Constant light exposure impairs embryonic development via disrupting oocyte cytoskeleton integrity and redox homeostasis

Abstract

Environmental constant light exposure could induce biological rhythm disruption, which negatively induces metabolic syndrome, mood disorders, cognitive declines, chronic disease risk, and reproduction defects. Previous studies indicated that constant light exposure affected ovary function and oocyte quality. Here, we reported that melatonin supplementation effectively alleviated the adverse effects of constant light exposure on embryonic development by improving oocyte quality. Our results showed that constant light exposure reduced blastocyst formation during mouse embryo development, and the levels of DNA and histone methylation modifications of embryos were altered. This might be due to the low oocyte quality since we observed aberrant spindle formation, which might be caused by HDAC6-medicated tubulin acetylation for microtubule stability, and this further induced K-MT attachment defects for aneuploidy. Besides, spindle migration was disturbed due to ARP2-based actin assembly defects. Light exposure also led to oocyte elevated ROS level, DNA damage, and then activated LC3-based autophagy and disrupted lysosome function. Notably, in vivo melatonin supplementation effectively rescued these defects in the oocytes and embryos of the mice under constant light exposure. Collectively, our results suggested that melatonin preserved embryo development potential and epigenetic modifications under constant light exposure by maintaining oocyte redox homeostasis and cytoskeletal dynamics.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12964-026-02879-z.

Introduction

Sexual reproduction gives rise to an embryo, which develops from a fertilized oocyte. Decreased oocyte quality greatly influences embryo developmental capacity and fetus health. Metabolic disorders associated with aging, obesity, and diabetes are mainly manifested as declines in oocyte quality, leading to female subfertility or infertility [1–3]. The beginning of oocyte maturation is marked by germinal vesicle breakdown, after which a meiotic spindle is organized by microtubules and follows migration to the cortex with help of actin filaments for asymmetric division [4]. Spindle assembly in oocytes is orchestrated by multiple microtubule regulatory factors, such as PLK1 and MAPK, which are involved in spindle pole organization, while tubulin acetylation plays an indispensable role for microtubule stability, thereby ensuring proper kinetochore-microtubule attachment. These processes monitor chromosome separation for preventing aneuploidy [5–7]. Meanwhile, the actin network is established by actin nucleation factors such as ARP2/3 complex and formin family, which greatly regulate the spindle position in oocytes [8]. Besides, oocyte cellular redox homeostasis protects genomic stability, molecular functions, and maintaining normal biological processes. Excessive reactive oxygen species (ROS) production with compromised antioxidants contributes to DNA damage and membrane organelles dysfunction, leading to autophagy and apoptosis [9, 10], which ultimately impairs oocyte maturation quality.

Biological rhythms are present in nearly all physiological processes and are synchronized with the earth’s light/dark cycle [11, 12]. Light information transmitted to the suprachiasmatic nucleus (SCN) coordinate body temperature, food intake, and hormone secretion [13]. However, in modern societies, prolonged night-time working hours and social activities extend the photophase, disrupting synchronization with the external light/dark cycle. Data from the 2015 National Health Interview Survey (n = 19,386 U.S. workers ≥ 18 years) indicated that more than 21 million people (14.2%) engaged in overnight work, and nearly 7% of daytime workers also reported working between 1:00 am and 5:00 am [14]. Moreover, according to Munich Chronotype Questionnaire, more than 44% of people experienced at least 1 h of social jetlag, while approximately 15% experience 2 h and more [15, 16]. Disruption of biological rhythm increases the risk of brain injuries, metabolic fatty liver, and atherosclerosis [17, 18]. Moreover, continuous night work has been shown to alter menstrual cycle regularity, leading to polycystic ovary syndrome (PCOS) and miscarriage in women [19, 20]. Similarly, in animal experiments, luteinization and luteal functions of the ovary were affected by continuous light exposure [21]. Impaired ovary development with apoptosis and DNA damage was shown after 24 h light treatment [22]. Furthermore, maternal circadian disruption before pregnancy resulted in compromised follicle development and reduced early embryonic competence in female offspring, suggesting potential risks to offspring fertility [23]. Therefore, it is essential to investigate the impact of light pollution on female mammalian reproduction and find an intervention to prevent these adverse responses.

Melatonin (MT) is an endogenous hormone principally secreted at night in the pineal gland, regulated by light information from SCN. Melatonin conveys central rhythmic signals to the peripheral, further mediating process like sleep, while it also feeds back and affects the circadian timing of the SCN [24]. Melatonin exerts its physiological effects by binding to its receptors MT1 and MT2 on the cell membrane, which are widely distributed in organs and tissues of the central nervous system, reproductive system, cardiovascular system, and others [25]. Besides, melatonin is also widely recognized as a potent natural antioxidant and free radical scavenger, regulating mitochondrial function, and promoting the expression of cellular superoxide dismutase (SOD) and glutathione peroxidase (GPx) [26]. In vitro experiments have shown that melatonin supplementation increases glutathione (GSH) levels in porcine oocytes under heat stress, significantly inhibiting oxidative stress and promoting oocyte maturation [27]. In vivo studies have also demonstrated that melatonin can reverse oxidative stress induced by exposure to the industrial dye Sudan I, thereby reducing apoptosis in mouse oocytes [28]. Furthermore, melatonin is considered as a neuroprotective factor that alleviates brain damage induced by light exposure by reducing microglial activation and inflammatory damage [29]. Therefore, melatonin may also act as an endogenous rhythmic regulator and antioxidant, playing a protective role in reproductive damage caused by negative environmental exposure.

In this study, we exposed the female mice to constant light and investigated its effects on mammalian early embryo development and the potential rescued activity of melatonin following biological rhythm disruption. Our results illustrated that melatonin effectively alleviated the impairment of embryo developmental potential caused by constant light exposure, which was approved by the epigenetic modification levels, and this may be due to the protective effects of melatonin on cellular redox homeostasis for cytoskeleton dynamics.

Materials and methods

Ethics statement and animals

All operations on mice were approved by Animal Research Committee of Nanjing Agriculture University, China (Ethics Approval NJAU.No20231207186). 10-week-old Institute of Cancer Research (ICR) male mice and 4-week-old ICR female mice were respectively obtained from Qinglongshan Animal Farm and Comparative Medical Center of Yangzhou University. Female mice were randomly divided into three groups: control, light-exposed, and melatonin 30 mg/Kg/day groups. The control mice were maintained under a normal light/dark cycle (L/D, 7:00 am to 7:00 pm), while experimental mice were housed under constant light exposure environment (L/L, 7:00 am to 7:00 am). To mimic potential effects of continuous night-time work or social activities on reproductive health, we set the illuminance at 300 lx, corresponding to the minimum recommended level for surrounding areas in general office environments according to ISO/CIE 8995-1:2025 (Lighting of indoor workplaces) and GB/T50034-2024 [30]. This intensity is comparable to standard indoor lighting. Melatonin (MT) was administered at a dose of 30 mg/Kg/day in drinking water for rescue treatment, based on previous studies [28, 31]. The average daily water intake per mouse was approximately 8 mL, as determined from Fig. S1, and the melatonin concentration was adjusted according to mean body weight. No significant differences in water consumption were observed among the groups. After 2 weeks, all the animals were kept in the standard L/D cycle with normal daily drinking water.

Antibodies and chemicals

Rabbit anti-H3K27me3 antibody (A2363), rabbit anti-H3K27ac3 antibody (A7253), rabbit anti-PLK1 antibody (A2548), and rabbit anti-HDAC6 antibody (A11259) were from Abclonal. Mouse anti-5 mC antibody (ab1085), rabbit γ-H2AX antibody (ab81299), rabbit anti-RAB10 antibody (ab104859), and rabbit anti-ARP2 antibody (ab128934) were from Abcam. Rabbit anti-tubulin antibody (11224-1-AP), and rabbit anti-GAPDH antibody (10494-1-AP) were from Proteintech. Rabbit anti-H3K4me3 antibody (9751T) and rabbit anti-LC3B antibody (2775 S) were from Cell Signaling Technology. Rabbit anti-5 hmC antibodies (39770) was from Active motif. Human anti-centrosome CREST (15-234-0001) was from Antibodies Incorporated. Rhodamine-phalloidin (FHDR1) was from Cytoskeleton. Dichlorodihydrofluorescein diacetate (DCFH-DA) Kit (S0033S-1), Annexin V-FITC/EGFP Apoptosis Detection Kit (C1062M), Lysosome-tracker (C1046), Horseradish peroxidase-conjugated goat anti-rabbit/mouse IgG antibodies (A0208/A0216), and Hoechst 33342 for live cells (C1027) were from Beyotime. Hoechst 33342 for dead cells (H3570) was from Life Technologies. Alexa Fluor 555 goat anti-human antibody (A21433) was from Invitrogen. Alexa Fluor 594 and 488 goat anti-rabbit antibody (ZF-0316; ZF-0511), Alexa Fluor 594 and 488 goat anti-mouse antibody (ZF-0313; ZF-0512) were from Zhongshan Golden Bridge Biotechnology. Mouse monoclonal anti-α-tubulin-FITC antibody (F2168), mouse monoclonal anti-acetylated tubulin antibody (T7451), melatonin (M5250), and all other unstated chemicals were from Sigma.

Oocyte and embryo harvesting and culture

The methods were due to our previous protocols [32]. Denuded germinal vesicle stage (GV) oocytes were obtained from chopped ovaries with a prefabricated glass tube. Three washes with M2 medium and the oocytes were cultured in M16 medium, sealed with paraffin oil at 37 ℃ in 5% CO₂ atmosphere for 2 h (germinal vesicle breakdown, GVBD), 9 h (metaphase Ⅰ, MⅠ), and 12 h (metaphase Ⅱ, MⅡ).

Mice after light exposure treatment were superovulated by injection of 10 IU PMSG, followed 48 h later by the injection of 10 IU hCG, and then mated with male mice. Cumulus-oocytes complexes (COCs) were collected by flushing oviducts with M2 medium 18–20 h after hCG injection, following 1 mg/mL hyaluronidase treatment for 5 min to remove cumulus cells and three washes with M2 medium. The fertilized eggs were cultured in KSOM medium under paraffin oil at 37 ℃ in a 5% CO₂ atmosphere for 24 h (2-Cell), 48 h (4-Cell), 72 h (Morula), and 96 h (Blastocyst).

Immunofluorescence staining and confocal microscopy

Oocytes or embryos were fixed in 4% paraformaldehyde (PFA) for 30 min and permeabilized with 0.5% Triton X-100 (in phosphate-buffered saline (PBS)) for 20 min at room temperature. After blocking in 1% bovine serum albumin (BSA) (in PBS) for 1 h at room temperature, the samples were incubated with primary antibodies at 4 ℃ overnight. Three washes in washing buffer and then labeled with the corresponding secondary antibodies for 1 h at room temperature. The primary antibodies used were anti-H3K27me3 (1:100), anti-H3K4me3 (1:800), anti-H3K27ac (1:100), anti-γ-H2AX (1:100), anti-LC3B (1:100; with sigma water), anti-α-tubulin-FITC (1:400), and anti-centrosome (1:100). The secondary antibodies used were Alexa Fluor 488/594 goat anti-rabbit (1:200), Alexa Fluor 488/594 goat anti-mouse (1:200), and Alexa Fluor 555 goat anti-human (1:800). Rhodamine-phalloidin (1:200) was used for actin staining for 1 h at room temperature and Hoechst 33342 (1:10,000) was used to stain chromosomes for 10 min at room temperature. Finally, oocytes were mounted on glass slides for examination by a confocal laser scanning microscope (Zeiss LSM 800 META, Jena, Germany).

For 5 mC and 5 hmC, the zona pellucida of zygotes was first removed by 10 mg/mL pronase (in M2 medium) for 5 min and fixed in PFA. Then the zona-free samples were denatured with 4 M HCl for 10 min and neutralized with 100 mM Tris-HCl (pH 8.5) for 10 min, followed by three washes and blocking in 1% BSA, 0.05% Tween20 with PBS for 15 min and 1 h at room temperature, respectively. Subsequently, samples were incubated with anti-5 mC (1:500) and anti-5 hmC (1:500) at 4 ℃ overnight and subsequently stained with appropriate secondary antibodies for 1 h at room temperature. The samples were finally mounted on glass slides and imaged as described above.

Cold treatment

For cold-stable microtubules visualization and kinetochore immunostaining, MⅠ stage oocytes were treated with pre-cooled M2 medium at 4 ℃ for 6 min before fixation. Then the samples were stained with anti-α-tubulin-FITC antibody or anti-centrosome antibody to detect microtubule stability or kinetochores and microtubule attachment detection as described above.

Annexin-V staining

To detect the externalization of phosphatidylserine in early apoptotic oocytes, Annexin V-fluorescein isothiocyanate (FITC) staining was performed using an Annexin V-FITC/EGFP Apoptosis Detection Kit. Oocytes were first incubated with 10 mg/mL pronase (in M2 medium) for 5 min to remove the zona pellucida and then stained with Annexin V-FITC (1:10, with M16 medium dilution) for 20 min. Three washes with M2 medium and then scanned the live oocytes with a laser confocal microscope by Zeiss LSM 800 META.

Chromosome spreading

For karyotype analysis of oocyte, MⅡ stage oocytes were first treated with 10 mg/mL pronase (in M2 medium) to remove zona pellucida. Then the oocytes were transferred to glass slides and fixed in distilled water solution containing 1% paraformaldehyde, 0.15% Triton X-100 and 3 mM dithiothreitol. The samples were dried slowly at room temperature for hours and then blocked in 1% BSA for 1 h at room temperature, subsequently stained with Hoechst33342 (1:10,000) for 10 min and examined under a laser scanning confocal microscope. The number of chromosomes of a single oocyte was counted.

Detection of lysosomes

Lysosome-tracker Red (1:10,000) was used for detection of lysosome distribution with M16 medium for 30 min at 37 ℃ and 5% CO₂. Three washes with M2 medium and then scanned the live oocytes with a laser confocal microscope by Zeiss LSM 800 META.

Fluorescence intensity analysis

ZEN lite 2011 and Image J software (National Institutes of Health, Bethesda, MD, USA) was used for fluorescence intensity measurement. The samples were performed in parallel and under identical conditions among groups. A region of interest on the target image was measured and the average values in each group were used to perform statistical analysis. The fluorescence intensity of the control group was normalized to 1. For 5 mC and 5 hmC quantification, the smaller nucleus was defined as the female pronucleus and the larger one as the male pronucleus. Fluorescence intensities of 5 mC and 5 hmC were measured separately in each pronucleus. The 5 mC/5 hmC ratio was calculated for each pronucleus and used for statistical analysis. For histone modifications analysis, fluorescence signals from both nuclei of 2 Cell were measured and analyzed. For abnormal γ-H2AX intensity calculations, the oocyte whose intensity quantified by Image J was above the average value plus SEM in each group was considered abnormal intensity.

RNA extraction and quantitative real time

Approximately 30 MⅠ stage oocytes were collected with a Dynabeads mRNA DIRECT™ Kit (Invitrogen, Dynal, Oslo, Norway), followed by cDNA synthesis according to cDNA synthesis kit (Takara Biomedical Technology, Dalian, China). QuantStudio 5 (Applied Biosystems, Carlsbad, CA, USA) was used to perform quantitative real-time polymerase chain reaction (RT-PCR) with ChamQ Universal SYBR qPCR Mster Mix (Q711, Vazyme, Nanjing, China).The relative quantitative analysis of genes were performed with the 2^─△△CT.

Western blot analysis

Approximately 150 living oocytes per group were lysed with NuPAGE LDS Sample Buffer and boiled at 100 ℃ for 10 min. The samples were treated with 4–20% SurePAGE (M00656, Nanjing GenScript Biotech Co., Nanjing City, China) at 160 V for 1 h and then transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) with eBlot™ L1 (L00686C, Nanjing GenScript Biotech Co., Nanjing City, China). Then the membranes were blocked in QuickBlock Blocking Buffer for 15 min at room temperature and subsequently incubated with anti-β-actin (1:1,000), anti-GAPDH (1:1,000), anti-tubulin (1:2,000), anti-RAD51 (1:1,000), anti-PLK1 (1:1,000), anti-ac-tubulin (1:2,000), anti-HDAC6 (1:1,000), and anti-ARP2 (1:1,000) at 4 ℃ overnight. After three washes in Tris Buffered Saline with Tween 20 (TBST) for 10 min, immunoblots were labeled with conjugated anti-mouse or anti-rabbit antibodies (1:2,000) for 1 h at room temperature. Finally, three washes in TBST were performed and the membranes were processed using the ECL Plus Western Blotting Detection System (Tanon-3900), and then Image J software was used to analyze the band intensity values.

Statistical analysis

All experiments were performed at least three biological replicates. Statistical analyses were conducted using GraphPad Prime 9.5.0 software (GraphPad, San Diego, CA). Data were analyzed using one-way ANOVA following Shapiro-Wilk test for normality. However, for western blot and qPCR results, data were normalized to the control groups to minimize technical variability. Comparisons between control and light-exposed groups were performed using one-sample t-test, and comparisons between light-exposed and rescue groups were analyzed using Student’s t-test. Data were presented as mean ± SEM, with the number of embryos/oocytes indicated in parentheses (n). The P-value < 0.05 was considered statistically significant. *, indicates the significance between control and other groups. #, indicates the significance between the light and light + MT groups.

Results

Melatonin rescues embryonic development potential under constant light exposure

To established constant light exposure model, female mice were kept under constant 300 lx light for two consecutive weeks, with the control group receiving a standard L/D cycle (7:00 am to 7:00 pm). Melatonin was simultaneously supplied in the drinking water as a rescue treatment (light + MT), and no significant change in daily water intake was observed under constant light exposure (Control: 40.75 ± 3.04, n = 15; Light: 38.75 ± 1.49, n = 15, P > 0.05; Light + MT: 38.00 ± 1.78, n = 15, P > 0.05) (Fig. S1). We first examined the developmental competency of preimplantation embryos. In the control and light + MT groups, most zygotes reached 2-Cell stage after 24 h culture, followed 48 h at 4-Cell, 72 h at morula, 96 h at blastocyst, while light exposure led to a considerably reduced formation rate of 2 Cell, 4-Cell, morula, and blastocyst (2-Cell: Control: 93.29 ± 4.55%, n = 105; Light: 64.39 ± 11.22%, n = 113, P < 0.05; Light + MT: 96.67 ± 2.08%, n = 133, P < 0.05; 4-Cell: Control: 92.83 ± 3.59%, n = 105; Light: 61.46 ± 11.13%, n = 113, P < 0.05; Light + MT: 90.09 ± 1.77%, n = 133, P < 0.05; Morula: Control: 91.25 ± 5.12%, n = 105; Light: 52.95 ± 8.33%, n = 113, P < 0.05; Light + MT: 83.67 ± 8.25%, n = 133, P < 0.05; Blastocyst: Control: 66.77 ± 6.95%, n = 105; Light: 40.31 ± 5.50%, n = 113, P < 0.05; Light + MT: 66.38 ± 6.88%, n = 133, P < 0.05) (Fig. 1A-B). Further analysis revealed abnormal mRNA expression of Cpeb3, Rad21, Espl1, and Cdc27, genes involved in maternal mRNA translation and chromosome segregation, which are closely associated with embryonic developmental competence (Cpeb3: Control: 1.00 ± 0.00; Light: 1.11 ± 0.03, P < 0.05; Light + MT: 0.91 ± 0.03; P < 0.01; Rad21: Control: 1.00 ± 0.00; Light: 0.90 ± 0.02, P < 0.05; Light + MT: 1.04 ± 0.05, P < 0.05; Espl1: Control: 1.00 ± 0.00; Light: 1.17 ± 0.02, P < 0.05; Light + MT: 0.91 ± 0.08, P < 0.05; Cdc27: Control: 1.00 ± 0.00; Light: 1.11 ± 0.05, P < 0.05; Light + MT: 0.90 ± 0.01, P < 0.05) (Fig. 1C). These results implied that melatonin could rescue mouse embryonic development potential after constant light exposure.

Fig. 1.

Melatonin rescues embryonic development potential under constant light exposure. A Representative images of zygote, 2-Cell, 4-Cell, morula, blastocyst in the control, light, and light + MT groups after culturing 0 h, 24 h, 48 h, 72 h, 96 h, respectively. Bar = 80 μm. B The percentage of 2-Cell, 4-Cell, morula, blastocyst in the control (n = 105), light (n = 113), and light + MT (n = 133) group cells after culturing 24 h, 48 h, 72 h, 96 h, respectively. The data were analyzed using one-way ANOVA test. C The mRNA level of genes Cpeb3, Rad21, Espl1, and Cdc27 in control, light, and light + MT oocytes. The mRNA level of the control group was normalized to 1. The data were analyzed using t-test. Data were presented as mean ± SEM. *, indicates the significance between the control and light groups. #, indicates the significance between the light and light + MT groups. *, P < 0.05. #, P < 0.05. ##, P < 0.01

Melatonin restores DNA methylation and histone modifications of embryos under constant light exposure

Dynamic epigenetic modifications change during early embryo development which could robustly determine the cell fate. We then examined DNA methylation and histone modification levels. As shown in Fig. 2A, weak 5 mC but strong 5 hmC signals were simultaneously found in the male pronucleus of control and light + MT groups, while 5 mC/5 hmC ratio was significantly increased after light exposure (Control: 0.5893 ± 0.0410, n = 47; Light: 1.280 ± 0.1408, n = 34, P < 0.001; Light + MT: 0.6706 ± 0.0493, n = 37, P < 0.05) (Fig. 2A). Similar findings were seen in the female pronucleus (Control: 1.3370 ± 0.0569, n = 47; Light: 2.0720 ± 0.1811, n = 34, P < 0.001; Light + MT: 1.4910 ± 0.0759, n = 39, P < 0.05) (Fig. 2A). In addition, significantly increased H3K27me3 and H3K4me3 fluorescence intensity were respectively observed in the 2-Cell after light treatment, while melatonin administration decreased these signals (H3K27me3: Control: 1.00 ± 0.00, n = 42; Light: 1.24 ± 0.02, n = 42, P < 0.01; Light + MT: 1.10 ± 0.01, n = 38, P < 0.01; H3K4me3: Control: 1.00 ± 0.00, n = 39; Light: 1.88 ± 0.17, n = 31, P < 0.05; Light + MT: 1.10 ± 0.04, n = 37, P < 0.05) (Fig. 2B-C). On the contrary, H3K27ac signals in the 2-Cell were showed no significant difference among these three groups (Control: 1.00 ± 0.00, n = 55; Light: 1.03 ± 0.02, n = 66, P > 0.05; Light + MT: 1.05 ± 0.05, n = 68, P > 0.05) (Fig. 2D). These results demonstrated that constant light exposure impairs embryo methylation modifications, while melatonin supplementation effectively ameliorates these defects.

Fig. 2.

Melatonin restores DNA methylation and histone modifications of embryos under constant light exposure. A Representative images and intensity ratio of 5 mC/5 hmC in male/female pronucleus from control (n = 47/47), light (n = 34/34), and light + MT (n = 37/39) groups. Red, 5 mC. Green, 5 hmC. Bar = 20 μm. The data were analyzed using one-way ANOVA test. B Representative images and relative intensity of H3K27me3 in 2-Cell of the control (n = 42), light (n = 42), and light + MT (n = 38) groups. Magenta, H3K27me3. Blue, DNA. Bar = 20 μm. C Representative images and relative intensity of H3K4me3 in 2-Cell of the control (n = 39), light (n = 31), and light + MT (n = 37) groups. Cyan, H3K4me3. Blue, DNA. Bar = 20 μm. D Representative images and relative intensity of H3K27ac in 2-Cell of control (n = 55), light (n = 66), and light + MT (n = 68) groups. Yellow, H3K27ac. Blue, DNA. Bar = 20 μm. Data were presented as mean ± SEM. For panels B-D, the fluorescence intensity of the control group was normalized to 1 and the data were analyzed using t-test. *, indicates the significance between the control and light groups. #, indicates the significance between the light and light + MT groups. ns, P > 0.05. *, P < 0.05.**, P < 0.01. ***, P < 0.001. #, P < 0.05. ##, P < 0.01

Melatonin restores microtubule stability for spindle assembly in oocytes under constant light exposure

Oocyte quality is a prerequisite for fertilization and embryo development. Accurate spindle formation effectively regulates chromosome segregation and prevents aneuploidy. We examined oocyte maturation to explore the potential cause for embryo defects. As shown in Fig. 3A, the oocytes in control and light + MT groups showed normal two poles and a barrel-shaped spindle after 9 h culture, while light exposure treatment caused aberrant spindle morphology. Statistical analysis suggested that the percentage of abnormal spindle in light exposure group was significantly higher than that in control group, which was rescued by melatonin supplementation (Control: 23.92 ± 3.05%, n = 45; Light: 40.38 ± 3.76%, n = 37, P < 0.05; Light + MT: 24.50 ± 4.48%, n = 45, P < 0.05). Spindle pole to pole distance (length, L) and the width across the middle of the spindle (width, D) were measured to further evaluate the spindle morphology. As shown in Fig. 3B, the ratio of the L/D in the light exposure oocyte was significantly decreased compared with the control oocytes, while melatonin supplementation dramatically recovered abnormal spindle morphology (Control: 1.4800 ± 0.0225, n = 53; Light: 1.3890 ± 0.0264, n = 54, P < 0.05; Light + MT: 1.500 ± 0.0242, n = 63, P < 0.01) (Fig. 3B). The expression level of PLK1, a critical spindle formation-related factor, was also significantly lower in MⅠ-stage oocyte exposed to light that in control and light + MT groups (Control: 1.00 ± 0.00; Light: 0.88 ± 0.01, P < 0.01; Light + MT: 1.03 ± 0.03, P < 0.05) (Fig. 3C). Karyotype analysis of MⅡ stage mouse oocytes were performed by chromosome spread. The results showed an abnormal chromosome number in oocytes from light exposure mice, with a higher incidence of aneuploidy than that in the control and light + MT groups (Control: 18.38 ± 2.38%, n = 38; Light: 48.72 ± 5.91%, n = 33, P < 0.05; Light + MT: 24.62 ± 6.89%, n = 31, P < 0.05) (Fig. 3D). Improper chromosome segregation indicates abnormal kinetochore-microtubule attachment (K-MT attachment). We stained kinetochore and microtubule simultaneously. Weak or barely microtubules contacted with kinetochores were observed in the light-exposed oocytes, but melatonin restored this, showing clear microtubules connected with kinetochores (Control: 17.04 ± 5.46%, n = 43; Light: 35.81 ± 3.44%, n = 53, P < 0.05; Light + MT: 18.38 ± 2.38%, n = 44, P < 0.05) (Fig. 3E). Microtubule stability contributes to K-MT attachment. Cold treatment was used to depolymerize unstable microtubules but reserve stable K-fibers. The results showed that microtubule signals were obviously decreased compared with control oocytes, while increased by melatonin administration, and fluorescence intensity analysis also confirmed this finding (Control: 1.00 ± 0.00, n = 45; Light: 0.80 ± 0.02, n = 36, P < 0.01; Light + MT: 0.96 ± 0.05, n = 37, P < 0.05) (Fig. 3F-G). Compared with the control and light + MT groups, light-exposed oocytes exhibited significantly decreased acetylated tubulin and increased HDAC6, a microtubule deacetylase, as determined by Western blot analysis (ac-tubulin: Control: 1.00 ± 0.00; Light: 0.90 ± 0.04, P < 0.05; Light + MT: 1.01 ± 0.01, P < 0.05; HDAC6: Control: 1.00 ± 0.00; Light: 1.17 ± 0.03, P < 0.05; Light + MT: 0.95 ± 0.06, P < 0.05) (Fig. 3H-I). These results illustrated that melatonin restores HDAC6-based microtubule acetylation for stability, which affects spindle assembly in mouse oocytes after constant light exposure.

Fig. 3.

Melatonin restores microtubule stability for spindle assembly in oocytes under constant light exposure. A Representative images and abnormal percentage of spindle in oocyte of the control (n = 45), light (n = 37), and light + MT (n = 45) groups. Green, α-tubulin. Blue, DNA. Bar = 10 μm. B Measurement of spindle morphology in the control (n = 53), light (n = 54), and light + MT (n = 63) group oocytes. We defined the distance of spindle pole to pole as L and the width across the middle of the spindle as D. The ratio of L/D was considerably reduced in the light exposure oocytes compared with control and light + MT groups. Cyan, α-tubulin. Bar = 10 μm. C Band intensity analysis of PLK1 in MⅠ-stage oocytes of control, light, and light + MT groups. A total of 150 live oocytes per group were collected for Western blot analysis. D Representative images of chromosome numbers and percentage of aneuploidy in control (n = 38), light (n = 33), light + MT (n = 31) oocytes of MⅡ stage. Blue, DNA. Bar = 5 μm. E Representative images and abnormal of attachments between kinetochore and microtubule/K-fibers (K-MT attachment) in oocyte from control (n = 43), light (n = 53), light + MT (n = 44) groups. Red, ACA. Green, α-tubulin. Blue, DNA. Bar = 5 μm. F Representative images of microtubule in oocyte from control, light, and light + MT groups after cold treatment. Black, α-tubulin. Blue, DNA. Bar = 10 μm. G Relative intensity of microtubule in oocyte from control (n = 45), light (n = 36), and light + MT (n = 37) groups after cold treatment. H Band intensity analysis of ac-tubulin in MⅠ stage oocytes of control, light, and light + MT groups. A total of 150 live oocytes per group were collected for Western blot analysis. I Band intensity analysis of HDAC6 in MⅠ stage oocytes of control, light, and light + MT groups. A total of 150 live oocytes per group were collected for Western blot analysis. Data were presented as mean ± SEM. For panels A, B, D, and E, the data were analyzed using one-way ANOVA test. For panels C, G, H, and I, the intensity of control group was normalized to 1, and these data were analyzed using t-test. *, indicates the significance between the control and light groups. #, indicates the significance between the light and light + MT groups. *, P < 0.05. **, P < 0.01. #, P < 0.05. ##, P < 0.01

Melatonin restores actin dynamics in mouse oocytes under constant light exposure

Besides, the spindle migration defect was observed in light-exposed oocytes. We measured the minimum vertical distance from the spindle pole to the cortex (length, L) and the diameter (D) of oocytes at the MⅠ stage. As shown in Fig. 4A, the ratio of L to D in the light exposure group was significantly increased compared with control and light + MT oocytes (Control: 0.1068 ± 0.0102, n = 38; Light: 0.1567 ± 0.0126, n = 41, P < 0.05; Light + MT: 0.1076 ± 0.0100, n = 41, P < 0.05). Actin filaments modulate oocyte spindle orientation. We then used phalloidin staining to label actin, and the results showed that the fluorescent signals of F-actin in the cortex of light exposure oocytes were significantly decreased compared to those in the control and light + MT groups (Control: 1.00 ± 0.00, n = 47; Light: 0.88 ± 0.00, n = 41, P < 0.001; Light + MT: 0.96 ± 0.01, n = 41, P < 0.01) (Fig. 4B). Similarly, reduced intensity of cytoplasmic actin filaments was seen in the oocytes from light exposure mice, which could be restored by melatonin supplementation (Control: 1.00 ± 0.00, n = 45; Light: 0.82 ± 0.04, n = 43, P < 0.05; Light + MT: 0.94 ± 0.05, n = 41, P < 0.05) (Fig. 4C). Moreover, compared with control and light + MT groups, the expression of ARP2, an essential actin nucleation factor, was significantly reduced in MⅠ-stage oocytes from mice exposed to constant light (Control: 1.00 ± 0.00; Light: 0.81 ± 0.06, P < 0.05; Light + MT: 1.01 ± 0.02, P < 0.05) (Fig. 4D). These results implied that melatonin administration reversed actin-related spindle migration in mouse oocytes after constant light exposure.

Fig. 4.

Melatonin restores actin dynamics in mouse oocytes under constant light exposure. A Measurement of spindle position to cortex in the control (n = 38), light (n = 41), and light + MT (n = 41) group oocytes. We defined the minimum vertical distance from the spindle pole to cortex as L and the dimeter of oocytes as D. The ratio of L/D was considerably increased in the light exposure oocytes compared with control and light + MT groups, and the data were analyzed using one-way ANOVA test. Cyan, α-tubulin. Bar = 20 μm. B Representative images and relative intensity of cortical actin filaments in control (n = 47), light (n = 41), light + MT (n = 41) oocytes of MⅠ stage. Black, Actin. Blue, DNA. Bar = 20 μm. C Representative images and relative intensity of cytoplasmic actin filaments in control (n = 45), light (n = 43), light + MT (n = 41) oocytes of MⅠ stage. Red, Actin. Blue, DNA. Bar = 10 μm. D Band intensity analysis of ARP2 in the MⅠ stage oocytes of control, light, and light + MT groups. A total of 150 live oocytes per group were collected for Western blot analysis. Data are presented as mean ± SEM. For panels B-D, the intensity of the control group was normalized to 1, and these data were analyzed using t-test. *, indicates the significance between the control and light groups. #, indicates the significance between the light and light + MT groups. *, P < 0.05. ***, P < 0.001. #, P < 0.05. ##, P < 0.01

Melatonin alleviates DNA damage and oxidative stress in oocytes under constant light exposure

Previous study showed DNA damage in mouse ovary after light exposure [22]. We also found a significantly increased intensity and percentage of abnormal high signals of γ-H2AX in oocytes of light exposure mice compared with control groups, which were alleviated by melatonin supplementation (γ-H2AX intensity: Control: 1.00 ± 0.00, n = 47; Light: 1.19 ± 0.03, n = 46, P < 0.05; Light + MT: 0.98 ± 0.05, n = 49, P < 0.05; percentage of abnormal γ-H2AX intensity: Control: 29.67 ± 8.61%, n = 47; Light: 59.87 ± 1.98%, n = 46, P < 0.05; Light + MT: 29.91 ± 7.27%, n = 49, P < 0.05) (Fig. 5A). Moreover, the expression of RAD51 was higher in the MⅠ stage oocytes from light exposure mice than that in control and light + MT groups (Control: 1.00 ± 0.00; Light: 1.32 ± 0.01, P < 0.001; Light + MT: 0.94 ± 0.12, P < 0.05) (Fig. 5B-C). Excessive ROS generation is commonly considered as cellular source of DNA damaging agent. In our results, we also found that ROS signals were apparently stronger in light exposure groups than that in the control and light + MT groups. Quantitative analysis of the percentage of oocytes with ROS intensity above the mean + SEM also confirmed our results (Control: 21.63 ± 6.50%, n = 63; Light: 51.09 ± 9.10%, n = 72, P < 0.05; Light + MT: 12.50 ± 4.55%, n = 70, P < 0.01) (Fig. 5D). Similarly, an increased percentage of aberrant ROS intensity was also found in MⅠ stage oocyte after light exposure (Control: 30.19 ± 4.38%, n = 53; Light: 47.61 ± 1.32%, n = 44, P < 0.05; Light + MT: 30.91 ± 4.59%, n = 54, P < 0.05) (Fig. 5E). Moreover, the expression of genes involved in anti-oxidative stress such as Sod1 and Sod2 was measured, and the results showed a significantly increased expression in light-exposed MⅠ stage oocyte compared with the control group, but this was rescued after melatonin administration (Sod1: Control: 1.00 ± 0.00; Light: 1.15 ± 0.04, P < 0.05; Light + MT: 0.74 ± 0.04, P < 0.001; Sod2: Control: 1.00 ± 0.00; Light: 1.30 ± 0.10, P < 0.05; Light + MT: 0.85 ± 0.07, P < 0.05) (Fig. 5F). These results declared that constant light exposure induced continuous DNA damage and oxidative stress in mouse oocytes, which could be alleviated by melatonin supplementation.

Fig. 5.

Melatonin alleviates DNA damage and oxidative stress in oocytes under constant light exposure. A Representative images and abnormal intensity of γ-H2AX in GV stage oocyte from the control (n = 47), light (n = 46), and light + MT (n = 49) groups. Red, γ-H2AX. Blue, DNA. Bar = 5 μm. The intensity of γ-H2AX that was above mean + SEM was considered abnormal. The results of abnormal γ-H2AX intensity were analyzed using one-way ANOVA test. B The expression of RAD51 in the MⅠ stage oocytes of control, light, and light + MT groups. A total of 150 live oocytes per group were collected for Western blot analysis. C Band intensity analysis of RAD51 in the MⅠ stage oocytes of control, light, and light + MT groups. D Representative images and abnormal ROS intensity in GV stage oocyte from the control (n = 63), light (n = 72), and light + MT (n = 70) groups. The intensity of ROS that was above mean + SEM was considered abnormal, and the data were analyzed using one-way ANOVA test. Green, ROS. Bar = 80 μm. E Representative images and abnormal ROS intensity in MⅠ stage oocyte from the control (n = 53), light (n = 44), and light + MT (n = 54) groups. The intensity of ROS that was above mean + SEM was considered abnormal, and the data were analyzed using one-way ANOVA test. Green, ROS. Bar = 80 μm. F The mRNA level of genes Sod1 and Sod2 in the control, light, and light + MT oocytes. Data are presented as mean ± SEM. For panels A, C, and F, the level of the control group was normalized to 1, and the data were analyzed using t-test. *, indicates the significance between the control and light groups. #, indicates the significance between the light and light + MT groups. *, P < 0.05. ***, P < 0.001. #, P < 0.05. ##, P < 0.01. ###, P < 0.001

Melatonin suppresses excessive autophagy and apoptosis in oocytes under constant light exposure

Prolonged oxidative stress is a key factor to trigger cell autophagy and apoptosis. We next evaluated the occurrence of autophagy and early apoptosis in mouse oocytes. As shown in Fig. 6A, the lysosome signals in the control and light + MT oocytes were weak and homogeneous, while light exposure led to apparent strong clusters in the cytoplasm. Statistical analysis suggested that the relative lysosome intensity in light exposure group was significantly higher than that in the control and light + MT groups (Control: 1.00 ± 0.00, n = 53; Light: 1.59 ± 0.12, n = 62, P < 0.05; Light + MT: 0.98 ± 0.05, n = 49, P < 0.01) (Fig. 6A). In addition, the percentage of abnormal LC3B distribution, characterized by several strong LC3B clusters rarely observed in the cytoplasm of control and light + MT oocytes, was significantly increased after light exposure (Control: 25.14 ± 3.80%, n = 65; Light: 48.62 ± 3.52%, n = 67, P < 0.01; Light + MT: 32.65 ± 2.64%, n = 62, P < 0.05) (Fig. 6B). Furthermore, as shown in Fig. 6C, Annexin-V staining showed that most oocytes in control and light + MT group had few fluorescence signals, whereas significantly positive signals were seen at the cell membrane in light-exposed mouse oocyte, which indicated the occurrence of early apoptosis. The statistical data confirmed that the apoptosis rate was significantly increased after light exposure, while it was dramatically reduced by melatonin supplementation (Control: 25.00 ± 1.61%, n = 35; Light: 47.61 ± 1.32%, n = 40, P < 0.001; Light + MT: 37.28 ± 0.22%, n = 35, P < 0.01) (Fig. 6C). Genes involved in apoptosis such as Bcl-2, Bax were also assessed by qRT-PCR. Compared with the control group, the level of Bcl-2 and the ratio of Bcl-2 to Bax were apparently increased, whereas no significant difference of BAX were seen in the light exposure oocyte (Bcl-2: Control: 1.00 ± 0.00; Light: 1.33 ± 0.05, P < 0.05; Bax: Control: 1.00 ± 0.00; Light: 1.17 ± 0.11, P > 0.05; Bcl-2/Bax ratio: Control: 1.00 ± 0.00; Light: 1.20 ± 0.06, P < 0.05) (Fig. 6D). Melatonin supplementation dramatically restored the transcription levels of Bcl-2, as well as the ratio of Bcl-2 to Bax (Light + MT: Bcl-2: 0.92 ± 0.07, P < 0.01; Bax: 0.99 ± 0.07, P > 0.05; Bcl-2/Bax ratio: 0.90 ± 0.04, P < 0.05) (Fig. 6D). These results suggested that the melatonin suppresses excessive autophagy and apoptosis in mouse oocytes after light exposure.

Fig. 6.

Melatonin suppresses excessive autophagy and apoptosis in oocytes under constant light exposure. A Representative images and relative lysosome intensity in oocyte from the control (n = 53), light (n = 62), and light + MT (n = 49) groups. Black, Lyso-tracker. Blue, DNA. Bar = 20 μm. B Representative images and abnormal distribution of cytoplasmic LC3B distribution in oocyte from control (n = 65), light (n = 67), and light + MT (n = 62) groups. The data were analyzed using one-way ANOVA test. Red, LC3B. Blue, DNA. Bar = 20 μm. C Representative images and rate of apoptosis in oocyte from the control (n = 35), light (n = 40), and light + MT (n = 35) groups using Annexin-V staining. Red, Annexin-V. Bar = 20 μm. The data were analyzed using one-way ANOVA test. D The mRNA level of genes Bcl-2, Bax, and Bcl-2/Bax ratio in the control, light, and light + MT oocytes. Data are presented as mean ± SEM. For panels A and D, the level of the control group was normalized to 1, and these data were analyzed using t-test. *, indicates the significance between the control and light groups. #, indicates the significance between the light and light + MT groups. ns, P > 0.05. *, P < 0.05. **, P < 0.01. ***, P < 0.001. #, P < 0.05. ##, P < 0.01

Discussion

In modern society, the widespread use of artificial indoor lighting and electronic devices (e.g., televisions, smartphones, and tablets), together with prolonged night-time working hours, expose individuals to increasing levels of artificial light at night. This allows people to actively extend the photophase and reduce the scotophase, leading to circadian misalignment that may negatively affect female reproduction. To this end, we investigated the effects of constant light exposure on embryo development and explored the rescuing effects of melatonin with a mouse model. We found that constant light exposure declined embryo developmental capacity and caused abnormal epigenetic modifications, which could be due to reduced oocyte quality. Melatonin supplementation effectively rescued these developmental defects by regulating oocyte cytoskeleton assembly and cellular redox homeostasis.

We first established a mouse model of constant light exposure. In vivo fertilization assays showed a significant decrease in preimplantation embryo development potential. Aberrant expression of maternal factors regulating mRNA translation, chromosome organization and separation, including Cpeb3, Rad21, Espl1, and Cdc27 [33–35], along with increased H3K27me3, H3K4me3, and 5 mC/5 hmC signals, indicated disrupted cell cycle control and epigenetic reprogramming during embryogenesis after mouse light exposure. These defects were ameliorated by melatonin supplementation. Since oocyte maturation is a prerequisite for embryo development potential, we further focused on oocyte quality. Accurate chromosome segregation and asymmetric division are two major goals of oocyte meiosis dependent on cytoskeleton organization [36]. Our results showed that spindle morphology was disrupted in light exposure oocyte, accompanied by chromosome separation errors. This finding is similar with a previous study that reported a high rate of oocyte aneuploidy and abnormal spindle assembly caused by constant light exposure [37]. Further analysis supported that melatonin could protect against these defects, which may be due to its rescued effects on PLK1 and HDAC6-based tubulin acetylation. PLK1 is a key factor participating in MTOCs decondensation and fragmentation, which is essential for bipolar spindle assembly and chromosome segregation in mouse oocyte [38, 39]. Meanwhile, acetylated tubulin makes microtubules more flexible to prevent mechanical breakage [40]. In addition to microtubule organization, actin assembly was also affected by light exposure, showing disturbed spindle migration and decreased expression of ARP2, an important actin assembly regulator. Spindle position is essential for asymmetric meiotic division that ensures the egg containing sufficient material for embryo development [36]. The disrupted spindle position caused by light exposure indicated the decreased oocyte quality for subsequent development. Notably, melatonin restored spindle position in light-exposed oocyte though its rescued effects on ARP2-based actin assembly. These results indicated that the dynamics and functions of cytoskeleton were affected by constant light exposure, which greatly decreased oocyte quality and further impaired embryo development potential. Melatonin supplementation can effectively alleviate these adverse effects caused by constant light exposure.

Previous studies have also reported the protective effects of melatonin on spindle assembly and position against mycotoxin exposure and oocyte aging through its antioxidant activity [41, 42]. Based on this evidence, we further examined redox homeostasis in oocytes following light exposure. Consistent with a previous report showing ovarian DNA damage in mice exposed to 250 lx for 12 days [22], we observed significantly increased γ-H2AX and elevated expression of homologous recombination-related protein RAD51, indicating enhanced DNA damage and activation of DNA damage repair pathways in light-exposed oocytes. As excessive ROS are well-recognized mediators of DNA damaging [43], we next assessed intracellular ROS levels and found a marked increase at both GV and MⅠ stages after constant light exposure. These findings suggested that continuous oxidative stress may contribute to the genomic instability observed in light-exposed oocyte. Notably, melatonin alleviated the imbalance of redox homeostasis in oocyte. Moreover, excessive free radicals may attack organelles and macromolecules, causing cellular oxidative injury, leading to autophagy and further apoptosis [10]. Immunofluorescence staining and q-PCR analysis demonstrated that the activation of autophagy and occurrence of apoptosis in light-exposed oocyte, which could be rescued by melatonin supplementation. These results suggested that light exposure led to continuous oxidative stress in oocytes and ultimately induced the occurrence of DNA damage, autophagy, and apoptosis, while melatonin could restore these defects. A previous study also demonstrated that a significant increment of DHE label in the retinal when rat exposed to 200 lx light-emitting diode (LED) for 5 days, which might be a key mechanism triggering retinal degeneration [44]. Besides, oxidative stress is the core mechanisms by which light pollution affects brain functions [45], and melatonin supplementation has been suggested to protect against light-induced brain damage by reducing oxidative stress and membrane changes [46]. Based on these findings, we proposed that melatonin may act as a reproductive-protector, safeguarding oocyte meiotic maturation by mitigating oxidative stress-induced damage. However, beyond its well-established ROS-scavenging activity, melatonin is also a key rhythmic regulator [47]. It is possible that melatonin may contribute to restoring disrupted clock signaling under constant light exposure, thereby indirectly improving oocyte quality. Although our current data mainly support an antioxidant-mediated mechanism underlying the protective effects of melatonin, potential interactions between redox homeostasis and circadian regulation need further study.

In summary, we identified the protective effects of melatonin against constant light exposure with mouse model. Our results showed that constant light exposure inhibited early embryo development and caused disturbed methylation modifications, which may be due to decreased oocyte quality, showing with aberrant cytoskeleton assembly and oxidative stress-induced cellular damage. Melatonin supplementation effectively alleviated these defects, primarily through its strong antioxidant activities in regulating cellular redox homeostasis. The protective molecular mechanism of melatonin in preventing oxidative stress caused by constant light exposure remains to be elucidated.

Authors’ contributions

SCS, PSL, CW conceived the study. PSL, ZJW, SLW, MXL performed the experiments. PSL analyzed the data. LLH, BYL, JS contributed to materials and agents. PSL, SCS, CW wrote the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (32571001, 82460314), the Fundamental Research Funds for the Central Universities of China (KJJQ2025001, RENCAI2025035), the National Key Research and Development Program of China (2023YFD1300502).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.