Expression of the melatonergic system during meiotic maturation of cynomolgus monkey cumulus–oocyte complexes

Melatonin is a multifunctional hormone synthesized in the pineal gland and peripheral reproductive tissues that regulates many biological processes. There is increasing evidence for a role of melatonin in oocyte maturation and embryonic development in various mammals. However, no study has reported evidence for the existence of melatonergic system, such as melatonin synthesis enzymes, melatonin membrane receptors, or melatonin binding sites in non‐human primate cumulus–oocyte complexes (COCs).

the expression of melatonin receptors in rat ovaries, 7 sheep cumulus cells and oocytes, 8 as well as higher melatonin levels in human ovarian follicular fluid than in blood. 9 In mammals, melatonin is also synthesized in extrapineal tissues and cells, including the retina, gut mucosa, liver, kidney, thymus, placenta, testis, ovary, and oocytes. 10,11 Melatonin synthesis begins with the N-acetylation of serotonin by arylalkylamine Nacetyltransferase (AANAT) to form N-acetylserotonin, which is subsequently methylated to melatonin by N-acetylserotonin Omethyltransferase. 12 AANAT, the penultimate enzyme, is ratelimiting in melatonin synthesis. 13 This endogenously produced melatonin can interact with several target proteins, including the G protein coupled membrane receptors MT1 14 and MT2. 15 Another target is a melatonin binding site that was first named ML2, then renamed MT3 in 1999. 16 Purification efforts indicated that MT3 is the cytosolic enzyme quinone reductase 2 (NQO2). 17 Finally, melatonin also binds to nuclear retinoic acid receptor-related orphan receptors alpha (RORα) and beta (RORβ), although these nuclear binding proteins have been a matter of debate. 18 Recent studies revealed the presence of transcripts and proteins of melatonergic system, such as AANAT, MT1, and MT2 in sheep 19 and mouse cumulus-oocyte complexes (COCs). 20 The existence of melatonergic system in COCs suggest a local role of melatonin in female reproductive processes, such as oocyte maturation. However, limited information was available on the existence of melatonergic system in human or non-human primate COCs. Because of the scarcity of oocyte samples available from women or monkeys, most studies on oocyte maturation mechanisms have been performed in other species such as mouse. However, individual oocytes from non-human primates now can be obtained through laparotomic or laparoscopic procedures for oocyte collection 21,22 and can be used to study mechanisms controlling oocyte maturation in human and non-human primates. 23 Therefore, this study examined the presence of transcripts and proteins of the rate-limiting enzyme for melatonin synthesis (AANAT), melatonin membrane receptors (MT1 and MT2), and a melatonin binding site (NQO2) in the COCs of the cynomolgus monkey, as a non-human primate model.

| Chemicals
Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich.

| Animals
Twenty-seven female cynomolgus monkeys with regular menstrual cycles were used as oocyte donors. They were caged individually and housed in a controlled environment (temperature 20-24°C, humidity 40%-60%, and 0800-2000 h light cycle), according to a previous study. 24 To detect the onset of menstruation, they were examined for

| RNA isolation and reverse transcription polymerase chain reaction (RT-PCR)
Poly(A) mRNAs were extracted from granulosa cells, GV-and MII-stage cumulus cells, and oocytes using the Dynabeads mRNA DIRECT Kit (Invitrogen), according to the manufacturer's protocol.
Briefly, after thawing, samples were lysed in 100 μL lysis/binding buffer at room temperature for 10 min, and 20 μL Dynabeads oligo(dT) 25 was added to each sample. The beads were hybridized  Table 1.

| Experimental design
In the first experiment to determine the presence of transcripts of the rate-limiting enzyme for melatonin synthesis (AANAT), melatonin membrane receptors (MT1 and MT2), and a melatonin binding site (NQO2) in cynomolgus monkey COCs, cumulus cells and oocytes derived from GV-and MII-stage COCs were sampled and used for RT-PCR analyses. GAPDH was used as an internal control.
In the second experiment to determine the presence of AANAT,

| DISCUSS ION
In the present study, transcripts and proteins of AANAT, MT1, MT2, and NQO2 were identified in the GV-and MII-stage COCs of cynomolgus monkeys using RT-PCR and immunocytochemistry. From these results, it could be speculated that melatonin and its binding proteins may have a local role during oocyte maturation in nonhuman primates.
In growing ovarian follicles, intercellular interactions between the oocyte and follicular somatic cell compartments such as cumulus, granulosa, and theca cells are involved in oocyte growth and meiotic maturation. 25 Among them, cumulus cells, the cells closest to the oocyte, play a crucial role during oocyte development, as they mediate gonadotropin-induced oocyte meiotic resumption, 26 supply messenger molecules 27 and nutrients for oocyte development. 28 Therefore, oocyte maturity is reflected in cumulus cell expansion. 29 Several in vitro studies have suggested that melatonin supplementation during in vitro oocyte maturation significantly enhances cumulus cell expansion and oocyte developmental competence in various species, including cattle, 10 pigs, 30 and mice. 31 Moreover, these stimulatory effects of melatonin on cumulus cell expansion and oocyte maturation are mediated by the membrane receptors MT1 or MT2 in cattle, 32 sheep, 8 and pigs. 33 In addition to these membrane receptors, NQO2 was demonstrated to be the third binding site of melatonin 34 and its presence was reported in bovine 35 and mouse oocytes. 36 However, existence of melatonergic system, such as melatonin synthesis enzymes, melatonin membrane receptors, or melatonin binding sites has never been reported in non-human primate COCs. In this study, our results revealed that two melatonin membrane receptors (MT1 and MT2) and a melatonin binding site (NQO2) are expressed in cynomolgus monkey cumulus cells and oocytes at both GV-and MII-stages (Figures 1 and 2). The existence of melatonin binding proteins during meiotic maturation of cynomolgus monkey COCs infers that melatonin could affect the maturation of cynomolgus monkey COCs via these receptors or a binding site.
Furthermore, our results suggest that AANAT, melatonin synthetic enzyme, is also expressed in cynomolgus monkey cumulus cells and oocytes at GV-and MII-stages (Figures 1 and 2  Although the existence of melatonergic system has not also been identified in human COCs, the beneficial effects of melatonin on human female reproduction have been well studied. Melatonin is used to improve ovarian function and oocyte quality in women experiencing infertility due to poor oocyte quality. 37 In women treated with melatonin, more oocytes per retrieval, mature oocyte, and good quality embryos per retrieved oocytes were obtained. 38 Melatonin treatment also increased the fertilization success of women with low fertilization rates in the prior in vitro fertilization treatment cycle. 39 Furthermore, melatonin supplementation during in vitro maturation of human oocytes promoted oocyte maturation rates, fertilization rates, and subsequent embryonic development after intracytoplasmic sperm injection. 40 These results suggest a functional role of melatonin in human oocyte maturation. However, in non-human primates, limited information was available on both the existence of melatonergic system and the effect of melatonin on oocyte maturation. Therefore, this study was conducted to elucidate the existence of melatonergic system in non-human primate COCs. Since we demonstrated the presence of an enzyme for melatonin synthesis (AANAT), melatonin membrane receptors (MT1 and MT2), and a melatonin binding site (NQO2) in cynomolgus monkey COCs, it is reasonable to assume that melatonin synthesis occurs in the COCs of non-human primates, and melatonin-binding proteins may mediate the effect of melatonin on non-human primate oocyte maturation. However, further study is needed to investigate whether melatonin membrane receptors or a melatonin binding site mediate the effects of melatonin on non-human primate oocyte maturation using melatonin-binding protein antagonists.

| CON CLUS ION
For the first time, we confirmed the existence of a rate-limiting