Gonadotropin-releasing hormone increases melatonin release in the pineal gland of the female rat in vitro.

To evaluate the effect of gonadotropin-releasing hormone (GnRH) on melatonin ( N-acetyl-5-methoxytryptamine) release and its synthesizing enzyme activities in pineal glands, pineals from adult female rats during diestrus were organ-cultured in a medium containing 10 -12, 10 -10, or 10 -8 M GnRH for 6 h. Melatonin release increased significantly in pineals cultured with 10 -10 and 10 -8 M GnRH compared to controls. However, in pineal glands that were organ-cultured in a medium containing 10 -12 to 10 -8 M GnRH, the activity of arylalkylamine N-acetyltransferase, which is the key regulatory enzyme in melatonin biosynthesis, showed no significant difference from controls. Likewise, GnRH at these concentrations had no significant effect on the activity of pineal hydroxyindole- O-methyltransferase, which catalyzes the final step of melatonin biosynthesis. These results show that GnRH stimulates pineal melatonin release, but suggest that GnRH does not affect its melatonin synthesis.

Stimulatory effects of LH on release of melatonin and activities of its synthesizing enzymes NAT and HIOMT in organ-cultured pineal glands of female rats.

The pineal hormone melatonin (N-acetyl-5-methoxytryptamine) exerts antigonadotropic effects in some mammalian species. To evaluate the effect of luteinizing hormone (LH) on melatonin release and its synthesizing enzyme activities in pineal glands, pineals of adult female rats undergoing diestrus were organ-cultured in a medium containing 10(-12), 10(-10) or 10(-8) M LH for 6 h. Melatonin release increased significantly in pineals cultured with 10(-12) and 10(-10) M LH, as compared to control values. Similarly, the activity of arylalkylamine N-acetyltransferase (NAT), the key regulatory enzyme in melatonin biosynthesis, was significantly higher in pineals cultured with 10(-12) and 10(-10) M LH for 6 h, while LH at 10(-8) M had no effect. Although LH at 10(-10) M increased pineal hydroxyindole-O-methyltransferase (HIOMT) activity, which catalyzes the final step of melatonin biosynthesis, LH at 10(-12) and 10(-8) M had no effect. These results demonstrate that at relatively low physiological levels, LH stimulates pineal melatonin synthesis and release, mainly by increasing NAT activity.

Effects of aggression and wing removal on brain serotonin levels in male crickets, Gryllus bimaculatus.

When pairs of adult male crickets (Gryllus bimaculatus) that had been housed individually for 7 days were placed together, they fought, and dominant-subordinate relationships were formed within 1min. Aggressive behavior by the dominant male was repeated during the period in which the two males were kept together. Immediately after 10min of aggressive interaction, brain serotonin (5-hydroxytryptamine, 5-HT) levels were unchanged in dominant males and significantly reduced in subordinate males. The emission of aggressive song by dominant males is known to be abolished by removal of the wings. All wings were thus removed from male crickets. After 7 days of isolation, pairs of wingless males were placed together. The wingless males fought and formed dominant-subordinate relationships within 1min. The wingless, dominant males displayed aggressive behavior. Brain 5-HT levels in the wingless males were reduced immediately after 10min of aggressive interaction, and no significant differences in brain 5-HT levels were detected between the dominant and subordinate males, unlike the case for intact males. These data indicate a difference in brain serotonergic activity between dominant and subordinate male crickets during aggressive interaction, and suggest that aggressive behavior by dominant male crickets rapidly reduce brain 5-HT levels in subordinate ones. Furthermore, the data suggest that aggressive song is responsible for the change in brain 5-HT levels.

Circadian clock controlling egg hatching in the cricket (Gryllus bimaculatus).

Adult crickets (Gryllus bimaculatus) were maintained under a 12-h light:12-h dark cycle (LD 12:12). After oviposition, their eggs were incubated under different lighting regimens at 23 degrees C, and temporal profiles of egg hatching were examined. When the eggs were incubated in LD 12:12 or in DL 12:12 with a phase difference of 12h from LD 12:12, throughout embryogenesis, 88% to 97% of hatching occurred within 3 h of the dark-light transition on days 17 and 18 of embryogenesis; the phases of the egg-hatching rhythms in the LD 12:12 and DL 12:12 groups differed by about 12 h. In eggs incubated in constant darkness (DD) throughout embryogenesis, a circadian (about 24 h) rhythm of hatching was found, and the phase of the rhythm was similar to that seen in eggs incubated in LD 12:12, but not DL 12:12, throughout embryogenesis. When eggs that had been incubated in DD after oviposition were transferred to DL 12:12 in the middle or later stages of embryogenesis and were returned to DD after three cycles of DL 12:12, the rhythm of hatching synchronized (entrained) to DL 12:12. However, when eggs in the earlier stages of embryogenesis were transferred from DD to DL 12:12 and returned to DD after three cycles, 52% to 94% of hatching did not entrain to DL 12:12. To determine whether photoperiodic conditions to which the parents had been exposed influenced the timing of egg hatching, adult crickets were maintained in DL 12:12, and their eggs were incubated in LD 12:12, DL 12:12, or DD throughout embryogenesis. The egg-hatching rhythm was also found in the eggs incubated under these three lighting regimens. In DD, the phase of the rhythm was similar to that seen in eggs incubated in DL 12:12, not LD 12:12, throughout embryogenesis. The results indicate that in the cricket, the timing of egg hatching is under circadian control and that the circadian rhythm of hatching entrains to 24-h light:dark cycles, but only if the light:dark cycles are imposed midway through embryogenesis. Therefore, by midembryogenesis, a circadian clock has been formed in the cricket, and this is entrainable to light:dark cycles. In addition, the photoperiodic conditions to which the parents (probably the mothers) have been exposed influence the timing of hatching, suggesting that maternal factors may regulate the timing of egg hatching.

Circadian rhythm of serotonin N -acetyltransferase activity in rat lens.

Activity of serotonin N -acetyltransferase (NAT), a key regulatory enzyme in melatonin biosynthesis, was detected in the rat lens. NAT activity in the lens showed significant diurnal variation in vivo and in vitro, peaking during the period of darkness, when the lenses were maintained under 14 hr light/10 hr dark cycle. Cultured lenses exhibited a circadian rhythm of NAT activity when maintained under constant darkness. However, the rhythm in vitro was not entrained when the light/dark cycle was delayed 8 hr from the cycle in intact animals. These data strongly suggest that the rat lens contains a circadian clock that controls NAT activity, although the circadian clock appears to lack a photic entrainment mechanism.

Melatonin secretion from organ-cultured pineal glands of rats: modulation by gonadectomy and gonadotropin-releasing hormone agonist administration.

The objective of the study was to evaluate the effect of pretreatments such as gonadectomy in male and female rats, and gonadotropin-releasing hormone agonist (GnRHa) administration in female rats, on levels of secretion of melatonin, using an organ culture of pineal glands. Gonadectomy 2 weeks before the animal was killed increased the amount of melatonin secreted into the medium by the pineal glands of female rats but not of male rats. The increase in in vitro melatonin secretion after ovariectomy in female rats was prevented by estrogen replacement. Ovariectomy 3 and 4 weeks before death also significantly increased the amount of melatonin secretion. Administration of GnRHa 2 weeks before decapitation significantly decreased serum estradiol concentrations and significantly increased melatonin secretion by the pineal glands of female rat. GnRHa administration 3 or 4 weeks before decapitation also significantly decreased serum estradiol concentrations, but did not increase pineal secretion of melatonin. The results indicate that ovariectomy increases melatonin secretion from organ-cultured pineal glands and that this increase is suppressed by estrogen in adult female rats. In contrast, orchiectomy in male rats does not influence in vitro secretion of melatonin. These results suggest that the GnRH-gonadotropin system may participate in the regulation of pineal melatonin secretion in adult female rats.

Circadian rhythm of serotonin levels in planarians.

In the freshwater planarian Dugesia japonica, which belongs to the most primitive metazoan phylum, serotonin (5-hydroxytryptamine) was detectable by both immunohistochemistry and high-performance liquid chromatography (HPLC) with fluorometric detection. Immunohistochemical studies showed that serotonin was localized primarily in the cephalic ganglion (brain), in the main nerve cords extending posteriorly from the brain and in the commissure axons connecting the main nerve cords. HPLC with fluorometric detection analysis revealed that the serotonin levels of planarians maintained under a 12:12h light:dark cycle showed significant diurnal variations with a trough in the middle of the dark phase. In constant darkness, the serotonin levels fluctuated with a circadian rhythm. These results demonstrate the existence of a circadian timekeeping mechanism in the planarian.

The effect of melatonin on in vitro fertilization and embryo development in mice.

To examine the effect of melatonin on in vitro fertilization and embryonic development, mouse embryos after insemination in vitro were cultured in a physiological medium with or without melatonin. Melatonin increased the fertilization rate significantly at a concentration between 10(-6) and 10(-4) M (27.6 vs. 43.9 or 40.4%, P < 0.01). Furthermore, a significant increase in the rate of embryos reaching the four-cell stage (16.0 vs. 26.7%, P < 0.01), the eight-cell stage (12.1 vs. 25.8 or 23.5%, P < 0.01), and blastulation (8.9 vs. 23.5 or 17.5%, P < 0.01) was observed when the embryos were cultured in a medium containing 10(-8) or 10(-6) M melatonin. These results demonstrate that melatonin supports fertilization and early embryo development after in vitro fertilization.

Noradrenaline concentrations in human preovulatory follicular fluid exceed those in peripheral plasma.

The concentrations of noradrenaline in preovulatory follicular fluid obtained from women undergoing in vitro fertilization were determined by high-performance liquid chromatography with fluorometric detection and compared with those in peripheral plasma obtained concurrently. All of the follicular fluid samples contained noradrenaline at concentrations substantially higher than those in the corresponding plasma samples. A positive correlation was found between noradrenaline levels in follicular fluid and plasma in each woman (n=11; r=0.952; p<0.001). However, there was no significant difference between noradrenaline concentrations in follicular fluid aspirated from follicles with or without an oocyte [mean+/-SEM, 0.207+/-0.002 ng/microl for follicular fluid samples with an oocyte (n=44), and 0.221+/-0.003 ng/microl for follicular fluid samples without an oocyte (n=13)]. The data indicate that noradrenaline accumulates in follicular fluid, supporting the physiological significance of noradrenaline in the local regulation of human ovarian functions.

Highly sensitive chelating agents for histochemical staining of rare earth metals.

In tissues, bromopyridylazo-diethylaminophenol has been found to be capable of staining very small amounts of rare earth metals, particularly praseodymium, terbium, dysprosium, holmium, ytterbium, and lutetium. Differentiation of a target metal from interfering metals was achieved using masking agents, polyphosphates and aminopolycarboxylic acids.

Circadian rhythms of melatonin-synthesizing enzyme activities and melatonin levels in planarians.

In most vertebrates and several insects, melatonin (N-acetyl-5-methoxytryptamine) is synthesized enzymatically from serotonin (5-hydroxytryptamine) by the sequential action of arylalkylamine N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT). In the freshwater planarian Dugesia japonica, which belongs to the most primitive metazoan phylum, activities of NAT and HIOMT, as well as melatonin, were found. The apparent Michaelis constants for substrates of NAT and HIOMT in the planarian were similar to those reported for the mammalian pineal gland and retina. When the planarians were maintained under a 12 h light:12 h dark cycle, the activities of NAT and HIOMT and melatonin levels exhibited a significant diurnal variation, peaking at the mid-dark time. In constant darkness, NAT activity and melatonin levels fluctuated with a circadian (about 24 h) rhythm. These data demonstrate that the planarian synthesizes melatonin through the same pathways as those in most vertebrates and several insects, and that its melatonin synthesis fluctuates in a circadian manner. Thus, it is strongly suggested that the planarian contains a circadian clock controlling melatonin synthesis.

Melatonin, its precursors, and synthesizing enzyme activities in the human ovary.

The presence of melatonin (N-acetyl-5-methoxytryptamine) and its precursors, serotonin (5-hydroxytryptamine) and N-acetylserotonin, was demonstrated in extracts of human ovary using reverse-phase high-performance liquid chromatography coupled with fluorometric detection. In addition, activities of two melatonin-synthesizing enzymes, arylalkylamine N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), were found in human ovary homogenates. The apparent Michaelis constants for the substrates of NAT and HIOMT in the human ovary were similar to those reported for the pineal glands of humans and other mammals. These findings strongly suggest that the human ovary, like the pineal gland, may synthesize melatonin from serotonin by the sequential action of NAT and HIOMT.

Detection of melatonin, its precursors and related enzyme activities in rabbit lens.

Melatonin (N -acetyl-5-methoxytryptamine) was detected in extracts of albino rabbit lens using radioimmunoassay. Furthermore, melatonin precursors, tryptophan and serotonin (5-hydroxytryptamine), were found in the lens extracts by high-performance liquid chromatography coupled with fluorometric detection. Also, activities of two melatonin-synthesizing enzymes, serotonin N -acetyltransferase (NAT) and hydroxyindole- O -methyltransferase (HIOMT), were found in the lens. The apparent Michaelis constants (K m) for substrates of NAT in the lens were similar to those reported for the pineal gland, although the apparent K m values for substrates of HIOMT in the lens were 10-fold higher than those in the pineal gland. When the rabbits were entrained to a 14-hr light: 10-hr dark cycle, melatonin levels and NAT activity in the lens showed significant day/night changes with high levels during the dark period, but HIOMT activity did not show these changes. These findings strongly suggest that the rabbit lens may synthesize melatonin from serotonin by the sequential action of NAT and HIOMT, and that the melatonin synthesis may fluctuate in a diurnal and/or circadian manner.

Circadian clock controlling arylalkylamine N-acetyltransferase-like activity in the cricket (Gryllus bimaculatus) egg.

When cricket (Gryllus bimaculatus) eggs were incubated under a 12-h light/12-h dark (LD) cycle for 6 days after oviposition at 24-26 degrees C and thereafter transferred to constant darkness (DD), arylalkylamine N-acetyltransferase (NAT)-like activity fluctuated in a circadian manner, peaking during the subjective dark period, and the rhythmic activity persisted during the 3rd day of incubation in DD. When the eggs were transferred from LD to a lighting regime in which the light and dark periods were reversed, the rhythm of NAT-like activity continued to oscillate in phase with the light/dark cycle. These data demonstrate that the cricket egg (probably the embryo) contains a circadian clock controlling NAT-like activity, and that the circadian clock entrains to environmental light/dark cycles.

Melatonin and serotonin N-acetyltransferase activity in developing eggs of the cricket Gryllus bimaculatus.

Melatonin (N-acetyl-5-methoxytryptamine) and serotonin N-acetyltransferase (NAT), a key regulatory enzyme in melatonin synthesis, are present in the adults and larvae of several insect species, as well as in vertebrates. To determine when melatonin and NAT first appear in insects ontogenetically, melatonin levels and NAT-like activity were measured in developing eggs of the cricket Gryllus bimaculatus. When the eggs were incubated under a 12-h light/12-h dark (LD) cycle at 24-26 degrees C, melatonin was detected in the egg extracts at all of the developmental stages examined. NAT-like activity was first found in the eggs 3 days after oviposition. From 5 to 11 days after oviposition, both NAT-like activity and melatonin levels showed significant day/night changes with the high levels occurring during the dark period of the LD cycle. By contrast, significant day/night changes were not detected in eggs just before hatching. To determine more detailed temporal changes, NAT-like activity was assayed in eggs 6 to 7 days after oviposition at 2- or 4-h intervals over a 48-h period. The activity in the eggs clearly exhibited a diurnal rhythm, peaking in the dark period of the LD cycle, and the rhythm persisted in constant darkness. These results suggest that the cricket egg (probably the embryo) synthesizes melatonin, and that its melatonin synthesis may fluctuate with a circadian rhythm. In addition, the results of the present study strongly suggest that a circadian clock controlling NAT activity functions in the cricket at the embryonic stage.

Histochemical staining of chromium with 2-(8-quinolylazo)-4,5-di-p-tolylimidazole.

A new highly sensitive staining agent for chromium (Cr) has been introduced. This staining agent, 2-(8-quinolylazo)-4,5-di-p-tolylimidazole (QTI), was found to be more than ten times as sensitive for Cr than the conventional staining agent, Chromazurol S, and QTI also stained cadmium, copper, lead and zinc. Differentiation between the staining of Cr and other metals was achieved by immersing the tissue sections in dilute alkylamine solutions before they were stained with QTI. Thus, it was possible to selectively stain for Cr by blocking other metals.

Hydroxyindole-O-methyltransferase activity in the silkworm (Bombyx mori).

Hydroxyindole-O-methyltransferase (HIOMT; EC 2.1.1.4) catalyzes the final step in the melatonin (N-acetyl-5-methoxytryptamine) biosynthetic pathway. HIOMT-like activity was detected in the head of fifth last-instar larvae of the silkworm (Bombyx mori), and the optimum pH for this activity was 7.9. The apparent Michaelis constants (K(m)) for S-adenosyl-L-methionine and N-acetylserotonin were 87.6 microM and 96.6 microM, respectively. When the silkworms were entrained to a 12 h light/12 h dark lighting schedule, the HIOMT-like activity showed a significant diurnal variation with high levels during the dark period. The diurnal variation in the activity persisted in constant darkness, but was suppressed by constant light. These findings demonstrate that HIOMT-like activity in the silkworm head occurs as a circadian rhythm and that the rhythm entrains to environmental light/dark cycles. The melatonin rhythm in the silkworm head may therefore be regulated not only by serotonin N-acetyltransferase (EC 2.3.1.87), but also by HIOMT.

Hydroxyindole-O-methyltransferase activity assay using high-performance liquid chromatography with fluorometric detection: determination of melatonin enzymatically formed from N-acetylserotonin and S-adenosyl-L-methionine.

A reliable, sensitive and rapid assay has been developed for determining the activity of hydroxyindole-O-methyltransferase (HIOMT; S-adenosyl-L-methionine:N-acetylserotonin-O-methyltransferase; EC 2.1.1.4), which catalyzes the final step in the melatonin (N-acetyl-5-methoxytryptamine) biosynthetic pathway. This method is based on the separation and detection of melatonin formed enzymatically from N-acetylserotonin and S-adenosyl-L-methionine, by high-performance liquid chromatography with fluorometric detection. The detection limit for melatonin formed per sample was as low as 150 fmol, indicating that the sensitivity of this assay was comparable to that of a radioisotopic assay. The assay was applied to the determination of HIOMT activity in rat pineal gland. The HIOMT activity obtained in this study was comparable with, or slightly lower than those reported previously using radioisotopic assays.

Detection of melatonin and serotonin N-acetyltransferase and hydroxyindole-O-methyltransferase activities in rat ovary.

Melatonin (N-acetyl-5-methoxytryptamine) and the activities of two melatonin-synthesizing enzymes, serotonin N-acetyltransferase (acetyl coenzyme A: arylalkylamine N-acetyltransferase EC 2.3.1.87; NAT) and hydroxyindole-O-methyltransferase (S-adenosyl-L-methionine: N-acetylserotonin-O-methyltransferase EC 2.1.1.4; HIOMT), were assayed in extracts of ovaries obtained from virgin Wistar-derived rats (7-9 week-old) during the light period of a 12 h light/12 h dark cycle. Melatonin was detected in the rat ovary using reverse-phase high-performance liquid chromatography (HPLC) coupled with fluorometric detection and radioimmunoassay (RIA). In addition, NAT and HIOMT activities were found in rat ovary. The apparent Michaelis constants (Km) for the substrates of NAT and HIOMT in the rat ovary were similar to those reported for the pineal gland and retina. These data suggest that the rat ovary, like the pineal gland and the retina, may synthesize melatonin from serotonin by the sequential action of NAT and HIOMT.

Histochemical staining of cadmium with 2-(8-quinolylazo)-4,5-diphenylimidazole.

In order to develop a stain with increased sensitivity and selectivity for cadmium (Cd), we synthesized and characterized 2-(8-quinolylazo)-4,5-diphenylimidazole (QAI). This chelating agent was more than twice as sensitive for Cd than the best conventional staining agents, including benzothiazolylazo-beta-naphthol. Differentiation between Cd and zinc (Zn) was achieved by immersing tissue sections in TRIS(2-aminoethyl)amine before they were stained with QAI. This pretreatment made it possible to selectively stain for Cd by blocking Zn.