ROLE OF MELATONIN ON REPRODUCTION IN SEASONAL BREEDING ANIMALS
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.
The seasonality of reproduction is an adaptive physiological process utilized by wild animals to deal with seasonal changes in temperature and food availability. Domestication has led to an almost complete loss of this adaptation in cattle and pigs but it is retained in most breeds of sheep, goats, horses and yaks originating from temperate latitudes (Wuliji et al., 2003). At these latitudes, photoperiod is the main environmental factor that determines the onset and duration of breeding seasons. The timing of the breeding season depends on the length of the gestation period so that parturition occurs in the spring. Thus, sheep and goats (5 month gestation period) are short-day breeders with conception occurring in autumn and winter, whereas horses (11 months gestation period) breed during the long days of spring (Ortavant et al., 1985). Photoperiodic information is integrated through a complex pathway involving both neural and humoral steps. Photoperiod is first perceived by the retina and transmitted via a multistep neural pathway, which involves the suprachiasmatic nuclei (SCN) and superior cervical ganglia, to the pineal gland where the message modulates the rhythm of melatonin secretion (Karsch et al., 1984). Melatonin is released only at night and, therefore, the duration of secretion differs between long and short days. This duration of melatonin secretion is then processed to regulate the activity of the hypothalamo hypophysial and gonadal axis (Karsch et al., 1988).
Melatonin or 5-methoxy-N-acetyltryptamine was first described from bovine pineal in the year of 1958 by Aaron Lerner Melatonin is the principal secretion of the pineal gland and it is regulated by the circadian rhythm. Initially, it was thought that it is unique to the pineal gland, but now it is known to be produced in many other tissues all over the body. The rhythmic release of melatonin acts as an endogenous synchronizer, modulating several physiological events like sleep-awake-cycle, blood pressure, body temperature, glucocorticoid or cortisol rhythm, and body defense mechanism, etc. Human melatonin has diurnal variations and its secretion gets elevated with the onset of darkness, reaches its peak concentrations in the mid-night followed by gradual decrease in the last part of the night. The gradual rise in melatonin level is seen in individuals from the birth and likewise, become peak during 2 to 4 years of age relatively lesser during the puberty and decreases gradually from the middle age. As the consequence of decline in levels of this natural ubiquitous molecule in old age or in pathological conditions, melatonin may modulate associated physiological functions.Melatonin is applied in combination therapie due to its negligible side-effects together with its ability to reduce the side effects and increase in the functional efficacy of large number of drugs, it finds applications.Melatonin is also well known for being a molecule with antioxidant properties and it also triggers the brain antioxidant defense mechanisms.
Melatonin has been associated with improved male reproductive functions. It has been shown that melatonin exposure is essential for reproductive functions in seasonal breeders. In humans, melatonin has been demonstrated to influence the anterior pituitary gonadotropins, gonadal steroids and testicular functions via specific receptors which are profusely expressed in the central nervous system as well as in the reproductive tissues. Moreover, the role of melatonin in protection against testicular pathogenesis and in the sustenance of normal spermatogenesis have been documented. But, the exact mechanism of its action other than its antioxidant properties in testicular cells, remains largely unknown. Attributing to its capability to cross physiological barriers, such as the blood- brain barrier, the blood-testis barrier without obvious toxic impact, melatonin finds high relevance as a therapeutic molecule in male reproductive disorders. In the present global scenario, where male fertility in terms of semen quality is showing a gradual decline molecules like melatonin with ameliorative effects on male reproductive functions should be explored further for therapeutic interventions. This article thus aims to review and present an updated report on the properties of melatonin and its receptors along with its versatile roles in the regulation of male reproductive functions.
Physiological functions of melatonin
Role of melatonin in the sleep-wakefulness cycle may be considered as its classical function.Melanopsin is the receptor for light that keeps the body in tune with external time. It contains illumination detecting retinal ganglion cells to signal for the absence or presence of light and it is mediated through the retinohypothalamic tract to the suprachiasmatic nucleus. Melatonin secretion increases near about 10 folds during darkness and falls towards low levels during appearance of the light of day these fluctuations in melatonin secretion help to entrain the body’s biological rhythms with the external light dark cues. It appears to enhance immunity and has been shown to reverse some of the age-related shrinkages of the thymus which is the source of T-lymphocytes. For some other mammals, it controls the seasonal breeding by targeting the hypothalamus.
Melatonin: As free radical scavenger
Free oxygen radicals are produced normally as byproducts during cellular metabolism that utilize oxygen. These radicals of oxygen contain unpaired free valence electrons. Oxygen is particularly susceptible to this conversion, as its outer pair of electrons spin is in the same direction, thus tending to accept electrons one at a time. This free radical form of oxygen is more reactive than the non-radical oxygen, such as the reactive oxygen species (ROS). These include superoxide anion, perhydroxyl radical, singlet oxygen, hydroxyl radical, hypochlorous acid, peroxyl, lipid peroxide, etc. Sometimes non-radical molecules are also termed as ‘ROS’ because of their oxidation nature like the hydrogen peroxide .Generally, ROS are essential for some normal physiological processes like spermatogenesis, sperm capacitation, hyperactivation and acrosome reaction, but an increased ROS production causes oxidative damage to the cells by inducing ‘oxidative stress’.
Endogenous anti-oxidative agents scavenge the reactive oxygen radicals and inhibit the oxidation of other molecules with ROS to prevent the cellular oxidative damage .Several endogenous antioxidant molecules like catalase, glutathione peroxidases, superoxide dismutase, thioredoxin, vitamin C, vitamin E, etc play vital role in maintaining the seminal redox homeostasis .Melatonin has also been considered as an important classical endogenous antioxidant molecule .due to its several distinguishable qualities. These include its amphiphilic nature enabling it to easily cross the blood-brain barrier or blood placental barrier and its interactions with nuclear receptor . Melatonin can mediate its antioxidant actions either in a receptor-dependent pathway as well as by directly scavenging the free radicals . Moreover, the ability of melatonin in upregulating antioxidant enzymes and downregulating prooxidant enzymes makes it a unique oxygen scavenger . Melatonin may interact with ROS indirectly by participating in redox cycling unlike vitamin C, glutathione, etc, and form several stable metabolites or end products through some add-on reactions in a deluge manner which can act as antioxidant too and also can easily be excreted via urine .For example, 3-hydroxymelatonin is more potent than its primary form in reducing hypervalent hemoglobin, on the other hand, N1-acetyl-5-methoxykynuramine may show powerful ROS scavenging capacity than its precursor and thus may prevent protein oxidation. Melatonin can also enhance the activities of some traditional antioxidants including alpha-tocopherol, glutathione peroxidase, ascorbate and superoxide dismutase, etc. It also may stimulate the mRNA levels of antioxidant enzymes including superoxide dismutase, glutathione peroxidase, and catalase.
Melatonin in male reproduction
In puberty and sexual maturation
During fetal life (midgestational fetus) the hypothalamic-pituitary-gonadal (HPG) axis is active but becomes quiescent towards the term due to the inhibitory feedback of placental hormones and regains at the birth time. The gonadotropin-releasing hormone (GnRH) and subsequently the gonadotropin levels gradually attain their peaks during the first three months of the life and again declines towards the sixth months remaining quiescent until puberty is attained. Its further reactivation depends on the gradual and continuous release of GnRH resulting in successive secretion of gonadotropins. From one experimental model it has been shown that melatonin secretion may inhibit the hypothalamic pulsatile release of GnRH in male Djungarian hamster. The melatonin levels attain their peak from two to four years of age, and on the other hand the reductions in nocturnal plasma melatonin occur around or prior to pubescence. Thus, it can be said that melatonin may play a role in inhibiting the hypothalamic GnRH activation. During puberty, body mass increases and this is inversely related to melatonin secretion leading to falling in its levels below the threshold concentrations, and so the GnRH pulse generator is re-activated and male reproductive functions are potentiated.Therefore, it is concluded that the decline of melatonin levels trigger puberty and high nocturnal level of melatonin secretion may be associated with delayed puberty in children. Relatively low levels of melatonin than the normal may be associated with precocious puberty in some clinical conditions like destructive pineal tumors in young boys.
In the year of 2005, kisspeptin was discovered as a most powerful activator of GnRH neuron located in the hypothalamus .Being a peptide, kisspeptin is expressed in the arcuate nucleus and in the anteroventral periventricular nucleus of the forebrain abundantly and it is encoded by Kiss1 gene and both estradiol and testosterone regulate the expression of the Kiss1 gene.It is well known to all that the mammalian reproductive system is controlled under the pulsatile release of GnRH and it is mediated by the effect of that kisspeptin. It is also noticed that, for the process of human puberty, all the GnRH neurons do not always get input from the kisspeptin neurons and some other neurons are also responsible like RFamide-related peptide (RFRP) neurons which secrete RFRP-1 and RFRP-3, a member of the RFamide peptide group, known to inhibit GnRH, and also it may play a role in between melatonin and GnRH release due to the effect of melatonin on RFRP neurons . Some animal studies reveal that followed by pinealectomy (a major source of endogenous melatonin), reduction in kisspeptin expression was observed under exogenous melatonin supplementation along with prolonged photoperiodic exposure. But depending on duration of exogenous melatonin, supplement kisspeptin is regulated; its acute supplementation may reduce the expression of kisspeptin gene at first but when it was used for longer time, it enhanced the kisspeptin gene expression which may again interfere in HPG axis to regulate the release of gonadotropins. Thus, different effects of melatonin on the reproductive system may be modulated by kisspeptin and which is also dependent on the duration of supplementation of the melatonin.
In a cross-sectional study, Waldhauser et al mentioned that as comparing to age group 1 year to 3 years, the mean nighttime serum melatonin level was dropping progressively throughout the adolescence age group of 15 to 20 years .It indicates that during the adolescence, decreased nocturnal melatonin levels are directly proportional to sexual maturation and inversely proportional with Tanner stage .When exogenous melatonin was used to treat animals, it would suppress the GnRH secretion as the emergence of exogenous melatonin administration may alter the sexual maturation. Nowadays, most important concern during the treatment of sleep disturbances is whether the very young or children or adolescents are being affected on their pubertal growth due to the exogenous melatonin supplementation.
Melatonin and HPG axis
Release of melatonin is utterly dependent on light and darkness cycle as well as seasonal variations. The breeding capacity of seasonal animals varies with these variations and this has already been linked with seasonal variations of melatonin .Melatonin, along with hypothalamic and pituitary hormones, plays prime functions in regulation of reproductive functions. The HPG axis is the principal reproduction regulatory axis but it may be modulated by several crosstalks with metabolic hormones growth factors, and other endogenous influencers thereby interfering with its effects on reproductive functions. Melatonin treatment to the pups of female rats showed disrupted melatonin profiles in growing pups indicating its maternal inheritance in the regulation of reproductive development. It has also been reported that delayed onset of pubertal changes due to reduced luteinizing hormone (LH) and prolactin profiles can be mitigated by melatonin treatment to the mother.Exogenous melatonin has been reported to show inhibitory effects on gonadotrophins secretions affecting the male reproductive development by interfering with follicle-stimulating hormone actions on Sertoli cells .It has been shown that in isolated pituitary cells melatonin administration decreases the effects of LH-releasing hormone .These inhibitory actions of melatonin are mediated by alterations in intracellular second messenger concentrations, specifically by increasing Ca2+ influx and cAMP accumulation inside the cells .These second messengers potentiate the effects of GnRH on LH secretion, but melatonin administrations affect their concentrations . Melatonin treatment also increases testosterone secretion that negatively regulates gonadotrophin secretions and thus sexual maturation.
Melatonin directly affects the neurons in suprachiasmatic nuclei and that secrete GnRH to mediate its effects. Its receptors have also been reported in pars tuberalis and pars distalis of the anterior pituitary .But, the actions of melatonin on sexual functions by hypothalamic and pituitary neurons are reported to be distinct. Inactivation of melatonin receptors has been also reported to disrupt diurnal rhythms and reproductive functions .Expression of melatonin receptors in hypothalamic and pituitary neurons potentiates the hypothesis of its roles in reproductive functions via GnRH pulse. Implantation of melatonin containing pellets in hypothalamus has been reported to reduce testicular weights in rats by 60% compared to control rats . Melatonin treatment to male mile over 10 days have shown a decrease in testicular and accessory organ weights along with reduced spermatogenesis. Successive administration of gonadotrophins has been reported to reverse these effects. Inhibitory actions of melatonin on GnRH secretions have been evidenced in isolated GnRH cells following melatonin administration.
Melatonin has also been reported to down-regulate the expression of LH-β and follicle-stimulating hormone-β as mediated by gonadotropin-inhibitory hormone (GnIH) to inhibit GnRH synthesis and secretion. In photoperiodic animals, pinealectomy has been found to disrupt reproductive functions due to the decreased melatonin secretion and altered GnIH production which have been reversed with exogenous melatonin administration[8l]. Under short-day conditions Siberian hamsters have exhibited higher GnIH expressions, indicating GnIH expression controls actions of melatonin. However, all these data postulate that GnRH alters the actions of melatonin and thereby its regulation over reproductive functions.
Melatonin and testicular steroidogenesis
Melatonin is also a key regulator of steroidogenesis and testicular and accessory sex organ development. Chronic exposure of light to hamsters have been found be related with altered tubular and interstitial structures in testis. Melatonin administration has also been reported to reduce mitochondrial and smooth endoplasmic reticular volume and surface area which are the key locations for testicular steroidogenesis.Moreover, testosterone biosynthesis is dependent on LH-mediated cAMP actions on Leydig cells .Rats treated with exogenous melatonin decreased Leydig cell cAMP concentrations and thus reduced LH signaling. Administration of luzindole, a melatonin receptor antagonist, has been reported to restore the cAMP concentrations in Leydig cells and thus stimulates LH functioning for testicular steroidogenesis. It is also found to reduce the LH-induced expression of steroidogenic acute regulatory protein by decreasing cAMP concentrations in Leydig cells .Other than cAMP-dependent actions of melatonin, it also decreases GnRH-dependent Ca2+ release and protein kinases activation which are associated with testosterone biosynthesis and secretion.
Along with reduction of LH- and GnRH-induced steroidogenesis, melatonin also affects testosterone production via acting through hypothalamic-pituitary-adrenal axis and also through the actions of corticotropin-releasing hormone, cortisol or corticosterone. The corticotropin-releasing hormone produced by Leydig cells acts as a prime autocrine regulator of GnRH-induced testicular steroidogenesis . Isolated Leydig cells from hamster testis, exposed to different photoperiods and melatonin concentrations showed decreased cAMP and androstane-3α, 17 β-diol levels with reduced expressions of steroidogenic acute regulatory protein (StAR), P450scc (side-chain cleavage), 3β- and 17β-hydroxysteroid dehydrogenase .Although it has been reported that melatonin upregulates mRNA expression of corticotropin-releasing hormone, use of corticotropin-releasing hormone antagonists can eliminate the effects of corticotropin-releasing hormone, or melatonin indicating melatonin pathway is independent of corticotropin-releasing hormone regulation.
Melatonin and oxidative stress in spermatozoa
In the sperm, melatonin reduces oxidative damage in the mitochondria, DNA fragmentation, lipid peroxidation of plasma membrane, and apoptotic markers by improving antioxidant activity of enzymatic systems and reducing ROS levels. In addition to the antioxidative properties of melatonin in seminal plasma. it has been also been demonstrated to have a direct action in sperm capacitation. Thus, high levels of seminal melatonin may protect sperm from oxidative damage, and prevent capacitation at the same time.Though there is less amount of melatonin found in ejaculated sperm in the female reproductive tract, the melatonin present in follicular fluid helps in the process of capacitation .It has been reported that melatonin regulates seminal calmodulin level and thus controls several reproductive functions, like hyperactivation, capacitation, and acrosome reaction. Melatonin, through its receptors on sperm membrane, MT1 and MT2, exert direct actions on spermatozoa .MT2 has been reported to be directly concerned with the process of capacitation .Melatonin has been reported to regulate secretion of bicarbonates and mobilization of calcium, and thus it may control the process of sperm capacitation by regulating the effects of calcium and bicarbonates on sperm.
Melatonin and semen quality
Spermatogenic disruption can be observed in testes exposed to endocrine disruptors or genital infections.Melatonin is well- known to protect testis from these reproductive disruptions .Lower seminal levels of melatonin have also been associated with male infertility .Declined seminal melatonin has also been reported to cause decreased sperm motility . These data point toward the role of melatonin in the regulation of semen quality. The receptor of the sperm membrane also indicated its role in sperm production and maturation. It has been reported that in rams reproductive functions are under the influence of photoperiod and semen quality decreases after the breeding season .Melatonin has been proven to improve semen quality in rams and male Damascus goats in non-breeding seasons. Isolated ram spermatozoa exposed to melatonin showed improved capacitation and phosphatidylserine translocation. These changes decline at lower dosage of melatonin .Melatonin due to its potential antioxidative properties has been reported to protect sperm cells from ischemia and improves sperm abnormalities. Sperm maturation medium with high melatonin has been found to increase sperm progressive motility, raise the number of motile sperms, improve sperm mitochondrial activity, along with decreasing endogenous nitric oxide levels. Cryopreserved sperms for insemination has shown an improvement in motility, straight-linear velocity and average path velocity when exposed to high levels of melatonin, even stored at 5°C and 17°C .The role of melatonin in amelioration of sperm damage is associated with various signaling cascades. It reduces sperm DNA fragmentation and apoptosis induced by hydrogen peroxide through MT1 and extracellular signal-regulated kinases.
Melatonin on sperm preservation
As discussed above, melatonin due to its antioxidative and antiapoptotic functions improves semen quality. It has been reported that boar spermatozoa treated with 100 nM melatonin improved sperm motility, sperm membrane integrity, sperm mitochondrial functions along with increased numbers of embryos produced through in vitro fertilization. Succu et al with different doses of melatonin showed increased sperm motility, sperm chromatin integrity and intracellular adenosine triphosphate (ATP) concentration in ram spermatozoa. Souza et al[l09] with various concentrations of melatonin also supported these observations in ram spermatozoa and reported higher sperm motility, sperm membrane and acrosome integrity, and mitochondrial activity in melatonin treated sperms. It has been proposed that improvement of sperm motility in melatonin-treated spermatozoa is attributed to its effects of sperm mitochondrial functions. Ashrafi et al .with different concentrations of melatonin to a freezing extender for bull spermatozoa reported better sperms functions with improved antioxidant profile in seminal plasma. El-Raey et al .with two different concentrations of melatonin (0.10 and 0.25 mM) to a freezing extender for buffalo spermatozoa have reported improved semen quality, plasma membrane and acrosome integrity, with higher conception rate after freeze-thawing. Lanconi et al[ll2] with 1 mM melatonin added to a freezing extender for horse spermatozoa reported same observations. For human spermatozoa, Karimfar et al .have reported the same with multiple doses of melatonin. Altogether, these reports reveal the semen quality- improving potential of melatonin and its beneficial effects on sperm cryo survival with fertility-enhancing actions in different species. However, in dogs, semen quality-improving and cryo survival- enhancing functions are not very much evident. Epididymal spermatozoa of German and Belgian Shepherd dogs incubated with 1 mM melatonin showed similar sperm motility, plasma membrane and acrosome integrity, capacitation status, and membrane fluidity after freeze-thawing compared to untreated sperms.
In some cases, the negative effects of the dissolving medium have been noted on semen quality. Gwayi and Bernard have reported negative effects of ethanol in rat spermatozoa.But, Martín- Hidalgo et al .did not find any detrimental effects of ethanol as a melatonin-dissolving medium on boar spermatozoa.
Studies have also reported negative effects of high levels of melatonin on cryopreserved sperms on its fertilizing potential. It has been hypothesized that excessive neutralization of ROS generation and inhibition of oxidative phosphorylation may reduce sperm viability as well as motility .Melatonin, which has been evident to facilitate sperm functions including capacitation, hyperactivation, and acrosome reaction, is a universal antioxidant, but its excessive dosage may hinder the normal functioning of ROS needed for these sperm functions. These reports indicate the importance of identification of optimal dosage of melatonin for cryopreservation in different species.
Melatonin
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.
Melatonin Receptors
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
1.Regulation of Seasonal Rhythms
Respond to the annual changes in photoperiod by adaptive alterations of their physiological state. Pineal gland is a neuroendocrine transducer receiving photoperiodic information from the retina and circadian SCN oscillator, and transmitting this to the reproductive system via melatonin (MEL) secretion (Foster et al., 1988) (Fig. 6 and 7).
2.Regulation of Circadian Rhythms MEL is synthesized during the dark phase of the light/dark cycle and is rapidly delivered to the body via the blood stream. Pinealectomy facilitates the resynchronization of the animal to a new photoperiod. The daily rhythm of MEL circadian mediator used by the endogenous SCN clock to deliver the circadian message to MEL target structures (containing MEL-R). “chronobiotic” effect by acting directly on the SCN, which contain MEL-R, to affect the circadian clock (Pevet et al., 2002). Exogenous MEL, applied directly into the SCN phaseadvances the endogenous MEL peak increases the amplitude of the MEL peak (Bothorel et al., 2002).
3.Role in seasonality and breeding The role of melatonin in the photoperiodism of sheep was clearly established in the early 80s. It is of interest that early breeding is achieved by feeding melatonin only before sunset. The nocturnal secretion of pineal melatonin provides information to vertebrates on changes in day length under the circumstances in which they live. In seasonal breeders, the secretion of melatonin is also a signal to the neural structures controlling the secretion of gonadotropins from the pituitary gland to drive their activity in accordance with the season of the year (Afify et al., 2004). The photoperiod drives the reproductive cycle, which comprises a season of high sexual activity during short days and an anestrous season that occurs during long days (Dahl and Petitclerc, 2003). Information on changes in the photoperiod is provided to the organism through nightly secretion of melatonin from the pineal gland. Thus, changes in the duration of melatonin secretion constitute a signal to the neural structures controlling the secretion of gonadotropins from the pituitary gland that a long duration is stimulatory and a short duration is inhibitory. Anestrous animals treated with exogenous melatonin, show a sustained high melatonin level in the organism led to the activation of the hypothalamo-pituitary GnRH/LH axis (Arendt et al., 1983; Bittman et al., 1983). Melatonin is able to stimulate LH secretion if it is delivered into this site. In response to a change in the photoperiod, the daily MEL profile displays substantial changes, primarily affecting the duration and/or the amplitude of the nocturnal peak. Distortion of the MEL message, in turn, has an impact on many physiological functions. How the organism “reads” the modifications of the MEL profile is still largely hypothetical and appears speciesdependent. The duration of the nocturnal peak seems to be an important parameter in many photoperiodic species. Photoperiodic dependent changes may rely on the absolute duration of the nocturnal MEL peak (A) or on the presence of a time-window of sensitivity to MEL (B). In addition, in some species, the amplitude of the nocturnal MEL peak may be an important parameter (C). a, amplitude of the nocturnal MEL peak; d, duration of the nocturnal peak of MEL; LP, long photoperiod; SP, short photoperiod (Adopted from: Simonneaux and Ribelayga, 2003). An increase in LH release is observed in luteal-phase ewes during the first hours of darkness. However, it appears that a central interaction between melatonin and estradiol is needed to sustain a high level of LH secretion during the reproductive period. Importantly, LH secretion is stimulated in all animals with microimplants placed correctly with respect to the higher binding area, suggesting that the PMH is an important target for melatonin in regulating reproductive activity in ewes. The easiness of diffusion of melatonin from the pineal recess of the third ventricle to its ventral part (Tricoire et al., 2002) indicates, in turn, that the pineal hormone may be caught by the PMH cells directly from the cerebrospinal fluid. Photoperiodic control of gonadal function is probably a result of changes in gonadotropin secretion. In both long-day and short-day breeding species, exposure to inhibitory photoperiods caused a decline in pituitary and blood levels of luteinizing hormone (LH) and follicle stimulating hormone ( FSH), while exposure to stimulatory day lengths caused opposite effects. Light exposure can regulate gonadotropin secretion by altering responsiveness of the hypothalamic – pituitary axis to the negative feedback actions of gonadal steroids. There is a negative relationship between gonadotropins and melatonin. This means that the increase of MLT concentration during winter months lead to poor reproductive in livestocks (Kassim et al., 2008). It indicates a high concentration of P4 and E2 at estrous stimulated by decline of MLT. Treatment of ruminants by extending light during autumn and winter (dark season) decrease serum concentration of MLT, particularly after puberty and increase the serum concentrations levels of P4, E2 and PGF2 (Sarkar and Prakash, 2005). Treatment of adult ewes with melatonin implants results in decreased prolactin secretion, apparently mimicking the prolactin changes observed when the daily photoperiod is shortened (Kennaway et al, 1982). In cattle, as in other species, there is dependence on the pineal for photoperiodic responses. Indeed, blinding and pinealectomy eliminate rhythmic patterns of melatonin release and, thus, photoperiodic responses (D’Occhio and Suttie, 1992). If light is perceived (i.e., low melatonin) approximately 15 h after dawn, a time termed the photosensitive phase, that is the cue for a long day. In contrast, the presence of darkness (i.e., high melatonin) during the photosensitive phase will be perceived as a short day (Daramola et al., 2006). Timed exogenous melatonin administration to anoestrous ruminants would promote an early onset of breeding activity (Afify et al., 2004). Exogenously administered melatonin could mimic the physiological effects of short daylength. When anoestrus ewes are treated with silastic envelopes containing melatonin the breeding season is advanced by five to 10 weeks (Dahl and Petitclerc, 2003).
Other Roles of Melatonin
Autocrine/Paracrine Effects:
In addition to the pineal gland, MEL is synthesized in several other structures (retina, Harderian gland, gut) where the genetic expression and biochemical activity of the MEL-synthesizing enzymes have been detected (Djeridane et al., 2000). In the retina, MEL is rhythmically synthesized in the photoreceptors in a circadian manner. MEL alters various aspects of retinal metabolism. Most of the retinal effects of MEL are indirect, and probably consist primarily in the inhibition of dopamine (DA) release from amacrine cells (Jaliffa et al., 2000; Tosini and Dirden, 2000).
Modulation of Neurotransmission:
MEL can alter the release of several neurotransmitters, especially DA, 5-HT, norepinephrine (NE), acetylcholine (ACh) and can modulate the postsynaptic response. MEL, through activation of its different receptor subtypes, can differentially modulate the function of type A gama amino butyric acid (GABAA ) receptors (Wan et al., 1999).
Effects on the Immune System:
In vivo, high exogenous doses of MEL show a general stimulation of the immune system (Reiter et al., 2000a). It increases T cell activity, lymphocyte growth, humoral responses, and may inhibit thymus involution with age (Maestroni, 2001). In vitro MEL also increases T helper and NK cell activities, the production of interleukin 2 and interferon gamma, and the expression of interleukin 1 mRNA in monocytes. In summary, there is an immuno-stimulating effect of MEL. These effects may occur via a direct action of MEL on its receptor since MEL-R have been identified in various tissues of the immune system, namely thymus, spleen, lymphocytes, and T helper cells. In addition, MEL acting as a chronobiotic may be involved in the circadian organization of the immune system (the number and activity of lymphocytes T, B, and NK displaying a daily rhythmicity). It has also been proposed that MEL may mediate seasonal changes in immune function, which is enhanced in short days with longer MEL peak duration (Nelson and Drazen, 2000).
Antioxidant/ Antiaging Property of Melatonin:
The lipophilic MEL diffuses into the cell cytosol and nucleus to protect cytosolic and nuclear macromolecules from free radical cytotoxicity (Reiter et al., 2000b). The use of oxygen in cell metabolism leads to the production of cytotoxic byproducts that are reactive free radical species (superoxide anion radical, peroxynitrite anion, hydrogen peroxide, nitric oxide, and the highly toxic hydroxyl radical), which destroy macromolecules like DNA, lipids, and proteins leading to cell death via apoptosis. High doses of MEL (in the micromolar range) are reported to neutralize most of these cytotoxic molecules, but especially the hydroxyl radical, which is scavenged in vivo by MEL, producing cyclic 3- hydroxyMEL excreted in the urine. In addition, MEL is reported to stimulate the activity of various antioxidant enzymes, like superoxide dismutase or glutathione peroxidase, but inhibits the pro-oxidant enzyme nitric oxide synthetase. MEL could be a very powerful antioxidant molecule, that the production of MEL decreases with age, and that the free radical effects are involved in the processes of aging and cancer, maintaining MEL at a high level could slow age- and cancer-related alterations. MEL also affects estrogen receptor transcriptional activity by regulating signal transduction pathways. In addition, MEL has been described as a potent supplement in the treatment or cotreatment of cancer: as an antioxidant, it may protect cell damage caused by carcinogens; as a chronobiotic, it may help determine optimum timing for carcinogen best efficiency; and it may act in synergy with the carcinogen retinoic acid to markedly reduce mammary tumor genesis in vivo (Kiefer et al., 2002).
Compiled & Shared by- Team, LITD (Livestock Institute of Training & Development)
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