Research Progress on the Function of Melatonin and Its Receptor in Animal Reproduction

Tian LIN Hefeng ZHANG Xiaoliang PEI Huina BO

Abstract Melatonin has been a research hotspot in the field of medicine and life sciences for more than 60 years since it was discovered. In order to accelerate the increase of economic animal productivity， a large number of studies also have focused on the regulation of melatonin and its receptors during animal reproduction in recent years. In this paper， the relevant characteristics of melatonin and the latest research progress in animal reproduction were discussed， and the biological functions of melatonin were reviewed from the aspects of germ cells， reproductive endocrine and embryo development， laying a theoretical foundation for further exploring the regulation mechanism of melatonin on reproductive process.

Key words Melatonin; Germ cells; Hormones; Embryo development

Received： October 9， 2021  Accepted： December 7， 2021

Tian LIN （1982-）， female， P. R. China， veterinarian， devoted to research about livestock inspection and testing.

*Corresponding author.

Melatonin is mainly an indoleamine hormone produced by the hypothalamic pineal gland of mammals. Its chemical component is N-acetyl-5-methoxytryptamine. In 1957， the dermatologists at Yale University separated and detected its molecular structure for the first time. Since then， melatonin has become a research hotspot in the fields of medicine and life sciences. Melatonin is an important regulator that mediates the effects of light on animal reproduction， and its synthesis and secretion show a rhythmic change of high during the day and low at night. For seasonal breeding animals， the duration of sunshine determines the activity of melatonin to inhibit or promote reproduction. In high latitudes， the photoperiod is the main environmental factor that determines the start and duration of the breeding season. The pineal gland controls reproductive activities by changing the duration of nocturnal secretion of melatonin[1]. In recent years， with the deepening of exploration， it has been discovered that melatonin plays an important role in animal sexual maturation， hormone secretion， gamete formation， early embryonic development， pregnancy and childbirth[2-3]. In modern animal husbandry， techniques such as cryopreservation of sperm， in-vitro maturation of oocytes and in-vitro development of embryos are the basis of artificial breeding. Improving the vitality of sperm after freezing and improving the maturation of oocytes in vivo and in vitro and the quality of embryonic development are of great significance to animal production. This paper mainly summarized related characteristics of melatonin and its latest research progress in animal reproduction， laying a theoretical foundation for the in-depth exploration of melatonin’s regulatory function on mammalian reproduction， the optimization of in vitro embryo production system， and the improvement of the economic benefits of animal husbandry production， and also providing a theoretical reference for human reproductive health.

Melatonin and Its Receptors

Melatonin （MEL） is an indoleamine hormone synthesis of which is regulated by light and which has obvious circadian rhythm. After the pineal gland cells take up tryptophan from the blood， they produce serotonin under the action of hydroxylase and decarboxylase. Serotonin produces N-acetylserotonin under the action of N-acetyltransferase （NAT）（Fig.1）. Then， melatonin is produced under the action of methylase， and this process requires the participation of hydroxyindole-O-methyltransferase （HIOMT）. HIOMT regulates the periodic secretion of melatonin， and the synthesis of melatonin in the pineal gland increases significantly after increasing the activity of HIOMT[4]（Fig.2）. The molecular weight of melatonin is 232.27. It has the characteristics of high lipophilicity， and being soluble in organic solvents and easily damaged by heat. Melatonin and its receptors are distributed in the retina， gastrointestinal tract， ovary and testis of male and female animals[5]， but melatonin is only periodically secreted in the pineal gland and retina. Studies have found that there are two types of receptors for melatonin. Membrane receptors include MT1， MT2 and possibly MT3， and another type of nuclear receptor， RZR/ROR， belongs to the orphan receptor subfamily. MT3 is mainly found in non-mammalian bodies， and mammals are only found in the liver and kidney of hamsters. Melatonin acts by binding to G-coupled protein receptors MT1， MT2 and nuclear receptor RZR/ROR in vivo[6]. Among them， MT1 and MT2 receptors can inhibit the activity of adenylate cyclase （cAMP）. The difference is that MT1 receptor can activate protein kinase C-β， while MT2 receptor can inhibit the activity of guanylate cyclase and stimulate protein kinase C[7]. MT1 receptor is a high-affinity receptor that can regulate ion flow and ion channels. MT2 receptor is a low-affinity receptor， which can regulate intracellular Ca2+ and inhibit the release of neurotransmitters[8]. Studies have shown that melatonin has a highly effective antioxidant effect and exerts a wide range of physiological functions in the body. Because of its hydrophilicity and lipophilicity， melatonin can easily pass through all physiological barriers， protect DNA， fatty acids and proteins in the nucleus and mitochondria from free radical damage， and reduce cell oxidative damage[9]. A large number of studies have confirmed that melatonin is involved in a variety of physiological processes， such as animal sleep， neuroendocrine， animal immune regulation[10]， species evolution[11]， anti-cancer[12]， anti-aging， skin color formation， cell growth cycle regulation， energy metabolism， angiogenesis[13]， neuroprotection and depression treatment[14]， and goat cashmere-producing performance[15-17]. Melatonin plays an active role in a variety of animal disease models and clinical research（Fig.3）.

Biological Function and Research Status of Melatonin in the Regulation of Animal Reproduction

Melatonin and its receptors with male animal germ cells

Melatonin and its receptors MT1 and MT2 are expressed in ram sperm[18]， indicating that melatonin may have a direct effect on sperm cells. Studies have shown that melatonin can significantly reduce the rate of sperm deformity and degree of lipid peroxidation and improve sperm stability， and can also improve the stability of mouse sperm DNA in the case of continuous or intermittent hypoxia in mouse sperm[19]. The antioxidant properties of melatonin can prevent the quality of epididymal sperm in the rat testicular ischemia-reperfusion model[20]. In the process of transplanting spermatogonial stem cells into the testis of azoospermic mice， melatonin can effectively reduce ROS and lipid peroxides， thereby increasing the efficiency of transplantation and improving the structural characteristics of testicular tissue[21]. For boar sperm for in-vitro fertilization， melatonin can affect its vitality and the stability of nucleoprotein independently of its antioxidant properties[22]. In addition， in the study of human sperm， it was found that melatonin was significantly related to sperm motility， and compared with the fertile group， the level of melatonin in the seminal plasma of the sterile group was significantly reduced[23]. Adding melatonin to diluted semen can increase the percentage of motile sperm and fast motile sperm[24].

In the process of sperm freezing， melatonin can protect sperm from damage. The study by Ashrafi et al. proved that melatonin could be added to a bovine sperm freezing liquid to offset the adverse effects on bovine sperm during the freezing and thawing process， and the total antioxidant capacity and antioxidant enzyme activity were improved after the addition of melatonin， resulting in a protection effect on frozen sperm. Melatonin can stimulate the activity of SOD， GPx and CAT and other enzymes involved in the elimination of ROS metabolism， thereby maintaining the fluidity of cell membranes[25]. In the process of dyeing， sorting and freezing， melatonin can protect buffalo sperm from the damage of active oxygen， and improve the quality of sperm after the process of freezing and thawing. During the cryopreservation of human sperm， melatonin can also effectively protect sperm from freezing damage through the PI3K/AKT signaling pathway[26].

The effect of melatonin on sperm is closely related to the added concentration of its external source. Martín-Hidalgo et al.[27] observed that although 1 μM melatonin could increase the proportion of live cells with intact acrosomes， it could not improve the function of pig semen stored at 17 ℃ for 7 d. Li et al.[28] found that 10-4 M melatonin could significantly improve the sperm quality of Holstein cattle. Micromolar concentrations of melatonin can regulate the capacitation of ram sperm induced by cAMP elevating agents by reducing the levels of ROS and cAMP， while at lower concentrations， melatonin changes the subpopulation of motile sperm. These findings provide a reference for further study of melatonin in controlling sperm capacitation in artificial insemination systems[29]. For human semen， after adding 2 mM melatonin， it was found that the proportions of motile sperm， forward motile sperm and fast motile sperm increased， the number of dead sperm decreased， and endogenous NO significantly decreased， but the ROS level did not change significantly. It shows that 2 mM melatonin can directly or indirectly remove excess NO， thereby protecting sperm. Thus， it can be inferred that different species have different sensitivity to melatonin， leading to differences in the concentration of melatonin that plays a beneficial role.

Melatonin with its receptors and oocytes

Melatonin has a positive effect on mammalian germ cells. The presence of melatonin membrane receptors and nuclear receptors was detected on mature oocytes， corpus luteum granulosa cells and membrane cells， suggesting that melatonin may directly affect the biological functions of the ovary. Studies have found that melatonin can be synthesized by the ovary and released into the follicular fluid， and accumulates concentration as the follicle develops， and the concentration of melatonin in large follicles is higher than that in small follicles[30]. It is currently hypothesized that serotonin synthesized in granulosa cells is used to synthesize melatonin， and melatonin acts as a calmodulin inhibitor and free radical scavenger in oocytes to participate in the growth and maturation of oocytes[31-32]. Melatonin can not only remove excessive free radicals produced by oocytes during ovulation and improve the nucleus and cytoplasm of oocytes[33]， but also can promote granular cell mitosis and follicular development by increasing the secretion of insulin-like growth factor IGF-1[34]. Many research reports have shown that high concentrations of melatonin in follicular fluid can inhibit follicular atresia and protect oocytes from free radical damage during ovulation， which is very important for the full development of oocytes[35]. Melatonin can significantly improve the nuclear maturation of oocytes in polyovarian syndrome. After melatonin treatment， the division rate of oocytes was significantly higher than that of the control group without melatonin， indicating that melatonin has the effect of inducing oocyte nuclear maturation and can ensure its fertilization ability[36].

Mammalian oocytes have a complex subcellular structure. When the temperature and osmotic pressure change slightly， oocytes will change. In the process of cryopreservation of oocytes， with the change of the external environment， the internal environment of cells will change significantly， and ROS will be generated at the same time， which will affect the balance between the redox reaction and the antioxidant system in cells， and the imbalance of the system will cause the occurrence of oxidation reactions， which will significantly reduce the viability of cells[37]. The addition of melatonin to the oocyte maturation media of porcine， bovine and sheep species can reduce the production of ROS， indicating that melatonin plays an important role in promoting oocyte maturation in vitro[38]. Supplementing a certain concentration of melatonin to the oocyte maturation fluids can promote the nuclear development from MI to MII stage， and increase the maturation rate of nucleus and the uniform distribution of cytoplasmic mitochondria[39]. In the in-vitro maturation systems of oocytes， in addition to being affected by the concentration， whether the added melatonin can play a beneficial role also depends on the oxygen concentration in the culture conditions. Under too high or too low oxygen conditions， the addition of melatonin may reduce blastocyst rate[40]. Therefore， to ensure that the melatonin added in vitro plays a positive and beneficial role， it is necessary to further explore the appropriate concentration of melatonin added to different species and suitable in-vitro culture conditions.

Tian LIN et al. Research Progress on the Function of Melatonin and Its Receptor in Animal Reproduction

Melatonin and its receptors with reproductive endocrine

Melatonin affects cell proliferation and energy metabolism， so melatonin is an important player in the regulation of reproductive hormone synthesis. The effects of melatonin on reproductive hormones depend on a variety of factors， including physiological conditions and animal species. For long-day seasonally breeding animals such as rodents， female rats experience anestrus and inhibited reproductive system under short-day cycles[41]. After melatonin injection， ovarian weight is reduced， and sexual maturation is delayed[42]. Melatonin reduces the expression of androgen receptor and testicular androgen-binding protein in male rats[43]. Injecting melatonin into the testes of male rats in the reproduction period results in reduced testicular volume and significantly lower testosterone levels[44]. Thus， melatonin has inhibitory effects on reproduction in long-day mammals. For short-day animals， implantation of melatonin in non-estrus doe can increase the content of follicle-stimulating hormone （FSH） and luteinizing hormone （LH） in vivo[45]. Melatonin is positively correlated with androgen levels in animals with short-day periods， and testosterone concentrations were twice as high in sheep treated with melatonin as in controls[46]. Sustained melatonin supplementation in short-day-reared animals can promote gonadal function[47-48]， long-term melatonin treatment can induce early testicular development in sika deer[49]， and subcutaneous melatonin injection increases testosterone concentrations in male goats[50]. The above results indicate that melatonin is an active factor in the reproduction of short-day animals.

Melatonin is the key to synergizing photoperiod with reproductive activity. Melatonin regulates the hypothalamic-pituitary-gonadal axis by activating the gonadotrophin releasing hormone （GnRH） receptors on hypothalamic neurons to regulate the secretion of FSH and LH， thereby affecting the secretion of gonadal reproductive hormones[51]. FSH and LH play an important role in animal reproduction. For female animals， FSH， LH and their receptors can regulate the level of cAMP in granulosa cells and theca cells， and cAMP can activate steroid synthase， thereby regulating the synthesis of progesterone， estradiol and so on. The LH peak before ovulation induces a high level of MT1 expression in granulosa cells， and at the same time， the expression level of the melatonin synthase SNAT gene in cumulus granulosa cells also increases significantly， resulting in an increase in the level of melatonin in the follicular fluid[52]，  which thereby regulates the luteinization of granulosa cells. Melatonin treatment can significantly increase the expression level of LH receptor in human granulosa cells， which can increase the likelihood of follicle ovulation[53]. In male animals， the long-term effects of exogenous melatonin in sheep can lead to the release of GnRH and LH， thereby activating reproductive activity[54].     There is evidence that melatonin has an inhibitory effect on reproduction in long-day animals， possibly through the inhibition of GnRH[55]. However， the exact mechanism by which melatonin regulates GnRH remains unclear. Kisspeptins （Kp） are upstream factors that promote GnRH secretion and are the key to transmitting melatonin messages[56]. It was found that photoperiod can modulate kiss1 signaling through melatonin to drive the reproductive axis[57]. After inducing zebrafish with melatonin， the Kiss1， kiss2 and GnRH3 genes in the brain and the expression of LHβ in the pituitary are enhanced， thereby promoting gonadal maturation and significantly improving the reproductive capacity of zebrafish[58]. Therefore， melatonin can cause changes in Kp expression， thereby affecting the reproductive system， but the relationship between melatonin and Kp still needs to be further explored.

Melatonin with embryonic development

Melatonin has antioxidant properties that decompose various free radicals and plays a key role in embryo transfer and embryonic development. High concentrations of maternal melatonin after fertilization can regulate the expression of antioxidant genes and antioxidant enzymes[59]. In addition to directly scavenging free radicals， it can also promote the secretion of progesterone by granulosa cells of the corpus luteum[60]， which is very important for the healthy development of embryos. During maternal pregnancy， melatonin can down-regulate the expression of the pro-apoptotic genes Bax and Caspase-3 and up-regulate the expression of the anti-apoptotic gene Bcl-2， increase the level of intracellular glutathione and reduce the level of ROS[61-62]， and in turn reduce the apoptosis rates of blastocysts and improve embryo quality and subsequent embryo implantation rate[63]. In order to meet the energy metabolism required for fertilization and early embryo development， there are a large number of lipid droplets in livestock embryos， especially in the process of in vitro preservation and maturation of gametes and embryos that are popular in modern farming. In this biochemical process， oxidative stress that damages embryos is unavoidable[64]. When gametes and embryos are exposed to relatively high oxidative stress conditions， intense ROS react with lipid droplets to damage critical cellular structures， disrupt cell membrane integrity， and alter the structure and function of nucleic acids and proteins[65]. Protein peroxidation leads to changes in protein structure and function， nucleic acid damage， and impacts on gene expression and genetic stability[66]. The free radical damage of the plasma membrane will affect the balance between the intracellular redox reaction and the intracellular antioxidant system， resulting in increased sensitivity to lipids. The external environment has undergone tremendous changes， which directly affect the function and viability of gametes and embryos[67]. Decreased antioxidant activity and elevated ROS in oocytes are the main causes of embryonic developmental defects， apoptosis， and embryonic developmental arrest in vitro[68]， of which apoptosis is considered to be one of the main factors for embryo loss and reduced viability of thawed embryos during in-vitro culture[69]. The addition of exogenous melatonin can improve the in-vitro oocyte maturation rate， blastocyst rate， embryo development and embryo survival rate of pigs， cattle， sheep and other livestock[70-72]， and significantly improve embryo development quality in in-vitro culture systems. In in-vitro culture system of mouse embryos， the addition of 10-6 melatonin can increase the fertilization rate， improve early blastocyst development， significantly improve the development of mouse pronuclear embryos and blastocyst hatching rate， and increase the implantation rate of embryos and pregnancy rate after transplantation. In pigs， melatonin at a concentration of 10-9 in vitro can significantly promote embryonic cleavage and blastocyst rates， but cells are damaged above or below this concentration[73]. In a 20% hyperoxia environment， adding appropriate melatonin to culture media can significantly reduce the effects of oxidative stress， thereby improving the blastocyst development rate and quality of bovine embryos in vitro. The addition of exogenous melatonin can improve the survival rate of goat embryos after thawing and the hatching rate of blastocysts after 24 h of culture， and reduce the rate of developmentally arrested embryos[74]. For embryos of different species， the concentration of melatonin added and the in-vitro culture conditions are different. Therefore， the establishment of an efficient and stable in-vitro culture system for different livestock still needs to be solved urgently.

Conclusions and Prospects

Based on previous research reports， a certain dose of melatonin can ensure normal body metabolism in mammals and improve reproductive performance. Scientists have been working on the direct regulation of melatonin in male sperm quality， oocyte maturation and embryonic development. In the body， as an antioxidant， melatonin reduces oxidative stress caused by reactive oxygen species generated during ovulation， reduces oxidative damage in follicles， and improves fertilization rate. In in-vitro sperm preservation and oocyte culture systems， adding a certain concentration of exogenous melatonin can reduce the oxidative stress caused by environmental changes， balance the relationship between the redox reaction and the antioxidant system in the system， and reduce damage to nucleic acid and protein structure and function from environmental shocks. As an antioxidant and endocrine hormone， the biological functions of melatonin in mammalian reproductive systems are obvious， and further studies on melatonin in regulating steroid hormone synthesis and exploring the mechanism of small molecule melatonin receptors may also contribute to the treatment of reproductive disorders and diseases caused by steroid hormone disorders. However， the effect of melatonin on sperm function， oocyte maturation and embryo development and its mechanism of action still need to be further understood， and how culture conditions and exogenous melatonin concentrations specifically affect the developmental capacity of oocytes and embryos also requires more research and exploration.

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Editor： Yingzhi GUANG  Proofreader： Xinxiu ZHU