Management of ovarian functions by melatonin

INTRODUCTION

Following the discovery of melatonin by Lerner et al., in 1958,^[1] there has been a considerable change in the understanding of the various functional aspects of melatonin. Apart from pineal, several extra pineal sites of melatonin biosynthesis in mammals have been identified including skin, retina, gut, ovary, and testis.^[2-5] Today, the most pertinent physiological actions of melatonin have been established to be in

1. Regulation of biological rhythms^[6] and reproduction in seasonal breeders,^[7]

2. Enhancing immune responses,^[8,9]

3. Prevention of carcinogenesis, and^[10]

4. As an endogenous antioxidant.^[11]

Regulation of seasonal reproduction is one of the initially established functions of melatonin. Within the environmental light dark cycle, circulatory melatonin peaks at night and decreases during the day. Hence, there is a significantly higher concentration of melatonin in blood during the longer nights of winter and vice versa in summer months.^[12] Endogenously released melatonin resulting from changes in day length modulates reproduction^[13] acts on the hypothalamic-pituitary-gonadal axis^[14] by activation of its receptors in hypothalamic gonadotropin-releasing hormone (GnRH) releasing neurons, pars tuberalis, gonadotrophs of pars distalis, testes, and ovaries.^[7,15] Melatonin regulates the pulsatile secretion of GnRH from the hypothalamus.^[15,16] Although the exact mechanism is still not clearly understood, it is believed not to be a direct effect on GnRH neurons considering there is no change in responsiveness to GnRH with changing photoperiods.^[17] It is predicted that two complementary mechanisms^[18] regulate seasonal GnRH secretion – a change in the steroid negative feedback on GnRH release^[19,20] and a direct modulation of GnRH secretion^[6,15,21] that is steroid-independent.

Cases of idiopathic infertility amongst women who have undergone pinealectomy^[22] and follicular atresia observed in pinealectomized rodents^[23] suggest a direct relationship between melatonin and the female reproductive system. Being lipophilic, melatonin can cross cellular barriers and scavenge free radicals directly and more effectively than any other dietary antioxidant. The downstream metabolites of melatonin generated in process also act as free radical scavengers,^[4] a property that is unique to melatonin. Although the foremost, the antioxidant effect of melatonin is just one aspect of the several impacts the molecule has on ovarian function.

Melatonin is established to promote luteinisation and progesterone synthesis.^[24] In buffalo granulosa cell cultures, melatonin, when added at a dose of 100 nM significantly increased luteinizing hormone (LH) receptor mRNA.^[25] Melatonin upregulates Steroidogenic Acute Regulatory protein (StAR) expression in primary cultures of human granulosa-lutein cells obtained from women undergoing in vitro fertilization (IVF).^[26] The stimulatory effect of melatonin on StAR expression mediated through both MT1 and MT2 melatonin receptors was demonstrated by use of pharmacological inhibitors. Further, melatonin exposure activates the PI3K/AKT signaling pathway and its inhibition attenuates the stimulatory effect of melatonin on StAR expression.^[27] There is no substantial evidence to support the capacity of melatonin to regulate follicular steroidogenesis before ovulation, but the fact that the melatonin content in the follicle increases as the cycle progresses cannot be ignored and is believed to be involved in granulosa cell proliferation which inadvertently influences steroid production.

MELATONIN AND SEASONALITY

Seasonal regulation of reproduction is one of the earliest observations in regard to effects of melatonin on mammalian physiology. Governed by photoperiodic changes, it is an outcome of the interactions between melatonin and several neural and neuroendocrine pathways in the hypothalamus. Daily changes in photic signals are relayed to the GnRH neurons through direct axonal projections of vasoactive intestinal peptide-expressing neurons of Supra Chaismatic Nucleus (SCN),^[28,29] and SCN arginine vasopressin-expressing neurons to kisspeptin neurons in the pre-optic area, while the RFRP-3/(Arg)(Phe) related peptide-3 (gonadotropin inhibitory hormone) neurons in the mediobasal hypothalamus receive both.^[30,31] Therefore, it can be said that the SCN neurons control the activity of GnRH neurons directly and indirectly by regulating the activity of POA kisspeptin neurons and DMH RFRP-3 neurons.

Since melatonin is the chemical relay of photic information between the environment and the SCN, it is the primary mediator of seasonal information that is integrated by these neuromodulators. Kisspeptins have a stimulatory effect on the reproductive axis while RFRP-3 is inhibitory in its pathway of action. Thus, melatonin is neither anti-gonadal nor pro-gonadal as previously believed^[12] but a chemical link between the environment and mammalian reproductive axis.

INTERACTIONS OF THE FEMALE REPRODUCTIVE SYSTEM WITH THE HYPOTHALAMO-PITUITARY-ADRENAL (HPA) AXIS: ROLE OF THE STRESS RESPONSE

The neuroendocrine axis of reproduction is sensitive toward changes in the environment, both external and physiological. In face of challenges, psychological, physical, or physiological,^[32,33] the body initiates a “stress response” that serves as an adaptive mechanism for the organism to return to the steady state. In mammals, reports suggest that there is a significant amount of cross-talk between the different components of the HPA axis with the center of reproduction that is typically antagonistic in nature.^[34,35] Interestingly, there is a strong sexual dimorphism in the stress response where the response in females is more pronounced due to the presence of estrogen responsive elements in the CRH gene as seen in case of humans,^[36,37] making females more vulnerable toward stress induced disruptive changes. The activation of the stress response has been found to decrease the pulsatile release of both GnRH and LH^[38,39] while glucocorticoids modulate levels of circulating gonadotropins by acting on hypothalamus and pituitary, affecting the follicular development in ovary.^[40,41] Glucocorticoid can also modulate levels of metabolic hormones and growth factors ^[42,43] that may lead to a decrease in estrogen production.^[25] Elevated levels of glucocorticoid seen during a stress response may inhibit progesterone synthesis^[44] and luteolysis by decreasing uterine prostaglandin F2α synthesis.^[45]

Generation of free radicals, especially reactive oxygen species (ROS), is another possible mechanism of glucocorticoid mediated inhibition of ovarian axis. Both acute and chronic elevation of glucocorticoid levels have been shown to cause oxidative stress.^[46-48] Moreover, the presence of glucocorticoid receptors in mitochondria suggests that they may play a role in the stress response^[49] by influencing both ATP and ROS production.^[50] Glucocorticoids have also been shown to increase both mitochondrial and non-mitochondrial sources of ROS production^[51] thereby making generation of ROS an important by-stander effect of the stress response.

OVULATION: THE ROLE OF GOOD ROS

ROS production is inevitable within the biological system as all metabolic organs produce high amount of free radicals. The ovary is no exception and association of ROS in maintaining the follicular fluid environment, folliculogenesis, and steroidogenesis is one of the benefits of endogenous ROS.^[52] Locally produced ROS is essential for follicle rupture and acts as second messengers, to modulate expression of genes involved in oocyte maturation.^[53-55] However, ROS in excess can lead to oxidative stress that can inflict damages to the oocyte and granulosa cells within a follicle. While ROS mediated oxidative damage to primordial follicles is ambiguous, antral follicles have been reported to be highly sensitive to oxidative stress-induced apoptosis of granulosa cells. As the recruited follicles enter the gonadotropin dependent stage, there is a marked increase in metabolic function of granulosa cells, especially the production of steroids through enhanced activity of cytochrome P450 enzymes.^[56] The electron transport associated with the P450 enzymes is a primary site of free-radical generation, suggesting that differentiation and functional activity of granulosa cells within the follicle experiences a surge in ROS production with a concomitant augmentation of prooxidants. The Graafian follicle is also a potential source of ROS due to the presence of large numbers of infiltrative macrophages, neutrophils, and metabolically active granulosa cells. The ROS produced within the pre-ovulatory follicle are essential for induction of ovulation. Interestingly, in dominant follicles an increase in ROS initiates the resumption of meiosis I of oocytes that have been found to be inhibited by antioxidants. The progression of meiosis II, on the other hand, is promoted by antioxidants,^[57] suggesting that there is a delicate balance between ROS and antioxidants in the ovary.^[58] Besides maintaining oocyte function ROS play an important role during luteolysis. Several antioxidants including melatonin are found in follicular fluid^[59-62] which protect oocytes from ROS-induced damage as its generation is an inevitable aspect of ovarian function.

ROLE OF ROS IN FEMALE INFERTILITY

Detailed understanding of the implications of ROS in female infertility is an area of great significance. The presence of oxidative and antioxidant systems in various female reproductive tissues suggests that infertility associated pathological conditions such as endometriosis and hydrosalpinx may be caused, at least in part, by oxidative stress.^[63-67] The potential negative effects of very high ROS concentrations are always a matter of concern, no matter what the benefits are.^[68] Oxidatively modified substances in peritoneal fluid and ectopic endometrial tissue have been found to be higher in case of patients with endometriosis^[69] which further supports the theory that ROS plays a major role in female reproductive pathophysiology.

MELATONIN AS AN ANTIOXIDANT IN THE OVARY

Melatonin reduces tissue oxidative stress by direct free-radical scavenging, in a non-receptor mediated manner as well as by upregulating the expression of genes coding for antioxidants such as SOD, catalase, and glutathione peroxidase. The gradual rise in melatonin concentration in ovarian follicles with a maximum at the prevoulatory stage^[70] is suggestive of the possible role of locally synthesized melatonin in maintenance of the pro-oxidant-antioxidant balance which protects the oocyte from peri-ovulatory increase in ovarian ROS. Accumulation of free radicals in the ovary has also been found to occur during activation of the stress response^[32] and due to exposure to chemicals and xenobiotics. Melatonin has been found to counteract the toxic effects of the herbicide, fenoxaprop-ethyl on the cytoskeletal integrity of oocytes during meiotic maturation^[71] caused due to oxidative stress. The chemical not only disrupted spindle assembly by affecting actin filaments but also increased ROS accumulation in the oocyte that led to apoptosis. Oxidative stress is a key role player in ovarian ageing^[72] and melatonin administration over a period from 10 to 43 weeks of age in mice was found to elevate 77 genes that decreased with age.^[73] Based on the results of pathway analysis, half of these genes were involved in ribosomal function and maintenance of the accuracy of protein synthesis suggesting that melatonin maintains ribosomal function, accuracies of gene translation, and protein synthesis, thereby slowing the processes of aging. Other genes identified by the authors to be regulated by melatonin include those involved in DNA repair and checkpoint functions. Melatonin also suppresses autophagy-related protein by enhancing intracellular pathways and had a stimulatory effect on antioxidative mechanisms. Moreover, the telomere length, which typically decreases during aging, and expressions of the sirtuin longevity genes (SIRT1 and SIRT3) were significantly higher in the melatonin-treated animals compared to the control mice. Collectively, the results suggest that, through various mechanisms, melatonin reduces at least some aging processes in the ovaries and oocytes. Another characteristic of ovarian ageing is the loss of integrity of the surface epithelium and a gradual decrease in the number of ovarian surface epithelial cells Melatonin has antagonistic effects against the oncogene-induced senescence of human ovarian surface epithelium cells by inhibiting the ROS-YTHDF2-MAPK-NF-kB pathway.^[74] A deeper understanding of the various pathways regulated by melatonin may provide key insights into the potential avenues for preventing and treating ovarian aging.

MELATONIN AND OOCYTE QUALITY

Apart from the known causes, there are many idiopathic cases of infertility wherein the exact cause cannot be determined and free-radical mediated injury might be an aspect that is often overlooked. The oocyte and the surrounding granulosa cells and feeder cells need significant care and protection from such damages. Melatonin, from the circulation or produced locally within the ovary and melatonin receptors in both the theca and granulosa cells^[72] perhaps preserves the oocyte from free-radical damages. Melatonin has been reported to improve the survival rate of mouse and goat preantral follicles cultured in vitro. In caprine oocytegranulosa cell complexes, melatonin upregulated expression of 75 genes, 12 of which have been identified to be potential regulators of metabolic pathways indicating that melatonin improves developmental competence of porcine oocytegranulosa cell complexes.^[73]

Oral administration of melatonin in mice reinstated oocyte meiotic maturation and fertilization ability of oocytes exposed to BPA that compromises the first polar body extrusion by disrupting normal spindle assembly, chromosome alignment, and kinetochore microtubule attachment and impairs oocyte quality. In the same study, BPA was also found to decrease the fertilization rate of oocytes by affecting interaction of the sperm with the zona pellucida proteins such as ovastacin and Juno, the sperm receptor on the egg membrane. Melatonin restored the above defects of fertilization proteins and events, improving oocyte quality through reduction of ROS levels and inhibition of apoptosis.^[74] In another study melatonin protected mouse oocytes from DNA damage induced by double strand breaks during prophase arrest^[75] and subsequently improved oocyte quality. It is suggested that DNA damage during meiotic prophase arrest prevented further maturation and deteriorated oocyte quality due to increase in chromosome fragmentation, spindle abnormality, mitochondrial aggregation, and oxidative stress. Melatonin treatment at this stage inhibited the accumulation of DNA damage by reducing the levels of phosphorylated histone, γλ H2AX. The authors have predicted that melatonin enhanced DNA repair through non-homologous end-joining pathway instead of directly downregulating kinases.

The oocyte is one of the sources of follicular melatonin biosynthesis. One study revealed that oocyte mitochondria are the major sites for melatonin production, contributing to the bulk of melatonin that is locally available during their maturation and promotes oocyte development.^[75] The study further explored that exogenous melatonin addition to oocyte cultures improved mitochondrial function by increasing copy of mitochondrial DNA, mitochondrial membrane potential, distribution, and ATP production. Further, melatonin enhanced the meiotic spindle assembly and inhibited 8-oxo-deoxyguanosine formation, thereby preventing potential DNA mutation from oxidative damage resulting in an improvement in oocyte quality. Such findings not only widen the scope of exogenous melatonin use especially in assisted reproductive techniques but also open a new horizon to explore the applications of endogenous or locally produced melatonin, especially with regard to female reproduction.

MELATONIN: AN ASSISTANT IN FEMALE REPRODUCTION

Oral supplementation of melatonin during ovarian stimulation of IVF cycle has been shown to reduce intrafollicular oxidative stress and increase fertilization rates and embryo quality.^[76] Women with sleep disturbances undergoing IVF when treated with 3 mg of oral melatonin at night till administration human chorionic gonadotropin (hCG) had increased number of metaphase II (MII) oocytes and percentage of Grade I embryos.^[77] Melatonin supplementation from the time of hCG trigger throughout the luteal phase increased progesterone levels in patients undergoing IVF-ET.^[64] Melatonin has also been observed to increase serum progesterone levels in patients with luteal phase defect.^[78]

Studies have demonstrated that low concentrations of melatonin in IVF culture media improved nuclear maturation rate of immature MI oocytes,^[79] implantation rate and an insignificant increase in clinical pregnancy rate in humans.^[80]

Daily melatonin intake at a dose of 3 mg/day along with Vitamin E (600 mg/day) has been found effective in yielding proper and good quality oocytes for IVF.^[64] The optimal melatonin concentration for human oocyte maturation in vitro was found to be 10⁻¹¹ M.^[81] The MII stage oocytes obtained from such treatments had a significantly higher fertilization rate and blastocyst formation rate compared to controls with greatly enhanced clathrin mediated endocytosis (CME) and decreased intra oocyte cAMP levels.^[82] Dynasore, an inhibitor of endocytosis increased intra oocyte cAMP level and blocked oocyte maturation, and melatonin were found to partially rescue oocyte maturation and significantly elevate the expression of clathrin and AP2 in the presence of dynasore suggesting that melatonin could promote human oocyte maturation and early embryo development through enhancing CME. Therefore, melatonin might be the future choice of medicine for IVF especially for women who undergo repeated failures due to poor oocyte quality.

CONCLUSION

The synergism of melatonin and the various factors governing female reproductive physiology has widened the scope of therapeutic approach to sustain the reproductive axis. The beneficial properties of melatonin are being used for the yield of good quality oocytes both in vitro and in vitro. The intervention of melatonin as an antioxidant to prevent ROS production during gamete/embryo handling and preservation, optimization of gamete quality, and viability has strengthened the ground for the use of melatonin in maintenance of normal outcome of assisted reproduction, beginning with quality of gametes, implantation, and maintenance of pregnancy, finally fetal development and parturition. Along with its oncostatic properties^[11] melatonin is also significantly helpful in counteracting reproductive carcinogenesis.

It was the year 1958, when melatonin was revealed with a plethora of biological functions and after 6 decades of its discovery, it is still emerging as a promising molecule that can benefit mankind in many ways.