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. 2026 Jan 30;21:25. doi: 10.1186/s13062-026-00733-3

Acrylamide disrupts meiotic G2/M transition and SAC activity during oocyte maturation

Shanshan Chen 1,2,#, Peng-Xia Wang 2,#, Yan-Ting Wang 1, Xing-He Ke 2,3, Minmin Ou 2,3, Shao-Chen Sun 2,✉, Bi-Yun Liao 3,✉, Xuanyi Chen 1,✉
PMCID: PMC12933907  PMID: 41618314

Abstract

Acrylamide is a synthetic material which is widely used in water treatment and paper manufacturing, and it is also generated from food formation, which shows neurological effects, skin irritation, or reproductive toxicity. Several studies focused on the effects of acrylamide on mitochondria and apoptosis in oocytes. In present study, we reported that acrylamide disturbed cell cycle progression of mouse oocyte meiosis. Our data showed that acrylamide caused both germinal vesicle (GV) breakdown and polar body extrusion defects. Further analysis indicated that acrylamide induced DNA damage in the GV oocytes, showing with increased γ-H2A.X expression, which active CHK2 for G2/M transition. This could be confirmed by the altered Cyclin B1 and CDK1 expression in oocytes. Besides, we found kinetochore-microtubule attachment was aberrant in metaphase I (MI) oocytes, which active spindle assembly checkpoint (SAC) for polar body extrusion, and this was confirmed by the consistent presentence of BubR1 and Bub3. This was due to the reduced tubulin acetylation-based microtubule stability, since HDAC6 and NAT10 expression was changed and cold treatment reduced the tubulin polymerization. Taken together, our study reported that acrylamide exposure disrupted cell cycle progression through DNA damage-based MPF activity and tubulin acetylation-based SAC activation in oocytes.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13062-026-00733-3.

Keywords: Oocyte, Meiosis, Cell cycle, DNA damage, Tubulin

Introduction

Acrylamide (ACR) is broadly used as a synthetic material in several industries and scientific processes, such as water treatment, cosmetics, papermaking and gel electrophoresis [1]. Acrylamide also can be formed through a glycation reaction (commonly called ‘Maillard reaction’) during the cooking of some several heat-treated carbohydrate-rich foods [2]. While it has been detected in baked foods [3], chips, breakfast cereals [4], coffee [5], various snacks and baby food [6]. Currently it has been proven that a broad range of population groups, ranging from babies to the elderly, are exposed to ACR in many countries all over the world. ACR is considered genotoxic, neurotoxic and a probable carcinogenic agent in humans [2]. Previous research has demonstrated that ACR can induce peroxidation of cholinergic transmitters in the nervous system, leading to a decrease in transmission ability and potential damage [7]. Moreover, when ACR enters the body through the respiratory tract, it can cause oxidative stress on lung epithelial BEAS-2B cells, resulting in morphological changes and even apoptosis [8]. Additionally, ACR has been associated with an increased risk of ovarian cancer by altering sex hormone levels [9]. In recent years, there has been an increasing number of reports on the impact of acrylamide on female reproductive health. Pregnant women consuming excessive amounts of ACR may experience inhibited fetal growth [10]. Additionally, studies have shown that ACR can result in dysfunction of mouse oocyte organelles [11]. Recent studies have shown that ACR exposure can disrupt oocyte maturation ability by affecting oxidative stress and apoptosis in mouse models [12]. It has also been observed that oral ACR intake can reduce ovarian weight and the number of GV oocytes in mice [13]. Besides, ACR exposure disrupted ZGA in mouse embryos by inducing mitochondrial oxidative stress, leading to DNA damage, abnormal histone modifications, and organelle disturbances [14].

The oocyte plays a crucial role in determining the developmental competence of embryos in women [15]. Oocyte maturation is a complex process that involves two asymmetric meiotic divisions. During these divisions, the oocyte undergoes a series of changes to acquire the necessary genetic material and cellular components for successful fertilization and embryonic development [16]. During cell cycle control of meiosis in oocytes, there are two important stages: meiosis resumption, also known as the G2/M transition, and the progression from metaphase I (MI) to metaphase II (MII) [17]. These meiotic stages play a crucial role in the development and maturation of oocytes. The G2/M transition in oocyte meiosis involves the activation of different signaling pathways and the coordination of cell cycle regulatory factors, such as CDK1 (Cyclin-dependent kinase 1), which are necessary for the transition from the G2 phase to the M phase. The successful progression of meiosis I in the cell cycle is dependent on the high activity of the metaphase promoting factor (MPF) [18]. MPF is a complex formed by the binding of Cyclin B1 and cyclin-dependent kinase (CDK1). The catalytic activity of MPF is mediated through CDK1, and it is worth noting that the binding of Cyclin B1 is essential for the activation of CDK1 [19].

While DNA damage will active the checkpoint activity, which is mediated by ATM-Chk2/ATR-Chk1 signaling pathway, therefore caused the G2/M transition defects, and arrested the oocytes at the GV stage [20]. Cells develop the mechanisms to detect and repair DNA damage in a timely manner: DNA damage such as single-stranded DNA (ssDNA) and double-stranded DNA breaks (DSBs) triggers the activation of the DNA damage response (DDR), which involves the activation of two key serine/threonine protein kinases, ATM and ATR [21]. Checkpoint kinases CHK1 and CHK2 function as downstream substrates of ATM and ATR, playing key roles in slowing or stopping cell cycle progression to allow cells to repair damaged DNA or prevent the inheritance of incompletely replicated DNA or damaged chromosomes. As the CDK1/Cyclin B1 complex acts as a key regulator of the G2/M DNA damage checkpoint and controls the transition from the G2 phase to the M phase [22].

While for the metaphase I arrest, spindle assembly checkpoint (SAC) is the main mechanism for this. After the germinal vesicle breakdown (GVBD), the chromosomes were driven by the microtubules to the metaphase plate, the microtubule-kinetochore attachment could be monitored by the spindle assembly checkpoint proteins. When the chromosomes are well-aligned, SAC activity is silenced and under the activation of APC/C, the oocytes enter anaphase and the chromosomes are separated. In oocytes, Bub1, Bub3, BubR1, Mad2 are shown to be the core SAC proteins, while chromosome passenger complex (CPCs) including Aurora B, survivin etc. also involves into this process [23]. Microtubule stability is critical for the microtubule-kinetochore attachment, while tubulin acetylation determines microtubule stability [24]. This posttranslational modification in oocytes is regulated by the several acetylases and deacetylases such as HDAC6, SIRT1, NAT10 etc., which is regulated by multiple factors such as motor proteins KIFs, PLK1 [25, 26].

Although the toxicity of ACR exposure on the female reproduction is reported, its effects on oocyte maturation, especially on the cell cycle regulation during meiosis is currently uncertain. The purpose of this study was to investigate the potential mechanisms of ACR on oocyte maturation from the perspective of the cell cycle. With the mouse model, we examined the effects and potential pathways underlying ACR toxic effects on oocytes, and the DNA damage, MPF activity, microtubule stability, kinetochore-microtubule attachment, and spindle assembly checkpoint (SAC) activity were examined. Our results indicate that ACR induces DNA damage for G2/M arrest, and ACR treatment reduces the microtubule stability for spindle assembly checkpoint, suggesting the effects of ACR on meiotic cell cycle in oocyte model.

Materials and methods

Antibodies and chemicals

Rabbit anti-gamma H2A.X antibody (ab81299), rabbit monoclonal anti-Cyclin B1 antibody (ab181593), rabbit monoclonal anti-Bub3 antibody (5319(2)), mouse monoclonal anti-CDK1 antibody (ab18), and sheep polyclonal anti-BUBR1 antibody (ab28193) were purchased from Abcam (Cambridge, UK). Alexa Fluor 594 goat anti-rabbit antibody, Alexa Fluor 488, and 594 goat anti-sheep antibody were purchased from Invitrogen (Carlsbad, CA, USA). Rabbit anti-Acetyl-α-Tubulin antibody (D20G3) and Mouse anti-β-actin antibody (8H10D10) were purchased from Cell Signaling Technology (Devers, MA, USA). Rabbit anti-CHK2(bs-1391R) were purchased from Bioss. Rabbit anti-NAT-10(13365_1_AP), Rabbit anti-GAPDH (10494_1_AP), Rabbit anti-αTubulin(11224-1_AP) were purchased from Proteintech. Horseradish peroxidase-conjugated goat anti- mouse/rabbit IgG antibodies were purchased from Beyotime (Nantong, China). All other unnoted chemicals were from Sigma (St. Louis, MO, USA).

Oocyte collection and culture

All were approved by the Animal Care and Use Committee of Nanjing Agricultural University and were performed in accordance with Animal Research Institute Committee guidelines.

All experiments on mice conducted in this study were followed the guidelines of the Animal Research Committee of Nanjing Agriculture University, China. The experiments were specifically approved by the committee of Animal Ethics and Farewell in Nanjing Agriculture University. Institute of Cancer Research (ICR) female mice between 4 and 6 weeks, were utilized in this study. Germinal vesicle stage oocytes were collected from ovaries by using a prefabricated glass tube. These oocytes were then cultured in M16 medium, under liquid paraffin oil, at a temperature of 37 °C in an atmosphere containing 5% CO2 for specific durations.

ACR treatment

ACR was dissolved in dimethyl sulfoxide (DMSO) and diluted in M16 medium to achieve final concentrations of 2mM, 3mM, and 4mM, which was based on our previous studies [27]. The DMSO concentration in the culture medium was less than 0.01%. For release groups, we washed the oocytes ten times (2 min each) in fresh M2 medium after 4 h, 8 h, 12 h culture with 3mM ACR and milrinone, Subsequently, the oocytes were transferred to fresh M16 medium and cultured until reaching the time point when the minority of oocytes underwent GVBD (germinal vesicle breakdown).

Immunofluorescence staining and confocal microscopy

For the detection of γ-H2AX, Cyclin B1, α-tubulin, Acetyl-α-Tubulin, the oocytes were fixed in 4% paraformaldehyde for 30 min at room temperature. Afterwards, they were permeabilized with 0.5% Triton X-100 for 20 min and blocked in 1% BSA-supplemented phosphate-buffered saline (PBS) for 1 h. To visualize kinetochore immunostaining and cold-stable microtubules, M2 medium was pre-cooled at 4 °C for at least 1 h. The oocytes were then transferred to the cooled medium for 6.5 min of cold treatment before fixation. For BUBR1 and Bub3 staining, the oocytes were first transferred to 0.5% Triton X-100 for 5 min, followed by three washes of 5 min each in washing buffer (0.1% Tween 20 and 0.01% Triton X-100 in PBS). After fixation with 4% paraformaldehyde for 20 min at room temperature and three additional washes in washing buffer, the oocytes were blocked for 1 h in 1% BSA in PBS. Subsequently, the oocytes were incubated with primary antibodies overnight at 4 °C, followed by three washes in washing buffer. The corresponding secondary antibodies were then applied and incubated with the samples for 1 h at room temperature. The primary antibodies used were anti-γ-H2AX (1:200), anti-Cyclin B1 (1:500), anti-α-tubulin-FITC (1:400), anti-Acetyl-α-Tubulin (1:500), anti-Bub3 (1:100), and anti-BUBR1 (1:50). The secondary antibodies used were Alexa Fluor 488/594 goat anti-rabbit or sheep IgG (1:200). After three washes in washing buffer, the oocytes were stained with Hoechst 33,342 for 15 min at room temperature and then mounted on glass slides for examination using a confocal laser scanning microscope (Zeiss LSM 800 META, Jena, Germany).

Western blot analysis

A total of 200 oocytes per group were collected and lysed with NuPAGE LDS Sample Buffer and boiled at 100 ℃ for 10 min. Then the proteins were separated by electrophoresis on a 10% Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 150 V for 1 h. Subsequently, the proteins were transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Following the transfer, the membranes were blocked with TBST solution containing 5% nonfat milk at room temperature for a minimum of 1 h. After a brief wash in TBST, the blocked membrane was incubated with the anti-β-actin antibody (dilution 1:2000), anti-gamma H2A.X (1:1000), anti-CDK1 (1:1,000), and anti-Cyclin B1 (1:1,000), anti-HDAC6(1:1000), anti-NAT10(1:1000), anti-CHK2 (1:500), anti-GAPDH (1:2000), anti-Acetyl-α-Tubulin (1:1000), anti-α-Tubulin(1:2000) at 4 ℃ overnight. After three consecutive washes in TBST for 10 min each, the membranes were then incubated with the HRP-conjugated secondary antibodies (dilution 1:2000) in TBST for 1 h. Finally, the membranes were washed three times in TBST and processed using the ECL Plus Western Blotting Detection System (Tanon-3900, China). The band intensity values were analyzed using Image J software.

Statistical analysis

At least three independent biological replicates were conducted for each treatment. Statistical comparisons were conducted by paired-wise t-test using GraphPad Prism 5 software (GraphPad, San Diego, CA). A significance level of P < 0.05 was considered statistically significant.

Results

Effects of ACR on G2/M transition and polar body extrusion in mouse oocyte meiosis

To investigate the toxic effect of acrylamide (ACR) exposure on oocytes, ACR was added to the mouse oocyte culture medium at final concentrations of 2 mM and 3 mM respectively. As shown in Fig. 1A, after 12 h of culture, there was no statistical significance in the polar body extrusion rate of oocytes between the 2 mM ACR treatment group and the control group. However, the polar body extrusion rate of oocytes in the 3 mM ACR treatment group was significantly lower than that in the control group (Control group: 64.35 ± 3.77%, n = 112; 2 mM ACR group: 45.95 ± 18.09%, n = 97, P > 0.05; 3 mM ACR group: 24.55 ± 5.26%, n = 110, P < 0.05) (Fig. 1B). We selected the 3 mM ACR concentration for subsequent studies, and we further examined the effect of ACR on the resumption of meiosis in mouse oocytes. To extend the exposure time of oocytes to ACR, milrinone was added to the culture medium to arrest oocytes at the germinal vesicle (GV) stage. Oocytes were released after being arrested for 4 h, 8 h, and 12 h, respectively. After release, the proportion of oocytes that underwent germinal vesicle breakdown (GVBD) was counted (Fig. 1C). As shown in Fig. 1D, the statistical results were as follows: at 4 h, Control group: 85.51 ± 7.90%, n = 122; 3 mM ACR group: 50.84 ± 16.32%, n = 121, P > 0.05; at 8 h, Control group: 77.47 ± 8.59%, n = 112; 3 mM ACR group: 39.87 ± 16.32%, n = 107, P < 0.01; at 12 h, Control group: 73.08 ± 8.97%, n = 126; 3 mM ACR group: 2.72 ± 2.87%, n = 120, P < 0.01. We found significant differences between the 8-hour and 12-hour ACR exposure groups and the control group, we selected 8 h for subsequent experiments. To further confirm our finding, we performed release experiment. After 8 h of ACR exposure, oocytes were released, washed, and then cultured in fresh ACR-free medium for another 4 h. A subset of oocytes was able to resume development and undergo GVBD (Fig. 1E). Compared with the continuous ACR exposure group, the percentage of GVBD in the release group (Control: 86.58 ± 3.13%, n = 122; 3 mM ACR: 43.20 ± 7.72%, n = 114; Release group: 54.14 ± 9.08%, n = 113) was significantly increased (Fig. 1E), further indicating that ACR exposure affects the cell cycle progression of mouse oocytes.

Fig. 1.

Fig. 1

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Effects of ACR on G2/M transition and polar body extrusion in mouse oocyte meiosis. (A) The typical image for the PB1 extrusion of control, 2 mM and 3 mM ACR-treated groups. (B) The rate of PB1 extrusion was significantly decreased in the 3mM ACR-treated group compared with the control. *, P < 0.05. (C) The typical picture for the oocyte GVBD after ACR exposure. (D) The oocyte GVBD rate at different durations of exposure to 3 mM ACR. **, P < 0.01. (E) The typical image for the GVBD of control, 3 mM ACR-treated and release from 3 mM ACR groups. (F) The oocyte GVBD rate of control, ACR-treated and release from 3 mM ACR groups. **, P < 0.01. Bar = 100 μm

Acrylamide affects cyclin B1/CDK1 during meiosis in mouse oocytes

Since ACR exposure significantly reduced the GVBD rate, we next examined the activity of maturation-promoting factor (MPF) to explore the potential mechanism for oocyte GV arrest. Since MPF is a complex composed of the catalytic subunit CDK1 and the regulatory subunit Cyclin B1, and plays a crucial role in the resumption of meiosis. Immunofluorescence staining of Cyclin B1 showed that the signal in the cytoplasm of GV-stage oocytes exposed to ACR was significantly weakened (Fig. 2A). Analysis of fluorescence intensity data further confirmed this result: compared with the control group, the level of Cyclin B1 in the ACR group was significantly decreased (Control group: 1.00 ± 0.00, n = 59; ACR group: 0.80 ± 0.08, n = 59, P < 0.05; Fig. 2B). Western blot results showed that the expression of Cyclin B1 protein in GV-stage oocytes exposed to ACR was lower than that in the control group (Fig. 2C), which was confirmed by band intensity analysis (Control group: 1.00 ± 0.00; ACR group: 0.83 ± 0.06, P < 0.05; Fig. 2D). Similar to Cyclin B1, the level of CDK1 protein in GV-stage oocytes exposed to ACR was also lower than that in the control group (Fig. 2E), which was also confirmed by band intensity analysis (Control group: 1.00 ± 0.00; ACR group: 0.75 ± 0.08, P < 0.05; Fig. 2F). These results indicate that ACR exposure impairs the MPF activity required for the G2/M transition during oocyte meiosis.

Fig. 2.

Fig. 2

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Acrylamide affects Cyclin B1/CDK1 during meiosis in mouse oocytes. (A) Typical image of Cyclin B1 fluorescence signal in oocytes after ACR exposure. White, Cyclin B1; blue, DNA. (B) The fluorescence intensity of Cyclin B1 in oocytes was significantly decreased after ACR exposure. *, P < 0.05. (C) Western blot results for Cyclin B1 expression in oocytes after ACR exposure. (D) Band intensity analysis of Cyclin B1 expression in ACR exposed group. Cyclin B1 expression was significantly decreased in ACR exposed group compared with the control group. *, P < 0.05. (E) Western blot results for CDK1 expression in oocytes after ACR exposure. (F) Band intensity analysis of CDK1 expression in ACR exposed group. Compared with the control group, the expression of CDK1 protein in ACR exposed group was significantly decreased *, P < 0.05. Bar = 20 μm

Acrylamide exposure induces DNA damage in mouse oocytes

Since DNA damage is one main cause of MPF activity loss, to investigate the potential mechanism of ACR exposure on G2/M transition defect, we conducted the DNA damage marker γ-H2A.X test. As shown in Fig. 3A, γ-H2A.X showed positive signals in the nuclei of GV-stage oocytes, and the fluorescence intensity in oocytes exposed to ACR was significantly higher than that in the control group. Analysis of fluorescence intensity data further confirmed this result: compared with the control group, the γ-H2A.X signal in oocytes exposed to ACR was significantly increased (Control group: 1.00 ± 0.00, n = 59; ACR group: 1.21 ± 0.03, n = 61, P < 0.01) (Fig. 3B). In addition, western blot results showed that the expression of γ-H2A.X in GV-stage oocytes exposed to ACR was higher than that in the control group (Fig. 3C), which was confirmed by band intensity analysis (Control group: 1.00 ± 0.00; ACR group: 1.13 ± 0.04, P < 0.05) (Fig. 3D). We also examined the activity of the DNA damage checkpoint protein CHK2. Similar to γ-H2A.X, the level of CHK2 protein in GV-stage oocytes exposed to ACR was higher than that in the control group (Fig. 3E), which was also confirmed by band intensity analysis (Control group: 1.00 ± 0.00; ACR group: 1.41 ± 0.10, P < 0.05) (Fig. 3F). These results indicate that ACR exposure causes DNA damage and triggers the activation of the DNA damage checkpoint at the G2 phase of mouse oocytes, thereby arresting oocyte maturation.

Fig. 3.

Fig. 3

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Acrylamide exposure induces DNA damage in mouse oocytes. (A) Typical image of γ-H2A.X fluorescence signal after ACR exposure. Red, γ-H2A.X; Blue, DNA. (B) The fluorescence intensity of γ-H2A.X was significantly enhanced after ACR exposure. **, P < 0.01. (C) Western blot results for γ-H2A.X expression after ACR exposure. (D) Band intensity analysis of γ-H2A.X expression in ACR exposed group. The γ-H2A.X protein expression was significantly increased in ACR exposed group compared with the control group. *, P < 0.05. (E) Western blot results for CHK2 expression after ACR exposure. (F) Band intensity analysis of CHK2 expression in ACR exposed group. Compared with the control group, the expression of CHK2 protein in ACR exposed group was significantly increased *P < 0.05. Bar = 10 μm

Acrylamide affects kinetochore-microtubule attachment for SAC during oocyte meiosis

Kinetochore-microtubule (K-MT) structures play a crucial role in chromosome alignment and segregation. The establishment of correct and stable bidirectional connections between kinetochores and microtubules is closely related to microtubule dynamic stability, monitored by spindle checkpoint proteins, and serves as a key mechanism regulating the metaphase-anaphase transition. Since ACR causes the failure of the metaphase-anaphase transition in oocytes, we hypothesized that this might occur by disrupting K-MT connections, activating checkpoint proteins, and thereby inhibiting cytokinesis. As shown in Fig. 4A, kinetochores of oocytes in the control group could form stable connections with microtubules, and chromosomes were well-aligned. In contrast, ACR exposure led to defects in kinetochore-microtubule attachment in oocytes, with only a small number of microtubules able to bind to centromeres, showing with partial attachment or no attachment. Statistical analysis showed that compared with the control group, the abnormal rate of K-MT attachment in the ACR exposure group was significantly increased (Control group: 25.50 ± 7.59%, n = 57; ACR group: 56.08 ± 0.92%, n = 57, P < 0.05) (Fig. 4B). Subsequently, we examined the checkpoint proteins BubR1 and Bub3. Staining was performed to evaluate the activity and localization of BubR1 and Bub3, which play crucial roles in activating the spindle assembly checkpoint (SAC) to halt cell division when errors in K-MT attachment are detected. We observed strong BubR1 and Bub3 signals at the centromeres of oocytes exposed to ACR, while only weak signals were detected in the control group (Fig. 4C and D). This indicates that ACR exposure leads to increased accumulation of BubR1 and Bub3 at centromeres and activation of the spindle assembly checkpoint (SAC).

Fig. 4.

Fig. 4

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Acrylamide affects kinetochore-microtubule attachment for SAC during oocyte meiosis. (A) In the control groups, a clear stable attachment between kinetochores and microtubules (K-MT) was observed, whereas there were barely microtubules attached to kinetochores after exposure to ACR. (B) The percentage of K-MT detachment in ACR-treated oocytes was significantly higher compared to the control group. *, P < 0.05. (C) Stronger BubR1 fluorescence signals were observed at the kinetochores of oocytes in the ACR- exposed group, whereas no significant signals were detected in the control group. (D) Stronger Bub3 fluorescence signals were observed at the kinetochores of oocytes in the ACR- exposed group, whereas no significant signals were detected in the control group. Bar = 10 μm

Acrylamide affects tubulin acetylation for microtubule stability in mouse oocytes

Microtubules are crucial for cell cycle progression during mitosis and meiosis. Since ACR disrupts the kinetochore-microtubule connection structure on which microtubule stability depends, we hypothesized that microtubule stability is also impaired. Consistent with this hypothesis, after cold treatment of oocytes to depolymerize unstable microtubules, we observed a significant reduction in microtubule signals in the ACR exposure group compared with the control group (Fig. 5A). Fluorescence intensity analysis further supported this finding (Control group: 1.00 ± 0.00, n = 46; ACR group: 0.62 ± 0.14, n = 46, P < 0.05) (Fig. 5B). These results indicate that ACR impairs microtubule stability during oocyte meiosis. We also performed acetylated tubulin (Ac-tubulin) immunofluorescence staining and found that the fluorescence signal in the ACR exposure group was significantly reduced compared with the control group (Fig. 5C). Statistical analysis further confirmed this observation (Control group: 1.00 ± 0.00, n = 61; ACR group: 0.71 ± 0.10, n = 52, P < 0.05) (Fig. 5D). We also conducted Western blot analysis on the expression of Ac-tubulin and found that the expression of Ac-tubulin in the ACR exposure group was significantly lower than that in the control group (Control group: 1.00 ± 0.00; ACR group: 0.66 ± 0.01, P < 0.001) (Fig. 5E). Considering the decrease in Ac-tubulin expression, we further examined the expression of HDAC6 and NAT10. We found that the expression of HDAC6 in the ACR exposure group was significantly higher than that in the control group (Control group: 1.00 ± 0.00; ACR group: 1.30 ± 0.04, P < 0.01) (Fig. 5F), while the expression of NAT10 was decreased (Control group: 1.00 ± 0.00; ACR group: 0.73 ± 0.03, P < 0.01) (Fig. 5G).

Fig. 5.

Fig. 5

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Acrylamide affects tubulin acetylation for microtubule stability in mouse oocytes. (A) After 8 h culture, the oocytes preformed 6.5 min cold treatment were used to test the stability of microtubules. After exposure to ACR, the microtubule signals were lower than that in the control group. Green, α-tubulin. Blue, DNA. (B) The analysis of fluorescence intensity revealed a significant decrease in the microtubule intensity in the ACR-exposed group compared to the control group. *, P < 0.05. (C) Exposed to ACR led to considerably decreased fluorescence signals of Acetyl-α-tubulin. Pink, Acetyl-α-tubulin. Blue, DNA. (D) The fluorescence intensity of Acetyl-α-tubulin was significantly reduced following ACR exposure. *, P < 0.05. (E) Western blot results for Acetyl-α-tubulin expression in the ACR-exposed groups, and the band intensity analysis for the protein expression of Acetyl-α-tubulin. Compared with the control group, the protein expression of Acetyl-α-tubulin was significantly decreased in the ACR- exposed group. ***, P < 0.001. (F) Western blot results for HDAC6 expression in the ACR- exposed groups, and the band intensity analysis for the protein expression of HDAC6. Compared with the control group, the protein expression of HDAC6 was significantly increased in the ACR- exposed group. **, P < 0.01. (G) Western blot results for NAT10 expression in the ACR-treated groups, and the band intensity analysis for the protein expression of NAT10. Compared with the control group, the protein expression of NAT10 was significantly decreased in the ACR- exposed group. **, P < 0.01. Bar = 10 μm

Discussion

For human, the acrylamide exposure in food industry and its toxicity are already widely known [28], and its relationship with the diseases such as cancers is well setup [29, 30], however, the data for reproductive disorders are still largely lack. It is necessary to clarify the reproductive toxicity of ACR. In present study we examined the effects of ACR exposure on oocyte maturation from cell cycle aspect. We found that ACR significantly affects oocyte G2/M transition and MI stage arrest, and this could be induced by DNA damage-based MPF activity, and tubulin acetylation-based SAC activation. Our data provided important insights for the ACR on cell cycle with mouse oocyte meiosis model, which will contribute to the understanding of ACR on female reproduction.

Previous studies showed that ACR exposure disturbed the zygotic genome activation in mouse embryos [27]. Recent study also showed that ACR could affect bovine oocyte maturation [31], and exposure to ACR could disrupt organelle distribution and functions in oocytes [32]. However, the effect of ACR exposure on the cell cycle regulation of oocyte maturation is still unclear. We first investigated its impact on two key markers of oocyte maturation: germinal vesicle breakdown (GVBD) and polar body extrusion (PBE). We showed that ACR exposure both affected meiosis resumption and polar body extrusion, these two are generally monitored by the G2/M transition and SAC-based MI-AI transition [33]. Therefore, we detected MPF activity, and the data suggested that CDK1 and Cyclin B1 expression were altered, which confirmed our hypothesis, indicating the effects of ACR on cell cycle.

The effect of ACR on the cell cycle of somatic cells has been reported in multiple models. For example, it is shown that ACR-induced senescence from ROS level, which is mainly through p38 and JNK to regulate ATF3-dependent p53 for cellular senescence in macrophages, which these molecules are mainly for cell cycle regulation [34]. This is mainly because of ACR on oxidative stress, since ACR inhibits the growth and metabolic activity of S. pombe cells, increases ROS level, and oxidative stress induced by ACR causes alterations in cell cycle regulation, resulting in chromosome segregation errors, showing sister chromatid non-disjunction during mitosis [35]. ACR reduces the viability of human chondrocytes and it also increases the expression of cell cycle arrest-related proteins in chondrocytes, including p53, CDK1 inhibitor 1, and CDK inhibitor proteins [36]. While DNA damage could active the checkpoint, and this is mainly through CHK2, our further analysis showed that γ-H2A.X fluorescence in the germinal vesicle increased, with increased CHK2 expression, together indicating the occurrence of DNA damage. Several negative environmental factors such as DEHP can cause DNA damage, which in turn activates the G2/M checkpoint and prevents cell cycle progression [37]. Studies have detected DNA damage in sperm from both ACR-exposed male mice and their male offspring [38], and ACR exposure has also been shown to cause DNA damage and ROS production in human colon adenocarcinoma Caco-2 cells, thereby inducing apoptosis [39]. These suggested that the observed decrease in CDK1-Cyclin B1 in our data may be a result of ACR-induced DNA damage. While environmental toxins are widely reported to affect cell cycle progression in oocytes, for example, gefitinib exposure affects GVBD and polar body extrusion for meiotic progression in mouse oocytes [40]. Propylparaben exposure also disturbs G2/M and metaphase-anaphase transition in oocyte meiosis [41].

Beyond arresting the G2/M transition, we also found that ACR causes failure of the metaphase-anaphase I transition in mouse oocytes. For the potential causes, we found that ACR affected kinetochore-microtubule (K-MT) attachment and activated SAC for metaphase I arrest, which was confirmed by the positive signals of BubR1 and Bub3 on the kinetochores. During meiosis, K-MT attachment is monitored by SAC activity: incorrect attachment leads to changes in tension, which activates checkpoints through kinetochores to halt the cell cycle until errors are resolved [42]. BubR1 and Bub3 are two key spindle checkpoint proteins, since the mitotic checkpoint complex (MCC) formed by them could inhibit the activity of APC/C (anaphase-promoting complex/cyclosome) until all chromosomes establish stable bidirectional connections with microtubules and are neatly arranged on the spindle equator [43]. Given that microtubule stability is crucial for correct kinetochore-microtubule connections, our further study showed that ACR disrupted microtubule stability due to the cold treatment experiment. We then analyzed microtubule acetylation: as a post-translational modification, acetyl-α-tubulin is present in various microtubule structures and maintains microtubule stability and normal spindle formation, with changes in its levels directly affecting microtubule stability [44]. It has been reported that changes in acetyl-α-tubulin levels in mouse oocytes lead to spindle assembly failure and chromosome misalignment due to the loss of TNKS activity during meiosis [45]. Our data showed that ACR altered tubulin acetylation level in the oocytes. While tubulin acetylation is mainly catalyzed by the acetyltransferase NAT10, while deacetylation is primarily carried out by deacetylases HDAC6 and SIRT2 [46]. Our further analysis showed that ACR exposure affected both HDAC6 and NAT10 protein expression for tubulin acetylation levels.

In future perspective, several issues should be clarified. For example, the current study used in vitro culture model to clarify the potential causes of ACR on oocytes, while it cannot simulate the complex endocrine environment and metabolic processes in vivo, and whether ACR metabolites (such as glycinamide) are involved in oocyte damage needs further verification; besides, the ACR concentration used in the experiment could not reflect the daily human exposure level, and the impact of actual exposure dose is needed from public health aspect; moreover, it will be interesting to check the effects of ACR on the oocytes from different ages.

In summary, our results indicate that ACR exposure affects the two critical cell cycle processes of oocyte meiosis: it induces DNA damage for MPF activity-based G2/M transition, and it also disrupts tubulin acetylation for kinetochore-microtubule attachment-based spindle assembly checkpoint, which ultimately disturb meiosis resumption and polar body extrusion.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (2.3MB, tif)

Acknowledgments

This study was supported by the Research Funding from Suzhou Clinical Center of TCM Reproduction (Szlcyxzx202107), and Suzhou Applied Basic Research Project (SYW2025148, SYW2024030).

Author contributions

XC, BYL, SCS conceived the study. PXW, SC performed the majority of the experiments. SC, PXW, SCS analyzed the data. YTW, XHK, MMO contributed to materials and agents. SC, SCS, PXW wrote the manuscript.

Funding

Research Funding from Suzhou Clinical Center of TCM Reproduction (Szlcyxzx202107), and Suzhou Applied Basic Research Project (SYW2025148, SYW2024030).

The authors declares that there are no competing interests.

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.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Shanshan Chen and Peng-Xia Wang contributed equally to this work.

Contributor Information

Shao-Chen Sun, Email: sunsc@njau.edu.cn.

Bi-Yun Liao, Email: yyfylby@163.com.

Xuanyi Chen, Email: chenxuanyi1212@163.com.

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Associated Data

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Supplementary Materials

Supplementary Material 1 (2.3MB, tif)

Data Availability Statement

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

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