Nature is home to a wide variety of melatonin-producing organisms, including unicellular organisms, plants, and animals. Pineal glands release melanin rhythmically according to the light–dark cycle, where light inhibits melatonin synthesis and release. The gonadotropin hormone is released from the hypophysis through melatonin receptors and thus maintains the ovarian function. Melatonin also acts as an antioxidant agent and is known to prevent damage from reactive oxygen species during embryonic growth and development. Application of melatonin implants to seasonal breeding animals improves fertility via enhancement of follicular development. Melatonin treatment also aids in the collection and maturation of oocytes, and in the formation of good quality embryos.
Melatonin was first isolated from the pineal gland of bovines in 1959. The pineal gland is located in epithalamus, at the junction where the two thalamus halves meet. The pineal gland contains two types of cells: pinealocytes, which produce indolamines (the most abundant being melatonin), and neuroglia. Furthermore, in small amounts it is produced by other organs such as the retina, liver, gastrointestinal tract, lymphocytes, and skin. Its presence has also been reported in other body fluids, like cerebrospinal fluid, follicular fluid, and seminal vesicles. Melatonin has the ability to easily pass through cell membranes. The pineal gland produces melatonin on a circadian rhythm, i.e. increased production at night while reduced production during the day. This shift in melatonin concentration is controlled by N-acetyl transferase (NAT) enzyme. The suprachiasmatic nucleus (SCN), a major circadian oscillator regulates melatonin production after receiving signal from the retina through retinohypothalamic tract. After its production in the pineal gland, the hormone is not stored, rather is released either into the blood or into the cerebrospinal fluid. The liver is primarily responsible for its metabolism. Besides this, melatonin is also believed to be responsible for detoxifying free radicals created during metabolic processes. There are reports stating involvement of melatonin in follicular growth, oocyte maturation and luteinization, reproductive development, parturition and maintenance of the gestation period.
Mammals have three subtypes of melatonin receptors: MT1 (formerly Mel 1a or ML1A), MT2 (formerly Mel 1b or ML1B), and MT3 (formerly ML2). MT1 and MT2 are G protein-coupled receptors on which melatonin appears to exert most of its cellular actions. The MT1 receptor length is 350 amino acids and its weight is 39,374 Da. This receptor is expressed in both the pars tuberalis and SCN of the hypothalamus in animals. The MT2 receptor is of 362 amino acids with a molecular weight of 40,188 Da and 60% homology with MT1 receptor. These receptors were found in a number of organs, including the retina, the brain, and the gastrointestinal tract. Furthermore, they are involved in inhibiting both soluble guanylyl cyclase and adenylyl cyclase. A common link between MT1 and MT2 receptors is the pertussis toxin-sensitive Gi protein, and the activation of this protein inhibits the AC/cAMP/PKA/CREB pathway. Through the MT2 receptor, inhibition of the GC/cGMP/PKG pathway can occur upon Gi activation. Both MT1 and MT2 receptors activate the phospholipase C pathway, which increases the level of 1,2-diacylglycerol (DAG) and inositol triphosphate (IP3). As a result of activation of MT1 receptors, melatonin induces multiple cellular responses that are mediated by both PTX-sensitive (Gi2 and Gi3) and PTX-insensitive (Gq/11) G proteins. Adenylate cyclase activity in target cells is inhibited by binding of melatonin to MT1 receptors. The MT2 receptors are low-affinity receptors that are coupled to phosphoinositol hydrolysis. MT1 receptor have greater sensitivity to 2-iodomelatonin than to melatonin, and even more stronger sensitivity to 6-chloromelatonin whereas affinity of MT2 receptor is similar for melatonin, 2-iodomelatonin, and 6-chloromelatonin. Ligands with higher affinity for the MT2 than for the MT1 melatonin receptor include luzindole (15 to 25 times higher), IIK7 (90 times higher), K185 (140 times higher) (Table 1). The nonindolic 4-phenylacetamidotetraline (4P-PDOT) is a selective MT2 melatonin receptor ligand. The rabbit retina was first shown to contain melatonin receptors that inhibit dopamine release using this melatonin receptor antagonist. MT3 is an active melatonin receptor found on the cytosolic enzyme quinine reductase 2 in mammals. The melatonin affinity of this receptor is generally low. It has also been reported that MT1 and MT2 are either expressed separately or in concert in the reproductive and cardiac tissues.
Biosynthesis and mechanism of action of melatonin
In the course of melatonin synthesis, tryptophan is converted to 5-hydroxy-tryptophan and subsequently to serotonin after decarboxylation. Acetyl group is then added to serotonin to form N-acetylserotonin and then after transfers of a methyl group from S-adenosylmethionine to the N-acetylserotonin to form melatonin. Breeding rhythms are regulated by melatonin in diverse ruminant species, such as goats and sheep (short-day species), and in horses (long-day species). The melatonin-mediated mechanisms control the GnRH pulsatility which, in turn, affects the activity of the reproductive neuroendocrine axis. This in-turn modulates prolactin secretion, an increase in the prolactin secretion leads to decrease in episodic LH secretion. Prolactin can alter the number of LH receptors in the ovary affecting steroidogenesis. This will ultimately led to decreased estrogen levels. Decrease in the level of thyroid hormone causes a rise in prolactin by increasing the thyroid-stimulating hormone. The negative feedback potency of estradiol is modulated by the action of melatonin at hypothalamic sites to elevate GnRH release, which act at hypothalamic and hypophysial loci to reduce LH secretion. The photoperiod affects melatonin levels and in turn melatonin modulates kisspeptin-1 expression, suggesting that kisspeptin pass on photoperiodic signals to the hypothalamic-hypophysial-ovarian (HPO) axis. By increasing the level of GnRH in the hypophysial portal blood, Kisspeptin increases LH secretion. Kisspeptin neurons show expression of estrogen and progesterone receptors which are regulated by positive and negative feedback to the pulsatile GnRH secretion response.
Effect of melatonin in reproductive cycle of buffaloes
The exogenous slow-release melatonin restored ovarian activity in summer anoestrus buffaloes. This was evident by 10-fold rise in plasma concentrations of GnRH and gonadotrophins after exogenous melatonin administration, which supplied the required boost for follicular development and ovulation. Melatonin activates the hypothalamus-pituitary-ovarian cascade, resulting in an early age onset of puberty. Application of melatonin implants in Murrah breed of buffalo leads to an earlier induction of cyclic ovarian activity which ultimately accelerated the puberty onset and improves conception rates. In anestrus buffaloes, melatonin injection induced increased progesterone (P4) level post artificial insemination when compared to the control group. This proposes that melatonin might have luteotrophic effect during summer season. It also ameliorates P4 receptors and their binding capacities in the uterus. In contrary to other animals, melatonin implants in buffaloes are responsible for increased estrogen levels in lactating buffaloes. Combined treatment of melatonin and Controlled Internal Drug Release (CIDR) gave better results in anestrus buffaloes. During out of breeding season, treatment of melatonin and CIDR causes significant increase in plasma levels of albumin, glucose, high-density lipoprotein (HDL), magnesium, calcium and alanine aminotransferase (ALT). Albumin, a major protein found in blood, could play a role as an antioxidant via carbonyl formation and thiol oxidation. Glucose plays a stimulatory role to follicular growth in ovary. This may be a possible reason for the difference in the glucose levels in different sized follicles as glucose is the primary source of energy for ovaries. In addition, it also acts as a signal for the release of GnRH. Hence, it can be concluded that melatonin and CIDR treatment improves reproductive performance of buffalo during out-of-breeding season.
Melatonin in Assisted Reproductive Technology (ART)
Assisted reproductive technologies involve handling of ovaries which creates exceeding stress to ovaries. Melatonin reduces ovarian oxidative stress by being a free radical scavenger as well as by enhancing the antioxidant enzyme activity. Melatonin increases the activity of antioxidant enzymes, such as glutathione peroxidase (GPX) and superoxide dismutase (SOD), in vitro decreasing the levels of blastocyst apoptosis. Melatonin is thought to produce its beneficial effects by activating the MTNR1A receptor on granulosa cells. Melatonin enhances eukaryotic initiation factor 2 (eIF2) signaling, which is essential to translation and protein synthesis. Melatonin also works on DNA damage-inducible 45 (GADD45), which is essential to DNA repair. A secondary effect of melatonin is to suppress autophagy-related proteins by stimulating intracellular pathways such as eIF2, GADD45, and alternative reading frames. A number of mechanisms contribute to the prevention of ovarian aging, including anti-oxidant action, DNA repair, maintenance of telomeres, enzyme activity and autophagy. Melatonin treatment also increases the number of oocyte collected, helps in maturation of oocyte, and production of good quality embryos. The supplementation of melatonin to in vitro maturation media improves fertilization rates of buffalo oocytes. Melatonin addition to cryopreservation medium for semen has protective effect on sperms. It also assists in in vitro sperm capacitation process in buffaloes.
Role of melatonin as an antioxidant in buffaloes
Factors like high temperature as in summer season and high milk yield etc. causes stress to buffaloes and hence affects their reproduction. This results in condition like anestrus, poor embryonic development and several other reproductive failures. The presence of high number of oxidative stress biomarkers in the follicular fluid of acyclic buffaloes is an indicative of the fact that stress causes reproductive troubles in buffaloes. Melatonin acts as a direct free radical scavenger via detoxification of hydroxyl radicals and inhibition of lipid peroxidation. Several findings support the notion that exogenous melatonin administration has a positive correlation with increased antioxidant capacity in buffaloes. Besides, free radical scavenging abilities, melatonin also expresses certain antioxidative enzymes such as SOD and GPX. SOD activities in corpus luteum helps in maintaining the progesterone concentrations in plasma. There have been a number of reports implicating melatonin in scavenging reactive oxygen species (ROS) and reactive nitrogen species (RNS). Melatonin associates with a variety of ROS to produce cyclic 3-hydroxymelatonin and other melatonin metabolites e.g., N1-acetyl-N2-formyl-5-methoxykynuramine and N-acetyl-5-methoxykynuramine. In terms of their ability to neutralize ROS, these metabolites are sometimes even more powerful than melatonin. Following ovulation LH surge releases ova, during this phase vascular endothelial cells and macrophages produce a great deal of ROS as neovascularization progresses in the follicles. Although, the production of ROS in minute quantities in follicles is the necessary stimulus for oocyte maturation and follicular rupture, excessive ROS might lead to cytotoxicity. Nitric oxide (NO), an intra-ovarian factor modulates the follicular development process through vasodilation, angiogenesis, steroidogenesis, normal follicular growth and development, etc. In condition of oxidative stress, NO is leveled up and all the aforementioned steps get affected. After melatonin treatment a profound decrease in NO level was observed, thus mitigating the stressful effects on follicular development. Unlike conventional antioxidants such as vitamin C, E, mannitol, or glutathione, melatonin is a direct free radical scavenger and has significantly greater antioxidant capacities.
A proper balance between the reproductive and productive health of animal is a crucial issue. These issues ultimately affect the economic status of farmers, as reproduction influences the progeny health and production impacts traits like milk production etc. During out of breeding season, buffalo exhibits anestrus symptoms which pose managemental difficulties to farmers. Melatonin modulates reproductive physiology in photoperiod dependent breeding animals especially buffaloes. In addition to scavenging free radicals, melatonin also stimulates production of antioxidative enzymes. It controls the follicular functions in ovaries and protects sperms quality in assisted reproductive technologies. Melatonin implants together with CIDR influences conception rates in anestrus buffaloes. Hence, melatonin implants and administration can help to solve reproductive problems in buffaloes to some extent together with existing therapies.
Suryaprakash Pannu1 and Mamta Pandey21PhD Research Scholar, Animal Reproduction Gynaecology and Obstetrics, ICAR-National Dairy Research Institute, Karnal, Haryana, India
2 Research Associate, Molecular Reproduction Lab, Animal Biotechnology Centre, ICAR-National Dairy Research Institute, Karnal, Haryana, India