Melatonin Alleviates Hypoxia-Induced Apoptosis of Granulosa Cells by Reducing ROS and Activating MTNR1B–PKA–Caspase8/9 Pathway

In mammalian ovaries, the avascular environment within follicular cavity is supposed to cause hypoxic status in granulosa cells (GCs), leading to apoptotic cell death accompanied by cumulative reactive oxygen species (ROS) production. Melatonin (N-acetyl-5-methoxytryptamine, MT), a broad-spectrum antioxidant that exists in porcine follicle fluid, was suggested to maintain GCs survival under stress conditions. In this study, using the established hypoxic model (1% O2) of cultured porcine GCs, we explored the effect of MT on GCs apoptosis. The results showed that MT restored cell viability and reduced the apoptosis of GCs during hypoxia exposure. In addition, GCs treated with MT exhibited decreased ROS levels and increased expression of antioxidant enzymes including heme oxygenase-1 (HO-1), glutathione S-transferase (GST), superoxide dismutase 1 (SOD1), and catalase (CAT) upon hypoxia incubation. Moreover, the hypoxia-induced expression of cleaved caspase 3, 8, and 9 was significantly inhibited after MT treatment. In contrast, blocking melatonin receptor 2 (MTNR1B) with a competitive antagonist 4-phenyl-2-propionamidotetralin (4P-PDOT) diminished the inhibitory effects of MT on caspase 3 activation. By detecting levels of protein kinase (PKA), a downstream kinase of MTNR1B, we further confirmed the involvement of MT–MTNR1B signaling in mediating GCs protection during hypoxia stress. Together, the present data provide mechanistic evidence suggesting the role of MT in defending GCs from hypoxia-induced apoptosis.


Introduction
In mammalian, more than 99% of the ovarian follicles undergo atresia during development [1,2]. Actually, apoptosis of ovarian granulosa cells (GCs) occurs much earlier than the morphological changes of follicular atresia, which could be observed only when GCs apoptosis reaches a certain level. Therefore, GCs apoptosis is considered to be the initiating factor leading to follicular atresia [3,4]. In atretic follicles, the GCs showed typical features of apoptotic death, such as nuclear pyknosis and DNA fragmentation [5]. The identification of internucleosomal DNA fragmentation during atresia in porcine ovaries suggests that apoptosis is a basic mechanism associated with ovarian follicular atresia in mammalian as well as avian species [5]. Hereafter, the phenomenon of GCs apoptosis has also been detected in atretic follicles of mice, pigs, cattle, sheep, and other animals [6][7][8][9].

Apoptosis by Flow Cytometry Analysis
The apoptosis rate of porcine GCs was measured using an Apoptosis Detection Kit (Vazyme Biotech) according to the manufacturer's protocol. In total, 2 × 10 4 cells were sorted by FACS (fluorescence activated Cell Sorting) using a cell counting machine (Becton Dickinson, Franklin, NJ, USA). The apoptosis rate was calculated using the following equation: (number of cells in the right upper quadrant + number of cells in the right lower quadrant)/(total number of cells).

Cell Viability Assay
The cell viability of GCs was measured using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, CK04), in which the tetrazolium salt (WST-8) is reduced by dehydrogenase activities in viable cells to generate a yellow water-soluble formazan dye. Therefore, the intensity of color is directly proportional to the number of living cells in culture. The experimental procedures were carried out following the manufacturer's directions. Briefly, GCs were seeded in 96-well plates and grown to 90% confluency for 3 days. After the indi-cated treatments, CCK-8 assay reagent (10 µL) was added to each well containing 100 µL medium and incubated in the dark for 2 h at 37 • C. The formation of formazan was assessed by determining the optical density (OD) at 450 nm under a microplate spectrophotometer (Thermo Fisher Scientific, Camarillo, CA, USA).

Detection of ROS by Immunofluorescence-Confocal Microscopy and Flow Cytometry Analysis
ROS (reactive oxygen species) levels were determined using the Fluorometric Intracellular ROS Kit (MAK145) according to the manufacturer's instructions. The Fluorometric Intracellular ROS Assay Kit provides a sensitive, one-step fluorometric assay to detect intracellular ROS (especially superoxide and hydroxyl radicals) in live cells within 1 h of incubation. ROS react with a cell-permeable sensor, resulting in a fluorometric product (lex = 520/lem = 605 nm) proportional to the amount of ROS present. Ready-to-use DAPI (4 ,6-Diamidino-2-phenylindole dihydrochloride, KGA215-50) were purchased from KeyGEN BioTECH (Jiangsu, China). The cells were imaged with a laser-scanning confocal microscope (Carl Zeiss, Zeiss LSM 710 META, Oberkochen, Germany). The results were calculated as fluorescence intensity in each GC by using the ImageJ 1.42q software (National Institutes of Health, Bethesda, MD, USA).
ROS levels were also detected by Flow Cytometry Analysis (FACS Calibur, American BD) according to the manufacturer's instructions. Briefly, add 100 µL/well (96-well plate) of Master Reaction Mix into the cell plate. Incubate the cells in a 5% CO 2 , 37 • C incubator for one hour. Treat cells with 20 µL/well of test compounds in suitable buffer. Measure the fluorescence intensity using a BD Accuri C6 flow cytometer. The data were analyzed using the FlowJo v7.6 software (Stanford University, Stanford, CA, USA).

Real-Time Quantitative RT-PCR Analysis
Total RNA was isolated with TRIzol reagent (Invitrogen, USA) and was immediately reverse-transcribed using Prime Script™ RT reagent Kit with gDNA Eraser (TaKaRa Bio Inc., RR047). The abundance of HO-1, TRX1, GST, SOD1, SOD2, CAT, and GPX4 mRNA molecules was measured by qRT-PCR. GAPDH and β-actin was used as housekeeping genes. The primers are shown in Table 1. Relative mRNA expression was calculated by the 2 −∆∆CT method.

PKA Detection
We used the PKA ELISA Kit (Shanghai, China) to detect the PKA content according to the manufacturer's instructions. Briefly, we added the sample to the pre-coated enzymelabeled wells; then we added the biotin-labeled recognition antigen and incubated at 37 • C for 30 min. Then, we washed the same with PBST (phosphate buffered saline with 0.05% Tween 20) to remove unbound biotin antigen; then, we added avidin-HRP and incubated at 37 • C for 30 min. After washing, the absorption peak at 450 nm was assessed under a microplate spectrophotometer (Thermo Fisher Scientific, Camarillo, CA, USA).
PKA activity assay. PKA activity was examined using the PKA Kinase Assay Kits, Type I (Immunechem) according to the manufacturer's protocol. In brief, porcine ovarian GCs were collected in 150 µL PBS and were lysed by repeatedly freezing and thawing. Then, we transferred the supernatant to an EP (Eppendorf) tube after centrifugation. The supernatant (30 µL/well) was added into the substrate plate containing kinase assay dilution buffer. Adenosine triphosphate (ATP) (10 µL/well) was added to initiate the kinase reaction at 30 • C for 90 min. After the reaction solution was removed, anti-p-substrate antibodies (40 µL/well) were incubated for 60 min at room temperature. Goat anti-rabbit IgG HRP was used as secondary antibody. TMB (3, 3 ,5, 5 -Tetramethylbenzidine) solution was used to develop the color, indicating reaction activity. OD450 was detected to calculate the relative kinase activity. The enzyme activity was normalized to the protein concentration, which was measured by using the BCA method.

Statistics Analysis
All experiments were repeated at least three times. Data were expressed as mean ± S.E.M. Statistical analyses were done using the univariate analysis of variance (ANOVA) followed by the Student t-test with SPSS 21.0 statistical software. p < 0.05 was considered significant, and p < 0.01 was considered highly significant.

Melatonin Reduced Apoptosis and Rescued Cell Viability of Porcine Granulosa Cells (GCs) in the Setting of Hypoxia
Using an established in vitro hypoxic model [30,31], we verified the hypoxia state by detecting the protein level of hypoxia-inducible factor 1α (HIF-1α), which showed a marked upregulation in GCs cultured with 1% O 2 ( Figure 1A). Concomitantly, the cell viability was significantly decreased after hypoxia incubation, along with an increased level of cleaved caspase-3 ( Figure 1B,C). To detect if melatonin could counteract the detrimental effect of hypoxia, porcine GCs were treated in normoxia and hypoxia (1% O 2 ) supplemented with different MT concentrations (0, 10 −9 , 10 −7 , 10 −5 , 10 −3 M). As shown in Figure 1B,C, MT concentration at 10 −9 , 10 −7 , and 10 −5 M significantly restored GCs viability and reduced the level of cleaved caspase-3 induced by hypoxia. Through flow cytometry analysis, we further affirmed that MT at 10 −7 M attenuated the apoptosis of porcine GCs triggered by hypoxia ( Figure 1D). level of cleaved caspase-3 ( Figure 1B,C). To detect if melatonin could counteract the detrimental effect of hypoxia, porcine GCs were treated in normoxia and hypoxia (1% O2) supplemented with different MT concentrations (0, 10 −9 , 10 −7 , 10 −5 , 10 −3 M). As shown in Figure 1B and C, MT concentration at 10 −9 , 10 −7 , and 10 −5 M significantly restored GCs viability and reduced the level of cleaved caspase-3 induced by hypoxia. Through flow cytometry analysis, we further affirmed that MT at 10 −7 M attenuated the apoptosis of porcine GCs triggered by hypoxia ( Figure 1D).  (D) Flow cytometry analysis of the apoptosis of porcine GCs. * p < 0.05 was considered significant; ** p < 0.01 was considered highly significant; *** p < 0.001 was considered very significant; **** p < 0.0001 was considered extremely significant; ## p < 0.01 was considered highly significant; & p < 0.05 was considered significant; ns p > 0.05 was considered no significant.

Melatonin Lowered ROS Level Caused by Hypoxia in Porcine GCs
Since hypoxia environment has been implicated in triggering ROS production, we next investigated whether MT functioned by decreasing ROS content in hypoxic GCs. As shown in Figure 2A, we found that hypoxia caused an increase in ROS level, which was reduced after MT treatment. Using flow cytometry, we further confirmed the role of MT in suppressing ROS levels during hypoxia ( Figure 2B).

Melatonin Lowered ROS Level Caused by Hypoxia in Porcine GCs
Since hypoxia environment has been implicated in triggering ROS production, we next investigated whether MT functioned by decreasing ROS content in hypoxic GCs. As shown in Figure 2A, we found that hypoxia caused an increase in ROS level, which was reduced after MT treatment. Using flow cytometry, we further confirmed the role of MT in suppressing ROS levels during hypoxia ( Figure 2B). MT. ** p < 0.01 was considered highly significant; **** p < 0.0001 was considered extremely significant. The flow cytometry analysis of ROS levels of porcine GCs treated in normoxia, hypoxia, and hypoxia plus 10 −7 M MT. ** p < 0.01 was considered highly significant; **** p < 0.0001 was considered extremely significant.

Melatonin Suppressed Hypoxia-Induced Apoptosis of Porcine GCs by Eliminating ROS and Increasing the Expression of Antioxidant Enzymes
MT could directly remove ROS or indirectly serve a function by increasing the expression of antioxidant enzymes and GSH [32,33]. GSH, the tripeptide, usually considered, has the most important functions for the removal of peroxides. Thus, we use GSH as the positive control of scavenging ROS. As shown in Figure 3A,B, both MT and GSH Antioxidants 2021, 10, 184 8 of 16 lowered ROS levels induced by hypoxia. MT treatment also revealed less inhibitory effect on caspase-3 activation compared with GSH ( Figure 3C), suggesting that MT-suppressed GCs apoptosis might be partially achieved via directly removing ROS. By determining the mRNA levels of antioxidant enzymes, we found that MT rescued the expression of heme oxygenase-1 (HO-1), glutathione S-transferase (GST), superoxide dismutase 1 (SOD1), and catalase (CAT) under hypoxia condition ( Figure 3D). MT could directly remove ROS or indirectly serve a function by increasing the expression of antioxidant enzymes and GSH [32,33]. GSH, the tripeptide, usually considered, has the most important functions for the removal of peroxides. Thus, we use GSH as the positive control of scavenging ROS. As shown in Figure 3A,B, both MT and GSH lowered ROS levels induced by hypoxia. MT treatment also revealed less inhibitory effect on caspase-3 activation compared with GSH ( Figure 3C), suggesting that MT-suppressed GCs apoptosis might be partially achieved via directly removing ROS. By determining the mRNA levels of antioxidant enzymes, we found that MT rescued the expression of heme oxygenase-1 (HO-1), glutathione S-transferase (GST), superoxide dismutase 1 (SOD1), and catalase (CAT) under hypoxia condition ( Figure 3D). . * p < 0.05 was considered significant; ** p < 0.01 was considered highly significant; *** p < 0.001 was considered very significant; **** p < 0.0001 was considered extremely significant; ns p > 0.05 was considered no significant.

Melatonin Alleviated Hypoxia-Induced Apoptosis by Deactivating Caspase-8 and Caspase-9
Apoptosis is a strictly regulated process activated by extrinsic or intrinsic pathways. Each pathway activates its own initiator caspase (8,9), which in turn will activate the executioner caspase-3 [34]. As shown in Figure 4A,B, hypoxia induced the expression of both caspase-8 and caspase-9, which was reversed following MT administration. To further assess whether caspase-8 and caspase-9 affected the pro-survival actions of MT under hypoxia conditions, GCs were treated with caspase-8 and caspase-9 inhibitor . * p < 0.05 was considered significant; ** p < 0.01 was considered highly significant; *** p < 0.001 was considered very significant; **** p < 0.0001 was considered extremely significant; ns p > 0.05 was considered no significant.

Melatonin Alleviated Hypoxia-Induced Apoptosis by Deactivating Caspase-8 and Caspase-9
Apoptosis is a strictly regulated process activated by extrinsic or intrinsic pathways. Each pathway activates its own initiator caspase (8,9), which in turn will activate the executioner caspase-3 [34]. As shown in Figure 4A Figure 4D-G, both LEHD and IETD decreased the level of cleaved caspase-3, and they displayed similar preventive effects with MT on hypoxiainduced apoptosis of porcine GCs. Using flow cytometry analysis, we obtained consistent results that MT, LEHD, and IETD decreased apoptosis rates in hypoxic GCs ( Figure 4H,I). We also examined the receptor and ligand of extrinsic apoptotic pathways using qRT-PCR. The results showed that MT decreased the expression of FASL and TRAIL induced by hypoxia ( Figure 4C).  Figure 4D-G, both LEHD and IETD decreased the level of cleaved caspase-3, and they displayed similar preventive effects with MT on hypoxia-induced apoptosis of porcine GCs. Using flow cytometry analysis, we obtained consistent results that MT, LEHD, and IETD decreased apoptosis rates in hypoxic GCs ( Figure 4H,I). We also examined the receptor and ligand of extrinsic apoptotic pathways using qRT-PCR. The results showed that MT decreased the expression of FASL and TRAIL induced by hypoxia ( Figure 4C).  (I) Statistics of the apoptosis of porcine GCs. * p < 0.05 was considered significant; ** p < 0.01 was considered highly significant; *** p < 0.001 was considered very significant; **** p < 0.0001 was considered extremely significant; ns p > 0.05 was considered no significant.

Melatonin Inhibited Hypoxia-Induced Apoptosis via MTNR1B-PKA Pathway
The physiological effects of MT are mediated through binding with its membrane receptors (MTNR1A, MTNR1B), nuclear binding sites (ROR), or non-receptor pathways. In current research, using MT membrane receptor antagonists (Luzindole and 4P-PDOT) and a selective RORα and RORγ inhibitor (SR1001), we further assessed the protective effects of MT on GCs under hypoxia. As shown in Figure 5A-C, 4P-PDOT, a competitive antagonist of MTNR1B, completely negated the action of MT on inhibiting caspase-3 cleavage during hypoxia. Since PKA is a downstream effector of MTNR1B, we then detected PKA activation after MT treatment. The result showed that the concentration and activity of PKA in hypoxic GCs was further increased after MT treatment ( Figure 5D,E). (I) Statistics of the apoptosis of porcine GCs. * p < 0.05 was considered significant; ** p < 0.01 was considered highly significant; *** p < 0.001 was considered very significant; **** p < 0.0001 was considered extremely significant; ns p > 0.05 was considered no significant.

Melatonin Inhibited Hypoxia-Induced Apoptosis via MTNR1B-PKA Pathway
The physiological effects of MT are mediated through binding with its membrane receptors (MTNR1A, MTNR1B), nuclear binding sites (ROR), or non-receptor pathways. In current research, using MT membrane receptor antagonists (Luzindole and 4P-PDOT) and a selective RORα and RORγ inhibitor (SR1001), we further assessed the protective effects of MT on GCs under hypoxia. As shown in Figure 5A-C, 4P-PDOT, a competitive antagonist of MTNR1B, completely negated the action of MT on inhibiting caspase-3 cleavage during hypoxia. Since PKA is a downstream effector of MTNR1B, we then detected PKA activation after MT treatment. The result showed that the concentration and activity of PKA in hypoxic GCs was further increased after MT treatment ( Figure 5D,E). and activity (E) in normoxia, hypoxia, and hypoxia plus 10 −7 M MT. * p < 0.05 was considered significant; ** p < 0.01 was considered highly significant; *** p < 0.001 was considered very significant; **** p < 0.0001 was considered extremely significant; ns p > 0.05 was considered no significant.

Discussion
The avascular environment within ovarian follicles limits oxygen supply for GCs, and the hypoxia status of the GCs are gradually intensified as the follicle develops, during which a large amount of growing follicles undergo degeneration via atresia. In fact, our previous studies have demonstrated that under severe hypoxia stimulation, increased apoptosis occurred in ovarian GCs [30,31], indicating that hypoxia may be an important factor in initiating follicular atresia. Interestingly, some of the follicles retain the potential to develop toward ovulation. However, it remains to be uncovered whether certain intra-ovarian factors preserved the viability of GCs during hypoxia stress. In this . * p < 0.05 was considered significant; ** p < 0.01 was considered highly significant; *** p < 0.001 was considered very significant; **** p < 0.0001 was considered extremely significant; ns p > 0.05 was considered no significant.

Discussion
The avascular environment within ovarian follicles limits oxygen supply for GCs, and the hypoxia status of the GCs are gradually intensified as the follicle develops, during which a large amount of growing follicles undergo degeneration via atresia. In fact, our previous studies have demonstrated that under severe hypoxia stimulation, increased apoptosis occurred in ovarian GCs [30,31], indicating that hypoxia may be an important factor in initiating follicular atresia. Interestingly, some of the follicles retain the potential to develop toward ovulation. However, it remains to be uncovered whether certain intraovarian factors preserved the viability of GCs during hypoxia stress. In this study, we found that (1) MT inhibited hypoxia-induced apoptosis of GCs. (2) MT mitigated oxidative stress, increased the expression of antioxidant enzymes, and reduced apoptosis in hypoxic GCs.
(3) MT acts through receptor MTNR1B to block the activation of caspase-8/9-caspase-3 axis, thereby alleviating the apoptosis of GCs. To our knowledge, these findings provide evidence suggesting a role of MT in maintaining GCs survival and follicular development under hypoxia.
MT is a ubiquitous hormone primarily produced by the pineal gland in the circulating system and locally synthesized in several cells and tissues [35,36]. In the ovary, granulosa cells, cumulus cells, and oocytes have been reported to synthesize melatonin [37][38][39], which might function in concert with circulating melatonin to maintain follicular health. He et al. revealed that MT could prevent the apoptosis of porcine GCs during follicular atresia. On the other hand, the atretic follicles exhibit decreased level of MT and increased GCs apoptosis [24]. During maternal aging, the loss of MT in follicular fluid is associated with oxidative stress, compromised oocyte quality, and impaired developmental capacity of ovarian follicles [40][41][42]. These studies suggest a close relationship among stress, MT, and survival of follicular cells. However, it remains unclear whether MT-mediated protection of GCs is disturbed under stress conditions. Many mammalian species from temperate zones display a seasonal pattern of reproduction controlled by the annual photoperiodic cycle [43]. Depending on the time of year when they are sexually active, species are characterized as short-day breeders, for instance, sheep, or long-day breeders, for instance, hamsters or horses [43]. MT, a pineal hormone that conveys photoperiodic information to the body physiology, has been shown to influence seasonal rhythms in reproductive function [44,45]. In winter, an extended duration of MT synthesis corresponding to prolonged nights stimulates the reproductive axis of short-day breeders and inhibit long-day breeders, illustrating the role that MT plays in a seasonal reproductive state [46,47].
Numerous evidences indicate that MT might improve cellular adaption to hypoxic conditions. Luo et al. reported that MT conferred cardioprotection by inhibiting apoptosis through the activation of the PI3K/Akt signaling pathway in hypoxic cardiomyocytes [48]. In the cytoskeleton, MT has been described to inhibit ROS production induced by hypoxia/reoxygenation (H/R) [49]. Ovarian follicles are a niche enriched with MT derived from the circulation system and follicular cells [38,50]. As such, MT augments the maturation efficiency of oocytes, and it protects the integrity of mouse GCs in pre-ovulatory follicles by reducing oxidative stress [51,52]. Therefore, it prompted us to inquire whether MT contributes to GCs survival in response to hypoxic conditions. Here, we provide the first evidence demonstrating the role of MT in inhibiting hypoxia-induced GCs apoptosis, suggesting that the level of MT in follicular fluid might determine the developmental fate of ovarian follicles.
Moreover, we found that MT protected GCs survival in a dose-dependent manner. MT in concentration at 10 −7 , 10 −5 M improved GCs viability significantly, while 10 −3 M suppressed the viability of GC ( Figure 1B), indicating that MT at high concentrations has a detrimental effect on GCs under normoxia. However, when GCs were cultured under hypoxia, cell viability was significantly increased by MT at concentrations that ranged from 10 −9 to 10 −3 M ( Figure 1B). Why did a high concentration (10 −3 M) of MT exhibit different effects in response to normoxia or hypoxia conditions? One possibility is that hypoxia induced the production of ROS, which reacted with MT; then, it consumed part of MT, hence reducing the cytotoxicity caused by excessive MT.
There are many methods for the detection of cell death. In this study, CCK-8 assay was employed to determine cell viability, Annexin V/PI (propidium iodide) staining was used to measure the cell apoptosis rate, and the cleavage activity of caspase-3 was quantified to assess the intensity of apoptosis. However, our data showed a discrepancy of death rate assessed by viability vs. AnnexinV/PI measurement and AnnexinV/PI staining vs. caspase-3 cleavage. Actually, CCK-8 assay is based on the colorimetric detection of dehydrogenases activity, which is directly proportional to the number of living cells. In another word, CCK-8 could reflect the death rate of cells. Indeed, several forms of cell death have been reported, including autophagic death, apoptosis, necrosis, anoikis, pyroptosis, etc. In addition to apoptosis, it is possible that other forms of cells death occur in GCs suffering hypoxia exposure. Hence, the results of CCK-8 cannot be simply equated with the rate of apoptosis. Although the level of cleaved caspase-3 is a well-described biomarker of apoptosis, the immunoblot technique used for quantitative assessment has limitations [53,54]. Therefore, the quantitative data of cleaved caspase-3 might not be proportional to apoptosis rates. Nevertheless, our results showed similar trend of alteration in apoptosis rate and the caspase-3 cleavage level in GCs with different set of treatments.
Hypoxia has been reported to induce the formation of ROS [55]. Studies revealed that mitochondrion, an organelle required for oxidative phosphorylation, produces a higher level of superoxide through the electron transport chain during hypoxia. In addition, NAD(P)H oxidases might be another generator of ROS under hypoxia conditions [56]. Actually, hypoxia is known to increase oxidative stress, which in turn activates the apoptotic mechanisms [57]. For instance, following cerebral hypoxia, the oxidative damage of mitochondria leads to the release of apoptotic proteins, such as cytochrome c and apoptosis inducible factor (AIF) into the cytosol [34], where cytochrome c binds with AIF and procaspase-9 to form an "apoptosome", which actives caspase-9 and subsequently caspase-3 cleavage for the execution of the apoptotic program [58]. MT is a well-described antioxidant that exerts its functions by directly scavenging ROS production or indirectly activating the endogenous antioxidant system. As expected, the present study showed that MT treatment could increase the expression of antioxidant enzymes in GCs, decrease the level of ROS, and inhibit hypoxia-induced apoptosis. Our results suggested that the inhibitory effect of MT on GCs apoptosis might partly rely on its antioxidant activity.
Apart from the antioxidative effects, MT has a bundle of means whereby it thwarts intracellular damage through activating effectors downstream of MT receptors [59]. Previous studies have found that melatonin receptors mainly consist of membrane receptors (MTNR1A, MTNR1B) and nuclear receptors (RZR/ROR, retinoid Z receptor/retinoid acid receptor related orphan receptor) [60]. In ovarian follicles, it has been demonstrated that MT mediated the proliferation, apoptosis, and steroidogenesis of GCs predominantly through the activation of MTNR1B [61,62]. In case of hypoxia, MTNR1B played a protective role in preventing primary cardiomyocytes against hypoxia/reoxygenation injury caused by myocardial ischemia/reperfusion [63]. Here, we described for the first time that in ovarian GCs, MT acts through MTNR1B to inhibit hypoxia-induced apoptosis via silencing the caspase 8/9-caspase 3 axis.
Canonical apoptosis pathways mainly include the mitochondrial apoptosis pathway, death receptor apoptosis pathway, and endoplasmic reticulum apoptosis pathway. Our previous data showed that hypoxia acted through the mitochondrial pathway to induce apoptosis in GCs [30,31]. Consistently, the present study revealed that the mitochondrial pathway, as evidenced by activation of the caspase-9-caspase-3 axis, is required for hypoxiainduced apoptosis in GCs. In agreement with our data, Wang et al. found that melatonin may inhibit GCs apoptosis by degrading Bim EL , which is a key component of the mitochondrial apoptosis pathway [64]. In addition, we found that the caspase-8-caspase-3 cascade is concomitantly activated during hypoxia incubation, suggesting that the death receptor apoptosis pathway might also be involved in triggering GCs apoptosis. In addition, the current results showed that MT inhibited the activation of both caspase-8 and caspase-9 upon hypoxia stimulation. Moreover, when GCs were treated with MT, antagonists against caspase-8 or caspase-9 could not further inhibit caspase-3 cleavage, indicating that MT can inhibit caspase-3 activation by inhibiting the mitochondrial apoptosis pathway and death receptor apoptosis pathway.

Conclusions
In conclusion, the current research uncovered a novel role of MT in repressing hypoxiainduced GCs apoptosis through scavenging cellular ROS, stimulating antioxidant enzymes expression, or inhibiting the caspase 8/9-caspase 3 axis via the MTNR1B-PKA pathway. These findings not only extended our understanding regarding the mechanism of follicular development under hypoxia but also provided potential avenues for improving animal reproductive performance by supplementing MT (Figure 6).
In conclusion, the current research uncovered a novel role of MT in repressing hypoxia-induced GCs apoptosis through scavenging cellular ROS, stimulating antioxidant enzymes expression, or inhibiting the caspase 8/9-caspase 3 axis via the MTNR1B-PKA pathway. These findings not only extended our understanding regarding the mechanism of follicular development under hypoxia but also provided potential avenues for improving animal reproductive performance by supplementing MT (Figure 6). Figure 6. Schematic illustration of the signaling pathway involved in MT-mediated GCs resistance to hypoxia-induced apoptosis. Hypoxia facilitates ROS accumulation and the cleavage of the initiator caspases (8,9), which in turn activates the executioner caspase 3, thereby inducing GCs apoptosis. In the presence of MT, the apoptosis of GCs is inhibited, possibly through the clearance of cellular ROS, activation of antioxidant enzymes expression, or suppression of Caspase 8/9-Caspase 3 axis via the MT-MTNR1B-PKA pathway.    (8,9), which in turn activates the executioner caspase 3, thereby inducing GCs apoptosis. In the presence of MT, the apoptosis of GCs is inhibited, possibly through the clearance of cellular ROS, activation of antioxidant enzymes expression, or suppression of Caspase 8/9-Caspase 3 axis via the MT-MTNR1B-PKA pathway.

Conflicts of Interest:
The authors declare no conflict of interest.