CCA-13965; No of Pages 6 Clinica Chimica Acta xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Clinica Chimica Acta
Invited critical review
2Q1
Melatonin and male reproduction
3Q2
Chunjin Li, Xu Zhou ⁎
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College of Animal Sciences, Jilin University, 5333 Xi'an Avenue, Changchun, Jilin Province 130062, PR China
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Article history: Received 9 March 2015 Accepted 14 April 2015 Available online xxxx
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Keywords: Male reproduction Melatonin Testis Antioxidant Sperm
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Melatonin is a neurohormone secreted by the pineal gland whose concentrations in the body are regulated by both the dark–light and seasonal cycles. The reproductive function of seasonal breeding animals is clearly influenced by the circadian variation in melatonin levels. Moreover, a growing body of evidence indicates that melatonin has important effects in the reproduction of some non-seasonal breeding animals. In males, melatonin affects reproductive regulation in three main ways. First, it regulates the secretion of two key neurohormones, GnRH and LH. Second, it regulates testosterone synthesis and testicular maturation. Third, as a potent free radical scavenger that is both lipophilic and hydrophilic, it prevents testicular damage caused by environmental toxins or inflammation. This review summarizes the existing data on the possible biological roles of melatonin in male reproduction. Overall, the literature data indicate that melatonin affects the secretion of both gonadotropins and testosterone while also improving sperm quality. This implies that it has important effects on the regulation of testicular development and male reproduction. © 2015 Published by Elsevier B.V.
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Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Melatonin secretion and its receptors . . . . . . . . . . . . . The roles of melatonin in male reproduction . . . . . . . . . . 3.1. The effects of melatonin on the hypothalamus and pituitary 3.2. Melatonin and testicular protection . . . . . . . . . . . 3.3. Melatonin and testosterone secretion . . . . . . . . . . 3.4. Melatonin and sperm quality . . . . . . . . . . . . . . 4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
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The seminiferous tubules and intertubular tissue are compartments of the testis that play essential roles in the production of sperm and testosterone. Various germ cells (spermatogonia, spermatocytes, spermatides, and spermatozoa) are localized in the seminiferous tubules, along with the Sertoli cells. The intertubular tissue consists of blood vessels and diverse cell types, including the Leydig cells responsible for testosterone synthesis [1]. The development of the testes and sperm production is mainly governed by a complex network of signaling processes involving the hypothalamic–pituitary–testicular axis. The Leydig and Sertoli cells are both targets of hormones released by the pituitary gland to regulate testicular development and spermatogenesis
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⁎ Corresponding author. Tel.:+86 431 87835142; fax: +86 431 87835142. E-mail address: [email protected] (X. Zhou).
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[2]. In addition to the established critical roles of hormones produced by the hypothalamus and pituitary gland, there is a growing body of evidence suggesting that the pineal gland is important in testicular development [3,4]. The pineal gland is situated within the brain, adjacent to the superior colliculi and behind the stria medullaris. It is mainly composed of pinealocytes, which produce hormones such as melatonin that regulate physiological processes [5]. Here, we review recent findings concerning melatonin's role in regulating male reproductive physiology.
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The mammalian pineal tissues are morphologically different both across and within species. These differences are thought to be associated with pineal functions and individual responses to environmental factors [6]. According to Vollrath's system of anatomical classification, the human pineal is of type A. Its most abundant cells are pinealocytes,
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The release of melatonin into the blood is regulated by the dark– light cycle. Because the relative duration of day and night changes with the seasons, the expression of melatonin also varies with the seasonal cycle. The reproductive capacity of seasonal animals varies with the seasonal cycle in a similar fashion, and it has been established that this variation is linked to the seasonal changes in melatonin secretion [23]. The neurohormones produced by the hypothalamus and pituitary are key regulators of the reproductive system, and there is a growing body of evidence suggesting that melatonin's effects on these tissues are responsible for much of its role in regulating reproduction. The
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Interest in the pineal gland's role in regulating the reproductive system has increased substantially in recent years. Melatonin is one of the most important hormones produced by the pineal. In female rats, treatment with melatonin inhibited ovarian development and delayed the onset of puberty [17], while male rats treated with exogenous melatonin exhibited reduced testis size [18,19]. Melatonin receptors have been detected in the human hypothalamus and pituitary, suggesting that melatonin may regulate the production of gonadotropin-releasing hormone (GnRH), FSH, and LH in these tissues [20,21]. It may also regulate testicular development directly by binding to specific receptors expressed in the testes [22].
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pups of female rats treated with melatonin during pregnancy exhibited delayed puberty and abnormal hormonal secretary patterns, suggesting that melatonin is transferred to offspring through maternal milk and influences their subsequent sexual development [24]. It was also found that the delayed onset of puberty following maternal melatonin treatment was caused by reductions in the concentration of LH and prolactin [25]. Melatonin treatment prevented the secretion of FSH and LH in male rats, which would affect their sexual maturation by reducing the stimulation of Sertoli cells by FSH [26]. An in vitro study on fetal rat pituitary cells showed that melatonin can significantly inhibit the induction of LH release by luteinizing hormone releasing hormone (LHRH) [27,28]. The decreased LH secretion from pituitary cells induced by melatonin may be associated with changes in the concentration of Ca2 + and cAMP accumulation [29,30]. Increased Ca2 + influxes or cAMP concentrations in pituitary cells potentiated the GnRH-induced release of LH but melatonin treatment partially suppressed this response, suggesting that the inhibition of LH release by melatonin may be mediated by reductions in the intracellular concentrations of these second messengers [29]. Another process that helps determine the onset of puberty is the negative feedback effect of testosterone on gonadotropin secretion. Melatonin treatment enhanced this effect, delaying sexual maturation in male rats [31]. The hypothalamic neurons in the suprachiasmatic nuclei (SCN) and the neurons that secrete gonadotropin-releasing hormone (GnRH) are the main targets of melatonin in the hypothalamus [32]. However, melatonin receptors are also expressed in the pars tuberalis and pars distalis regions of the anterior pituitary [20]. The reproductive actions mediated by the melatonin receptors in the hypothalamic neurons and anterior pituitary are different. The phase-changing effects on the circadian rhythms are mainly controlled by melatonin receptors in the SCN. Genetic inactivation of both MT1 and MT2 receptors in mice can cause changes in the circadian rhythm [33]. However, the roles of melatonin receptors in the anterior pituitary are time-dependent due to the postnatal decline in the abundance of melatonin receptors in the par distalis [21]. The presence of melatonin receptors in the hypothalamus and pituitary is consistent with the hypothesis that melatonin's influence on reproduction is due to its effects on the secretion of GnRH and pituitary hormones. The implantation of melatonin-containing pellets into the GnRH neuronal system of the hypothalamus reduced testis weight by up to 60% in male mice compared to implant-free mice [34]. Although melatonin increases the number of GnRH-secreting cells, it does not significantly affect their size or morphology [34]. These results indicate that melatonin's effects in the hypothalamus stem from suppressing the release of GnRH rather than its synthesis. Progressive treatment of male mice with melatonin over 10 days caused significant reductions in testicular and seminal vesicle mass, and also reduced or completely suppressed sperm production [35]. Subsequent treatment with exogenous gonadotropins reversed these changes in the mass of the testes and accessory sex organs [36]. The inhibition of GnRH release by melatonin was confirmed by in vitro studies on immortalized GnRH-secreting neurons [37]. Recent findings show that GnRH synthesis and release can be regulated by a neuropeptide named gonadotropin-inhibitory hormone (GnIH), which was first discovered in quails by a Japanese group in 2000 [38]. GnIH analogues have since been identified in diverse avian species and mammals [39]. The main function of GnIH in animal physiology is to inhibit GnRH synthesis and release by acting on the GnRH neurons [40]. It may also target the anterior pituitary, where the GnIH-receptor is widely distributed [41]. The expression of LH-b and FSH-b was down-regulated in sheep treated with GnIH [39]; similarly, cows treated with GnIH exhibited a reduced LH pulse frequency [42]. The synthesis and release of GnIH in the hypothalamus are regulated by multiple factors including the photoperiod, stress, and internal signals [39]. Melatonin may have profound effects on one or more of these factors. In photoperiodic animals, melatonin is mainly synthesized by the pineal gland and eyes. Quails that have undergone pinealectomy
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which constitute the pineal gland and produce pineal hormones including melatonin [7]. Although some photoreceptors have been found in pineal glands, mammalian pinealocytes are not capable of directly producing hormones in response to light treatment [8]. The indoleamine N-acetyl-5-methoxytryptamine was the first product of the pineal gland to be identified, being isolated from bovine pineal samples in 1958. Because of its role in frog skin melanophores and its similar chemical structure to serotonin, this compound was named melatonin. Extensive subsequent studies demonstrated it to be the most important pineal hormone [9]. The mechanism of melatonin biosynthesis in pinealocytes has been determined by Axelrod [10]. It begins with the uptake of tryptophan from the circulatory system by pinealocytes. The tryptophan is then converted into serotonin via 5-hydroxytryptophan, and serotonin is transformed into melatonin by a two-step process catalyzed by the enzymes N-acetyl transferase (NAT) and hydroxyindoleO-methyl transferase (HIOMT); the NAT-catalyzed step is rate limiting [11]. The genes encoding these enzymes are expressed weakly during the day and strongly during the night, so pineal melatonin secretion exhibits a circadian rhythm [6]. Melatonin can also be synthesized in epithelial cells, bone marrow cells, and lymphocytes, and locally synthesized melatonin is probably important in various physiological processes [12]. Melatonin secretion is mainly regulated by the light/dark cycle, which is controlled by an endogenous clock located in the suprachiasmatic nuclei of the hypothalamus. In mammals, most of the circulating melatonin originates from the pineal gland. This melatonin rapidly reaches all of the body's tissues, passing directly across cell membranes to interact with intracellular receptors due to its high lipid- and watersolubility [13]. However, some functions of melatonin are mediated by interactions with specific membrane-bound receptors [14]. Western blotting, RT-PCR, and in situ hybridization studies on mammalian tissues have identified two high-affinity melatonin receptors (MT1 and MT2) from the G-protein coupled receptor superfamily that are expressed in the brain and peripheral organs [15]. The third known melatonin receptor is named MT3, and was isolated from hamster brain samples; it is not a G-protein coupled receptor and has a comparatively low melatonin affinity [16]. The local production of melatonin throughout the body and the widespread distribution of its receptors suggest that it contributes to the regulation of diverse physiological processes.
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In addition to its role in regulating hormone release from the hypothalamus and pituitary in male animals, melatonin can also affect the testes directly by binding to specific receptors. Semi-quantitative RT-PCR experiments showed that both melatonin receptor 1 and melatonin receptor 2 were expressed in the testes of juvenile and adult rats [22]. Sixteen month old mice intraperitoneally injected with 10 mg/kg melatonin daily for 14 days had seminiferous tubules with a wide lumen lined by a low height germinal epithelium, indicating that melatonin has adverse effects on the seminiferous tubules in the testes of aged mice [46]. Histological and ultrastructural analyses showed that melatonin-treated rats had small testes and low numbers of spermatids [47]. Despite these adverse effects, there is also evidence that melatonin plays a protective role in testicular development. Compounds with antioxidant effects help to protect the testes from environmental damage, the side effects of cancer therapy, and other toxic molecules. Melatonin is a powerful antioxidant and was shown to be more effective at removing radicals than vitamin E [48]. Testicular torsion is a form of genital trauma that occurs frequently during the peripubertal period. It must be diagnosed accurately and rapidly in order to avoid damage arising from abnormal hormone production, subfertility, and potentially even complete infertility [49]. Studies on animal models have shown that the damage caused by testicular torsion is related to the duration of ischemia–reperfusion (I/R) [50]. The pathological effects of testicular torsion are largely due to the formation of reactive oxygen species (ROS) during ischemia–reperfusion [51]. ROS can cause DNA damage, impairment of protein function and peroxidation of lipids [52]. The mammalian testes are rich in polyunsaturated fatty acids that are readily attacked by ROS [53]. Several studies have shown that antioxidants are effective at reducing the damage caused by testicular torsion due to their ability to “mop up” excess ROS. Melatonin is a lipophilic and hydrophilic compound that readily passes through cellular membranes and is also a potent antioxidant [54]. Treatment with 50 mg/kg melatonin significantly reduced levels of oxidative stress indicators and lipid peroxidation in a rat model of testicular torsion [55]. In addition, histopathological studies revealed that tissue sections from the testes of rats with induced torsions contained cytoplasmic residues from disrupted sperm and large vacuole-like structures whereas sections from a melatonin-treated group with torsion injuries induced in the same way exhibited microstructures similar to those of uninjured control animals [56]. In a rat model with an artificially induced varicocele, melatonin treatment reduced the severity of the damage sustained by the epithelium and seminiferous tubules while also increasing antioxidant enzyme activity and reducing the levels of nitric oxide (NO), which can impair sperm function [57]. Melatonin can also increase the responsiveness of the Sertoli cells to FSH during testicular development, which may help prevent testis damage [58]. The protective roles of melatonin in testicular torsion suggest that it may have clinical applications as a free radical scavenger and indirect antioxidant in the treatment of testicular damage. This could be
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particularly important in the context of cancer treatment because some widely used anticancer drugs exhibit significant toxicity, including testicular toxicity. In animal models with testicular damage induced by certain anticancer drugs (e.g. doxorubicin, cisplatin, or cyclophosphamide), melatonin treatment helped protect against drug-induced testicular toxicity [59–62]. Various environmental toxicants have also been shown to cause reproductive damage by increasing levels of oxidative stress in the testes. For example, exposure to cadmium, fluoride, or ochratoxin A induced cellular stress in the testes and germ cell apoptosis in animal models, but it was shown that these effects could be alleviated by treatment with melatonin [63–65]. Interestingly, melatonin can also be used to prevent oxidative damage to the testes induced by electromagnetic radiation [66]. Melatonin is thought to improve lipid profiles via its powerful antioxidant activity and regulatory role in cholesterol metabolism, both of which may be important in the lipid-rich testes [67]. In men, poor semen quality was observed in some patients with hyperlipidemia [68]. Melatonin has been shown to effectively protect against testicular damage induced by hyperlipidemia in ApoE-knockout male mice fed on a high-fat diet [69], and clinical observations indicate that infertile men with reduced sperm motility, leucocytospermia, varicocele and nonobstructive azoospermia all exhibit unusually low melatonin levels, which may be linked to circadian variation in the concentrations of gonadotropins and melatonin in the plasma or seminal plasma [70].
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and orbital enucleation exhibit significantly reduced expression of GnIH precursor and the mature GnIH peptide relative to control birds; the expression of these peptides is restored in a dose-dependent fashion by treatment with exogenous melatonin [43]. Melatonin also appears to regulate the expression of GnIH in photoperiodic mammals. For example, Siberian hamsters exhibit stronger GnIH mRNA expression under short day conditions than under long day conditions. However, the opposite was true in hamsters that had been treated with exogenous melatonin or undergone pinealectomy [44]. The effects of melatonin on GnIH synthesis may be directly mediated by melatonin receptor 1, which has been identified in the GnIH neurons [45]. These results demonstrate that GnIH is a modulator that mediates melatonin signaling and thereby enables its regulation of animal reproduction.
As discussed above, pineal hormones are involved in reproductive regulation in seasonal animals. Adult male hamsters exposed to short day photoperiods for 8–16 weeks exhibited pronounced gonadal regression, which entails significant morphological changes in the tubular and interstitial compartments of the testes [71]. In addition, melatonin treatment significantly reduced the absolute volume and surface area of the mitochondria and smooth endoplasmic reticulum in the Leydig cells of mice, which is notable because these organelles are the primary locations of enzymes involved in androgen biosynthesis [72]. The mechanism of testosterone synthesis is very complex and regulated by multiple factors. Testosterone production is mainly dependent on cAMP signaling, which is stimulated by LH [73]. When rat Leydig cells were exposed to melatonin, testosterone release and cAMP production were obviously inhibited in a dose-dependent manner. The inhibitory effects of melatonin on LH or cAMP-stimulated testosterone production were abolished by luzindole, a melatonin receptor antagonist, but 22R-hydroxycholesterol reversed melatonin's inhibitory effects. These results imply that melatonin did not suppress the activity of P450scc, an enzyme critical for steroid hormone synthesis [74]. However, it did significantly reduce the stimulation of steroidogenic acute regulatory (StAR) protein expression by LH or cAMP [74]. In addition to the cAMP-dependent pathway, melatonin's effects on cAMP-independent pathways of testosterone production have been investigated. There is some evidence indicating that GnRH may increase cytosolic Ca2 + concentrations and activate protein kinase C, which is probably associated with testosterone production [73]. Studies using a fluorescent Ca2+ indicator showed that melatonin reduced GnRH-induced testosterone secretion suppressing the GnRH-dependent release of Ca2 + from intracellular stores and thereby reducing cellular Ca2 + levels [75]. In addition to suppressing LH- and GnRH-induced testosterone synthesis in the Leydig cells, melatonin also regulated testosterone production by interacting with the corticotropin-releasing hormone (CRH) system in the testes [76]. Corticotropin-releasing hormone was first isolated from sheep hypothalami and is mostly synthesized in the neurons of the paraventricular nucleus. It is a major physiological regulator of the pituitary–adrenocortical axis that acts by interacting with specific membrane-bound receptors in the corticotrophs of the anterior pituitary gland and thereby activating a cAMP-dependent signaling pathway [77]. In addition to being secreted by the hypothalamus, CRH is
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Melatonin is known to regulate seasonal and circadian rhythms in mammals. However, a large body of experimental evidence indicates that it also has beneficial effects on reproduction in both male and female animals. Its biological roles in reproductive regulation are mediated by binding to specific receptors, which have been detected in the hypothalamic–pituitary–gonadal axis. Melatonin primarily influences testicular development by regulating the secretion of neurohormones (particularly GnRH) and testosterone. The mechanisms by which it regulates testosterone secretion are very complex, including both indirect pathways involving the hypothalamic–pituitary–gonadal axis and also direct effects on the Leydig cells of the testes (Fig. 1). As is the case for other hormones involved in reproductive regulation, abnormal serum levels of melatonin can cause male infertility. While the pineal gland is the main site of melatonin synthesis, it is also produced by many other tissues and has been shown to protect diverse cell types including immune cells and neurons. In addition to regulating hormone secretion, the antioxidant activity of melatonin allows it to help prevent testicular damage caused by toxic environments and testicular inflammation (Fig. 1). However, in some cancer cells, melatonin appears to promote apoptotic death. The mechanisms by which
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Spermatogenesis is easily disrupted when the testes are exposed to toxic environments or by testicular inflammation. Melatonin has been shown to help prevent sperm damage in the testes both in vitro and in vivo. In men, abnormal levels of melatonin in semen are associated with infertility [70]. High endogenous semen melatonin levels have been associated with mild oligozoospermy and azoospermy. However, low endogenous semen melatonin levels are associated with abnormal sperm progression [80]. These results suggest that melatonin is involved in spermatogenesis. The presence of melatonin receptors in the sperm of some species suggests that it may have a direct physiological role in regulating sperm functions [81,82]. In addition, testicular activity in rams is regulated by the photoperiod, whose effects are mediated by melatonin. Although semen production continues throughout the year in rams, sperm quality is lower outside the breeding season [83]. It has been reported that melatonin treatment improves the testicular development and semen quality of rams and male Damascus goats during the non-breeding season [84,85]. In vitro, exposure of ram spermatozoa to melatonin has direct effects: capacitation and phosphatidylserine translocation are reduced at high melatonin concentrations but shortterm capacitation is increased by exposure to low concentrations of melatonin, leading to increased oocyte fertilization rates [86]. The increased cleavage of oocytes fertilized with melatonin-treated sperm may be linked to melatonin-induced increases in the hyaluronidase activity of semen [87]. In a study on 8 healthy men, melatonin administration for 1700 h caused no discernible changes in semen quality or serum and seminal plasma hormone levels in six cases but appreciably reduced sperm concentrations in the remaining two men, possibly as a consequence of the inhibition of aromatase at the testicular level [88]. In rats with testicular ischemia/reperfusion injuries, melatonin significantly reduced the frequency of sperm abnormalities, possibly because of its antioxidative properties [89]. In vitro studies demonstrated that the use of a melatonin-containing incubation medium increased the percentage of motile, progressive, and rapid sperm cells, increased mitochondrial activity, and decreased endogenous NO levels relative to those observed in sperm cultivated in melatonin-free media [90]. These effects were attributed to melatonin's antioxidant properties. Sperm storage in vitro is important in artificial insemination. Unfortunately, sperm deteriorates rapidly at room temperature and also when stored at lower temperatures or in liquid nitrogen. The augmentation of semen extender media with melatonin significantly improved the motility parameters of ram semen (e.g. its straight-linear velocity and average path velocity) stored at 5 °C or 17 °C [91]. Melatonin also improved the quality of thawed bovine serum by reducing its levels of lipid peroxidation and increasing the activity of antioxidant enzymes [92]. The prevention of sperm damage by melatonin may be associated with its induction of various signaling processes. Notably, melatonin exposure significantly reduced caspase activity and DNA fragmentation
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induced by H2O2 in sperm, and both of these effects depended on the 391 expression of melatonin receptor 1 and extracellular signal-regulated 392 kinases [93,94]. 393
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produced in the testes [78]. CRH secreted by the Leydig cells is an important negative autocrine modulator of gonadotropin-induced testosterone production [78]. In Leydig cells from hamster testes subjected to different photoperiods, melatonin significantly reduced the gonadotropinstimulated production of cAMP and androstane-3α,17β-diol [79], along with the expression of proteins and enzymes that are essential for testosterone synthesis including StAR, P450scc, 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase [76]. However, melatonin significantly increased CRH mRNA levels in Leydig cells [76]. Both melatonin and CRH regulated testosterone production by activating tyrosine phosphatases and thereby reduced the phosphorylation levels of extracellular signal-regulated kinase (erk) and c-jun N-terminal kinase (jnk). This in turn down-regulated the expression of c-jun, c-fos and StAR [79]. Treatment with a selective CRH antagonist abolished all of these effects in Leydig cells treated with either CRH or melatonin, indicating that melatonin acts indirectly via the CRH system [79].
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Fig. 1. The role of melatonin in the regulation of male reproduction. Melatonin secreted by the pineal gland indirectly influences male reproduction by binding to specific receptors and thereby inhibiting the production of both GnRH and LH. In the hypothalamus, melatonin can also inhibit GnRH release by increasing GnIH production. In the testes, it inhibits testosterone production but may also protect against damage caused by toxic environments or testicular inflammation, and can improve sperm quality due to its antioxidant activity.
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melatonin can contribute to both protection and apoptosis in different cell types remain to be clarified. Overall, however, the literature data strongly suggest that melatonin plays vital but complex roles in the regulation of male reproduction.
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Contents lists available at ScienceDirect
Clinica Chimica Acta
Invited critical review
2Q1
Melatonin and male reproduction
3Q2
Chunjin Li, Xu Zhou ⁎
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College of Animal Sciences, Jilin University, 5333 Xi'an Avenue, Changchun, Jilin Province 130062, PR China
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Article history: Received 9 March 2015 Accepted 14 April 2015 Available online xxxx
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Keywords: Male reproduction Melatonin Testis Antioxidant Sperm
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Melatonin is a neurohormone secreted by the pineal gland whose concentrations in the body are regulated by both the dark–light and seasonal cycles. The reproductive function of seasonal breeding animals is clearly influenced by the circadian variation in melatonin levels. Moreover, a growing body of evidence indicates that melatonin has important effects in the reproduction of some non-seasonal breeding animals. In males, melatonin affects reproductive regulation in three main ways. First, it regulates the secretion of two key neurohormones, GnRH and LH. Second, it regulates testosterone synthesis and testicular maturation. Third, as a potent free radical scavenger that is both lipophilic and hydrophilic, it prevents testicular damage caused by environmental toxins or inflammation. This review summarizes the existing data on the possible biological roles of melatonin in male reproduction. Overall, the literature data indicate that melatonin affects the secretion of both gonadotropins and testosterone while also improving sperm quality. This implies that it has important effects on the regulation of testicular development and male reproduction. © 2015 Published by Elsevier B.V.
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Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Melatonin secretion and its receptors . . . . . . . . . . . . . The roles of melatonin in male reproduction . . . . . . . . . . 3.1. The effects of melatonin on the hypothalamus and pituitary 3.2. Melatonin and testicular protection . . . . . . . . . . . 3.3. Melatonin and testosterone secretion . . . . . . . . . . 3.4. Melatonin and sperm quality . . . . . . . . . . . . . . 4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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The seminiferous tubules and intertubular tissue are compartments of the testis that play essential roles in the production of sperm and testosterone. Various germ cells (spermatogonia, spermatocytes, spermatides, and spermatozoa) are localized in the seminiferous tubules, along with the Sertoli cells. The intertubular tissue consists of blood vessels and diverse cell types, including the Leydig cells responsible for testosterone synthesis [1]. The development of the testes and sperm production is mainly governed by a complex network of signaling processes involving the hypothalamic–pituitary–testicular axis. The Leydig and Sertoli cells are both targets of hormones released by the pituitary gland to regulate testicular development and spermatogenesis
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⁎ Corresponding author. Tel.:+86 431 87835142; fax: +86 431 87835142. E-mail address: [email protected] (X. Zhou).
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[2]. In addition to the established critical roles of hormones produced by the hypothalamus and pituitary gland, there is a growing body of evidence suggesting that the pineal gland is important in testicular development [3,4]. The pineal gland is situated within the brain, adjacent to the superior colliculi and behind the stria medullaris. It is mainly composed of pinealocytes, which produce hormones such as melatonin that regulate physiological processes [5]. Here, we review recent findings concerning melatonin's role in regulating male reproductive physiology.
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2. Melatonin secretion and its receptors
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The mammalian pineal tissues are morphologically different both across and within species. These differences are thought to be associated with pineal functions and individual responses to environmental factors [6]. According to Vollrath's system of anatomical classification, the human pineal is of type A. Its most abundant cells are pinealocytes,
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3.1. The effects of melatonin on the hypothalamus and pituitary
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The release of melatonin into the blood is regulated by the dark– light cycle. Because the relative duration of day and night changes with the seasons, the expression of melatonin also varies with the seasonal cycle. The reproductive capacity of seasonal animals varies with the seasonal cycle in a similar fashion, and it has been established that this variation is linked to the seasonal changes in melatonin secretion [23]. The neurohormones produced by the hypothalamus and pituitary are key regulators of the reproductive system, and there is a growing body of evidence suggesting that melatonin's effects on these tissues are responsible for much of its role in regulating reproduction. The
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Interest in the pineal gland's role in regulating the reproductive system has increased substantially in recent years. Melatonin is one of the most important hormones produced by the pineal. In female rats, treatment with melatonin inhibited ovarian development and delayed the onset of puberty [17], while male rats treated with exogenous melatonin exhibited reduced testis size [18,19]. Melatonin receptors have been detected in the human hypothalamus and pituitary, suggesting that melatonin may regulate the production of gonadotropin-releasing hormone (GnRH), FSH, and LH in these tissues [20,21]. It may also regulate testicular development directly by binding to specific receptors expressed in the testes [22].
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pups of female rats treated with melatonin during pregnancy exhibited delayed puberty and abnormal hormonal secretary patterns, suggesting that melatonin is transferred to offspring through maternal milk and influences their subsequent sexual development [24]. It was also found that the delayed onset of puberty following maternal melatonin treatment was caused by reductions in the concentration of LH and prolactin [25]. Melatonin treatment prevented the secretion of FSH and LH in male rats, which would affect their sexual maturation by reducing the stimulation of Sertoli cells by FSH [26]. An in vitro study on fetal rat pituitary cells showed that melatonin can significantly inhibit the induction of LH release by luteinizing hormone releasing hormone (LHRH) [27,28]. The decreased LH secretion from pituitary cells induced by melatonin may be associated with changes in the concentration of Ca2 + and cAMP accumulation [29,30]. Increased Ca2 + influxes or cAMP concentrations in pituitary cells potentiated the GnRH-induced release of LH but melatonin treatment partially suppressed this response, suggesting that the inhibition of LH release by melatonin may be mediated by reductions in the intracellular concentrations of these second messengers [29]. Another process that helps determine the onset of puberty is the negative feedback effect of testosterone on gonadotropin secretion. Melatonin treatment enhanced this effect, delaying sexual maturation in male rats [31]. The hypothalamic neurons in the suprachiasmatic nuclei (SCN) and the neurons that secrete gonadotropin-releasing hormone (GnRH) are the main targets of melatonin in the hypothalamus [32]. However, melatonin receptors are also expressed in the pars tuberalis and pars distalis regions of the anterior pituitary [20]. The reproductive actions mediated by the melatonin receptors in the hypothalamic neurons and anterior pituitary are different. The phase-changing effects on the circadian rhythms are mainly controlled by melatonin receptors in the SCN. Genetic inactivation of both MT1 and MT2 receptors in mice can cause changes in the circadian rhythm [33]. However, the roles of melatonin receptors in the anterior pituitary are time-dependent due to the postnatal decline in the abundance of melatonin receptors in the par distalis [21]. The presence of melatonin receptors in the hypothalamus and pituitary is consistent with the hypothesis that melatonin's influence on reproduction is due to its effects on the secretion of GnRH and pituitary hormones. The implantation of melatonin-containing pellets into the GnRH neuronal system of the hypothalamus reduced testis weight by up to 60% in male mice compared to implant-free mice [34]. Although melatonin increases the number of GnRH-secreting cells, it does not significantly affect their size or morphology [34]. These results indicate that melatonin's effects in the hypothalamus stem from suppressing the release of GnRH rather than its synthesis. Progressive treatment of male mice with melatonin over 10 days caused significant reductions in testicular and seminal vesicle mass, and also reduced or completely suppressed sperm production [35]. Subsequent treatment with exogenous gonadotropins reversed these changes in the mass of the testes and accessory sex organs [36]. The inhibition of GnRH release by melatonin was confirmed by in vitro studies on immortalized GnRH-secreting neurons [37]. Recent findings show that GnRH synthesis and release can be regulated by a neuropeptide named gonadotropin-inhibitory hormone (GnIH), which was first discovered in quails by a Japanese group in 2000 [38]. GnIH analogues have since been identified in diverse avian species and mammals [39]. The main function of GnIH in animal physiology is to inhibit GnRH synthesis and release by acting on the GnRH neurons [40]. It may also target the anterior pituitary, where the GnIH-receptor is widely distributed [41]. The expression of LH-b and FSH-b was down-regulated in sheep treated with GnIH [39]; similarly, cows treated with GnIH exhibited a reduced LH pulse frequency [42]. The synthesis and release of GnIH in the hypothalamus are regulated by multiple factors including the photoperiod, stress, and internal signals [39]. Melatonin may have profound effects on one or more of these factors. In photoperiodic animals, melatonin is mainly synthesized by the pineal gland and eyes. Quails that have undergone pinealectomy
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which constitute the pineal gland and produce pineal hormones including melatonin [7]. Although some photoreceptors have been found in pineal glands, mammalian pinealocytes are not capable of directly producing hormones in response to light treatment [8]. The indoleamine N-acetyl-5-methoxytryptamine was the first product of the pineal gland to be identified, being isolated from bovine pineal samples in 1958. Because of its role in frog skin melanophores and its similar chemical structure to serotonin, this compound was named melatonin. Extensive subsequent studies demonstrated it to be the most important pineal hormone [9]. The mechanism of melatonin biosynthesis in pinealocytes has been determined by Axelrod [10]. It begins with the uptake of tryptophan from the circulatory system by pinealocytes. The tryptophan is then converted into serotonin via 5-hydroxytryptophan, and serotonin is transformed into melatonin by a two-step process catalyzed by the enzymes N-acetyl transferase (NAT) and hydroxyindoleO-methyl transferase (HIOMT); the NAT-catalyzed step is rate limiting [11]. The genes encoding these enzymes are expressed weakly during the day and strongly during the night, so pineal melatonin secretion exhibits a circadian rhythm [6]. Melatonin can also be synthesized in epithelial cells, bone marrow cells, and lymphocytes, and locally synthesized melatonin is probably important in various physiological processes [12]. Melatonin secretion is mainly regulated by the light/dark cycle, which is controlled by an endogenous clock located in the suprachiasmatic nuclei of the hypothalamus. In mammals, most of the circulating melatonin originates from the pineal gland. This melatonin rapidly reaches all of the body's tissues, passing directly across cell membranes to interact with intracellular receptors due to its high lipid- and watersolubility [13]. However, some functions of melatonin are mediated by interactions with specific membrane-bound receptors [14]. Western blotting, RT-PCR, and in situ hybridization studies on mammalian tissues have identified two high-affinity melatonin receptors (MT1 and MT2) from the G-protein coupled receptor superfamily that are expressed in the brain and peripheral organs [15]. The third known melatonin receptor is named MT3, and was isolated from hamster brain samples; it is not a G-protein coupled receptor and has a comparatively low melatonin affinity [16]. The local production of melatonin throughout the body and the widespread distribution of its receptors suggest that it contributes to the regulation of diverse physiological processes.
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In addition to its role in regulating hormone release from the hypothalamus and pituitary in male animals, melatonin can also affect the testes directly by binding to specific receptors. Semi-quantitative RT-PCR experiments showed that both melatonin receptor 1 and melatonin receptor 2 were expressed in the testes of juvenile and adult rats [22]. Sixteen month old mice intraperitoneally injected with 10 mg/kg melatonin daily for 14 days had seminiferous tubules with a wide lumen lined by a low height germinal epithelium, indicating that melatonin has adverse effects on the seminiferous tubules in the testes of aged mice [46]. Histological and ultrastructural analyses showed that melatonin-treated rats had small testes and low numbers of spermatids [47]. Despite these adverse effects, there is also evidence that melatonin plays a protective role in testicular development. Compounds with antioxidant effects help to protect the testes from environmental damage, the side effects of cancer therapy, and other toxic molecules. Melatonin is a powerful antioxidant and was shown to be more effective at removing radicals than vitamin E [48]. Testicular torsion is a form of genital trauma that occurs frequently during the peripubertal period. It must be diagnosed accurately and rapidly in order to avoid damage arising from abnormal hormone production, subfertility, and potentially even complete infertility [49]. Studies on animal models have shown that the damage caused by testicular torsion is related to the duration of ischemia–reperfusion (I/R) [50]. The pathological effects of testicular torsion are largely due to the formation of reactive oxygen species (ROS) during ischemia–reperfusion [51]. ROS can cause DNA damage, impairment of protein function and peroxidation of lipids [52]. The mammalian testes are rich in polyunsaturated fatty acids that are readily attacked by ROS [53]. Several studies have shown that antioxidants are effective at reducing the damage caused by testicular torsion due to their ability to “mop up” excess ROS. Melatonin is a lipophilic and hydrophilic compound that readily passes through cellular membranes and is also a potent antioxidant [54]. Treatment with 50 mg/kg melatonin significantly reduced levels of oxidative stress indicators and lipid peroxidation in a rat model of testicular torsion [55]. In addition, histopathological studies revealed that tissue sections from the testes of rats with induced torsions contained cytoplasmic residues from disrupted sperm and large vacuole-like structures whereas sections from a melatonin-treated group with torsion injuries induced in the same way exhibited microstructures similar to those of uninjured control animals [56]. In a rat model with an artificially induced varicocele, melatonin treatment reduced the severity of the damage sustained by the epithelium and seminiferous tubules while also increasing antioxidant enzyme activity and reducing the levels of nitric oxide (NO), which can impair sperm function [57]. Melatonin can also increase the responsiveness of the Sertoli cells to FSH during testicular development, which may help prevent testis damage [58]. The protective roles of melatonin in testicular torsion suggest that it may have clinical applications as a free radical scavenger and indirect antioxidant in the treatment of testicular damage. This could be
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particularly important in the context of cancer treatment because some widely used anticancer drugs exhibit significant toxicity, including testicular toxicity. In animal models with testicular damage induced by certain anticancer drugs (e.g. doxorubicin, cisplatin, or cyclophosphamide), melatonin treatment helped protect against drug-induced testicular toxicity [59–62]. Various environmental toxicants have also been shown to cause reproductive damage by increasing levels of oxidative stress in the testes. For example, exposure to cadmium, fluoride, or ochratoxin A induced cellular stress in the testes and germ cell apoptosis in animal models, but it was shown that these effects could be alleviated by treatment with melatonin [63–65]. Interestingly, melatonin can also be used to prevent oxidative damage to the testes induced by electromagnetic radiation [66]. Melatonin is thought to improve lipid profiles via its powerful antioxidant activity and regulatory role in cholesterol metabolism, both of which may be important in the lipid-rich testes [67]. In men, poor semen quality was observed in some patients with hyperlipidemia [68]. Melatonin has been shown to effectively protect against testicular damage induced by hyperlipidemia in ApoE-knockout male mice fed on a high-fat diet [69], and clinical observations indicate that infertile men with reduced sperm motility, leucocytospermia, varicocele and nonobstructive azoospermia all exhibit unusually low melatonin levels, which may be linked to circadian variation in the concentrations of gonadotropins and melatonin in the plasma or seminal plasma [70].
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and orbital enucleation exhibit significantly reduced expression of GnIH precursor and the mature GnIH peptide relative to control birds; the expression of these peptides is restored in a dose-dependent fashion by treatment with exogenous melatonin [43]. Melatonin also appears to regulate the expression of GnIH in photoperiodic mammals. For example, Siberian hamsters exhibit stronger GnIH mRNA expression under short day conditions than under long day conditions. However, the opposite was true in hamsters that had been treated with exogenous melatonin or undergone pinealectomy [44]. The effects of melatonin on GnIH synthesis may be directly mediated by melatonin receptor 1, which has been identified in the GnIH neurons [45]. These results demonstrate that GnIH is a modulator that mediates melatonin signaling and thereby enables its regulation of animal reproduction.
As discussed above, pineal hormones are involved in reproductive regulation in seasonal animals. Adult male hamsters exposed to short day photoperiods for 8–16 weeks exhibited pronounced gonadal regression, which entails significant morphological changes in the tubular and interstitial compartments of the testes [71]. In addition, melatonin treatment significantly reduced the absolute volume and surface area of the mitochondria and smooth endoplasmic reticulum in the Leydig cells of mice, which is notable because these organelles are the primary locations of enzymes involved in androgen biosynthesis [72]. The mechanism of testosterone synthesis is very complex and regulated by multiple factors. Testosterone production is mainly dependent on cAMP signaling, which is stimulated by LH [73]. When rat Leydig cells were exposed to melatonin, testosterone release and cAMP production were obviously inhibited in a dose-dependent manner. The inhibitory effects of melatonin on LH or cAMP-stimulated testosterone production were abolished by luzindole, a melatonin receptor antagonist, but 22R-hydroxycholesterol reversed melatonin's inhibitory effects. These results imply that melatonin did not suppress the activity of P450scc, an enzyme critical for steroid hormone synthesis [74]. However, it did significantly reduce the stimulation of steroidogenic acute regulatory (StAR) protein expression by LH or cAMP [74]. In addition to the cAMP-dependent pathway, melatonin's effects on cAMP-independent pathways of testosterone production have been investigated. There is some evidence indicating that GnRH may increase cytosolic Ca2 + concentrations and activate protein kinase C, which is probably associated with testosterone production [73]. Studies using a fluorescent Ca2+ indicator showed that melatonin reduced GnRH-induced testosterone secretion suppressing the GnRH-dependent release of Ca2 + from intracellular stores and thereby reducing cellular Ca2 + levels [75]. In addition to suppressing LH- and GnRH-induced testosterone synthesis in the Leydig cells, melatonin also regulated testosterone production by interacting with the corticotropin-releasing hormone (CRH) system in the testes [76]. Corticotropin-releasing hormone was first isolated from sheep hypothalami and is mostly synthesized in the neurons of the paraventricular nucleus. It is a major physiological regulator of the pituitary–adrenocortical axis that acts by interacting with specific membrane-bound receptors in the corticotrophs of the anterior pituitary gland and thereby activating a cAMP-dependent signaling pathway [77]. In addition to being secreted by the hypothalamus, CRH is
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Melatonin is known to regulate seasonal and circadian rhythms in mammals. However, a large body of experimental evidence indicates that it also has beneficial effects on reproduction in both male and female animals. Its biological roles in reproductive regulation are mediated by binding to specific receptors, which have been detected in the hypothalamic–pituitary–gonadal axis. Melatonin primarily influences testicular development by regulating the secretion of neurohormones (particularly GnRH) and testosterone. The mechanisms by which it regulates testosterone secretion are very complex, including both indirect pathways involving the hypothalamic–pituitary–gonadal axis and also direct effects on the Leydig cells of the testes (Fig. 1). As is the case for other hormones involved in reproductive regulation, abnormal serum levels of melatonin can cause male infertility. While the pineal gland is the main site of melatonin synthesis, it is also produced by many other tissues and has been shown to protect diverse cell types including immune cells and neurons. In addition to regulating hormone secretion, the antioxidant activity of melatonin allows it to help prevent testicular damage caused by toxic environments and testicular inflammation (Fig. 1). However, in some cancer cells, melatonin appears to promote apoptotic death. The mechanisms by which
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Spermatogenesis is easily disrupted when the testes are exposed to toxic environments or by testicular inflammation. Melatonin has been shown to help prevent sperm damage in the testes both in vitro and in vivo. In men, abnormal levels of melatonin in semen are associated with infertility [70]. High endogenous semen melatonin levels have been associated with mild oligozoospermy and azoospermy. However, low endogenous semen melatonin levels are associated with abnormal sperm progression [80]. These results suggest that melatonin is involved in spermatogenesis. The presence of melatonin receptors in the sperm of some species suggests that it may have a direct physiological role in regulating sperm functions [81,82]. In addition, testicular activity in rams is regulated by the photoperiod, whose effects are mediated by melatonin. Although semen production continues throughout the year in rams, sperm quality is lower outside the breeding season [83]. It has been reported that melatonin treatment improves the testicular development and semen quality of rams and male Damascus goats during the non-breeding season [84,85]. In vitro, exposure of ram spermatozoa to melatonin has direct effects: capacitation and phosphatidylserine translocation are reduced at high melatonin concentrations but shortterm capacitation is increased by exposure to low concentrations of melatonin, leading to increased oocyte fertilization rates [86]. The increased cleavage of oocytes fertilized with melatonin-treated sperm may be linked to melatonin-induced increases in the hyaluronidase activity of semen [87]. In a study on 8 healthy men, melatonin administration for 1700 h caused no discernible changes in semen quality or serum and seminal plasma hormone levels in six cases but appreciably reduced sperm concentrations in the remaining two men, possibly as a consequence of the inhibition of aromatase at the testicular level [88]. In rats with testicular ischemia/reperfusion injuries, melatonin significantly reduced the frequency of sperm abnormalities, possibly because of its antioxidative properties [89]. In vitro studies demonstrated that the use of a melatonin-containing incubation medium increased the percentage of motile, progressive, and rapid sperm cells, increased mitochondrial activity, and decreased endogenous NO levels relative to those observed in sperm cultivated in melatonin-free media [90]. These effects were attributed to melatonin's antioxidant properties. Sperm storage in vitro is important in artificial insemination. Unfortunately, sperm deteriorates rapidly at room temperature and also when stored at lower temperatures or in liquid nitrogen. The augmentation of semen extender media with melatonin significantly improved the motility parameters of ram semen (e.g. its straight-linear velocity and average path velocity) stored at 5 °C or 17 °C [91]. Melatonin also improved the quality of thawed bovine serum by reducing its levels of lipid peroxidation and increasing the activity of antioxidant enzymes [92]. The prevention of sperm damage by melatonin may be associated with its induction of various signaling processes. Notably, melatonin exposure significantly reduced caspase activity and DNA fragmentation
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induced by H2O2 in sperm, and both of these effects depended on the 391 expression of melatonin receptor 1 and extracellular signal-regulated 392 kinases [93,94]. 393
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produced in the testes [78]. CRH secreted by the Leydig cells is an important negative autocrine modulator of gonadotropin-induced testosterone production [78]. In Leydig cells from hamster testes subjected to different photoperiods, melatonin significantly reduced the gonadotropinstimulated production of cAMP and androstane-3α,17β-diol [79], along with the expression of proteins and enzymes that are essential for testosterone synthesis including StAR, P450scc, 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase [76]. However, melatonin significantly increased CRH mRNA levels in Leydig cells [76]. Both melatonin and CRH regulated testosterone production by activating tyrosine phosphatases and thereby reduced the phosphorylation levels of extracellular signal-regulated kinase (erk) and c-jun N-terminal kinase (jnk). This in turn down-regulated the expression of c-jun, c-fos and StAR [79]. Treatment with a selective CRH antagonist abolished all of these effects in Leydig cells treated with either CRH or melatonin, indicating that melatonin acts indirectly via the CRH system [79].
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Fig. 1. The role of melatonin in the regulation of male reproduction. Melatonin secreted by the pineal gland indirectly influences male reproduction by binding to specific receptors and thereby inhibiting the production of both GnRH and LH. In the hypothalamus, melatonin can also inhibit GnRH release by increasing GnIH production. In the testes, it inhibits testosterone production but may also protect against damage caused by toxic environments or testicular inflammation, and can improve sperm quality due to its antioxidant activity.
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This work was supported by the National Natural Science Foundation of China (3A413X686604, 3A413X486604).
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melatonin can contribute to both protection and apoptosis in different cell types remain to be clarified. Overall, however, the literature data strongly suggest that melatonin plays vital but complex roles in the regulation of male reproduction.
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