Melatonin protects against oxybenzone-induced deterioration of mouse oocytes during maturation

Effects of melatonin on in vitro development of OBZ-exposed mouse oocytes and embryos

To investigate the toxic effects of OBZ exposure on the in vitro maturation (IVM) of immature oocytes retrieved from ICR mice, we first cultured oocytes with increasing concentrations (2, 50, 250, 500, and 1000 nmol/L) of OBZ. As shown in Figure 1A, 1B, in the control group, most of the oocytes released the first polar body (82.45 ± 8.54%, n = 367). However, OBZ exposure significantly decreased the PBE rate of mouse oocytes (25 nM, 74.99 ± 9.38%, n = 391, P < 0.05; 50 nM, 71.11 ± 11.87%, n = 346, P < 0.05; 100 nM, 71.19 ± 4.89%, n = 334, P < 0.05; 250 nM, 69.41 ± 8.59%, n = 314, P < 0.05; 500 nM, 55.87 ± 10.19%, n = 351, P < 0.05; and 1000 nM, 54.21 ± 12.68%, n = 343, P < 0.05; Figure 1B). When the OBZ concentration was 2 nM, there was no significant difference in the maturation rate between the 2 nM OBZ-exposed oocytes and untreated oocytes (76.84 ± 12.84%, n = 360 vs 82.45 ± 8.54%, n = 367, P = 0.10). These results suggested that OBZ exposure inhibited the meiotic maturation of mouse oocytes in a dose-dependent manner. Based on the previous study [10], 500 nmol/L OBZ was used in further treatment experiments.

Next, to investigate whether MT has protective effects against OBZ-induced meiotic arrest, MT was dissolved in absolute ethanol and then diluted in 500 nmol/L OBZ IVM medium to yield a final concentration of 1 × 10⁻⁹ mol/L, 1 × 10⁻⁷ mol/L, and 1 × 10⁻⁵ mol/L, respectively. The quantitative results showed that 1 × 10⁻⁷ mol/L MT not only significantly increased the proportion of PBE in OBZ-exposed oocytes (76.33 ± 8.34%, n = 241, P < 0.05; Figure 1C, 1D) compared to that of the treatment with OBZ alone (51.62 ± 11.59%, n = 245), but the 1 × 10⁻⁷ mol/L MT treatment results were similar to those of the treatment with MT alone (83.03 ± 8.64%, n = 245) and untreated control group (80.72 ± 10.76%, n = 262). Higher concentrations of MT did not obviously improve oocyte maturation in the OBZ exposure group (57.40 ± 13.95%, n = 254). Although 1 × 10⁻⁹ mol/L MT significantly increased the rate of PBE (69.09 ± 7.01%, n = 220) compared to that of the group treated with only OBZ, it was significantly lower than that of the control group. Based on these results, we chose to use 1 × 10⁻⁷ mol/L MT for treatment in the subsequent study.

We then asked whether OBZ exposure affected early embryonic development in mice and evaluated the effects of MT on the in vitro development of OBZ-treated embryos. As shown in Figure 1E–1G, the proportion of embryos that developed to the blastocyst stage was significantly lower in the group treated with 500 nM OBZ than it was in the control untreated group (41.25 ± 7.04%, n = 191 vs 84.52 ± 7.08%, n = 184, P < 0.05). As expected, 1 × 10⁻⁷ mol/L MT supplementation effectively improved the developmental competence of OBZ-exposed embryos (87.54 ± 6.78%, n = 217). However, there were no significant differences in the cleavage rates of embryos (87.20 – 90.71%) among the different groups.

Effects of melatonin on histone methylation levels in mouse oocytes exposed to OBZ

Histone modifications play functional roles in oocyte meiotic maturation, fertilization, and further embryonic development in mammals. To determine whether melatonin reversed the OBZ-induced decrease in mouse oocyte maturation by altering histone modifications, the global H3K4me3 and trimethylation of lysine 36 on histone H3 (H3K36me3) levels were examined by immunofluorescence staining. OBZ-exposed oocytes presented significantly lower H3K4me3 fluorescence intensity levels than did those in the control group (P < 0.05; Figure 2A, 2B), while no statistically significant differences were found in the average fluorescence intensity of H3K36me3 among the control, OBZ, and OBZ+MT groups (Figure 2A, 2C). As expected, the detrimental effects of OBZ could be rescued by MT; coincubation with MT effectively increased H3K4me3 fluorescence intensity (P < 0.05). These results suggested that OBZ exposure demethylated trimethyl-histone H3K4 in in vitro matured oocytes and that MT could restore these changes.

To further unravel the possible mechanism underlying the beneficial effects of MT on histone H3K4 trimethylation in OBZ-exposed oocytes, we detected the mRNA levels of Kdm5a, Kdm5b, and Kdm5c, which are H3K4-specific histone demethylases. We found that the transcript levels of Kdm5a and Kdm5b were significantly increased in OBZ-exposed oocytes compared with their controls, but OBZ exposure did not change the expression level of Kdm5c (Figure 3A). In the OBZ+MT group, Kdm5b and Kdm5c mRNA transcript levels were lower than they were in the OBZ group. Somewhat unexpectedly, MT supplementation significantly upregulated Kdm5a gene expression compared with that of the untreated oocytes.

Effects of melatonin on oxidative stress and early apoptosis in mouse oocytes exposed to OBZ

To investigate whether OBZ exposure induced oxidative stress, we detected the ROS levels in each group. As shown in Figure 4A, 4B, the ROS signal was significantly higher in the OBZ exposure group than they were in the control group (P < 0.05), whereas there was little ROS signal in the OBZ+MT group. Because glutathione (GSH) is the predominant antioxidant system in mammalian oocytes, we next detected intracellular GSH in different groups. We found that the GSH level was significantly decreased in oocytes exposed to OBZ (P < 0.05), while MT supplementation significantly increased the GSH level (Figure 4A, 4C). Furthermore, the expression levels of antioxidative stress genes (Sod1 and Gpx1) were detected by qRT-PCR. Remarkably, OBZ-exposed oocytes decreased the expression of antioxidative stress genes; however, the downregulation could be significantly reversed to a level comparable to that of the control group with MT treatment (Figure 3B).

Abnormal ROS and GSH levels could induce the occurrence of early apoptosis in oocytes, and we examined early apoptosis by Annexin-V staining assay in different groups. Our results showed that the percentage of Annexin-V-positive oocytes in the OBZ exposure group significantly increased compared with that in the control group (P < 0.05), while the increased percentage was significantly reversed by MT supplementation (Figure 4D, 4E). Moreover, we detected the relative mRNA expression of apoptosis-related genes in different groups. As shown by qRT-PCR analysis, OBZ exposure significantly downregulated the expression of the anti-apoptosis gene Bcl2 (Figure 3C), but it exhibited significant upregulation of pro-apoptosis genes, including Bax, Caspase-3, and P53 (Figure 3D). These transcriptional changes were reversed when MT was added to the IVM medium with OBZ.

Effects of melatonin on mitochondrial characterization in mouse oocytes exposed to OBZ

Mitochondrial distributions are critical indicators of the cytoplasmic maturation of oocytes, so we then examined their changes in different groups. Oocytes exposed to OBZ showed a significantly increased percentage of maturated oocytes with aberrant mitochondrial distribution, while treatment with MT led to a significant improvement in the homogeneous distribution of mitochondria (Figure 5A, 5B).

Next, we investigated the mitochondrial ΔΨm using a mitochondrial membrane potential assay kit for detecting JC-1. The ratio of the intensity of rhodamine isothiocyanate to fluorescein isothiocyanate fluorescence, which reflects the mitochondrial ΔΨm, was significantly decreased in mouse oocytes exposed to OBZ compared to the control (P < 0.05), whereas the reduction could be significantly reversed to a level comparable to that of the control group with MT treatment (Figure 5C, 5D). In addition, the immunofluorescence staining results showed that OBZ exposure significantly decreased the average fluorescence intensity of ATP5A compared to the untreated group (Figure 2A, 2D). As expected, MT supplementation could restore the abnormal global levels of ATP5A. However, no statistically significant differences were found in the average fluorescence intensity of ATP5B among the control, OBZ, and OBZ+MT groups (Figure 2A, 2E).

Effects of melatonin on spindle configuration in mouse oocytes exposed to OBZ

As meiotic arrest is largely due to abnormal spindle assembly, we then examined spindle morphologies by immunostaining with anti-α-tubulin-FITC antibody, and chromosome alignment was visualized by counterstaining with DAPI. Our results showed that most oocytes in the control group exhibited a typical barrel-shaped spindle apparatus with well-aligned chromosomes on the equatorial plate; however, spindle formation was severely disrupted, and chromosomes were disorganized in the OBZ exposure group, while melatonin supplementation relieved the disruption caused by OBZ (Figure 6A). The proportion of oocytes with aberrant spindle morphology in the OBZ-treated group was significantly higher than that in the control group (P < 0.05), while there was no difference between the OBZ+MT group and the control group (Figure 6B). Furthermore, we found that the average intensity of fluorescence labeling of α-tubulin in the OBZ-exposed oocytes was much weaker than it was in the untreated oocytes (P < 0.05), while there was no statistically significant difference between the control group and OBZ+MT group (Figure 6C).

Effects of melatonin on in vivo development of OBZ-exposed mouse oocytes and embryos

To corroborate the in vitro data, we then performed in vivo experiments. Our results showed that after 28 days of OBZ treatment with or without MT, no statistically significant differences were found in body weight or ovary weight among the different groups (Figure 7A, 7B). We then investigated for the first time the meiotic maturation of oocytes acquired from in vivo experiments. Similar to the IVM results, OBZ exposure in mice significantly decreased the PBE rate of oocytes compared with the control oocytes (59.58 ± 9.92%, n = 301 vs 81.51 ± 8.51%, n = 252, P < 0.05), while MT exposure significantly reversed oocyte maturation to a level that was comparable to that of the control group (71.16 ± 12.01%, n = 267, P = 0.26; Figure 7C). Next, we detected the whole blood concentrations of OBZ in different groups. As shown in Figure 7D, the average level of OBZ in the OBZ group was significantly higher than that in the control group (0.21 ± 0.07 ng/mL, n = 5 vs 0.05 ± 0.03 ng/mL, n = 5, P < 0.05), but it was similar to that of the OBZ+MT group (0.15 ± 0.07 ng/mL, n = 5). Intriguingly, the median concentration of OBZ in the control group was significantly lower than it was in the OBZ+MT group.

Next, we explored the early embryo developmental potential of embryos acquired from in vivo experiments. As shown in Figure 7F, the percentage of mouse embryos that reached the blastocyst stage was significantly lower in the OBZ group than it was in the OBZ+MT and control groups (68.53 ± 26.25%, n = 180 vs 84.10 ± 11.98%, n = 183; 78.89 ± 6.29%, n = 186, P < 0.05). However, the cleavage rates were similar in the three different groups (87.01 – 91.90%; Figure 7E).

Effects of melatonin on oocyte quality in OBZ-exposed mouse models

We then asked whether OBZ administration could affect histone H3K4me3 in in vivo matured oocytes. As shown in Figure 8A, the level of histone H3K4me3 in the OBZ group was also significantly decreased compared with that of the control group (P < 0.05). In contrast with the in vitro results, MT administration did not significantly improve the global histone H3K4me3 level compared with that of the OBZ group (Figure 8A, 8B). Moreover, histone H3K4me3 demethylases were investigated by qRT-PCR. Our results showed that there was no difference in Kdm5a mRNA expression among the three different groups (Figure 8C). In the OBZ group, the Kdm5b transcript level was significantly increased compared with that in the control group (P < 0.05), while the abnormal increase could be reversed by melatonin administration (Figure 8C). Unexpectedly, the expression of Kdm5c was significantly downregulated in the OBZ and OBZ+MT groups (Figure 8C).

To further verify the results of in vitro experiments, the intracellular ROS levels, GSH levels, early apoptotic phenomena, and mitochondrial characterization were investigated. In the OBZ group, the ROS signal (Figure 9A, 9B) and rate of early apoptosis in oocytes (Figure 9A, 9D) were markedly increased compared to the control (P < 0.05), while the level of GSH was significantly decreased (P < 0.05; Figure 9A, 9C). As expected, MT significantly counteracted oxidative damage and inhibited early apoptosis (Figure 9A–9D). The rate of oocytes with aberrant mitochondrial distribution was not only increased in the OBZ group (P < 0.05; Figure 9E), but the mitochondrial ΔΨm was significantly decreased when compared with their controls (P < 0.05; Figure 9F). Similar to the in vitro results, melatonin administration significantly rescued aberrant mitochondrial characterization (Figure 9E, 9F).

Next, we investigated spindle morphologies and chromosome alignment through immunofluorescence staining. Our results showed that control oocytes usually exhibit a typical barrel-shaped spindle and well-aligned chromosomes at the equator (Figure 10A). In contrast, many disorganized spindle morphologies and misaligned chromosomes were observed in the OBZ group (Figure 10A). We found a higher percentage of spindle defects and chromosome congression failure in OBZ-treated oocytes than we found in their controls (P < 0.05; Figure 10B). Moreover, the average fluorescence intensity of α-tubulin in the untreated group was much higher than it was in the OBZ group (P < 0.05; Figure 10C). It was noted that the defects of the spindle could be restored by MT treatment (Figure 10A–10C).

Mice and chemicals

Institute of Cancer Research (ICR) mice were used in this study. All experiments were approved by the Animal Ethics Committee of the Third Affiliated Hospital of Guangzhou Medical University and were performed in accordance with the guidelines of Animal Care and Use of the Third Affiliated Hospital of Guangzhou Medical University (TAHGMUACC-2019-0126).

Unless otherwise specified, all chemicals and reagents used in this study were purchased from Sigma Chemical Company (St. Louis, MO, USA). OBZ was purchased from Selleck Chemicals (Houston, TX, USA). A rabbit anti-H3K4me3 antibody and a rabbit anti-H3K36me3 antibody were purchased from Cell Signaling Technology (Devers, MA, USA). Mouse anti-ATP5A and mouse anti-ATP5B antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 594 goat anti-mouse antibodies were purchased from Invitrogen (Carlsbad, CA, USA).

Oocyte/zygote collection and culture

For the in vitro experiment, 6- to 10-week-old female ICR mice were intraperitoneally injected with 5 international units (IU) of pregnant mare serum gonadotropin (PMSG). Forty-six to 48 hours later, oocytes with intact germinal vesicle were collected from the ovaries of mice, washed three times in M2 medium, and then cultured in M16 medium under paraffin oil at 37° C in a humidified incubator with 5% CO₂, 5% O₂, and 90% N₂.

For the in vivo experiment, 90 female mice (6 weeks old) from the ICR were distributed equally into 3 groups at random: control, OBZ (drinking water with 500 nM OBZ per day for 28 days), OBZ+MT (drinking water with 500 nM OBZ and MT 15 mg/kg body weight per day for 28 days). For metaphase II (MII) oocyte collection, mice were superovulated by the injection of 5 IU of PMSG, which was followed by administration of 5 IU of human chorionic gonadotropin (hCG) after 48 hours. Cumulus-oocyte complexes were collected from the oviduct ampullae 14 to 16 hours post-hCG, and denuded MII oocytes were obtained by removing the cumulus mass in medium containing 0.5 mg/mL hyaluronidase, which took place for 2 min at 37° C in a 5% CO₂ atmosphere.

For zygote collection, ICR females were primed with intraperitoneal injection of 5 IU each if PMSG and hCG 48 h apart. The superovulated female mice were mixed with males in the same cage at a 1:1 ratio. Zygotes were collected from oviducts and were cultured in KSOM medium at 37° C in a humidified incubator with 5% CO₂, 5% O₂, and 90% N₂.

OBZ and MT treatment

OBZ was dissolved in DMSO to produce a 50 mmol/L stock solution and then was diluted in M16 medium to produce final concentrations of 2, 50, 250, 500, and 1000 nmol/L. The final concentration of DMSO was less than 0.1% of the culture medium.

Melatonin was dissolved in absolute ethanol to 0.1 mol/L and then diluted in 500 nmol/L OBZ-containing M16 medium to yield final concentrations of 1 × 10⁻⁹ mol/L, 1 × 10⁻⁷ mol/L, and 1 × 10⁻⁵ mol/L. The final concentration of ethanol was less than 0.1% of the culture medium.

Immunofluorescence staining

Mouse oocytes were washed three times in 1% PVA-supplemented PBS (PBS-PVA) and then were fixed with 4% paraformaldehyde in PBS (PBS-PFA) for 30 min. These oocytes were permeabilized using PBS containing 1% Triton X-100 for 20 min and then were blocked in PBS containing 2% bovine serum albumin (PBS-BSA) for 2 h at room temperature. After blocking in PBS-BSA, oocytes were stained by incubation with different primary antibodies (1:400 for H3K4me3; 1:800 for H3K36me3; 1:200 for ATP5A and ATP5B; and 1:300 for α-tubulin) at 4° C overnight. After extensive washing, the samples were then incubated with Alexa Fluor 488 goat anti-rabbit antibody (1:500) and Alexa Fluor 594 goat anti-mouse antibody at room temperature for 1 hour. Oocytes were counterstained with DAPI for 10 min after three washes in PBS-BSA. Finally, oocytes were mounted on glass slides and were viewed under a confocal laser scanning microscope (Nikon A1 R, Tokyo, Japan). The fluorescence intensities of oocytes were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Each experiment was repeated at least 3 times with at least 30 oocytes.

Detection of reactive oxygen species (ROS), glutathione (GSH), and early apoptosis

Matured oocytes from different groups were analyzed for their intracellular ROS and GSH levels using 2’,7’-dichlorodihydrofluorescein diacetate (DCHFDA) and a ThiolTracker™ Violet GSH detection reagent (Invitrogen/Molecular Probes, Carlsbad, CA, USA), respectively. In brief, oocytes were incubated with 100 μmol/L DCHFDA or 20 μmol/L prewarmed ThiolTracker™ Violet dye solution in the dark for 30 min at 37° C, washed three times in PBS-PVA, and viewed under an epifluorescence microscope (Nikon, Tokyo, Japan). The fluorescence intensities of oocytes were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Each experiment was repeated at least 3 times with at least 30 oocytes.

The early apoptosis of mouse oocytes was evaluated by an Annexin-V-FITC kit (Life Technologies Inc., Grand Island, NY, USA). According to the manufacturer’s instructions, samples were washed with 1× annexin-binding buffer for 5 min and incubated in 490 μL 1× annexin-binding buffer supplemented with 10 μL annexin-V conjugate for 15 min at room temperature in the dark. Then, oocytes were moved to glass slides three times with PBS-PVA. The fluorescence signal of oocytes was examined immediately under an epifluorescence microscope (Nikon, Tokyo, Japan). Each experiment was repeated at least 3 times with at least 30 oocytes.

Mitochondrial staining

Mitochondrial distribution was detected using MitoTracker Deep Red (Invitrogen, Paisley, UK). Matured oocytes were incubated with 200 nmol/L of MitoTracker probe in M2 medium for 30 min at 37° C. Then, oocytes were washed three times with PBS-PVA and fixed in PBS-PFA for 40 min at room temperature. Fluorescence signals were detected using an epifluorescence microscope (Nikon, Tokyo, Japan). Each experiment was repeated at least 3 times with at least 30 oocytes.

Mitochondrial membrane potential (ΔΨm) was determined using a MitoProbe™ JC-1 Assay Kit (Life Technologies Inc, Grand Island, NY, USA) according to the manufacturer’s instructions. Briefly, oocytes were incubated in M2 medium containing 4 μmol/L JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolyl-carbocyanine iodide) at 37° C for 30 min in the dark. The oocytes were then washed three times with PBS-PVA, mounted on slides under cover slips, and examined immediately under an epifluorescence microscope (Nikon, Tokyo, Japan). Microphotographs were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Mitochondria ΔΨm was expressed as the ratio of red fluorescence, which corresponded to activated mitochondria (J-aggregates), to green fluorescence, which corresponded to less-activated mitochondria (J-monomers). Each experiment was repeated at least 3 times with at least 30 oocytes.

Measurement of OBZ concentration

Whole blood samples were collected into sterile BD Vacutainer^® plastic lithium heparin tubes and then were stored at 4° C until analysis. Total (free plus conjugated species) concentrations of OBZ in samples were measured using a previously described method [65]. In brief, whole blood samples were incubated in 1 mol/L ammonium acetate buffer solution for hydrolyzation with β-glucuronidase/sulfatase. After hydrolysis, phenols were extracted and preconcentrated with solid phase extraction and then were further detected using ultrahigh-performance liquid chromatography-electrospray ionization tandem mass spectrometry (Waters, Milford, MA, USA). The detection was performed in negative ion mode by multiple reaction monitoring. The limit of detection (LOD) was 0.04 ng/mL. Strict quality control was conducted during the analysis. CR data were collected using an automated chemistry analyzer (Hitachi 7020, Tokyo, Japan), which were obtained for correcting the phenol concentrations caused by blood concentration and dilution. The experiment for the detection of OBZ levels in each group was repeated 5 times.

Quantitative real-time PCR (qRT-PCR)

To investigate the mRNA expression levels of histone H3K4me3 demethylation-related genes (Kdm5a, Kdm5b, and Kdm5c), antioxidative stress genes (SOD and GPx1), and apoptosis-related genes (Bcl2, Bax, Caspase-3, and P53), oocytes from different groups were collected. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous reference gene. Total RNA was extracted from the oocytes using a Qiagen RNeasy Mini Kit (Qiagen, Toronto, Canada); then, the RNA was reverse transcribed to generate cDNA and was stored at -20° C until use. Real-time PCR amplification was conducted with a One Step SYBR PrimeScript RT-PCR Kit (TaKaRa Bio. Inc., Tokyo, Japan) on an ABI-7900 SDS instrument (Applied Biosystems, Foster City, CA, USA). Each real-time PCR consisted of 10 μL of Advanced SYBR Green PCR Master Mix, 4.8 μL of nuclease-free water, 4 μL of cDNA sample, and 1.2 μL of gene-specific primers. The amplification protocol comprised an initial denaturation step at 95° C for 30 s, which was followed by 40 cycles of denaturation at 95° C for 5 s, annealing at 60° C for 30 s and extension at 72° C for 1 min. All oligonucleotide primer sequences are presented in Supplementary Table 1. The expression of each target gene was quantified relative to that of an internal reaction control gene (GAPDH) using the equation R = 2^{-[ΔCt sample-ΔCt control]}. For ease of comparison, the average mRNA transcript level of each gene from the control group was set as one.

Statistical analysis

For each treatment, there were at least 3 biological replicates, and no fewer than 30 oocytes were examined; results are expressed as the mean values ± standard error of the mean (SEM). The data were analyzed with univariate analysis of variance (ANOVA) followed by Tukey’s test using SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA). Differences in gene expression and fluorescence intensity were compared by Student’s t test. P values < 0.05 were regarded as statistically significant.