Masanori T. Itoh

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.

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.

Melatonin and arylalkylamine N-acetyltransferase activity in the silkworm, Bombyx mori

Melatonin (N-acetyl-5-methoxytryptamine) was identified in the head and hemolymph of the silkworm, Bombyx mori, using reversed-phase high-performance liquid chromatography coupled with fluorometric detection and radioimmunoassay. In addition, evidence of arylakylamine (serotonin) N-acetyltransferase (NAT) a key enzyme controlling the synthesis of melatonin in vertebrates, was found in the head of the silkworm. Melatonin levels in the head and hemolymph and the NAT activity in the head were significantly higher during the dark period than during the light period of a 12-h light/12-h dark cycle. The day-night changes persisted in constant darkness but were suppressed by constant light. The results suggest that the synthesis and release of melatonin in the silkworm head occur as a circadian rhythm that is entrained by environmental light/dark cycles, as it is in the pineal gland of vertebrates. Melatonin in the silkworm head may function as a neurochemical mediator of photoperiodic control of developmental events such as molting, eclosion and diapause.

Autophagy impairment stimulates PS1 expression and gamma-secretase activity.

γ-Secretase plays an important role in the development of Alzheimer disease (AD). γ-Secretase activity is enriched in autophagic vacuoles and it augments amyloid-β (Aβ) synthesis. Autophagy-lysosomal dysfunction has been implicated in AD, but whether γ-secretase activity is affected by autophagy remains unclear. Here we report that γ-secretase activity is enhanced in basal autophagy-disturbed cells through the α subunit of eukaryotic translation initiation factor 2 (eIF2α) kinase, general control nonderepressible 2 (GCN2). Presenilin-1 (PS1) expression was increased even in the presence of nutrients in autophagy-related 5 knockdown (Atg5KD) human embryonic kidney (HEK293) cells expressing a short hairpin RNA as well as in chloroquine-treated HEK293 cells. However, PS1 expression induction was prevented in GCN2KD and ATF4KD cells. Furthermore, Atg5KD cells showed an increase in Aβ production and Notch1 cleavage. These were reduced by an autophagy inducer, resveratrol. Thus, we conclude that the autophagy-lysosomal system regulates γ-secretase activity through GCN2.

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.

The rat oocyte synthesises melatonin

Melatonin (N-acetyl-5-methoxytryptamine) is an indoleamine originally identified in the pineal gland, where it is synthesised enzymatically from serotonin (5-hydroxytryptamine) by the sequential action of arylalkylamine N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT; also known as hydroxyindole O-methyltransferase). Melatonin directly affects ovarian functions and previous studies have suggested that melatonin is synthesised in the ovary. In the present study, we examined whether AANAT and ASMT are expressed in the adult rat ovary. Reverse transcription-polymerase chain reaction analyses demonstrated that both AANAT and ASMT mRNAs are expressed in the ovary. Western blotting for AANAT protein showed that the ovary, like the pineal gland, contains this enzymatic protein with a molecular mass of 24kDa. Immunohistochemistry revealed that the AANAT protein is localised to the oocyte, corpus luteum and medulla, including mast cells. AANAT protein was found in oocytes at all stages of follicular development, and its levels in oocytes increased progressively throughout follicular development. Furthermore, isolated oocytes metabolised exogenous serotonin to melatonin. These findings demonstrate that melatonin is synthesised from serotonin in oocytes. Melatonin synthesised in the oocyte may be implicated in its own growth or maturation, for example, by acting as a calmodulin antagonist or an antioxidant.

Day‐night changes in melatonin levels in different organs of the cricket (Gryllus bimaculatus)

Abstract: Day‐night levels of melatonin (N‐acetyl‐5‐methoxytryptamine) were determined in different organs of adult female crickets (Gryllus bimaculatus) exposed to a 12/12 light/dark cycle, using reversed‐phase high performance liquid chromatography coupled with fluorometric detection. Melatonin levels in the compound eye, brain, and palp were significantly higher during the dark period than during the light period, suggesting that a diurnal rhythm of melatonin levels exists in these organs of crickets, with a peak during the dark period. Conversely, melatonin levels were significantly higher during the light period than the dark period in the cercus, ovipositor, antenna, hind‐leg and ovary. No significant day‐night difference was found in the fore‐ and mid‐legs, Malpighian tube, and digestive tube. Thus, these organs may have different melatonin‐metabolizing systems compared to those found in the compound eye, brain, and palp. Differences in the phasing of the melatonin rhythm in various organs of the cricket suggest possible differences in melatonin function in these organs.

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 florometric 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 12:12 h light:dark cycle showed significant diurnal variations with a trough in the middle of the dark phase. In constant darkness, the serotonin levels gluctuated with a circadian rhythm. These results demonstrate the existence of a circadian timekeeping mechanism in the planarian.

Expression and cellular localization of melatonin‐synthesizing enzymes in the rat lens

Abstract:  Melatonin (N‐acetyl‐5‐methoxytryptamine) prevents oxidative stress‐induced cataract development, and previous studies have suggested that the ocular lens synthesizes melatonin. In the present study, we examined whether two key enzymes in melatonin biosynthesis, arylalkylamine N‐acetyltransferase (AANAT) and hydroxyindole‐O‐methyltransferase (HIOMT), are expressed in the lens of adult male rats. Reverse transcriptase‐polymerase chain reaction analyses demonstrated that both AANAT and HIOMT mRNAs are expressed in the lens. Western blotting for AANAT protein showed that the lens, like the pineal gland, contains this enzyme protein with a molecular mass of 24 kDa. Immunohistochemistry revealed that AANAT protein is localized to the lens cortical fiber cells. Serotonin, which is a substrate for AANAT and a melatonin precursor, was also found in this region. These findings demonstrate that the lens expresses AANAT and HIOMT, and suggest that the cortical fiber cells are the main melatonin‐synthesizing sites in the lens. Locally synthesized melatonin in the lens cortical fiber cells may protect the lens itself from cataract development.

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°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 12 h 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 DL12:12, 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 DL12:12. 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 DL12:12, 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.