Introduction
Follicle development accompanied by changes in granulosa cell function and oocyte maturation underlies the physiology of female fertility. Mammalian follicle development exhibits cyclical changes and consists of three main stages: follicle recruitment and priming, selection and dominance, and maturation and ovulation (Edson et al., Reference Edson, Nagaraja and Matzuk2009). During follicle development, the developmental capacity of oocytes is closely related to the function of granulosa cells (Boucret et al., Reference Boucret, Chao de la Barca, Morinière, Desquiret, Ferré-L’Hôtellier, Descamps, Marcaillou, Reynier, Procaccio and May-Panloup2015; Shen et al., Reference Shen, Liu, Li and Zhang2021). In the initial stage of follicle development, a group of primordial follicles in the ovary is recruited and starts to grow at the same time as the degeneration of the corpus luteum. With the help of a series of cytokines and gonadotropins, the granulosa cells proliferate rapidly; this promotes follicle growth and, eventually, the follicular cavity appears. As the follicle develops, granulosa cells produce large amounts of oestrogen and transport ATP and small molecule metabolites to the oocyte for its development and maturation (Monniaux, Reference Monniaux2016). Therefore, the follicle provides an ideal microenvironment for oocyte development and maturation, such as the hypoxic environment within the follicular cavity, which is necessary for egg maturation and ovulation (Redding et al., Reference Redding, Bronlund and Hart2008; Tam et al., Reference Tam, Russell, Peet, Bracken, Rodgers, Thompson and Kind2010; Lim et al., Reference Lim, Thompson and Dunning2021).
About 140 million people live in areas higher than 2500 m, and about 40 million people travel to areas above 2500 m yearly (Moore et al., Reference Moore, Niermeyer and Zamudio1998). The hypoxic stress caused by high altitude has been proven harmful to non-adapted humans (Moore et al., Reference Moore, Zamudio, Zhuang, Sun and Droma2001). Notably, the effect of hypoxia on female fertility has been described by various authors who have indicated that females exposed to hypoxia exposure experienced deficiency of pre-ovulatory follicle development, birth weight reduction, embryo loss, and intrauterine growth restriction (Parraguez et al., Reference Parraguez, Atlagich, Díaz, Bruzzone, Behn and Raggi2005, Reference Parraguez, Atlagich, Díaz, Cepeda, González, De los Reyes, Bruzzone, Behn and Raggi2006, Reference Parraguez, Diaz, Cofré, Urquieta, De Los Reyes, Astiz and Gonzalez-Bulnes2014; Hartinger et al., Reference Hartinger, Tapia, Carrillo, Bejarano and Gonzales2006). Only a few studies have shown that follicular growth is restricted when female follicles are exposed to hypoxic conditions in vitro (Connolly et al., Reference Connolly, Kane, Quinlan, Dockery and Hynes2017; Ma et al., Reference Ma, Wang, Gao, Liu, Zheng, Gao, Liu and Jiang2019). Although hypoxic environmental stress has been shown to be detrimental to female fertility, the mechanism and how to release this effect still needs further investigation. A previous study demonstrated that hypobaric hypoxia would cause oxidative damage, which could be alleviated by antioxidants (Farias et al., Reference Farias, Puebla, Acevedo, Tapia, Gutierrez, Zepeda, Calaf, Juantok and Reyes2010). Numerous studies have shown that the antioxidant activity of melatonin secreted by the pineal gland plays a positive role in regulating the reproductive process of female animals.
Melatonin plays a vital role in regulating circadian rhythms, pubertal development, and seasonal adaptation in animals (Reiter et al., Reference Reiter, Tamura, Tan and Xu2014; Han et al., Reference Han, Wang, Li, Li, Ge, Reiter and Wang2017; Jiang et al., Reference Jiang, Shi, Liu, Zhao and Zhao2021b; Cipolla-Neto et al., Reference Cipolla-Neto, Amaral, Soares, Gallo, Furtado, Cavaco, Gonçalves, Santos and Quintela2022). In recent years, its critical role in animal reproductive activity has been widely demonstrated. Melatonin regulates gonadal hormone levels through the hypothalamic–pituitary–gonadal axis or targets the gonads to regulate germ cell development (Berlinguer et al., Reference Berlinguer, Leoni, Succu, Spezzigu, Madeddu, Satta, Bebbere, Contreras-Solis, Gonzalez-Bulnes and Naitana2009; Cipolla-Neto et al., Reference Cipolla-Neto, Amaral, Soares, Gallo, Furtado, Cavaco, Gonçalves, Santos and Quintela2022). In female reproduction, melatonin could relieve oxidative stress (Zou et al., Reference Zou, Chen, Ding, Gao, Chen, Liu, Hao, Zou, Ji, Zhou, Wei, Cao and Zhang2020), delay ovarian ageing (Tong et al., Reference Tong, Sheng, Sun, Li, Li, Zhang and Chen2017), reduce granulosa cell apoptosis (Jiang et al., Reference Jiang, Shi, Liu, Zhao and Zhao2021b), and promote egg maturation and embryonic development (Liu et al., Reference Liu, Ji, Liu, Wang, Xie, Zhang, Wei, Zhou and Cao2019b). Melatonin can remove or inhibit the production of ROS, reducing the damage of hypoxic stress to animals (Poeggeler et al., Reference Poeggeler, Saarela, Reiter, Tan, Chen, Manchester and Barlow-Walden1994; Amaral and Cipolla-Neto, Reference Amaral and Cipolla-Neto2018; Gonzalez-Candia et al., Reference Gonzalez-Candia, Veliz, Carrasco-Pozo, Castillo, Cárdenas, Ebensperger, Reyes, Llanos and Herrera2019). Melatonin can also improve fetal pulmonary circulation and cardiac function by increasing the activity of antioxidant enzymes and resisting the fetal pulmonary hypertension syndrome caused by perinatal hypoxia in high-altitude animals (Reiter et al., Reference Reiter, Tan, Rosales-Corral, Galano, Zhou and Xu2018; Gonzaléz-Candia et al., Reference Gonzaléz-Candia, Arias, Aguilar, Figueroa, Reyes, Ebensperger, Llanos and Herrera2021). All these suggest that melatonin has a promising application against hypoxic stress.
In this study, we used hypoxic stress mice as an animal model to study the mechanism of hypoxic stress affecting female reproduction. The melatonin-hypoxia group was used to assess the role of melatonin in alleviating hypoxic reproductive stress in females. Alleviating the effect of melanin on the reproductive damage of hypoxic stress mice provides a theoretical reference for further research and improvement of the effect of hypoxic stress on the reproductive activity of female animals.
Materials and methods
Animals
CD-1 mice were purchased from the Charles River Laboratory Animal Centre (Beijing, China) and housed in a controlled environment with food and water ad libitum. Female CD-1 mice, 4–6 weeks of age, were divided randomly into the following treatments:
Control: The control group were maintained in normal conditions.
Hypoxia: The hypoxia group was housed in a hypobaric hypoxic chamber and treated with vehicles for 4 weeks.
Hypoxia+MT: Melatonin with hypoxia group housed under the same hypoxia conditions, and that received melatonin powder (Sigma, M5250) dissolved in corn oil at a one-time oral dose of 30 mg/kg body weight daily for 4 weeks.
All animals used in the experiment were conducted under the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Welfare and Ethics Committee at West Anhui University.
Hypoxia treatment evaluation
Mouse heart tissue was removed immediately after 4 weeks of hypoxic treatment and rinsed with normal saline, and then moisture was adsorbed onto a filter paper. The weights of the right ventricle (RV) and left ventricle plus ventricular septum (LV+S) were recorded to calculate RV/[LV+S] (Chen et al., Reference Chen, Shen, Zhu, Yang, Ye, Liu, Gu and Chen2019).
Ovary histological analysis
The histological analysis of ovaries followed a previous study (Xu et al., Reference Xu, Wei, Yang, Jia, Ma, Yang, Jun and Zhang2020). Briefly, mouse ovarian tissues were fixed in 4% PFA for 2 h and then dehydrated using a series of graded ethanol (30%, 50%, 70%, 95%, and 100%). The tissues were embedded in paraffin (Leica, German) and cut into 5-µm sections (Leica RM2235, German) with serial section. After drying at 42°C, the sections were rehydrated and stained with haematoxylin and eosin (H&E). Images were acquired using a microscope (Nikon ECLIPSE E200, Japan) with a charge-coupled device (CCD; MshOt MS60, China). The classification of follicles was based on the shape of the granulosa cell and the number of layers surrounding the oocyte as described by Hadek (Reference Hadek1965). Finally, each ovary was cut sequentially into 10 sections, and the proportions of preantral, antral, mature follicles, and corpus luteum were counted in each section.
Granulosa cell proliferation assay
For immunohistochemistry, ovary sections were rehydrated and endogenous peroxidase activity was blocked in 3% H2O2 for 10 min at room temperature (RT). Rehydrated tissues were washed three times with phosphate-buffered saline (PBS: NaH2PO4, Na2HPO4, NaCl; pH 7.4). Sections were blocked in 10% goat serum for 1 h and incubated with a primary antibody against proliferating cell nuclear antigen (PCNA; 1:200, Ruiying Technology, RLM3031) in antibody dilution buffer overnight at 4°C. Sections were washed three times with PBS and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:200, Proteintech, SA00013–6). Sections were washed three times with PBS and visualized using 3,3-diaminobenzidine (DAB; ab64238) for 3 min. Sections were counterstained with haematoxylin and observed under a microscope (Nikon, E200, Japan). Image Pro Plus software was used to calculate the percentage of PCNA-positive cells in total granulosa cells in preantral and antral follicles.
TUNEL assay
Mouse ovarian tissue sections were rehydrated in an ethanol gradient and then stained using a TUNEL apoptosis detection kit (Beyotime Biotechnology, C1090) to detect apoptosis. Briefly, the tissue sections were treated with proteinase K (20 μg/ml) without DNase at 37°C for 15 min. Sections were washed three times with PBS and incubated with TUNEL detection solution for 1.5 h at RT. Sections were washed three times with PBS and stained with H33342 (1 μg/ml) for 1 min. After washing, samples were mounted with the anti-fluorescence quencher, and digital images were captured on a Leica DMR fluorescence microscope (Mannheim, Germany). The number of apoptotic granulosa cells was counted in the antral follicle using Image Pro Plus software.
In vitro fertilization and embryo culture
The in vitro fertilization (IVF) procedure was performed as described previously (Chen et al., Reference Chen, Shen, Zhu, Yang, Ye, Liu, Gu and Chen2019). Briefly, matured (MII) oocytes from the oviduct ampulla were inseminated with capacitated sperm and incubated with HTF medium at 37°C in 5% CO2 in air. After 4 h post-fertilization, oocytes were washed and cultured in KSOM medium at 37°C in 5% CO2 in air. Proportions of 2-cell embryos were assessed at 24 h post-fertilization.
Measurement of intracellular ROS level
To measure intracellular ROS levels, matured (MII) oocytes were incubated in M2 solution containing 20 µM, 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA; Life Technologies, Invitrogen TM, C6827) for 30 min (37°C, 100% humidity, and 5% CO2 concentration), and then washed three times in M2 solution containing 3 g/l BSA for 5 min each. Finally, oocytes were placed under a fluorescence microscope and measured at 460 nm excitation with a filter, and fluorescence images were recorded as TIFF files. After deducting the background value, fluorescence intensities were quantified using ImageJ software (Version 1.48; National Institutes of Health, Bethesda, MD, USA; Zou et al., Reference Zou, Chen, Ding, Gao, Chen, Liu, Hao, Zou, Ji, Zhou, Wei, Cao and Zhang2020).
Statistical analysis
All quantitative data are presented as the mean ± standard error of the mean (SEM) for at least three biological replicates. Data were analyzed using one-way analysis of variance (ANOVA) followed by a Duncan multiple comparison test using SPSS 21.0 software (SPSS Inc.). Next, 10 sections were used to count the proportions of corpora luteum and different types of follicles; at least 10 follicles were used for cell apoptosis detection. Differences between means were considered significant at a P-value < 0.05.
Results
Hypoxia exposure impairs the normal follicle development of mice
To study the effect of hypoxic stress on reproductive fertility, female mice were exposed to hypoxia for 4 weeks to simulate hypoxia exposure to explore changes in fertility. The ratio of RV/[LV+S] was used to assess the success of the mouse model. As shown in Figure 1, RV/[LV+S] was higher in the hypoxia exposure group (Hypoxia) compared with the control group (P <0.05). This result indicated that the hypoxic stress mouse model was successfully established.
Figure 1. Effect of acute hypobaric hypoxia and melatonin on right ventricular hypertrophy index in female mice. Mice were submitted to acute hypobaric hypoxia with or without treatment of melatonin. Control: Mice were treated with normal environment. Experiment: 4 weeks treatment. At least four mice were counted per group. Significance between different groups is represented by different letters (P < 0.05).
As a basis for female fertility, we first examined the follicle development of oogenesis. After hypoxia exposure, the normal oogenesis process was impaired with the increased preantral (primordial, primary, and secondary follicles) and antral follicle proportion in the ovary (Figure 2). While there was little change in corpora luteum and mature follicle proportion, which indicated that normal folliculogenesis was impaired by hypoxia exposure.
Figure 2. Quantification of preantral, antral, mature follicles and corpora lutea in mouse ovaries from control, Hypoxia, and Hypoxia+MT animals. (A) Classification of different developmental stage follicles in mouse ovaries. (B) The proportion of different developmental stage follicles per ovarian cross-section from control, Hypoxia, and Hypoxia+MT female mice. At least 10 ovary sections were counted per mouse. Significance between different groups is represented by different letters (P < 0.05). Black scale bar = 50 μm.
Melatonin reverses the disorder of follicle development caused by hypoxia exposure
as melatonin is often used to relieve oxidative stress, we wanted to examine whether melatonin could relieve the disorder in female follicle development caused by hypoxic stress. The increased RV/[LV+S] caused by hypoxia exposure could be reversed by melatonin feeding (P > 0.05). There was no significant difference between the control and Hypoxia+MT groups (Figure 2). This result supported the cardioprotective effects of an oral administration of melatonin in female mice that suffer from hypoxia exposure.
Then, we further explored if melatonin feeding could rescue the impaired folliculogenesis induced by hypoxia exposure. The follicle counting results showed that the preantral and antral follicle proportion in the Hypoxia+MT group was significantly lower than that in the Hypoxia group but had no difference from the control group. The results suggested that melatonin can improve follicular dysplasia caused by hypoxic stress.
Melatonin relieves the hyper-proliferation of granulosa cells induced by hypoxia exposure
Follicles are composed of oocytes and granulosa cells, and the development of follicles is mainly mediated by the proliferation and renewal of granulosa cells (Monniaux, Reference Monniaux2016). Next, we analyzed the proliferation activity of granulosa cells under hypoxic stress. We chose PCNA as a proliferation cell marker in this assay. The result showed that PCNA-positive granulosa cells were significantly higher in the Hypoxia group than the other groups (P < 0.05; Figure 3B). These results demonstrated that hypoxic stress induced hyper-proliferation of granulosa cells, which would trigger the disorder of folliculogenesis.
Figure 3. Proliferation activity of granulosa cells in follicles from control, Hypoxia, and Hypoxia+MT female mice. (A) Representative images of immunohistochemical staining of the proliferation cell marker PCNA. (B) Proliferation activity differences of granulosa cells in growing follicles (preantral and antral follicles) from the control, Hypoxia and Hypoxia+MT female mice. At least 10 follicles were counted per mouse. Significance between different groups is represented by different letters (P < 0.05). The red arrows indicate high proliferative granulosa cells. The black arrows indicate low proliferative granulosa cells. Black scale bars = 10 μm.
Furthermore, we also analyzed whether the addition of melatonin affected the hyper-proliferation of granulosa cells induced by hypoxic exposure. As shown in Figure 3B, compared with the Hypoxia group, PCNA-positive granulosa cells in the Hypoxia+MT group were significantly reduced (P < 0.05). These results indicated that hypoxic exposure induced the proliferation of granule cells in mouse follicles. At the same time, melatonin partly overcame the excessive proliferation of granulosa cells caused by hypoxia exposure during follicle development.
Melatonin reduces apoptosis of granulosa cells in antral follicles of hypoxic mice
The above result showed that, although hypoxia induced an increasing proportion of antral follicles on the ovary, it did not change the rate of mature follicles. To further analyze the reasons for this phenomenon, we analyzed the apoptosis of granulosa cells in antral follicles. We examined the apoptosis of granulosa cells in follicles from control, Hypoxia, and Hypoxia+MT groups using TUNEL-staining targeting DNA fragments. The apoptosis rate of granulosa cells in antral follicles in the Hypoxia group was significantly increased than that in other groups (P < 0.05; Figure 4B). However, there was no obvious difference between the Hypoxia+MT group and the control group in the apoptosis rate of granulosa cells (P > 0.05; Figure 4B). These results suggested that hypoxic stress-induced follicular developmental disorders may be caused by the apoptosis of granulosa cells. Melatonin could rescue the apoptosis of granulosa cells during follicle development caused by hypoxia exposure.
Figure 4. Apoptosis rate of granulosa cells in antral follicles from control, Hypoxia, and Hypoxia+MT female mice. (A) Representative images of fluorescence staining of DNA fragment marker TUNEL. (B) Apoptosis rate differences of granulosa cells in antral follicles from control, Hypoxia, and Hypoxia+MT female mice. At least 10 follicles were counted per group. Significance between different groups is represented by different letters (P < 0.05). The white arrows indicate apoptotic granulosa cells in the follicle.
Melatonin rescues the hypoxic stress-induced decline in early embryonic development
To further confirm the effect of hypoxia exposure on early embryonic development, we carried out superovulation and in vitro culture experiments. After hCG injection, the number of MII oocytes obtained from these three groups showed no difference, which indicated that hypoxic stress did not affect ovulation (Figure 5A). During the subsequent embryo development, the cleavage rate in Hypoxia was significantly lower compared with the control group (P < 0.001), whereas melatonin feeding could rescue the decreased embryo development ability induced by hypoxia exposure (Figure 5B). These data indicated that hypoxia exposure was detrimental to early embryo development. At the same time, melatonin improved the developmental competence of oocytes caused by hypoxia exposure.
Figure 5. Effect of acute hypobaric hypoxia with or without melatonin on ovulation and early embryo development. (A) The three experimental groups had no differences on the number of ovulation. (B) The difference of 2-cell rates from control, Hypoxia, and Hypoxia+MT groups. At least 60 oocytes were used per group. Bars with different letters indicate significant difference (P < 0.001).
Melatonin attenuates oxidative stress in oocytes induced by hypoxic exposure
Because the developmental competence of the MII oocytes was severely impaired by hypoxia exposure, ROS levels in the oocytes were measured to evaluate intracellular oxidative stress in the control, Hypoxia, and Hypoxia+MT groups. The ROS level in oocytes from the Hypoxia group was significantly higher than that of the corresponding control group (P < 0.05; Figure 6A,B). However, when melatonin was fed, intracellular ROS levels (MII oocytes) were decreased, and showed no difference when compared with the control group (P > 0.05). Therefore, melatonin acts against ROS accumulation in MII oocytes from the oxidative stress caused by hypoxia exposure.
Figure 6. Effect of acute hypobaric hypoxia with or without melatonin on ROS levels in MII oocytes. (A) Representative images of fluorescence staining of ROS marker H2DCFDA in MII oocytes from control, Hypoxia, and Hypoxia+MT female mice. (B) The three experimental groups had differences in ROS levels (H2DCFDA fluorescence). At least 20 oocytes were counted per group. Significance between different groups is represented by different letters (P < 0.05).
Discussion
Our study describes the effects of melatonin administration on follicle development and oocyte quality in a mice model of hypoxia stress. In this study, we provided strong evidence that acute hypoxic stress caused the accumulation of growing follicles in the ovaries of mice, increased proliferation and apoptosis of granulosa cells in the follicle, and induced ROS levels to increase in oocytes, which ultimately led to the reduction of oocyte developmental potential. However, feeding antioxidant melatonin effectively inhibited abnormal follicle development and excessive proliferation and apoptosis of granulosa cells, and reduced the ROS level in eggs, therefore alleviating the fertility damage caused by hypoxia stress.
The abnormal granulosa cell cycle under an hypoxic environment resulted in the accumulation of growing follicles in the ovary. Previous studies have shown that a basal hypoxic microenvironment can limit the growth of antral follicles and reduce the diameter of the follicle in vitro (Connolly et al., Reference Connolly, Kane, Quinlan, Dockery and Hynes2017; Ma et al., Reference Ma, Wang, Gao, Liu, Zheng, Gao, Liu and Jiang2019). In this work, the proportion of antral follicles increased significantly in the Hypoxia group, but there was no significant difference in corpora lutea and mature follicle proportion. The findings of these analyses uncovered that hypoxia causes antral follicle atresia. The poor GC number and function led to decreased production of steroid hormones and may trigger follicular atresia (Shen et al., Reference Shen, Lin, Zhang, Tang, Chen and Liu2012; Zhang et al., Reference Zhang, Chen, Li, Chen, Liu, Wu, Li, Li, Cao, Zhou, Shen, Liu and Tao2022). Previous studies have reported that oxidative stress caused by ovarian hypoxia is the main cause of ovarian ageing and GC dysfunction (Shen et al., Reference Shen, Cao, Jiang, Wei and Liu2018; Jiang et al., Reference Jiang, Shen, Chen, Wei, Tao and Liu2021a). Oxidative stress also alters the expression of more than 70 downstream genes, which leads to the overproduction of ROS and initiation of apoptotic cell death in ovarian follicles and granulosa cells (Lim and Luderer, Reference Lim and Luderer2011). In this study, the proliferation and apoptosis of granulosa cells were increased in the antral follicles, and there was no difference in the number of ovulations under hypoxia exposure. Therefore, oxidative stress injury of granulosa cells induced by hypoxia is the main cause of antral follicular accumulation in the ovary.
Hypobaric hypoxia causes ROS, a significant increase in mature oocytes, and results in the developmental failure of early embryos. Previously, it has been demonstrated that hypoxia modifies the activity of the cytochrome chain responsible for mitochondrial oxidative phosphorylation, resulting in a decrease in ATP synthesis and increased ROS (Turrens, Reference Turrens2003), at the same time as a decrease in the activity of the cellular antioxidant system, which may lead to oxidative injury and apoptosis (Adam-Vizi, Reference Adam-Vizi2005; Ramanathan et al., Reference Ramanathan, Gozal and Siegel2005; Coimbra-Costa et al., Reference Coimbra-Costa, Alva, Duran, Carbonell and Rama2017). Under normal conditions, the cell produces a ROS level that acts beneficially for tissue regeneration, intracellular redox regulation, and embryogenesis. However, an excess of ROS can oxidize cellular molecules, such as lipids, carbohydrates, amino acids, and nucleic acids, modifying their functions and compromising cellular viability by producing lipid peroxidation, mitochondrial damage, and apoptosis. This impairs oocyte nuclear and cytoplasmic maturation, and subsequent embryo development and quality (Ambrogi et al., Reference Ambrogi, Dall’Acqua, Rocha-Frigoni, Leão and Mingoti2017). In the present study, we showed a high ROS level in MII oocytes along with the decreasing embryo cleavage rate in the Hypoxia group. We proposed that hypoxia may induce excessive ROS accumulation in mature oocytes, resulting in oocyte oxidative injury and poor developmental ability.
Several studies have demonstrated the beneficial effects of melatonin against oxidative stress-stimulated granulosa cell damage. Melatonin treatment can markedly prevent the apoptosis of porcine granulosa cells during follicular atresia via its membrane receptors and its free-radical scavenging activity in vitro (He et al., Reference He, Deng, Jiang, Li, Shi, Chen and Han2016). It was proposed that melatonin alleviates hypoxia-induced apoptosis of granulosa cells by reducing ROS production and activating the MTNR1B–PKA–Caspase8/9 pathway in porcine (Tao et al., Reference Tao, Zhang, Zhou, Li, Yang, Liu, Zhang, Deng, Zhang, Shen, Liu and Liu2021). In addition, several studies have demonstrated that melatonin is against hypoxia-induced proliferation of cancer cells for preventing tumour progression (Chuffa et al., Reference Chuffa, Fioruci-Fontanelli, Mendes, Ferreira Seiva, Martinez, Fávaro, Domeniconi, Pinheiro, Delazari Dos Santos and Martinez2015; Bastani et al., Reference Bastani, Akbarzadeh, Rastgar Rezaei, Farzane, Nouri, Mollapour Sisakht, Fattahi, Akbarzadeh and Reiter2021; Estaras et al., Reference Estaras, Martinez, Garcia, Ortiz-Placin, Iovanna, Santofimia-Castaño and Gonzalez2022). In our study, melatonin relieved hypoxia-induced proliferation and the apoptosis of granulosa cells, which in turn led to a significant reduction in the proportion of antral follicles compared with the Hypoxia group. Findings from these data suggested that melatonin effectively modulated granulosa cell cycle and restored follicular development under hypoxia environment.
Melatonin alleviated the limit of follicle growth and improved oocyte quality under hypoxia exposure in female animals. Previous studies have shown that acute hypoxic stress caused follicle development, arrest, LH secretion deficiency, and luteal area reduction in sheep, and antioxidants partially alleviated the reproductive damage caused by hypoxic stress (Parraguez et al., Reference Parraguez, Urquieta, Pérez, Castellaro, De los Reyes, Torres-Rovira, Aguado-Martínez, Astiz and González-Bulnes2013, Reference Parraguez, Diaz, Cofré, Urquieta, De Los Reyes, Astiz and Gonzalez-Bulnes2014). In this study, we showed that the process of follicle development and ROS level in the oocyte returned to normal while feeding melatonin in hypoxia-exposure female mice. Melatonin has a positive regulatory effect on the secretion of oestrogen and reducing of ROS in the ovary or oocytes. On the one hand, melatonin combined with melatonin receptors on the membrane of granulosa cells to activate the activity of aromatase CYP19A1 and to promote oestrogen synthesis and antral follicle maturation (Liu et al., Reference Liu, Yang, Li, Ao, Zhang, Zhou and Li2019a; Cheng et al., Reference Cheng, Fang, Li, Wang, Li, Yan, Jia, Wu, Wang, Han and Sun2020). On the other hand, melatonin is known as an antioxidant or free-radical scavenger by reducing the excessive production of ROS, thereby protecting against cardiac dysfunction under hypoxia (Poeggeler et al., Reference Poeggeler, Saarela, Reiter, Tan, Chen, Manchester and Barlow-Walden1994; Gonzaléz-Candia et al., Reference Gonzaléz-Candia, Arias, Aguilar, Figueroa, Reyes, Ebensperger, Llanos and Herrera2021). Previous studies have suggested that adding melatonin (MT) for in vitro maturation improves the developmental competence of oocytes by scavenging reactive oxygen species (ROS) through melatonin receptor 2 in the cumulus–oocyte complex (Kim et al., Reference Kim, Ridlo, Lee and Kim2020; Zhu et al., Reference Zhu, Guan, Lv, Zhao, Yan, Shi, Ji, Zhang and Liu2021). Melatonin acts on the Nrf2 signalling pathway through its receptors to induce the expression of downstream antioxidant proteins, thereby reducing the accumulation of ROS and enhancing the in vitro development potential of oocytes under stress (Guo et al., Reference Guo, Yang, Qin, Qazi, Pan, Zang, Lv, Deng, Fang and Zhou2021). A recent study confirmed that the expression of the melatonin receptor 2 (MT2) gene was identified in granulosa cells and oocytes (Jin et al., Reference Jin, Sun, Jiang, Cui, Bian, Lee, Zhang, Lee and Liu2022). Based on these studies, we suggested that melatonin may directly act on the oocyte itself or indirectly through granulosa cells. Furthermore, melatonin improved the adaptation of follicular cell response to the hypoxic environment and against oxidative damage induced by hypoxia (Tanabe et al., Reference Tanabe, Tamura, Taketani, Okada, Lee, Tamura, Maekawa, Asada, Yamagata and Sugino2015; Liu et al., Reference Liu, Yang, Li, Ao, Zhang, Zhou and Li2019a).
In summary, the present study showed that melatonin plays an important role in alleviating female fertility damage caused by acute hypoxic stress in mice, such as adjusting follicle development, maintaining the balance of proliferation and apoptosis in granulosa cells, decreasing ROS accumulation, and improving egg quality. Therefore, our findings support melatonin as a plausible treatment for reproductive diseases that coexist with hypoxia and oxidative stress. Future studies are required to understand the protection mechanism of melatonin against reproductive damage caused by hypoxic stress in female animals.
Acknowledgements
This research was funded by the Natural Science Foundation of Anhui Province of China (2108085QC136), Ruina Zhang was supported by the ‘Talent Introduce’ Programme (WGKQ202001009), Postdoctoral Science Foundation (WXBSH2020002) and Domestic Visiting Programme (wxxygnfx2023001) of West Anhui University.
Competing interests
The authors declare no conflicts of interest.
Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
