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.

Rhythmicity in testicular melatonin and its correlation with the dynamics of spermatogenic cells in an annual reproductive cycle of Clarias batrachus under natural photo-thermal conditions.

Melatonin, the pineal hormone, is synthesized and secreted rhythmically in accordance with various environmental cues especially photo-thermal conditions. The reproductive physiology of seasonal breeders is synchronized with the surroundings by melatonin as a neuroendocrine mediator to acts as an important factor in fish reproduction. However, the data on the participation of melatonin in male reproduction and the putative interaction with the process of spermatogenesis in fish is scarce till date. So, major objectives of the current study are to determine for the first time, the relationship, if any, between seasonal levels of melatonin and testicular development and maturation of the germ cells, and also the involvements of specific meteorological parameters in spermatogenesis under natural photo-thermal conditions. We measured the concentration of circulatory and testicular melatonin; value of gonadosomatic index (GSI), relative percentages of different developing spermatogenic cells, area and perimeter (size and shape) of seminiferous lobules along with the level/duration of rainfall, water temperature and day length in six reproductive phases throughout an annual cycle in adult male catfish (Clarias batrachus). Intra-testicular and serum melatonin concentration showed a similar seasonal pattern with a peak during "functional maturity" phase and trough during "slow spermatogenesis" phase. Correlation as well as regression analyses also supported this positive relationship. Interestingly, intra-testicular melatonin also showed a significant positive correlation with GSI and relative percentage as well as lobular size of mature stages (spermatid and spermatozoa) of germ cells in an annual cycle. Furthermore, meteorological factors exhibited as critical cues to regulate the dynamics (in %) of spermatogenic cells and the level of testicular melatonin throughout the annual gonadal cycle. Our results corroborated by principal component (PC) analysis and showed very clearly that active "functional maturity" state is characterized by GSI, testicular melatonin, relative abundance and lobular size of mature spermatogenic stages as key internal oscillators; and studied environmental variables as the external clues for the regulation of spawning process. Collectively, the present data revealed that there is a relationship between melatonin levels and testicular growth and development of germ cells in Clarias batrachus under natural photo-thermal conditions.

In silico study to unravel molecular networking of melatonin in the regulation of gametogenesis.

Melatonin, a pineal hormone, has potential role on steroidogenesis, growth and maturation of sperm and ovum during gametogenesis. The possible use of this indolamine as an antioxidant in the production of good quality gametes opens up a new area of current research. Nowadays, a large number of reproductive dysfunctions like infertility and failure in fertilization due to gametic malformations are major concern worldwide. So, understanding molecular mechanisms including interacting genes and their action is a prerequisite to the therapeutic approach against these issues. The aim of present bioinformatic study is the detection of molecular network concerning therapeutic potential of melatonin in gametogenesis. It includes target genes identification, gene ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, network analysis, prediction of signalling pathways and molecular docking. We obtained common top 52 targets of melatonin in the process of gametogenesis. They are involved in biological processes related to the development of gonads and primary sexual characteristics and sex differentiation. We took top 10 pathways out of total 190 enriched pathways for further analysis. Subsequently, principal component analysis also revealed that among top ten hub targets (TP53, CASP3, MAPK1, JUN, ESR1, CDK1, CDK2, TNF, GNRH1 and CDKN1A), only TP53, JUN and ESR1were significantly interacted with melatonin on the basis of squared cosine value. So, present in silico investigation provides considerable information on the interactive network between therapeutic targets of melatonin along with the involvement of intracellular signalling cascade regulating biological processes associated with the gametogenesis. This novel approach may be pertinent in improving modern research on reproductive dysfunctions associated abnormalities.

Is divalent magnesium cation the best cofactor for bacterial ß-galactosidase?

ß-Galactosidase is a metal-activated enzyme, which breaks down the glucosidic bond of lactose and produces glucose and galactose. Among several commercial applications, preparation of lactose-free milk has gained special attention. The present objective is to demonstrate the activity kinetics of ß-galactosidase purified from a non-pathogenic bacterium Arthrobacter oxydans SB. The enzyme was purified by DEAE-cellulose and Sephadex G-100 column chromatography. The purity of the protein was checked by high-performance liquid chromatography (HPLC). The purified enzyme of molecular weight ~95 kDa exhibited specific activity of 137.7 U mg-1 protein with a purification of 11.22-fold and yield 12.42%. The exact molecular weight (95.7 kDa) of the purified protein was determined by MALDI-TOF. Previously, most of the studies have used Mg+2 as a cofactor of ß-galactosidase. In this present investigation, we have checked the kinetic behavior of the purified ß-galactosidase in presence of several bivalent metals. Lowest Km with highest substrate (orthonitrophenyl- ß-galactoside or ONPG) affinity was measured in presence of Ca2+ (42.45 µM ONPG). However, our results demonstrated that Vmax was maximum in presence of Mn+2 (55.98 µM ONP produced mg-1 protein min-1), followed by Fe=2, Zn+2, Mg+2, Cu+2 and Ca+2. A large number of investigations reported Mg+2 as potential co factor for bgalacosidase. However, ß-galactosidase obtained from Arthrobacter oxydans SB has better activity in the presence of Mn+2 or Fe2+.

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 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.