Beneficial role of melatonin in protecting mammalian gametes and embryos from oxidative damage

PANG Yun-wei, JlANG Xiao-long, ZHAO Shan-jiang, HUANG Zi-qiang, ZHU Hua-bin

Embryo Biotechnology and Reproduction Laboratory, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences,Beijing 100193, P.R.China

Abstract Mammalian gametes and embryos are particularly vulnerable to oxidative stress-induced damage, which is mainly caused by reactive oxygen species (ROS) originating from normal metabolism and/or the external environment. Several researchers have implicated the role of oxidative stress in the activation of apoptosis, causing peroxidative damage to sperms/oocytes and inducing embryo fragmentation, arrest, or demise. Melatonin is a tryptophan derivative that is known for its powerful free radical-scavenging activity and broad-spectrum antioxidant property. Numerous studies have shown that melatonin and its metabolic derivatives can sequentially detoxify ROS in an antioxidant cascade, and modulate various antioxidant enzymes via its receptors to prevent radical-mediated damage. The identification of melatonin receptors in cumulus/granulosa cells,oocytes, and epididymal tissues implies that melatonin has protective actions on gametes and embryos. Enriching the semen extender or culture medium with melatonin significantly benefits sperm characteristics, improves oocyte maturation potential and quality, and enhances the developmental competence of preimplantation embryos. Certainly, further comparative studies are needed to show the unique antioxidant role and the advantage of melatonin in this field. This review summarizes the harmful effects of ROS and the beneficial role of melatonin against oxidative damage of gametes and embryos.

Keywords: reactive oxygen species, oxidative stress, melatonin, sperm, oocyte, embryo development

1. lntroduction

Oxygen radicals or reactive oxygen species (ROS) such as superoxide anion radicalhydrogen peroxideand hydroxyl radical (·OH) are unstable and highly reactive molecules that remove the electron from other molecules.Cellular ROS are commonly generated via mitochondrial oxidative phosphorylation (OXPHOS) or as a response following exposure to stress-related factors or exogenous toxins (Ray et al. 2012). ROS are second messengers in cellular functions by activation of cell-signaling pathways(Agarwal et al. 2008). However, excessive ROS may attack lipids, proteins, and nucleic acids, impair the energy metabolism process, and lead to oxidative stress-related cellular damage (McCormack et al. 2005; Niso-Santano et al. 2011; Tamura et al. 2012). Gametes and embryos have evolved an antioxidant defense system that includes enzymatic and non-enzymatic mechanisms to protect themselves against ROS-induced damage. Enzymatic antioxidants include superoxide dismutase (SOD), catalase(CAT), glutathione peroxidase (GPX), and peroxiredoxins.Non-enzymatic antioxidants are also known as free radical scavengers, and include vitamin C, vitamin E, glutathione(GSH), hypotaurine, selenium, and melatonin (Agarwal et al.2005; Dumollard et al. 2009).

Melatonin (N-acetyl-5-methoxytryptamine) was discovered as a secretory product of the pineal gland and is now known to be generated in many, perhaps all,organs in the body (Reiter et al. 2013, 2014). Melatonin has been found to have multiple physiological functions;it exhibits a synchronous circadian rhythm and is involved in sleep modulation (Arendt 2000; Fisher et al. 2008),seasonal reproductive regulation (Reiter et al. 2009b), blood pressure reduction (Reiter et al. 2009a), immune stimulation(Carrillo-Vico et al. 2006), inflammatory inhibition (Manka and Majewska 2016), and oncostatic/cytoprotective actions(Pacini and Borziani 2016). The lipophilic and hydrophilic properties of melatonin permit it to rapidly pass through all morphophysiological barriers with ease and distribute in other organs and fluids (Tamura et al. 2012; Cruz et al.2014a). Melatonin is well known to be a powerful free radical scavenger and broad-spectrum antioxidant (Reiter et al. 2005; Zhang and Zhang 2014). What sets melatonin apart from other antioxidants is that in addition to melatonin itself, its metabolic derivatives formed during antioxidant reactions, including cyclic 3-hydroxymelatonin (3-OHM),N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), and N1-acetyl-5-methoxykynuramine (AMK), are all excellent free radical scavengers that contribute to the reduction of oxidative stress (Galano et al. 2013; Tamura et al. 2013;Reiter et al. 2014). Numerous studies have shown that melatonin plays a protective role in the reproductive system,as melatonin receptors have been identified in cumulus cells, granulosa cells, oocytes, and epididymal tissues(Frungieri et al. 2005; El-Raey et al. 2011; Tian et al. 2014).Moreover, melatonin is effective in promoting early embryo development and subsequent implantation (Rodriguez-Osorio et al. 2007; Pang et al. 2013; Wang et al. 2014a;He et al. 2015). The current review summarizes the role of melatonin in protecting gametes and embryos from free radical-induced oxidative stress.

2. Production and deleterious effects of ROS

2.1. ROS production in gamates and embryos

The development of mammalian oocytes and embryos during preimplantation stages is dependent on energy supply (Lopes et al. 2010). Molecular oxygen, a key energy substrate, is mostly consumed by mitochondria via OXPHOS(Balaban et al. 2005). Inhibiting OXPHOS can reduce ROS concentrations, implying that this metabolic reaction leads to the generation of ROS (Agarwal et al. 2008). At several sites along the electron transport chain (ETC), namely, complexes I(where NADH dehydrogenase acts) and III (where ubiquinol is converted to ubiquinone), some electrons leak out and combine with oxygen or other electron acceptors to create free radicals (Agarwal et al. 2008; Ramalho-Santos et al.2009). This “escape” of electrons from the ETC is a major source of ROS in aerobic cells, including oocytes (Seidler and Moley 2015). Other researchers have found that mitochondrial dysfunction in oocytes may be associated with increased generation of ROS (Wang et al. 2009; Ou et al. 2012). Velez-Pardo et al. (2007) demonstrated that oxygen radicals and H2O2levels are significantly higher in non-competent embryos than those in competent embryos,which are correlated with mitochondrial damage. In a study in bovine zygotes, increased ROS production occurred around the time of fertilization, during which mitochondrial activity is stimulated by sperm entry (Lopes et al. 2010). Studies have demonstrated that spermatozoa are also capable of ROS generation (Ramalho-Santos et al. 2009; Benkhalifa et al. 2014). The plasma membranes of mammalian spermatozoa are heavily endowed with polyunsaturated fatty acids (Aitken and Fisher 1994; Sariozkan et al.2010). In humans, the free unsaturated fatty acid content of spermatozoa was found to be positively correlated with superoxide generation in mitochondria (Koppers et al. 2010).Disruption of the mitochondrial electron transport flow in human spermatozoa induces ROS generation at complex I,leading to peroxidative damage to the midpiece and a loss of sperm movement, and disruption of the electron flow at complex III leads to rapid H2O2release into the extracellular space (Koppers et al. 2008).

Exogenous sources may also enhance ROS production in gametes and embryos. High ex vivo oxygen (O2) tensions can induce oxidative stress due to the increased intracellular ROS levels (Bain et al. 2011). Embryos cultured under atmospheric oxygen concentrations were found to exhibit significantly elevated ROS production in both porcine(Kitagawa et al. 2004) and bovine embryos (Favetta et al.2007b). Enzymatic activity for the formation of superoxide radicals is enhanced in conditions of hyperoxia (Agarwal et al. 2008). Apart from the in vitro oxygen tension,various culture microenvironments could give rise to ROS,such as the presence of excess glucose, metallic ions,ultraviolet light, serum amino oxidases, and the freeze-thaw process (Guerin et al. 2001). When medium for oocyte maturation contained excessive glucose, the developmental competence of bovine oocytes is impaired due to the increase of ROS and the decrease in the intracellular glutathione content that acts to protect cells against oxidativestress (Hashimoto et al. 2000). In living cells, metallic cations such as copper (Cu) and iron (Fe) act as catalysts in ROS generation via the Haber-Weiss reaction (Guerin et al.2001; Roychoudhury et al. 2016). During the development of porcine parthenotes, redundant iron impairs mitochondrial function, resulting in a high concentration of ROS (Zhao et al. 2015). However, metallic ion chelators such as ethylenediaminetetraacetic acid (EDTA) can chelate divalent cations in culture media, preventing them from participating in the generation of ROS (Kim et al. 2006). Another factor that promotes ROS formation is ultraviolet irradiation,which results in ROS production in an orderly manner via dismutation and Fenton reactions in mitochondria (Feng et al. 2012). High ROS levels are primarily found in dead and morphologically abnormal spermatozoa, as well as in the leukocytes presented in ejaculates (Agarwal et al. 2006).Additionally, excess ROS production has been detected during the freeze-thaw or centrifugation process (Tasdemir et al. 2013; Sapanidou et al. 2015) (Fig. 1).

2.2. Deleterious effects of ROS on gamates and embryos

Fig. 1 Endogenous and exogenous sources of reactive oxygen species (ROS) in gametes and embryos.

ROS-mediated oxidative stress has been involved in activating the apoptosis cell signaling, inflicting peroxidative damage to the sperm membrane or to the oocyte and inducing embryo fragmentation, arrest, or demise(Dumollard et al. 2007; Agarwal et al. 2008). ·OH radicals,which are generated by the H2O2-driven Fenton reaction,disrupt metaphase-II (MII) mouse oocyte microtubules and chromosomal alignment, which may account for poor oocyte quality (Shaeib et al. 2013). In rats, ROS has been reported to cause apoptosis of granulosa cells, reduce estradiol 17-β concentration, and decrease oocyte quality by inducing apoptosis of oocytes (Tripathi et al. 2013). Alvarez et al. (2015) illustrated that exposure of porcine oocytes to exogenousandduring in vitro maturation(IVM) strongly restrains their ability to form pronuclei after fertilization. Moreover, oxidative stress can trigger the destabilization of maturation-promoting factor (MPF); perturb calcium homoeostasis; cause mitochondrial dysfunction;and induce oxidative damage to the intracellular components such as lipids, proteins, and DNA of the oocyte (Lord and Aitken 2013). Spermatozoa are particularly sensitive to oxidative attack due to their limited content of cytoplasm,which is mostly eliminated during the final stages of spermiogenesis (Aitken and Fisher 1994; Lane et al. 2014).Accumulating evidence suggests that the decreased motility and fertilization potential of spermatozoa are often correlated with high ROS production and lipid peroxidation (LPO) levels in these cells (Baker and Aitken 2004; Fujii and Tsunoda 2011; Cassina et al. 2015). The high concentrations of intracellularand· in sperm show deleterious effects on the mitochondrial membrane potential (MMP) of sperm,and H2O2may interfere in pronucleus formation (Ghaleno et al. 2014). Studies conducted on bovine spermatozoa have shown that an increased susceptibility of spermatozoa to oxidative stress compromised sperm DNA integrity and impaired oxidative and capacitation status, adversely affected embryo quality (Simoes et al. 2013; de Castro et al. 2016). Spermatozoa that undergo oxidative stress delay subsequent embryonic development (Lane et al.2014), resulting in early developmental anomalies, which ultimately lead to embryonic and fetal demise (Burruel et al.2014). Additionally, vitrification and thawing cycles, which significantly reduce the antioxidant levels found in seminal plasma, also potentially intensify oxidative damage of sperm(Bilodeau et al. 2000).

In vitro production of mammalian embryos is associated with a high frequency of developmental abnormalities owing to development retardation or apoptosis caused by oxidative stress (Betts et al. 2014). Superproduction of ROS could directly damage the mitochondrial potential,with subsequent activation of proteases/caspases,thereby leading to chromatin fragmentation/condensation,apoptosis, impairment of cell division, and developmental arrest in bovine embryos (Velez-Pardo et al. 2007). A direct relationship between the increased H2O2level and apoptosis has been documented in human fragmented embryos (Yang et al. 1998). Favetta et al. (2007b) found that embryos cultured in high oxygen tension exhibited an increased incidence of oxidative stress, and markedly decreased the embryonic developmental abilities compared to embryos cultured in low oxygen tension. They also demonstrated that the oxidative stress-induced developmental failure is positively correlated with the expression of the stress adaptor protein p66shc. This protein is known to regulate intracellular ROS metabolism and apoptosis (Favetta et al. 2004; Giorgio et al. 2005; Favetta et al. 2007b; Betts and Madan 2008). P66shc knockdown embryos showed a reduced incidence of arrest, increased manganesesuperoxide dismutase (MnSOD) levels, reduced intracellular ROS levels, and reduced DNA damage, which correlate with enhanced developmental potential (Favetta et al.2007a; Bain et al. 2013; Betts et al. 2014). Early mouse embryos exposed to ROS for short time intervals presented with embryo arrest at the first cellular cycle (2-cell) and disrupted preimplantation development, differentiation, and morphogenesis (Cebral et al. 2007). In mouse zygotes,H2O2treatment impaired the in vitro developmental competence; decreased the blastocyst formation rates; and increased the occurrence of fragmentation, apoptosis, and retardation in blastocysts (Yu et al. 2014). Another study found that mouse embryos exposed to oxidative stress exhibited rapid reduction of MMP and the appearance of the DNA damage marker γH2AX, which may suggestthe compromised development of these embryos (Qian et al. 2016). Nevertheless, it should be borne in mind that the role of ROS is reflected in two sides: “positive” and“negative”. On the one hand, they function as signaling molecules and mediate capacitation, hyperactivation, and acrosome reaction that are crucial to the acquisition of fertilizing ability. On the other hand, excessive ROS impair the intracellular environment, endanger cell survival, and damage the gamete and embryo development (Agarwal et al. 2005) (Fig. 2).

Fig. 2 Schematic representation of the deleterious effects of reactive oxygen species (ROS) on gametes and embryos. MPF,maturation-promoting factor.

3. Protective roles of melatonin on gametes and embryos

Melatonin is a known potent and efficient endogenous radical scavenger, as the pineal indoleamine could directly detoxify the highly reactive ·OH and protect biomolecules within every cellular compartment against oxidative damage(Poeggeler et al. 1993; Reiter et al. 1994). Hydrogen atom transfer, single electron transfer, as well as radical adduct formation are all considered to be the primary mechanisms involved in determining the free radical-scavenging activity of melatonin (Galano et al. 2011). In fact, melatonin serves as a precursor for a multitude of free radical scavengers that can sequentially scavenge ROS in an antioxidant cascade to prevent radical-mediated damage (Reiter et al.2014; Zhang and Zhang 2014). In addition, melatonin can indirectly limit oxidative stress by stimulating the activities of several antioxidant enzymes. It is reported that the indole causes an increase in GSH reductase activity in both the liver and kidney cortex in a diabetic rabbit model (Winiarska et al. 2006). Preincubation with melatonin enhances the expression of antioxidant enzymes CAT, GPX, and SOD at both the gene and protein levels (Fischer et al. 2013).The melatonin-mediated changes in the expression levels of antioxidant enzymes occur in a dose-dependent manner(Rodriguez et al. 2004; Zhang and Zhang 2014).

Melatonin employs multiple mechanisms to modulate cellular physiology, including via membrane receptors,nuclear binding sites, and interaction with cytosolic molecules (Reiter et al. 2010). Melatonin exerts most of its major physiological actions by reacting with the two membrane receptors MT1 and MT2, which belong to the G protein-coupled receptor superfamily containing sevenpass transmembrane domains (Reiter et al. 2010; Chan and Wong 2013; Jockers et al. 2016). These two receptor subtypes show identical ligand-binding properties, and link to activate a great deal of signaling pathways, such as inhibiting adenylyl cyclase activities, provoking the expression of phospholipase C, and modulating of several effector molecules (Chan and Wong 2013). The third membrane-associated receptor MT3 is a cytosolic quinone reductase (QR2). Previous literature has demonstrated that MT3 exerts its protective role by blocking electron transfer from quinones (Chen et al. 2013). However, there is no consensus about the statement that MT3 and QR2 are the same molecule (Cutando et al. 2011). The nuclear binding sites for melatonin include members of the retinoic acid receptor superfamily, namely, the retinoid Z receptor (RZR)and retinoid acid receptor-related orphan receptor (ROR)(Reiter et al. 2010; Chan and Wong 2013). It has been proposed that the stimulatory effects of melatonin on ROS-detoxifying enzymes are likely modulated via its interaction with calmodulin, which conversely activates the expression of the nuclear melatonin receptor RORα (Tomas-Zapico and Coto-Montes 2005).

Research within the last decade has indicated that melatonin has hitherto unknown beneficial effects on the animal reproduction process. Melatonin implants not only increase superovulation and transplantation competence of transgenic embryo in sheep (Zhang et al. 2013), but also ameliorate the superovulation ability of the aged Rasa Aragonesa ewes during the seasonal anestrous period(Forcada et al. 2006). More importantly, melatonin plays a key role in protecting mammalian gametes and embryos from oxidative damage (Lanoix et al. 2012; Cruz et al.2014b; Reiter et al. 2014).

3.1. Melatonin and sperm quality

Oxidative stress is one of the most important factors contributing to poor semen quality. The direct action of melatonin on sperm cells is linked to its free radicalscavenging properties and its ability to cross the plasma membrane, resulting in reduced oxidative damage to sperm cells both intracellularly and in extracellular media (Cebrian-Perez et al. 2014). Intensive research has revealed that melatonin directly benefits sperm characteristics. In studies on male rats, it was found that exposure of sperm cells to melatonin prevents oxidative stress-induced damage by impeding the decrease in antioxidant enzyme activities and testosterone level (Sonmez et al. 2007; Othman et al.2016), decreasing sperm abnormalities (Kurcer et al. 2010),protecting the sperm chromatin condensation process,and promoting the production of functional sperm (Othman et al. 2016). During the non-reproductive season, Rasa Aragonesa rams that received melatonin implants exhibited modified seminal plasma hormonal profile, elevated testosterone and 17-β estradiol levels, increased percentage of progressive motile spermatozoa, and improved fertilization parameters (Casao et al. 2010b; Casao et al. 2013). Furthermore, ram spermatozoa exposed to melatonin were directly affected, as evidenced by decreased capacitation and phosphatidylserine translocation at a concentration of 10–6mol L–1, and increased short-term capacitation and in vitro fertilization (IVF) at a concentration of 10–10mol L–1(Casao et al. 2010a). However, Succu et al. (2011) reported that melatonin added to ram semen freezing extender improved postthawing sperm function,as evidenced by their post-thaw viability rates, motility parameters, intracellular ATP concentrations, DNA integrity,and fertilizing ability, and 10–3mol L–1melatonin was found to be the most effective concentration. Jang et al.(2010) investigated the effect of melatonin on boar sperm characteristics in the presence or absence of H2O2. They observed that melatonin improved semen quality during in vitro storage and increases the developmental potential of IVF embryos (Jang et al. 2010). Contrarily, Martin-Hidalgo et al. (2011) revealed that 10–6mol L–1melatonin increased the proportion of viable sperm with an intact acrosome, but this treatment did not affect other characteristics of boar semen stored at 17°C for 7 days.

A previous study in mice demonstrated that melatonin could protect spermatozoa against diazinon-induced DNA damage, and promote the compaction of chromatin(Sarabia et al. 2009). In addition, melatonin reduces the degree of LPO and improves the stability of sperm DNA in mice under intermittent hypoxia (Vargas et al. 2011). In a recent study, Li et al. (2016) observed that pretreatment with melatonin significantly alleviated the toxic effects of cadmium exposure in male mice, evidenced by increased motility parameters and MMP, reduced DNA damage and apoptotic rate, and ameliorated serum sex hormone levels,and its mechanism may involve inhibiting malondialdehyde(MDA) level, restoring GSH and SOD activities, and downregulating proinflammatory cytokine levels. Melatonin has been reported to be present in human seminal fluid,but it is not necessarily associated with sperm motility(Bornman et al. 1989). However, du Plessis et al. (2010)demonstrated that melatonin benefits human spermatozoa by enhancing motility parameters and simultaneously reducing nonviable spermatozoa. This is consistent with the paper of Ortiz et al. (2011), in which it was suggested that short-term in vitro exposure of human sperm to 10–3mol L–1melatonin improves several sperm motility parameters.The beneficial roles of melatonin have also been observed in flow cytometry-sorted buffalo sperm, where melatonin was found to exert a protective effect against the oxidative damage induced by staining, sorting, and freezing;moreover, melatonin treatment increased semen quality after the freeze-thaw processes (Li et al. 2012). The effects of melatonin on bovine spermatozoa are controversial.Cheuqueman et al. (2015) supplemented IVF culture media with different concentrations of melatonin and found a significant increase in DNA fragmentation and oxidation in spermatozoa that were cultured with 10–3mol L–1melatonin,but this DNA damage did not impair fertilization. However,Ashrafiet al. (2013) demonstrated that supplementation of freezing medium with 2× or 3×10–3mol L–1melatonin improves the semen quality, including motility, viability,normal morphology, and plasma membrane integrity of cryopreserved bull spermatozoa, probably by reducing LPO and elevating the total antioxidant capacity and antioxidant enzyme activities. Similar to the findings of Ashrafiet al.(2013), our results indicate that 10–3mol L–1melatonin improves plasma membrane integrity, mitochondrial activity,and acrosome integrity in bovine sperm samples, leading to improved embryonic development and blastocyst quality(Pang et al. 2016a).

The presence of melatonin receptors (MT1 and MT2) in rat epididymis tissue (Shiu et al. 2000) and in human (van Vuuren et al. 1992) and ram spermatozoa (Casao et al.2012) suggests that the role of melatonin on sperm cells might be more complicated than that previously proposed and more likely to be receptor mediated (Cebrian-Perez et al. 2014). In the rat corpus epididymis, changes in the binding characteristics of these receptors in response to castration and steroid hormones indicate that MT1 and MT2 are biologically functional receptors whose activities are differentially modulated by testosterone and hydrocortisone.Furthermore, these membrane receptors are coupled to pertussis toxin (PTX)-sensitive Giprotein and are possibly involved in androgenic and adrenergic regulation of rat corpus epididymal epithelial cell functions (Shiu et al. 2000).Frungieri et al. (2005) detected the MT1 receptor in Leydig cells of hamster testes, and indicated that melatonin may act as a local inhibitor of human chorionic gonadotropin(hCG)-stimulated cyclic adenosine monophosphate (cAMP)and androgen secretion through MT1. Both MT1 and MT2 have been identified in ejaculated ram spermatozoa, but the distribution of MT1 and the density of MT2 are variable(Casao et al. 2012). Contrary to previous reports, da Silva et al. (2011) showed that neither MT1 nor MT2 was present in stallion spermatozoa, and all the beneficial effects can be directly attributed to the intrinsic antioxidant scavenging activity of melatonin. In a recent study, Gonzalez-Arto et al.(2016) demonstrated for the first time that both MT1 and MT2 exist in the spermatozoa of all species, regardless of type of reproduction or sperm source. The distribution and density of melatonin receptors may be affected by the physiological status of spermatozoa, such as maturation,capacitation, and apoptosis. Previously, Fujinoki (2008)studied hamsters and showed that stimulation and inhibition of the MT1 receptor regulate melatonin-enhanced sperm hyperactivation. However, the modulating effect of melatonin on ram sperm functionality during in vitro capacitation was shown to be primarily mediated via the MT2 receptor (Gonzalez-Arto et al. 2014). Melatonin is suggested to regulate capacitation through its receptors via calcium signaling (Cebrian-Perez et al. 2014). Melatonin can interact with intracellular proteins such as calmodulin,calreticulin, and tubulin, and antagonize the binding of Ca2+to calmodulin (Pandi-Perumal et al. 2008). Critical data on this point are yet to be elucidated.

3.2. Melatonin and oocyte maturation

The identification of melatonin receptors in rat antral follicles and corpus luteum (Soares et al. 2003) as well as in human(Niles et al. 1999) and bovine granulosa cells (GCs) (Wang et al. 2012) indicates that melatonin is a direct modulator of ovarian function. Woo et al. (2001) reported that melatonin enhances the effects of hCG-stimulated progesterone production, increases the mRNA levels of luteinizing hormone (LH) receptor, and decreases the expression levels of GnRH and GnRH receptor genes via melatonin receptors in human granulosa luteal cells, supporting the notion that melatonin plays a direct role in ovarian physiology. In bovine GCs, both MT1 and MT2 are expressed and localized to their respective target sites, and exogenous melatonin acts via these receptors to inhibit apoptosis, stimulate progesterone production, and suppress estradiol production (Wang et al.2012). In addition to these receptor-mediated actions,melatonin has been reported to affect the physiology of all ovarian cellular components via its multifaceted free radical scavenging properties (Reiter et al. 2013). Tanavde et al.demonstrated that in pigs, melatonin modulates ovarian theca cell steroidogenesis at the molecular level, which may be mediated by the downregulation of CYP11A and CYP17 genes; these genes contribute to the production of the steroidogenic progesterone and androstenedione,respectively (Tanavde and Maitra 2003). Recently, Lima et al. (2015) reported that melatonin decreases the expression of the cAMP responsive element modulator(Crem), which represses Cyp17a expression. This result further proves that melatonin controls ovarian function by affecting steroidogenic enzymes.

Melatonin is also detectable in ovarian follicular fluid(Reiter et al. 2013). The concentration of melatonin in follicular fluid is positively correlated with progesterone concentrations. When patients who had luteal phase defect were given melatonin (3 mg d–1at 22:00) throughout the luteal phase, the serum progesterone concentrations were improved after daily melatonin treatment. The result demonstrates that melatonin contributes to luteinization for progesterone production during ovulation (Taketani et al. 2011). A previous finding reported by Abecia et al.(2002) also provided evidence for an effect of melatonin on progesterone secretion in vivo. Intrafollicular concentrations of 8-hydroxy-2´-deoxyguanosine (8-OHdG, a biomarker of damaged DNA products) in women with poor oocyte quality were significantly higher than those with normal oocyte quality in patients undergoing IVF-embryo transfer (IVF-ET).Administration of melatonin with a 3 mg tablet significantly reduced intrafollicular concentrations of 8-OHdG, and the intrafollicular melatonin concentrations are four times higher than patients without melatonin treatment, which indicates that exogenous melatonin is effective for ameliorating oocyte quality (Tamura et al. 2008). Preovulatory follicular fluid contains higher melatonin concentrations than plasma,and the melatonin concentration increases with follicular growth in humans but decreases in pigs (Shi et al. 2009;Tamura et al. 2009; Cruz et al. 2014b). He Y et al. (2016)studied pigs and found that the intrafollicular melatonin level decreases whereas the percentage of apoptotic granulosa cells increases with progression of follicular atresia. Previously, the melatonin precursors serotonin and N-acetylserotonin and the activities of two key melatoninsynthesizing enzymes arylalkylamine N-acetyltransferase(AANAT) and acetylserotonin O-methyltransferase (ASMT)have been identified in human ovarian extracts (Itoh et al.1999). Both Aanat and Asmt mRNAs were also detected in rat ovaries. Furthermore, AANAT protein was detected in oocytes at all stages of follicular development, and its concentration progressively increases throughout follicular development, demonstrating that the melatonin synthesized in the oocyte may be implicated in its own growth or maturation (Sakaguchi et al. 2013). El-Raey et al. (2011)detected the expression of the ASMT gene in bovine oocytes and cumulus cells, suggesting that both oocyte and cumulus cells might function together in melatonin biosynthesis. Additionally, He C et al. (2016) demonstrated that mitochondria are the major sites for melatonin synthesis in mice oocytes, where large concentrations of melatonin is synthesized during oocyte maturation.

Several studies have noted that melatonin enhances oocyte maturation in vitro in a wide range of species,including cows (El-Raey et al. 2011; Tian et al. 2014;Rodrigues-Cunha et al. 2016), pigs (Kang et al. 2009; Li et al.2015), mice (Nikmard et al. 2016), and humans (Tamura et al. 2008). During IVM, supplementing oocyte maturation medium with melatonin reduces DNA damage in cumulus cells from bovine cumulus-oocyte complexes (COCs), but this treatment does not influence embryo developmental competence (Takada et al. 2012). Supplementation of IVM medium with 10 and 50 ng mL–1melatonin promotes oocyte nuclear maturation and cumulus cell expansion, and modifies the mitochondrial distribution patterns (El-Raey et al. 2011). Endogenous melatonin effectively improves in vitro bovine oocyte maturation efficiency by increasing the expression levels of oocyte maturation-associated genes(GDF9 and MARF1) and cumulus cell expansion-related genes (PTX3 and HAS1/2) (Tian et al. 2014). To maximize the beneficial properties of melatonin, Remiao et al. (2016)encapsulated melatonin into lipid-core nanocapsules and used them during in vitro oocyte maturation. They found this approach to be more effective than conventional melatonin supplementation for decreasing the ROS levels and the apoptosis index, increasing the cleavage and blastocyst formation rates, upregulating the GPX1 and SOD2 genes, and downregulating the CASP3 and BAX genes (Remiao et al. 2016). Melatonin supplementation in a defined maturation medium is able to stimulate meiosis resumption in bovine COCs and improves subsequent embryo development in vitro (Rodrigues-Cunha et al. 2016).Zhao et al. (2016) administered melatonin to vitrification solution or to IVM and vitrification solution, and found that addition of 10–9mol L–1melatonin reduced the ROS level and prevented apoptotic events of vitrified bovine oocytes,resulting in improved developmental potential.

Supplementation of the in vitro medium with 10 ng mL–1melatonin resulted in a greater proportion of nuclear and cytoplasmic matured oocytes, and the MT1 receptor transcript detected in porcine cumulus cells and granulosa cells implied that melatonin may directly act on oocytes for maturation (Kang et al. 2009). Under heat stress conditions, application of 10–7mol L–1melatonin not only increased the polar body and blastocyst rates, but also maintained normal steroid hormone levels. Furthermore,melatonin addition during IVM under heat stress reduces ROS formation, increases GSH production, inhibits cell apoptosis, and promotes the expression of genes associated with mitochondrial function (Li et al. 2015). Just recently,Jin et al. (2017) reported that melatonin treatment results in increased content and reduced size of lipid droplets,observed in combination with enhanced transcripts of genes related to lipogenesis and lipolysis. Melatonin also increases the fatty acid, mitochondrial, and ATP contents,and simultaneously upregulates the genes associated with β-oxidation and mitochondrial biogenesis in porcine oocytes,indicating that melatonin promotes lipid metabolism, thereby providing an essential energy source for oocyte maturation and subsequent embryonic development (Jin et al. 2017).

In mice, addition of melatonin to IVM medium alleviates oxidative stress-induced damage, induces nuclear maturation, and guarantees fertilization potential in oocytes in polycystic ovarian syndrome (Nikmard et al. 2016).Aging adversely affects female fertility, with a decline in oocyte quality being the major contributing factor (Tamura et al. 2016). Several studies have reported that melatonin administration in mice delays ovarian aging, as indicated by substantial increases in the quantity and quality of ovulated oocytes, pool of follicles, telomere length, and developmental competence (Song et al. 2016; Tamura et al.2016). These results provide evidence that melatonin exerts protective effects against aging-associated fertility decline by maintaining a mitochondrial redox balance, enhancing Sirt expression and ribosome function, increasing the activities of antioxidant enzymes, and reducing autophagy.Lord et al. (2013) showed that melatonin efficiently relieves aging mouse oocytes of oxidative stress in vitro by delaying the onset of apoptosis and preventing fragmentation.Importantly, melatonin-treated oocytes experience an increased optimal window for fertilization and generate embryos of improved quality compared to their untreated aged counterparts. In a goat model, melatonin application prior to oocyte collection causes more follicular waves,accelerates the timing of embryo development, and improves blastocyst yield (Berlinguer et al. 2009).

3.3. Melatonin and embryo development

The ability of melatonin to improve embryonic development in different species has been extensively reported. Papis et al. (2007) observed a significant association between oxygen tension and melatonin treatment in bovine presumptive zygotes. A relatively short period (48 h) of in vitro culture with melatonin under high concentration of atmospheric oxygen (20%) was reported to enhance the final developmental rate and the cell number of bovine embryos, which could be attributed to the free radicalscavenging property of melatonin (Papis et al. 2007).Supplementing culture medium with 10–7mol L–1melatonin accelerates the development of bovine embryos in vitro,promotes blastocyst yield, and enhances the quality of the resulting blastocysts (Wang et al. 2014b). Both MT1 and MT2 were detected in bovine blastocysts produced in vitro, and MT1 mediated the beneficial effect of melatonin on embryo development (Wang et al. 2014a). A recent research proved that exogenous application of melatonin ameliorates the production efficiency of bovine cloning.Melatonin supplementation reduces apoptosis and ROS,increases the ratio of inner cell mass (ICM) to total cells, and improves the development of bovine embryos generated via somatic cell nuclear transfer (SCNT) both in vitro and in vivo.Moreover, the global H3 acetyl lysine 9 (H3K9ac) level is significantly elevated in the melatonin-treated SCNT group,indicating that the addition of melatonin may affect nuclear reprogramming, resulting in improved embryo quality (Su et al. 2015). Nevertheless, the specific mechanism by which melatonin enhances the developmental potential of bovine preimplantation embryos under oxidative stress remains largely unclear. Our latest results show that pretreatment with melatonin can counteract the toxic effect of paraquat on the developmental failure in bovine preimplantation embryos, and melatonin promotes embryonic survival via modulation of p38 mitogen-activated protein kinase (MAPK)signaling (Pang et al. 2016b).

Melatonin has been found to efficiently support preimplantation development of both mouse IVF and SCNT embryos in vitro (Ishizuka et al. 2000; Salehi et al.2014). Asgari et al. (2012) found that both 10–8and 10–7mol L–1melatonin promoted the development of 2-cellstage embryos to blastocysts and hatching blastocysts,increasing the total cell, trophoectoderm, and ICM numbers and implantation rates. Enriching the culture medium with melatonin improved the quality and developmental rate of early mouse embryos, protecting them from the harmful effects of apoptotic inducers such as actinomycin-D and tumor necrosis factor alpha (TNF-α) (Niknafs et al. 2014).At physiological concentrations (10–7mol L–1), melatonin not only promotes the in vitro development of murine pronuclear embryos, but also increases the efficiency of embryo implantation, offspring number, and postnatal survival of the pups. These changes could be partially attributed to the ability of melatonin to decrease the expression of the pro-apoptotic genes Trp53, Bax, and Casp3 and increase the expression of the anti-apoptotic gene Bcl2 (Wang et al.2013). Tan et al. (2015) demonstrated that supplementing the culture medium with 10–9mol L–1melatonin reverses the disrupted expression patterns of genes associated with actin organization, ameliorating the distribution and organization of actin filaments (F-actin) from the 8-cell stage onwards,indicating that melatonin restores impaired embryonic development by improving actin organization. More recently,Ren et al. (2015) confirmed that melatonin could rescue several mitochondrial dysfunctions in mouse IVF embryos,including damaged mitochondrial biogenesis, dysregulated GSH homeostasis, and free radical-induced apoptosis.During early gestation, the expression of melatonin’s ratelimiting enzyme Aanat gradually enhanced in the uterus, and only the membrane receptor MT2 is expressed on day 2 of gestation. Data indicate that melatonin can be synthesized in the early pregnant uterus and influences early gestation via MT2 (He et al. 2015).

Rodriguez-Osorio et al. (2007) investigated the effects of melatonin on porcine preimplantation embryos in vitro.Addition of 10–9mol L–1melatonin to culture medium had a positive effect on the cleavage rates and blastocyst total cell numbers of fertilized porcine embryos, and these protective properties were further confirmed under heatstress conditions. Similarly, 10–10mol L–1melatonin resulted in increased cleavage and blastocyst formation rates,higher total cell number, and decreased ROS levels and apoptotic rates in porcine parthenogenetic embryos. The beneficial effects of melatonin on embryo development are accompanied by altered expression of the apoptosis-related genes BCL2L1 and BAX and the pluripotency marker gene OCT4. Furthermore, melatonin significantly enhanced the developmental competence of porcine SCNT embryos(Choi et al. 2008). In contrast to previous reports, Nakano et al. (2012) showed that melatonin treatment reduced the ROS levels in 4-cell parthenogenetic and SCNT embryos but did not affect the potential of embryos to develop into blastocysts. However, Pang et al. (2013) demonstrated that donor cells treated with melatonin exhibited enhanced developmental potential into porcine cloned embryos, and an optimized regime is established by combined donor and embryo treatment with melatonin, which improves blastocystformation and embryo quality in porcine SCNT embryos by inhibiting the p53-mediated apoptotic pathway.

Melatonin treatment was shown to improve the survival of thawed ovine embryos, enhance the embryonic hatchability after 24 h of culture, and decrease the proportion of degenerated embryos at the end of incubation (Abecia et al.2002). Exogenous melatonin has a beneficial effect on in vivo embryo viability in Rasa Aragonesa ewes, as subcutaneous implants of melatonin at lambing during the anestrous period significantly improved the number of fertilized embryos/corpus luteum (CL), number of viable embryos/CL, viability rate and pregnancy rate, particularly in undernourished ewes(Vazquez et al. 2010). In the study by Succu et al. (2014),low concentration (10–9mol L–1) of melatonin was shown to be beneficial in restoring embryo function after vitrification and warming, as indicated by increased re-expansion and hatching rates, higher total cell number, and decreased apoptotic and oxidative index. On the other hand, a higher dose of melatonin (10–3mol L–1) added during the post-warming period is toxic to embryo development (Succu et al. 2014). In buffaloes, supplementation with 10–5and 5×10–5mol L–1melatonin in the culture medium results in a higher transferable embryo yield, thereby improving the in vitro embryo production efficiency (Manjunatha et al.2009). In rabbit species, enriching the culture medium with 10–6mol L–1melatonin enhanced the development and hatchability rates of embryos recovered at 48 h post-insemination, while the addition of 10–3mol L–1melatonin was found to improve the development of rabbit morulae but adversely affected the development of earlier embryos(Mehaisen and Saeed 2015). Mehaisen et al. (2015)revealed that exogenous melatonin enhances embryonic development in fresh and vitrified embryos via multiple mechanisms, including modifying the expressions of several key genes in embryonic development (Gja1, Pou5f1 and Nanog), increasing the activities of glutathione-S-transferase and SOD, and reducing the levels of the oxidative substrates LPO and nitric oxide, as well as modulating the expression of oxidative stress response-related genes (Nfe2l2, Sod1,and Gpx1) (Mehaisen et al. 2015).

There is evidence that melatonin has some potential effects on human, especially in clinical trial for infertility patients (Tamura et al. 2012). Tamura et al. (2008)investigated the effect of melatonin treatment on clinical outcome of IVF-ET. After the first IVF-ET cycle, the non-pregnant patients with a low fertilization rate (＜50%)were administrated with melatonin during the next IVFET procedure. Results showed that the fertilization and pregnancy rate are higher in patients with melatonin treatment than those without melatonin treatment (Tamura et al. 2008). When the infertility women were given 3 mg of melatonin orally at 22:00 from day 5 of the previous menstrual cycle until the day of oocyte retrieval, the treatment group yields a higher proportion of good embryos (Tamura et al.2012). Batioglu et al. (2012) demonstrated that melatonin administration in women underwent IVF cycles did not affect the mean number of oocytes and fertilization rate, but the treatment group gained a higher number of class 1 embryos.These data suggest that melatonin treatment may become a new cure choice for benefitting women who suffer from infertility. However, whether the reported antioxidant actions of melatonin in protecting gametes and embryos from free-radical-induced oxidative stress is unique and superior to other antioxidants remains to be established.

4. Conclusion

A large number of researches carried out with rats, human and in vitro have provided convincing evidences to suggestthat melatonin might play an important role in protecting gametes and embryos from free radical-mediated damage.It was shown that when added to semen extender or culture medium, melatonin could directly improve sperm characteristics, promote oocyte maturation, and enhance the developmental potential of early embryos. However,the widely reported beneficial effects of melatonin on gametes and embryos seem to be dose-dependent.The vast majority of studies have demonstrated that physiological concentrations of melatonin are very effective in ameliorating sperm function and oocyte quality, and in embryo development, while high concentrations of melatonin can exert some toxic effects on gametes and preimplantation embryos. Additional details will be acquired to ascertain how the concentration of melatonin influences the competence of gametes and embryos, and the mechanisms by which melatonin affects gametes and embryos in a dosedependent manner. More importantly, further in vivo studies are needed to investigate the efficacies of melatonin in protecting embryos of agricultural animals from oxidative damage in comparison with other antioxidants.

Acknowledgements

This work was supported by the grants from the Agricultural Science and Technology Innovation Program, China (ASTIPIAS06) and the earmarked fund for China Agriculture Research System (CARS-36).