densities (Bmax) of [125I]iodomelatonin binding sites in the kidneys of guinea pig, duck and chicken ranged from 1.07 to 6.43 fmol/mg protein and the equilibrium dissociation constants (Kd) from 19.2 to 44.6 pmol/l at the middle of the light period (mid-light). It appears that [125I]iodomelatonin binding in the kidneys of mammalian species may have lower densities compared with birds. In the mammals studied, the guinea pig kidney showed the highest [125I]iodomelatonin binding. Pharmacological data indicated that the [125I]iodomelatonin binding to kidneys of guinea pig, duck and chicken was highly specific to melatonin, 2-iodomelatonin and 6-chloromelatonin. Diurnal variations in the Bmax of [125I]iodomelatonin binding sites were detected in the kidneys of duck and chicken with no difference in affinity. However, there was no diurnal variation in the Kd or Bmax in the guinea pig kidneys. The density of [125I]iodomelatonin binding sites in the cortex of guinea pig kidney was more than 8-fold higher than the binding in the medulla. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 10 mumol/l) reduced the Bmax and increased the Kd of [125I]iodomelatonin binding sites in the chicken kidney. However, in the membrane preparations of the guinea pig kidney, co-incubation with GTP gamma S (15 mumol/l) increased the Kd with no effect on the Bmax of the [125I]iodomelatonin binding. The effects of GTP gamma S on the kidney [125I]iodomelatonin binding suggest that these binding sites may couple to a G protein. The identification of [125I]iodomelatonin binding sites in the kidneys of mammals and birds supports the possibility of melatonin acting directly on the renal system.

Effects of guanosine 5'-O-(3-thiotriphosphate) on 2-[125I]iodomelatonin binding in the chicken lung, brain and kidney: hypothesis of different subtypes of high affinity melatonin receptors.

Effects of 10 mumol/l guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a non-hydrolyzable analog of guanosine 5'-triphosphate, on 2-[125I]iodomelatonin binding were investigated. In the chicken lung, 10 mumol/l GTP gamma S significantly increased (p < 0.05) the equilibrium dissociation constant (Kd) values, but did not affect the maximum number of binding sites (Bmax). Conversely, in the chicken brain, GTP gamma S significantly depressed (p < 0.05) the Bmax, but did not change the Kd of the 2-[125I]iodomelatonin binding sites in the brain tissue. A third variation was observed in the chicken kidney with GTP gamma S altering (p < 0.05) both the Kd and the Bmax of 2-[125I]iodomelatonin binding sites. The reason underlying the different effects of GTP gamma S on 2-[125I]iodomelatonin binding in the tissue preparations is not clear. However, we would like to hypothesize that they may represent distinct subtypes of the ML-1-type melatonin receptor with different receptor-G-proteins-effector complex. The group represented by the 2-[125I]iodomelatonin binding sites found in the chicken lung, which is downregulated by GTP gamma S with a consequent increase in the Kd value, has been designated ML-1 alpha. The second group, exemplified by the brain 2-[125I]iodomelatonin binding sites, which respond to GTP gamma S with a change in Bmax, has been labelled ML-1 beta. The third group, characterized by GTP gamma S-mediated alterations in both Bmax and Kd and found in the chicken kidney, has been called ML-1 gamma. Different subtypes of melatonin receptors may address the issue of the different physiological actions of melatonin reported in individual tissues within the same species or similar tissues but different species. Specialized responses could be generated depending on the predominant subtype of ML-1 receptors associated with the target tissue.

The identification and characterization of 125I-labelled iodomelatonin-binding sites in the duck kidney.

The melatonin-binding sites in membrane preparations of duck kidney were demonstrated by utilizing 125I-labelled iodomelatonin as a radioligand. Binding at these sites was found to be reversible, saturable, specific and of high affinity. Scatchard analysis of the specific binding revealed an equilibrium binding constant (Kd) of 44.6 +/- 4.4 pmol/l (n = 6) and a total number of binding sites (Bmax) of 6.43 +/- 0.60 fmol/mg protein (n = 6) at the mid-point of the light period (mid-light). The Hill coefficient approached 1.0, suggesting a single class of 125I-labelled iodomelatonin-binding site in the duck kidney. Diurnal variation in 125I-labelled iodomelatonin binding showed that the Bmax was 53.4% higher at mid-light than at mid-point of the dark period (P < 0.05), with no significant variation in Kd. The Kd value determined from kinetic analysis was 22.5 pmol/l in birds at mid-light, which was comparable with values determined from equilibrium studies. The order of pharmacological affinity for 125I-labelled iodomelatonin-binding sites in the duck kidney membrane preparations was: 2-iodomelatonin > melatonin > 6-chloromelatonin > 6-hydroxymelatonin > N-acetylserotonin >> 5-methoxytryptamine, 5-methoxytryptophol, 5-hydroxytryptamine, tryptamine, 1-acetylindole-3-carboxaldehyde, 5-hydroxyindole-3-acetic acid, L-tryptophan, 5-methoxyindole-3-acetic acid, 3-acetylindole, acetylcholine, epinephrine, norepinephrine and harmaline. The pharmacological characteristics indicated that 125I-labelled iodomelatonin-binding sites are highly specific for melatonin. Our finding of 125I-labelled iodomelatonin-binding sites in the kidney suggests that melatonin may regulate kidney function.(ABSTRACT TRUNCATED AT 250 WORDS)

The identification of 125I-labelled iodomelatonin-binding sites in the testes and ovaries of the chicken (Gallus domesticus).

The existence of 125I-labelled iodomelatonin-binding sites in chicken ovaries and testes was investigated. The specific binding of 125I-labelled iodomelatonin to chicken ovarian and testicular tissue satisfies all the criteria for a binding site. It was rapid, stable, saturable, reversible, specific and of high affinity. Equilibrium studies showed that total and non-specific binding increased over a range of 5-150 pmol 125I-labelled iodomelatonin/l tested, with specific binding reaching saturation towards the middle range of radioligand concentrations. Scatchard analyses indicated a dissociation constant (Kd) of 36.5 +/- 5.3 pmol/l (means +/- S.E.M.) in the membrane preparations of chicken testes at the middle point in the period of light and a maximum number of binding sites (Bmax) of 0.93 +/- 0.40 fmol/mg protein (n = 6). In membrane preparations of chicken ovaries, the Kd was 102.2 +/- 27.3 pmol/l and the Bmax was 2.77 +/- 0.38 fmol/mg protein (n = 6). Equilibrium and kinetic dissociation constants in the picomolar range indicate high-affinity and physiologically relevant 125I-labelled iodomelatonin-binding sites. Competitive inhibition studies determined the following order of relative potency for inhibition of 125I-labelled iodomelatonin-binding to chicken gonadal membranes: 6-chloromelatonin greater than melatonin greater than N-acetylserotonin much much greater than 5-hydroxytryptamine, tryptamine, 5-methoxytryptophol, 1-acetylindole-3-acetic acid, 5-hydroxyindole-3-acetic acid and L-tryptophan. The presence of 125I-labelled iodomelatonin-binding sites suggests a direct pineal-gonadal connection in the chicken.