Nivalenol affects Cyclin B1 level and activates SAC for cell cycle progression in mouse oocyte meiosis

Abstract Objectives Nivalenol (NIV) is a secondary metabolite of type B trichothecene mycotoxin produced by Fusarium genera, which is widely found in contaminated food and crops such as corn, wheat and peanuts. NIV is reported to have hepatotoxicity, immunotoxicity, genotoxicity, and reproductive toxicity. Previous studies indicate that NIV disturbs mammalian oocyte maturation. Here, we reported that delayed cell cycle progression might be the reason for oocyte maturation defect caused by NIV exposure. Methods and Results We set up a NIV exposure model and showed that NIV did not affect G2/M transition for meiosis resumption, but disrupted the polar body extrusion of oocytes. Further analysis revealed that oocytes were arrested at metaphase I, which might be due to the lower expression of Cyclin B1 after NIV exposure. After cold treatment, the microtubules were disassembled in the NIV‐exposed oocytes, indicating that NIV disrupted microtubule stability. Moreover, NIV affected the attachment between kinetochore and microtubules, which further induced the activation of MAD2/BUBR1 at the kinetochores, suggesting that spindle assemble checkpoint (SAC) was continuously activated during oocyte meiotic maturation. Conclusions Taken together, our study demonstrated that exposure to NIV affected Cyclin B1 expression and activated microtubule stability‐dependent SAC to ultimately disturb cell cycle progression in mouse oocyte meiosis.


| INTRODUCTION
Food safety is a global concern especially in the situation of high mycotoxin contamination occurrence. 1 About 25% of the crops in the world have been contaminated by molds and fungi growth, which may further aggravate under the on-going global warming. 2 Mycotoxins are a group of low molecular compounds with structural diversity produced by different types of fungi. 3 One of the most common fungi found worldwide is Fusarium genera. 4 Nivalenol (NIV) is considered to be an important secondary metabolite of trichothecene mycotoxin produced by Fusarium species since it is frequently found in contaminated crops such as corn, wheat and peanuts. 5 Previous studies have demonstrated that NIV is obviously toxic to digestive and immune systems. Animal experiments show that NIV not only significantly decreases red and white blood cell count, but also affects the lymph follicle germ centers of spleen, lymph node, thymus and bone marrow in mice. 6,7 NIV also accelerates J774A.1 macrophages apoptosis and shows anti-proliferative effects with a dose-dependent manner. 8 In addition, 0.5 μg/ml NIV induces HeLa cells G1/S and G2/M transition failure by means of autoradiography. 9 Recent evidences have identified that NIV is also potentially toxic to reproductive system. In males, testis damage and reduction on spermatogenic cells number are observed after NIV exposure. 10 In females, subchronic toxicity study has suggested that 100 ppm NIV increases ovarian follicular atresia and inhibits corpus luteum development in F344 rats. 11 Besides, our previous results show that NIV disrupts mitochondria function and induces apoptosis during oocyte maturation. 12,13 Oocyte meiotic maturation is a prerequisite for successful fertilization and subsequent embryo development. Meiosis resumption (G2/M transition) and the progression to metaphase II (MII) are two major stages in oocyte meiosis. 14 Oocyte is arrested in germinal vesicle (GV) stage until luteinizing hormone (LH) surge stimulation, quickly followed by germinal vesicle breakdown (GVBD) which marks the beginning of prometaphase I (pro-MI). 15 After oocyte enters pro-MI, acentriolar microtubule organizing centers (MTOCs) near the nuclear envelope are fragmented and highly dynamic microtubules capture the condensed chromosome to build attachment between kinetochore and microtubules (K-MT), indicating that the initiation of bipolar spindle formation and chromosome transport to the cell equator. 16 The microtubules which encounter and build correct attachment with a kinetochore become stabilized, whereas are depolymerized soon. 17 Then oocyte reaches metaphase I (MI) when chromosomes align at the spindle equator. At MI stage, the spindle migrates to the oocyte cortex along its long axis, waiting the signal of metaphase-anaphase transition to progress through MI and extrude the first polar body (PB1). 18 Finally, oocyte is arrested at MII stage until fertilization.
The successful cell cycle progression of meiosis I is depended on high metaphase promoting factor (MPF) activity. 19 MPF, a complex of Cyclin B1 and cyclin-dependent kinase (CDK1), shows its catalytic activity though CDK1 and it is noted that binding to Cyclin B1 is essential for CDK1 activation. 20 When all chromosomes build stable attachment with microtubules and align at the spindle plate, Cyclin B1 is dramatically degraded by an APC/C-dependent process and subsequently inactivate MPF. 21 Besides, proper anaphase onset and chromosome segregation are also critical to ensure oocyte maturation quality. Correct and stable bipolar K-MT attachment is monitored by the meiotic spindle-assembly checkpoint (SAC) which could produce a "wait-anaphase" signal to delay anaphase for faithful chromosome segregation during oocyte maturation. 17 Several core components of SAC proteins, such as MAD1, MAD2, BUB1, BUB2, and BUBR1 (MAD3 in yeast) are reported to regulate metaphase I arrest in meiosis. 22 It is suggested that unattached kinetochores can recruit MAD2 to bind CDC20, the co-activator of APC/C, and further associate with BUBR1 and BUB3 to form the mitotic checkpoint complex (MCC) for cell cycle control. 23 Although the toxicity of NIV on oocyte and sperm are reported, the mechanism is still unclear. In this study, we investigated the effects of NIV on cell cycle control during oocyte meiotic maturation.
Surprisingly, our results showed that NIV did not affect G2/M transition; however, a significant reduction in Cyclin B1 level, the disrupted microtubule stability and K-MT attachment indicated that NIV induced metaphase I arrest in mouse oocytes. Our data provided an explanation for the NIV effects on oocyte maturation quality from cell cycle aspect.

| Antibodies and chemicals
Rabbit polyclonal anti-gamma H2AX antibody (ab2893), rabbit monoclonal anti-Cyclin B1 antibody (ab181593), mouse monoclonal anti-  Denuded germinal vesicle stage oocytes were obtained from chopped ovaries with a prefabricated glass tube and then cultured in M16 medium under liquid paraffin oil at 37 C in 5% CO 2 atmosphere for specific times.

| Nivalenol treatment
The nivalenol (NIV) was dissolved in dimethyl sulfoxide (DMSO) to a 50 mM reserve solution. For high-dose groups, 50 mM NIV was directly diluted to final concentration of 100 and 200 μM with M16 medium. For low dose groups, 50 mM NIV was first diluted to a median concentration of 2 mM with DMSO and then produced the final concentrations of 3 and 5 μM in M16 medium, which was based on previous studies on oocytes. 24 The final DMSO concentration administered to oocytes was <0.4%. For release groups, we washed the oocytes 10 times (2 min each) in fresh M2 medium after 2 h culture with 5 μM NIV, the time point when most oocytes underwent GVBD. Then these oocytes were transferred to fresh M16 medium and cultured another 10 h under liquid paraffin oil at 37 C in 5% CO 2 atmosphere, since most oocytes reached MII stage after 12 h culture.

| Immunofluorescence staining and confocal microscopy
For detections of γ-H2AX, Cyclin B1, α-tubulin and MAD2, the oocytes were fixed in 4% paraformaldehyde for 30 min at room temperature, followed by permeabilization with 0.5% Triton X-100 for 20 min and blocking in 1% BSA-supplemented phosphate buffer saline (PBS) for 1 h.
For kinetochore immunostaining and cold-stable microtubules visualization, M2 medium was pre-cooled at 4 C for at least 30 min and oocytes were transferred to the medium for 5 min cold treatment before fixation.
For BUBR1 staining, oocytes were first transferred to 0.5% Triton X-100 for 5 min and then washed three times (5 min each) in washing buffer

| Fluorescence intensity analysis
Image J software (National Institutes of Health, Bethesda, MD) was used for fluorescence intensity measurement. The control and treated oocytes were placed in different area on the same glass slide and scanned in the same environment using the same parameters. A region of interest on the target image was detected and the average of all mean values between the control and treated groups were used to perform statistical analysis. After three washes in TBST (10 min each), immunoblots were labeled with conjugated anti-mouse or anti-rabbit antibodies (1:1000) 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, China) and then Image J software was used to analyze the band intensity values.

| Statistical analysis
At least three independent biological replicates were performed for each treatment. Statistical comparisons were conducted by pairedwise t-test or ANOVA using GraphPad Prism 5 software (GraphPad, San Diego, CA). Data were expressed as mean ± SEM and the number of oocytes observed (n) was put in parentheses. p < 0.05 was considered statistically significant.

| NIV causes metaphase I arrest in oocyte meiosis
We next adopted NIV release approach to check the effects of NIV on oocyte cytokinesis. As shown in Figure 2A Figure 2C). Further analysis for pro-MI and MI stage distribution showed that the oocytes exposed to NIV mostly delayed to reach MI compared with the control groups, since the percentage of pro-MI oocytes was much higher in the NIV-treated group (Control: 4.16 ± 2.13%, n = 166; NIV group: 19.21 ± 3.45%, n = 137, p < 0.05; Figure 2D). We also measured the distance from the spindle pole to the cortex (length, L) and the diameter (D) of oocytes to check early MI and late MI stage distribution. As shown in Figure 2E, the ratio of the spindle migrated to the cortex (L/D) in the NIV-treated group was significantly increased compared with the control oocytes (Control: 0.09 ± 0.02, n = 43; NIV group: 0.21 ± 0.02, n = 47, p < 0.01). These results indicated that NIV induced metaphase I block during oocyte meiosis.

| NIV affects Cyclin B1 expression for cell cycle progression in oocytes
To explore how NIV induced oocyte cell cycle progression defects, we next examined the activity of MPF, a complex of the catalytic subunit CDK1 and the regulatory subunit Cyclin B1, as it plays a vital role after meiosis resumption. We first quantified the CDK1 and Cyclin B1 level after 9 h culture in NIV-treated oocytes and found that NIV did not affect the expression of CDK1, but the Cyclin B1 level was dramatically lower than control oocytes ( Figure 3A). Statistical analysis showed a significant lower intensity of Cyclin B1 in the NIV exposure oocytes after 9 h culture (Control: 1.00 ± 0.00; NIV group: 0.37 ± 0.01, p < 0.001; Figure 3B). We also stained Cyclin B1 and the immunofluorescence results showed that the signals of Cyclin B1 at MI stage were much weaker in the NIV exposure oocytes ( Figure 3C).
The fluorescence intensity analysis data also confirmed this finding (Control: 1.00 ± 0.00, n = 57; NIV group: 0.58 ± 0.04, n = 60, p < 0.01; Figure 3D). Since Cyclin B1 is rapidly accumulated after GVBD and continuously degraded by an APC/C-dependent manner before anaphase I onset, we asked whether NIV might induce Cyclin B1 accumulation defect when oocyte re-enters meiosis. The Cyclin B1 level in the NIV-treated group after 3 h and 6 h culture was investigated subsequently. Western blotting results showed that the Cyclin B1 expression increased at 6 h culture compared with 3 h in the control group, while it was markedly decreased after NIV treatment in 6 h culture oocytes ( Figure 3E), which was confirmed by the band intensity analysis (Control: 1.00 ± 0.00; NIV group: 0.42 ± 0.02, p < 0.01; Figure 3F). Therefore, we assumed that NIV affected Cyclin B1 expression for the cell cycle progression of mouse oocytes.

| NIV affects microtubule stability in oocyte meiosis
Microtubules are crucial for cell cycle progression in both mitosis and meiosis, we subsequently explored the effects of NIV on microtubule stability. As shown in Figure 4A, the oocytes in the control groups showed normal two poles and barrel-shaped spindles after 9.5 h culture, while NIV treatment caused the aberrant spindle morphology.
However, a higher abnormal spindle rate after NIV exposure was found (Control: 21.09 ± 2.22%, n = 45; NIV group: 53.50 ± 3.29%, n = 36, p < 0.05; Figure 4B). While we performed cold treatment to depolymerize the unstable microtubules after 9.5 h culture, we found that the spindle area and microtubule signals were both markedly decreased than the control oocytes ( Figure 4C). The area calculation (Control: 1.00 ± 0.00, n = 33; NIV group: 0.81 ± 0.03, n = 32, p < 0.05) and fluorescence intensity analysis (Control: 1.00 ± 0.00, n = 33; NIV group: 0.61 ± 0.08, n = 32, p < 0.05) also confirmed this finding ( Figure 4D), indicating that the microtubule stability was destroyed by NIV in oocyte meiosis. Green: α-tubulin; Blue: DNA; Bar = 20 μm. The proportion of ATI/MII stage was much lower but higher for MI stage after NIV treatment. *p < 0.05. (D) The rate of pro-MI stage oocyte was much higher after exposure to NIV than the control group. Blue, DNA; Bar = 5 μm; *p < 0.05. (E) Measurement of spindle position to cortex. We defined the distance from the spindle pole to cortex as L and the dimeter of oocytes as D. The ratio of L/D was considerably increased after NIV exposure (Control: n = 43; NIV group: n = 47). Green, α-tubulin; Blue, DNA; Bar = 20 μm; **p < 0.01.

| NIV affects K-MT attachment for SAC in oocyte meiosis
K-MT attachment contributes to chromosome alignment and segregation, which depends on microtubule stability. Due to the fact that NIV induced microtubule depolymerization by cold treatment, we presumed that NIV might affect the stabilization of K-MT attachment.
We performed cold treatment to better visualize the stable connection of K-MT. As expected, exposure to NIV resulted in K-MT detachment of oocytes, showing with barely microtubules which could attach with kinetochores; while stable microtubules connected with kinetochores in the control oocytes were observed after cold treatment ( Figure 5A). The statistical analysis showed that the percentage of abnormal K-MT attachment was much higher in the NIV exposure group (Control: 20.54 ± 3.13%, n = 52; NIV group: 51.99 ± 3.60%, n = 60, p < 0.01; Figure 5B). Since improper K-MT attachment signal can activate SAC for MI arrest, we therefore tested the activity/ localization of MAD2 and BUBR1, two important regulators of SAC.
After culturing oocytes with 9.5 h, strong MAD2 signals were still found at the kinetochores of NIV-treated oocytes, whereas few signals could be detected in the control oocytes ( Figure 5C); a similar result was also observed for the BUBR1 signals ( Figure 5D). These findings indicted that NIV caused the continuous SAC activation even after 9.5 h culture, which might be due to K-MT attachment defect in oocyte meiosis.

| DISCUSSION
Mycotoxins have been attracted great attention since they are widely found worldwide and toxic to humans and animals. Many studies have focused on the oxidative stress, DNA damage and apoptosis to clarify the adverse effects of mycotoxin exposure, but few studies focus on cell cycle aspect. In present study, we used NIV, a typical secondary metabolite of trichothecene mycotoxin, to investigate its effects and mechanisms on cell cycle of oocyte meiosis. Our results indicated that Our results suggested that no change of γ-H2AX expression after NIV exposure, which confirmed our findings for the negative effects of NIV on oocyte meiosis resumption.
We next analyzed the meiotic progression after GVBD. Our results showed that NIV decreased the oocyte ability to extrude PB1 but it could be rescued after releasing from NIV exposure, which were consistent with previous studies. 13 Cell cycle analysis indicated that NIV exposure induced pro-MI or MI stage arrest, suggesting that the harmful effects of NIV on cell cycle control during oocyte meiosis. Cyclin B1, a regulatory subunit of MPF, synthesizes from prophase and associates with CDK1 to form an inactive pre-MPF before oocyte meiotic resumption. A low level of Cyclin B1 at GV stage and during GVBD is sufficient for mouse oocyte to re-enter meiosis without de novo protein synthesis, 29 and Cyclin B1 is dramatically high in pro-metaphase with translation of Ccnb1 until APC/C activation. 30 Our results showed low Cyclin B1 level after 3 h culture in both control and treatment groups. Whereas, a significant decreased Cyclin B1 expression was observed after 6 h and 9 h culture in NIV-exposed groups compared with control  Taken together, our results indicated that NIV did not affect meiotic resumption but reduced Cyclin B1 expression and activated SAC proteins with unstable microtubules and K-MT attachment, ultimately led to oocyte meiosis arrest.