Response of gastrointestinal melatonin, antioxidants, and digestive enzymes to altered feeding conditions in carp (Catla catla).

The purpose of present study was to ascertain whether the response of gastrointestinal (gut) melatonin to altered feeding conditions was related to the levels of different antioxidants and digestive enzymes in the same gut tissues of a sub-tropical carp (Catla catla). Accordingly, the fish were subjected to food deprivation for 4 or 8 days and separately to re-feeding for 4 or 8 or 12 days after deprivation of food for 8 days, and their gut tissue homogenates were used to measure the levels of melatonin, both enzymatic [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST)] and non-enzymatic [reduced glutathione (GSH)] antioxidants, as well as different digestive enzymes (α-amylase, cellulase, protease, and lipase). Notably, the gut levels of melatonin, SOD, CAT, GPx, and GST underwent gradual increase with the progress of food deprivation, but a sudden fall after restoration of food supply for 4 days and a rise thereafter. Conversely, the activity of all the digestive enzymes significantly decreased after deprivation of food, but started increasing when food supply was reinforced. Gut melatonin concentrations by showing a positive correlation with the titers of different antioxidants (in both food-deprived and re-fed fish groups) and a negative (in food-deprived fish) or a positive (in re-fed fish) correlation with the activity of each digestive enzyme underlined possible physiological interplay between them. Collectively, our findings lend support to the hypothesis that gut melatonin response to altered feeding conditions in carp might be associated with the oxidative status as well as the digestive functions of the gastrointestinal tissues itself.

Influences of exogenous melatonin on the oocyte growth and oxidative status of ovary during different reproductive phases of an annual cycle in carp Catla catla.

The present study aimed to evaluate antioxidant role of melatonin in determining seasonality of ovarian growth in adult carp Catla catla. Accordingly, an identical regimen of exogenous melatonin administration (100 µg/100 g body weight per day for 15 days) was followed during the preparatory, prespawning, and spawning phases of an annual reproductive cycle. The study did not include postspawning phase, when the ovaries were completely regressed and devoid of any healthy growing follicles. The ovarian response was evaluated by determining relative number of developing oocytes as well as measuring the levels of melatonin, oxidative stress (using malondialdehyde [MDA] as the marker), both enzymatic (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GPx], and glutathione S-transferase [GST]) and nonenzymatic (reduced glutathione [GSH]) antioxidants in the ovarian homogenates. Due to melatonin treatment, oocyte growth was accelerated in the preparatory phase but retarded in the prespawning and spawning phases of annual cycle. Conversely, melatonin administration in each reproductive phase led to a significant reduction of MDA and elevations of SOD, CAT, GPx, GST, GSH, as well as melatonin levels in the ovary. As a result, melatonin titers in the ovary always reported a negative correlation with MDA and a positive correlation with SOD, CAT, GST, GPx, as well as GSH levels. However, melatonin content of ovary and the values of gonosomatic index in melatonin-treated carp displayed a positive correlation in the preparatory phase and a negative correlation in the remaining parts of reproductive cycle. Thus, it seems likely that melatonin by acting as an antioxidant reduces intraovarian oxidative stress throughout the seasons of follicular growth, whereas exogenous melatonin administration exerts progonadal influences during the preparatory phase, but antigonadal effects during the prespawning and spawning phases of reproductive cycle.

The Role of Melatonin as a Hormone and an Antioxidant in the Control of Fish Reproduction.

Reproduction in most fish is seasonal or periodic, and the spawning occurs in an appropriate season to ensure maximum survival of the offspring. The sequence of reproductive events in an annual cycle is largely under the control of a species-specific endogenous timing system, which essentially relies on a well-equipped physiological response mechanism to changing environmental cues. The duration of solar light or photoperiod is one of the most predictable environmental signals used by a large number of animals including fish to coordinate their seasonal breeding. In vertebrates, the pineal gland is the major photoneuroendocrine part of the brain that rhythmically synthesizes and releases melatonin (N-acetyl-5-methoxytryptamine) into the circulation in synchronization with the environmental light-dark cycle. Past few decades witnessed an enormous progress in understanding the mechanisms by which melatonin regulates seasonal reproduction in fish and in other vertebrates. Most studies emphasized hormonal actions of melatonin through its high-affinity, pertussis toxin-sensitive G-protein (guanine nucleotide-binding protein)-coupled receptors on the hypothalamus-pituitary-gonad (HPG) axis of fish. However, the discovery that melatonin due to its lipophilic nature can easily cross the plasma membrane of all cells and may act as a potent scavenger of free radicals and stimulant of different antioxidants added a new dimension to the idea explaining mechanisms of melatonin actions in the regulation of ovarian functions. The basic concept on the actions of melatonin as an antioxidant emerged from mammalian studies. Recently, however, some new studies clearly suggested that melatonin, apart from playing the role of a hormone, may also be associated with the reduction in oxidative stress to augment ovarian functions during spawning. This review thus aims to bring together the current knowledge on the role of melatonin as a hormone as well as an antioxidant in the control of fish reproduction and shape the current working hypotheses supported by recent findings obtained in carp or based on knowledge gathered in mammalian and avian species. In essence, this review highlights potential actions of melatonin as a hormone in determining temporal pattern of spawning and as an antioxidant in regulating oocyte maturation at the downstream of HPG axis in fish.

Melatonin actions on ovaprim (synthetic GnRH and domperidone)-induced oocyte maturation in carp.

The major objective of the present study was to demonstrate the actions of exogenous melatonin on ovaprim (synthetic GnRH and domperidone)-induced final oocyte maturation focusing on the oxidative status of pre-ovulatory follicles in the carp Catla catla. Accordingly, gravid carp during the early spawning phase of the reproductive cycle were injected with melatonin and/or ovaprim at different time intervals or luzindole (a pharmacological blocker of melatonin receptors) before their administration. We studied their effects on the latency period, the rate of germinal vesicle breakdown (GVBD; a visual marker of final oocyte maturation) in oocytes, and the levels of maturation-promoting factor (MPF), as well as oxidative stress, different antioxidants, melatonin and MT1 melatonin receptor protein in the extracts of pre-ovulatory follicles. Notably, melatonin treatment 2 h before the injection of ovaprim resulted in the shortest latency period as well as the highest rate of GVBD and MPF formation. Exogenous melatonin, irrespective of the injection schedule, caused a significant reduction in intra-follicular oxidative stress and an increase in the levels of both enzymatic and non-enzymatic antioxidants, melatonin and its receptor protein. Concentrations of ovarian melatonin in each fish exhibited a significant negative correlation with the level of oxidative stress, but a positive correlation with the rate of GVBD and the activity/level of different antioxidants. However, no significant effects of melatonin and/or ovaprim were detected in luzindole-pretreated carp. Collectively, the present study provides the first evidence that melatonin pretreatment in carp ameliorates ovaprim actions on the process of final oocyte maturation by the formation of MPF and alleviates oxidative stress in pre-ovulatory follicles by stimulating different antioxidants.

Gut melatonin response to microbial infection in carp Catla catla.

The purpose of present study was to demonstrate the response of gut melatoninergic system to Aeromonas hydrophila infection for 3 or 6 days and search for its correlation with the activity of different antioxidative and digestive enzymes to focus their interplay under pathophysiological conditions in carp (Catla catla). Microscopic study of gut in infected fish revealed degenerative changes in the tunica mucosa and lamina propria layers with sloughed off epithelial cells in the lumen. The activity of each digestive enzyme was reduced, but the levels of melatonin, arylalkylamine-N-acetyl transferase protein, the key regulator of melatonin biosynthesis, and different enzymatic antioxidants in gut were gradually and significantly increased with the progress of infection. Gut melatonin concentrations in A. hydrophila challenged carp by showing a positive correlation with the activity of each antioxidative enzyme, and a negative correlation with different digestive enzymes argued in favor of their functional relation, at least, during pathological stress. Moreover, parallel changes in the gut and serum melatonin titers indicated possible contribution of gut to circulating melatonin. Collectively, present carp study provided the first data to suggest that endogenous gut melatonin may be implicated to the mechanism of response to microbial infections in any fish species.

Effects of starvation, re-feeding and timing of food supply on daily rhythm features of gut melatonin in carp (Catla catla).

Influences of starvation, re-feeding and time of food supply on daily rhythm features of melatonin (5-methoxy-N-acetyltryptamine) and its key regulator AANAT (arylalkylamine N-acetyltransferase) protein in the gut tissues were separately evaluated in carp Catla catla. The first experiment was aimed at demonstration of duration dependent effects of starvation and re-feeding after starvation on the daily profiles and rhythm features of gut melatonin and AANAT. Accordingly, juvenile carp were randomly distributed in three groups, which were (a) provided with balanced diet daily at a fixed time, that is, 10:00 clock hour or zeitgeber time (ZT) 4 (control), or (b) starved (for 2-, 4-, 6- or 8 days), or (c) initially starved for 8 days and then re-fed (for 2-, 4-, 6-, 8-, 12- or 16 days) daily with the same food and at the time (ZT4) used for control fish. The carp in each group were sampled for collection of gut tissues at six different time points at a regular interval of 4 h in a daily cycle. In another experiment, the influences of timing of food supply were separately examined in four fish groups, which were provided with a fixed amount of food once daily either at 06:00 or 12:00 or 18:00 or 24:00 clock hour corresponding to ZT0 or ZT6 or ZT12 or ZT18, respectively, for 7 days before sampling at 12 different time points with a regular interval of 2 h in a 24-h cycle. The study revealed a gradual increase in the mesor and amplitude values of melatonin and AANAT in gut with the progress of starvation till their values reached maximum at day-6 and remained steady thereafter. In contrast, re-feeding of 8-day starved fish resulted in a sharp decrease in their mesor and amplitude values after 2 days and then followed by a steady-state increase till re-attainment of their values close to control fish at the end of 16 days. The acrophase of these gut variables in each control, starved and re-fed fish was noted mostly at midday or ZT6. However, the results of another experiment demonstrated that a shift of food supply time led to a shift in their acrophase. The amount of residual food in the gut lumen in each, but not starved, fish by showing a significant positive correlation independently with the gut levels of melatonin and AANAT also indicated possible role of food as the synchronizer for their daily rhythms. Collectively, it appears reasonable to argue that daily profiles of gut melatonin and AANAT are strongly influenced by the availability of food, while their daily rhythm features seem to be dependent mostly on the time of food supply in carp.

Gut Melatonin in Vertebrates: Chronobiology and Physiology.

Melatonin, following discovery in the bovine pineal gland, has been detected in several extra-pineal sources including gastrointestinal tract or gut. Arylalkylamine N-acetyltransferase (AANAT) is the key regulator of its biosynthesis. Melatonin in pineal is rhythmically produced with a nocturnal peak in synchronization with environmental light-dark cycle. A recent study on carp reported first that melatonin levels and intensity of a ~23 kDa AANAT protein in each gut segment also exhibit significant daily variations but, unlike pineal, show a peak at midday in all seasons. Extensive experimental studies ruled out direct role of light-dark conditions in determining temporal pattern of gut melatoninergic system in carp, and opened up possible role of environmental non-photic cue(s) as its synchronizer. Based on mammalian findings, physiological significance of gut-derived melatonin also appears unique because its actions at local levels sharing paracrine and/or autocrine functions have been emphasized. The purpose of this mini review is to summarize the existing data on the chronobiology and physiology of gut melatonin and to emphasize their relation with the same hormone derived in the pineal in vertebrates including fish.

Melatonin concentrations in relation to oxidative status and oocyte dynamics in the ovary during different reproductive phases of an annual cycle in carp Catla catla.

The present study on carp Catla catla is the first attempt to search for a relationship between the concentrations of melatonin, oxidative status, and oocyte dynamics in the ovary of any fish. We measured the levels of melatonin, different antioxidative agents, and the marker of intracellular stress along with the profiles of different developmental stages of oocyte in the ovary of adult carp during four distinct phases in an annual reproductive cycle. Ovarian melatonin titers displayed significant seasonal variations with a peak during spawning and nadir during post-spawning, and thereby underlined its proximity to the course of ovarian development. A significant positive correlation was found between the ovarian levels of melatonin and the activity of superoxide dismutase (SOD), catalase (CAT), and glutathione transferase (GST), although each of them showed a negative correlation with the level of malondialdehyde (MDA)-a faithful indicator of intracellular stress. However, ovarian melatonin titers did not exhibit any correlation with the levels of reduced glutathione (GSH) and the activity of glutathione peroxidase (GPx). Collectively, our findings suggest that melatonin measured in carp ovary may be associated with an enhanced activity/level of selective antioxidative agents for reduction in oxidative stress to augment ovarian functions during the spawning.

Melatonin: a potent candidate in the regulation of fish oocyte growth and maturation.

Recent studies on several fish species, especially carp, implicated pineal hormone melatonin (N-acetyl-5-methoxytryptamine) as a potent candidate in the regulatory mechanism of seasonal reproduction. Under natural conditions, the temporal pattern of serum melatonin varied with daily light-dark cycle and the reproductive status of the fish as well. Carefully controlled study revealed that exogenous administration of melatonin may result in stimulation or inhibition or no influences at all on the gonadal functions depending on the reproductive status of fish. Cross-talk between the melatonin and ovarian steroid has been evident from in vitro study, in which melatonin accelerated the action of 17α,20ß-dihydroxy-4-pregnen-3-one or maturation inducing hormone (MIH) on meiotic cell cycle resumption in carp oocytes by formation of maturation promoting factor (MPF) - a complex of two proteins, cyclin B and cyclin dependant kinase Cdk1. While several lines of evidence suggest melatonin effects on hypothalamo-hypophyseal-gonadal axis, localization and dynamics of a 37-kDa melatonin receptor protein in carp oocytes argued in favor of extra-hypothalamic direct action of melatonin on fish reproduction. A recent study in carp indicated that influences of an identical regimen of photoperiods in different parts of annual cycle on ovarian functions vary in relation to the profiles of serum melatonin, but not to any rhythm parameters of MT1 or MT2 receptors on the gonad or brain. The purpose of this short review is to bring together the current knowledge on the biological effects of melatonin on fish reproduction mainly focusing the recent findings on carp.

Influence of altered photoperiods on serum melatonin and its receptors (MT1 and MT2) in the brain, retina, and ovary in carp Catla catla.

Daily variation in melatonin receptor (MT1 and MT2) density in three specific tissues-brain, retina, and ovary-and its temporal relationship with serum melatonin were evaluated for the first time in a freshwater teleost, the carp Catla catla, under natural as well as altered photoperiods in different reproductive phases of the annual cycle. Cosinor analysis was used to determine rhythmic features of the serum melatonin and receptors (MT1 and MT2) in different tissues. In each photoperiodic group, irrespective of season, the daily minimum serum melatonin level was noted at midday. However, the daily peak value of melatonin varied in relation to both photo-schedules and reproductive phases. Under natural photoperiods (NPs; duration varied with seasons) and short photoperiods (SPs; light [L]:dark [D] 8:16), it occurred in the late dark phase during the preparatory phase, and at midnight in the remaining parts of the annual cycle. On the other hand, in each reproductive phase, compared to corresponding NP carp, the daily melatonin peak under long photoperiods (LPs; L:D 16:8) exhibited a phase delay of ∼2-3 h (occurring during the late dark phase). The melatonin levels at each sampling point were highest during the postspawning phase and lowest during the spawning phase, irrespective of the photoperiodic history of the fish. In each tissue, Western blot analysis revealed a band at ∼37 kDa and a band at ∼36 kDa corresponding to the molecular weights of native MT1 and MT2 receptor proteins, respectively, with the band intensity of MT1 always being higher than that of a 36-kDa protein. The content of both melatonin receptor proteins varied significantly according to the studied tissue (being highest in the retina, intermediate in the brain, and lowest in the ovary), time in the daily cycle (peak at midnight and fall at midday), and reproductive phase in the annual cycle (highest in the spawning phase and lowest in the postspawning phase). Remarkably, no significant effects of altered photoperiod were detected on any rhythm parameters of either MT1 or MT2 in any of the studied tissues. Collectively, the results of the present study suggest a role of photoperiod in determining daily and seasonal profiles of serum melatonin, but not its receptor proteins, on the ovary or on any nongonad tissues in carp.

Neural regulation of dark-induced abundance of arylalkylamine N-acetyltransferase (AANAT) and melatonin in the carp (Catla catla) pineal: an in vitro study.

In all the vertebrates, synthesis of melatonin and its rhythm-generating enzyme arylalkylamine N-acetyltransferase (AANAT) reaches its peak in the pineal during the night in a daily light-dark cycle, but the role of different neuronal signals in their regulation were unknown for any fish. Hence, the authors used specific agonist and antagonists of receptors for different neuronal signals and regulators of intracellular calcium (Ca(2+)) and adenosine 3',5'-cyclic monophosphate (cAMP) in vitro to study their effects on the abundance of AANAT and titer of melatonin in the carp (Catla catla) pineal. Western blot analysis followed by quantitative analysis of respective immunoblot data for AANAT protein, radioimmunoassay of melatonin, and spectrophotometric analysis of Ca(2+) in the pineal revealed stimulatory effects of both adrenergic (α(1) and ß(1)) and dopaminergic (D(1)) agonists and cholinergic (both nicotinic and muscarinic) antagonists, inhibition by both adrenergic and dopaminergic antagonists and cholinergic agonists, but independent of the influence of any agonists or antagonists of α(2)-adrenergic receptors. Band intensity of AANAT and concentration of melatonin in the pineal were also enhanced by the intracellular calcium-releasing agent, activators of both calcium channel and adenylate cyclase, and phophodiesterase inhibitor, but suppressed by inhibitor of calcium channel and adenylate cyclase as well as activator of phophodiesterase. Moreover, an inhibitory effect of light on the pineal AANAT and melatonin was blocked by both cAMP and proteasomal proteolysis inhibitor MG132. Collectively, these data suggest that dark-induced abundance of AANAT and melatonin synthesis in the carp pineal are a multineuronal function, in which both adrenergic (α(1) and ß(1), but not α(2)) and dopaminergic signals are stimulatory, whereas cholinergic signals are inhibitory. This study also provides indications, though arguably not conclusive evidence, that in either case the neuronal mechanisms follow a signal-transduction pathway in which Ca(2+) and cAMP may act as the intracellular messengers. It also appears that proteasomal proteolysis is a conserved event in the regulation of AANAT activity in vertebrates.

Importance of light in temporal organization of photoreceptor proteins and melatonin-producing system in the pineal of carp Catla catla.

The importance of light in the temporal organization of photoreceptor proteins and melatonin-producing system has been investigated for the first time in the pineal of a tropical fish. In this study, an identical experimental paradigm was followed during the four distinct phases of an annual cycle in adult carps (Catla catla) maintained either under natural photoperiod (NP) or continuous illumination (LL) or darkness (DD) for 30 days. At the end of each experiment, the pineal from fish in each experimental group was collected either at 06:00, 12:00, 18:00, or 24:00 in a daily cycle and assessed by Western blot analysis for pineal rod-like opsin, alpha-transducin, and AANAT. The same animals were also used for measurement of serum melatonin levels, and the serum as well as intra-pineal Ca(++) levels at each timepoint. The study revealed a daily rhythmicity with a peak at 12:00 h and nadir at 24:00 h in the band intensity of pineal rod-like opsin and alpha-transducin in NP fish, while the band intensities of these photo-pigment proteins remained high under LL and low under DD, irrespective of clock hour during the 24 h cycle. The band intensity of pineal AANAT, levels of serum melatonin, and both serum Ca(++) and intra-pineal Ca(++) were maximum at 24:00 h and minimum at 12:00h in NP fish, and they were significantly lower under LL and higher under DD at each point of study. The results showed loss of daily rhythm in each studied variable in both LL and DD carps, suggesting that their circadian organization is dependent on the external light-dark conditions, rather than an endogenous circadian oscillator in the pineal.

Neuronal regulation of photo-induced pineal photoreceptor proteins in carp Catla catla.

In the present in vitro study on the pineal in carp Catla catla, specific agonist and antagonists of receptors for different neuronal signals and regulators of intra-cellular Ca(++) and cAMP were used to gather basic information on the neuronal signal transduction cascade mechanisms in the photo-induced expression of rod-like opsin and alpha-transducin-like proteins in any fish pineal. Western-blot analysis followed by quantitative analysis of respective immunoblot data for both the proteins revealed that photo-induced expression of each protein was stimulated by cholinergic (both nicotinic and muscarinic) agonists and a dopaminergic antagonist, inhibited by both cholinergic antagonists and a dopaminergic agonist, but not affected by any agonists or antagonists of adrenergic (alpha(1), alpha(2) and beta(1)) receptors. Moreover, expression of each protein was stimulated by voltage gated L type calcium channel blocker, adenylate cyclase inhibitor and phosphodiesterase activator; but suppressed by the activators of both calcium channel and adenylate cyclase, and by phosphodiesterase inhibitor. Collectively, we report for the first time that both cholinergic and dopaminergic signals play an important, though antagonistic, role in the photo-induced expression of photoreceptor proteins in the fish pineal through activation of a signal transduction pathway in which both calcium and cAMP may act as the intracellular messengers.

Photoreceptor proteins and melatonin rhythm generating AANAT in the carp pineal: Temporal organization and correlation with natural photo-thermal cues.

We studied temporal organization of both the photoreceptor (rod-like opsin, alpha subunit of the G protein transducin or alpha-TD) and melatonin generating (AANAT) proteins in the same pineal of a tropical surface dwelling free-living carp Catla catla, and analyzed possible correlation between them as well as with natural photo-thermal variables in an annual cycle. The pineal from individual fish was collected at four different time points (06.00 h, 12.00 h, 18.00 h, and 24.00 h) in a 24.00 h cycle and the same was repeated in four distinct seasons in an annual reproductive cycle to study each protein following Western blot and densitometric analyses of respective immunoblots. The rod-like opsin was represented by four distinct bands, a closely spaced doublet of 39 kDa and bands of 78 and 115 kDa. Two separate bands, one at 43 kDa and another at 65 kDa, were detected for alpha-TD, and a single band at 23 kDa for AANAT. Both the pineal photoreceptor proteins exhibited an identical pattern of diurnal variations with a peak at midday (12.00 h) and fall at midnight (24.00 h), while maximum band intensity of AANAT was noted in midnight (24.00 h) and minimum at midday (12.00 h) depicting a significant negative correlation (p<0.001) between them. Likewise, in an annual cycle, a significant (p<0.01) negative correlation was found between the expression of each pineal photoreceptor protein (being highest during the spawning phase) and AANAT (maximum during the post-spawning phase). Seasonal fluctuations of both the photoperiod and water temperature exhibited a significant (p<0.01) positive correlation with the expression of pineal photoreceptor proteins and a significant (p<0.05) negative correlation with the pineal AANAT. Collectively, the present phenological study is the first report on temporal organization of pineal photoreceptor proteins and their correlation with the melatonin rhythm-generating enzyme AANAT as well as environmental photo-thermal cues depicting their integrative role in the synthesis of proteins in the pineal in any fish. Nonetheless, importance of further experimental studies on carp is emphasized for a conclusive evidence of functional relationship between the studied variables in the pineal and the components of environment in which the fish live in.

Localization and dynamics of Mel(1a) melatonin receptor in the ovary of carp Catla catla in relation to serum melatonin levels.

We studied the localization, sub-cellular distribution and daily rhythms of a 37 kDa melatonin receptor (Mel(1a)R) in the ovary to assess its temporal relationship with the serum melatonin levels in four different reproductive phases in carp Catla catla. Our immunocytochemical study accompanied by Western blot analysis of Mel(1a)R in the ovary revealed that the expression of this 37-kDa protein was greater in the membrane fraction than in the cytosol. Ovarian Mel(1a)R protein peaked at midnight and fell at midday in each reproductive phase. Conversely, serum melatonin levels in the same fish demonstrated a minimum diurnal value at midday in all seasons, but a peak at midnight (during pre-spawning, spawning, and post-spawning phases) or at late dark phase (during preparatory phase). In an annual cycle, band intensity of Mel(1a)R protein showed a maximum at night in the spawning phase and a minimum in the post-spawning phase, demonstrating an inverse relationship with the levels of serum melatonin. Our data provide first evidence of the presence of Mel(1a) melatonin receptor in carp ovary and offer interesting perspectives especially for the study of the mechanisms of the control of its rhythmicity and its response to external factors.

Melatonin: fifty years of scientific journey from the discovery in bovine pineal gland to delineation of functions in human.

Melatonin (N-acetyl-5-methoxytryptamine) was first purified and characterized from the bovine pineal gland extract by Aron Lerner and co-workers in 1958. Since then, a plethora of information has piled up on its biosynthesis, metabolism, time-bound periodicity, physiological and patho-physiological functions, as well as its interactions with other endocrine or neuro-endocrine organs and tissues in the body. Melatonin has wide range of applications in physiology and biomedical fields. In recent years, a significant progress has been made in the understanding mechanism of its actions at the cellular and molecular levels. Consistent efforts have uncovered the mystery of this indoleamine, and demonstrated its role in regulation of a large as well as diverse body functions in different groups of animals in general, and in humans in particular. Current review, in commemoration of 50 years of discovery of melatonin, while revisiting the established dogmas, summarizes current information on biosynthesis, secretion, metabolism and molecular mechanism of action of melatonin at cellular level and highlights the recent research on its role in human physiology and clinical biology.

Influence of serotonin on the action of melatonin in MIH-induced meiotic resumption in the oocytes of carp Catla catla.

The influences of serotonin (5-hydroxytryptamine) on the action of melatonin (N-acetyl-5-methoxytryptamine) in MIH (maturation inducing hormone)-induced meiotic resumption were evaluated in the oocytes of carp Catla catla using an in vitro model. Oocytes from gravid female carp were isolated and incubated separately in Medium 199 containing either (a) only melatonin (MEL; 100 pg/mL), or (b) only serotonin (SER; 100 pg/mL), or (c) only MIH (1 microg/mL), or (d) MEL and MIH (e) or MEL (4 h before) and MIH, or (f) MEL and SER, (g) or SER and MIH, or (h) SER (4 h before) and MIH, or (i) luzindole (L-antagonist of MEL receptors; 10 microM) and MEL, or (j) MEL, L and MIH, or (k) MEL (4 h before), L and MIH, or (l) metoclopramide hydrochloride (M-antagonist of SER receptors; 10 microM) and SER, or (m) M, MEL, SER, or (n) M, SER and MIH, or (o) M, SER (4 h before) and MIH, or (p) M, MEL SER and MIH, or (q) MEL, L, SER and M, or (r) MEL, L, SER, M, and MIH, or (s) MEL, SER, L and MIH. Control oocytes were incubated in the medium alone. Oocytes were incubated for 4, or 8, or 12, or 16 h and effects were evaluated by considering the rate (%) of germinal vesicle breakdown (GVBD). At the end of 16 h incubation, 93.24+/-1.57% oocytes underwent GVBD following incubation with only MIH, while incubation with only MEL or only SER resulted in 77.15+/-1.91% or 14.42+/-0.43% GVBD respectively. Interestingly, incubation with MEL 4 h prior to addition of MIH in the medium, led to an accelerated rate of GVBD (92.58+/-1.10% at 12 h). In contrast, SER, irrespective of its time of application in relation to MIH, resulted in a maximum of 64.57+/-0.86% GVBD. While L was found to reduce the stimulatory actions of melatonin, M suppressed the inhibitory actions of serotonin. In each case, both electrophoretic and immunoblot studies revealed that the rate of GVBD was associated with the rate of formation of maturation promoting factor (a complex of two proteins: a regulatory component--cyclin B and the catalytic component--Cdk1 or cdc2). Collectively, the present study reports for the first time that SER not only inhibits the independent actions of MIH, but also the actions of MEL on the MIH-induced oocytes maturation in carp.

Testicular functions and serum titers of LH and testosterone in methyl parathion-fed roseringed parakeets.

Adult male roseringed parakeets were fed concentrations (0, 10 or 20 microg/100g body wt./day) of methyl parathion (MP) for 5 or 10 days. There were four sampling periods for each treatment group. The first two sampling periods were after 5 or 10 days of daily dosing. In two other sampling periods, birds were given daily doses for 10 days, and sampling occurred at 5 or 10 days after the end of treatment. A significant dose- and duration-dependent reduction in the paired testicular weight, seminiferous tubular diameters, the number of tubules with healthy germ cells, plasma acetylcholinesterase (AChE) activity and plasma levels of luteinizing hormone (LH) and testosterone occurred in MP-fed birds. The inhibitory influences of MP persisted till day-5 and followed by recovery from the gonado-suppressive effects of MP at day-10 after the end of last treatment for 10 days. These findings provide the first experimental evidence that MP-induced testicular dysfunctions in parakeets possibly results from an impaired activity of hypophysial-gonadal axis. Moreover, it is evident that the organophosphorous (OP)-induced changes in the avian testes are reversible.

Melatonin in the regulation of annual testicular events in carp Catla catla: evidence from the studies on the effects of exogenous melatonin, continuous light, and continuous darkness.

The physiological significance of melatonin in the regulation of annual testicular events in a major carp Catla catla was evaluated through studies on the effects of graded dose (25, 50, or 100 microg/100 g body wt.) of melatonin exogenously administered for different durations (1, 15, or 30 days) and manipulation of the endogenous melatonin system by exposing the fish to constant darkness (DD) or constant light (LL) for 30 days. An identical experimental schedule was followed during the preparatory (February-March), pre-spawning (April-May), spawning (July-August), and post-spawning (September-October) phases of the annual cycle. Irrespective of the reproductive status of the carp, LL suppressed while DD increased the mid-day and mid-night values of melatonin compared to respective controls. Influences of exogenous melatonin varied in relation to the dose and duration of treatment and the reproductive status of the carp. However, testicular response to exogenous melatonin (at 100 microg, for 30 days) and DD in each reproductive phase was almost identical. Notably, precocious testicular maturation occurred in both DD and melatonin-injected fish during the preparatory phase and in LL carps during the pre-spawning phase. In contrast, testicular functions in both the melatonin-treated and DD fish were inhibited during the pre-spawning and spawning phases, while the testes did not respond to any treatment during the post-spawning phase. In conclusion, this study provided the first experimental evidence that melatonin plays a significant role in the regulation of annual testicular events in a sub-tropical surface-dwelling carp Catla catla, but the influence of this pineal hormone on the seasonal activity of testis varies in relation to the reproductive status of the concerned fish.

Development of dual-enzyme-based simultaneous immunoassay for measurement of progesterone and human chorionic gonadotropin.

The development of a simultaneous multianalyte immunoassay for the detection of progesterone and human chorionic gonadotropin (hCG) in serum is described. In this simultaneous multianalyte assay, two different enzymes, viz. horse radish peroxidase (HRP) and alkaline phosphatase (ALP), were used as markers. To the simultaneous immobilized progesterone and hCG antibody microwells, 50 microL of different concentrations of combined standards or serum samples was added in duplicate and then 100 microL of combined conjugate reagent, composed of 17-alpha-OH-P-ALP and hCG-biotin was added to all the wells and incubated for 1h at 37 degrees C. After incubation, the contents of the wells were decanted and washed thoroughly with running tap water. After washing, 100 microL alkaline phosphatase substrate along with streptavidin-horseradish peroxidase was added to all the wells and incubated for 0.5 h at 37 degrees C. After incubation, the developed color was measured at 405 nm. The absorbency at this stage provides the result for the progesterone assay. The contents of the wells were decanted and washed. In the next step, 100 microL of tetramethylbenzidene/H2O2 reagent was added to all the wells. After 15 min of incubation, 100 microL of 0.5 M H2SO4 was added to all the wells and the color was read at 450 nm. The absorbency at this stage provides the result for the hCG assay. Sensitivity of the progesterone and hCG assays were 0.118 ng/ml and 0.124 IU/ml respectively. Intra- and inter assay percentage coefficients of variation ranged from 1.8 to 7.1 and 9.1 to 11.5 for progesterone and from 2.1 to 10.4 and 7.2 to 11.3 for hCG. There was good correlation between the discrete and the simultaneous assays. For progesterone assay, R2 was 0.99 and for hCG R2 was also 0.99. The developed dual assay for progesterone and hCG may be useful for the diagnosis of abnormal pregnancies such as miscarriages and ectopic pregnancies.