Exposure to Aroclor-1254 Impairs Spindle Assembly During Mouse Oocyte Maturation Shu-Zhen Liu,1 Ze-Feng Wei,2 Xiao-Qian Meng,1 Xiao-Ying Han,1 Dong Cheng,3 Tao Zhong,1 Tian-Liang Zhang,3 Zhen-Bo Wang4 1
Key Laboratory of Animal Resistance Research, College of Life Science, Shandong Normal University, Jinan, China
2
Affiliated Hospital of Jining Medical University, Jining, China
3
Department of Toxicology, Shandong Center for Disease Control and Prevention, Jinan, China
4
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
Received 29 March 2015; revised 17 June 2015; accepted 27 June 2015 ABSTRACT: Polychlorinated biphenyls (PCBs), as typical environmental estrogen disruptors, are a structurally-related group of halogenated aromatic hydrocarbons that are composed of 209 isomers and present as a mixture in the environment. PCBs congener with different numbers and positions of chlorine atoms substituted on the biphenyl moiety. Aroclor-1254 is a mixture of more than 60 PCB congeners. Previous studies have provided the evidence that PCBs have severe negative effects on reproductive functions, but the effects of PCBs on spindle assembly during mouse oocyte maturation in vitro have not been reported. In the present study, female ICR mouse immature oocytes were cultured in M2 medium with 1 and 10 lg mL21 Aroclor-1254 separately in vitro. The percentage of germinal vesicle breakdown (GVBD) and the first polar body extrusion were recorded. The results showed no significant difference in the percentage of GVBD or the first polar body extrusion between control oocytes and Aroclor-1254treated oocytes. Further studies showed that the normal localization of g-tubulin and Aurora-A kinase was interfered and a-tubulin assembling into spindle was affected when mouse oocytes were exposed to Aroclor-1254. The length of spindle from 10 lg mL21 Aroclor-1254-treated oocytes was longer than that from control oocytes, and the spindle area in the Aroclor-1254-treated groups were decreased. Furthermore, the percentage of DNA damage in cumulus cells revealed an increase after exposed to Aroclor1254. These results will provide the important reference for the prevention of reproductive disorders C 2015 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2015. caused by PCBs. V Keywords: Aroclor-1254; mouse; oocyte; spindle; g-tubulin; Aurora-A
INTRODUCTION Correspondence to: S.-Z. Liu; e-mail: [email protected] Contract grant sponsor: National Nature Science Foundation of China. Contract grant numbers: 31101068, 31101034. Contract grant sponsor: Shandong Province Postdoctoral Innovation Project. Contract grant number: 201103102. Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22169
Polychlorinated biphenyls (PCBs), as typical environmental estrogen disruptors, are manufactured in the 1960s and widely used in industrial production for its chemical stability. Although PCBs are banned to be produced and used in the 1980s, PCBs are difficult to be degraded, stable, fatsoluble, and easy to assemble with food chains so that there is a lot of residual PCBs in the natural environment.
C 2015 Wiley Periodicals, Inc. V
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During oocyte maturation, oocytes develop to meiosis II after a series of biological events such as germinal vesicle breakdown (GVBD), chromosome condensation and spindle assembly. Because of going through cell division, the spindle plays an important role during oocyte maturation. g-tubulin is a very conserved protein, and it can be detected in almost eukaryotic cells. g-tubulin, one of pericentriolar materials (PCMs), plays a key role during the positioning of spindle (Brinkley, 1985; Joshi, 1994). Many studies on g-tubulin have demonstrated that it is usually gathered on the bipolar of spindle during mitosis (Schiebel, 2000; Moritz and Agard, 2001; Raynaud-Messina and Merdes, 2007). g-tubulin not only gathers on the bipolar of spindle, but also has a dot in cytoplasm during the meiosis of mouse oocytes (Gueth-Hallonet et al., 1993; Palacios et al., 1993; Combelles and Albertini, 2001). Spindle cannot assemble normally and chromosomes cannot aggregate adequately when the expression of g-tubulin is inhibited (Oakley et al., 1990). Many investigations on the function of g-tubulin in spindle assembly especially in meiosis have been conducted; however, the effects of PCBs on g-tubulin in spindle assembly during meiosis have not been reported. The Aurora family kinase is essential for the formation of bipolar spindle, the segregation of chromosomes and the completion of cytokinesis (Carmena and Earnshaw, 2003). Aurora-A kinase can help g-tubulin ring complex (g-TuRC) to target to centrin, and then influences the replication and maturity of centrosome (Terada et al., 2003). Aurora-A kinase also plays a key role in the segregation of centrosomes, and the overexpression of Aurora-A can lead to centrosome amplification (Dutertre et al., 2002; Barr and Gergely, 2007). Some reports have confirmed that the abnormal expression of Aurora-A kinase can lead to the abnormal segregation of centrosomes, thus resulting in the decrease in the length of the spindle or the formation of monopolar spindle (Glover et al., 1995). It has been proved that Aurora-A kinase can be co-localized with the dipolar of centrosome and spindle (Kimura et al., 1997). The abnormal expression of Aurora-A kinase also can lead to the formation of multipole or monopole spindle, because the instability of AuroraA kinase can result in the impact on the replication and segregation of centrosomes (Roghi et al., 1998). The studies on Aurora-A kinase in somatic cells suggests that it is essential for spindle assembly in mitosis, and it is necessary for the spindle assembly during meiosis (Castro et al., 2003; Crane et al., 2004; Yao et al., 2004). Therefore, the purpose of this study is to investigate the effect of Aroclor-1254 on g-tubulin, Aurora-A kinase and a-tubulin in mouse oocytes, and to explore the effect of Aroclor-1254 on the spindle assembly in mouse oocytes in vitro. Several reports have described that PCBs can be accumulated in human follicular fluid and the concentration of PCBs can be ranged from micrograms to milligrams per milliliter (Trapp et al., 1984; Jarrell et al., 1993; Petro et al.,
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2012). In in vitro study, low concentration of Aroclor-1254 exposure has been shown to disrupt oocyte maturation, fertilization, and developmental competence in pigs and cattles (Campagna et al., 2001; Pocar et al., 2001). In addition, Aroclor-1254 can affect in vitro fertilization and embryonic development of mice (Kholkute et al., 1994a). The concentrations of Aroclor-1254 in this study are 0.01, 0.1, 1.0, and 10.0 lg mL21, and the minimum effective dose (0.1 lg mL21) is the average level detected in human follicular fluid (Younglai et al., 2002). These studies have provided the evidence that PCBs have a severe negative effect on reproductive function, but the effects of PCBs on spindle assembly in mouse oocytes have not been reported. It is the first time to explore the effect of Aroclor-1254 on spindle assembly in mouse oocytes in vitro. Our study has found that Aroclor-1254 could disturb the normal movement of g-tubulin and Aurora-A kinase, and impair spindle assembly.
MATERIALS AND METHODS Animals Animal care and use were conducted in accordance with the Animal Research Institute Committee Guidelines of the Ethics Committee of Shandong Normal University, China. Mice were maintained on a strict 12 h light/dark cycle, temperature was maintained at 21–258C and the mice were given free accessibility to food and water. The food was provided by key laboratory for animals in Shandong Normal University. Female ICR mice with the age of 6- to 8-weeks old and body weight of 25–28 g were provided by the Beijing HFK Bio-Technology (Beijing, China). The mice were allowed to adapt for at least 7 days in experimental conditions before experiments.
Chemicals Aroclor-1254 was purchased from Accustandard (USA). Pregnant mare serum gonadotropin (PMSG) was purchased from Ningbo Second Hormone Factory (China). All other reagents were obtained from Sigma Chemical Co, unless indicated otherwise.
Collection of COCs and Treatment The healthy female mice (not in estrus period) were super-ovulated with an intraperitoneal injection of 10 IU PMSG. After PMSG injection for 46 h, the mice were killed by cervical dislocation. Ovarian was harvested from the reproductive system of mouse and put it in M2 medium. Under a dissecting microscope, follicles were punctured by sterile syringe. Cumulus oocyte complexes (COCs) were collected with homemade mouth straw, and then cleaned for follow-up study.
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TABLE I. Effect of Aroclor-1254 on the maturation of COCs in vitro Group(lg mL21)
No. of oocytes (n)
No. of GVBD(n)
No. of PBI (n)
GVBD(%)
PB I(%)
238 227 248
233 220 241
200 182 200
97.78 6 0.86 96.87 6 0.98 97.06 6 0.92
86.12 6 1.96 82.67 6 2.76 83.24 6 2.56
0 1 10
Aroclor-1254 was dissolved in absolute ethanol at the concentration of 500 mg mL21 and serially diluted in M2 medium to the desired concentrations. The COCs were divided into three groups, the experimental group with 1 and 10 lg mL21 of Aroclor-1254 in M2 medium, the control group with M2 medium directly culturing at 378C CO2 incubator for 8 or 14 h.
Immunofluorescence After culturing, oocytes were collected and fixed with 4% (w/v) paraformaldehyde in PBS (pH 7.4) for 30 min at room temperature. Fixed samples were permeabilized in incubation buffer (0.5% Triton X-100 in 20 mM HEPES, pH 7.4, 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose) for 30 min. After washing three times in PBS containing 0.01% TritonX100, samples were incubated in block solution (PBS containing 1% BSA) for 1 h at room temperature. Then, the samples were incubated with primary antibody for 1 h. After washing three times in PBS containing 0.01% Triton-X100, the samples were incubated with second antibody for 1 h. Nuclear status of oocytes was evaluated by staining with 1 lg mL21 propidium iodide (PI) or 4’,6-diamidino-2-phenylindole (DAPI) in PBS for 10 min. Finally, the samples were examined under a Zeiss confocal laser scanning microscope. The length and the area of the spindle were recorded using Image ProPlus software, the percentage of abnormal arranged chromosome is the ratio of the oocytes of abnomal arranged chromosome to the total oocytes. Moreover, the DNA damage in cumulus cells was evaluated, the percentage of DNA damage is the ratio of labeled cells to total cells.
Statistical Analysis Data were analyzed using SPSS 18.0 statistical software. At least three replicates were conducted for each treatment. Data were analyzed using chi-square test and ANOVA, and presented as Mean 6 SD. Statistical significance was determined when P value was 0.05).
The Effect of Aroclor-1254 on the Spindle Morphology of Mouse Oocytes Mouse cumulus oocyte complexes were cultured for 8 and 14 hs in M2 medium with different concentrations of Aroclor-1254 during in vitro maturation. The spindles in metaphase meiosis II were detected by the localization of atubulin (Green) and could be divided into three kinds (bipolar, multipolar, and nonpolar) of spindles depending on the shape. The effect of Aroclor-1254 on the spindle shape of mouse oocytes was shown in Figure 1(a). In the control group, the percentages of bipolar, multipolar, and nonpolar spindles were 63.9, 25.89, and 10.18%, respectively. In 1 lg mL21 Aroclor-1254 treated group, the percentages of bipolar, multipolar and nonpolar spindles were 17.78, 60.56, and 21.67%; and in 10 lg mL21 Aroclor-1254 treated group, percentages of bipolar, multipolar and nonpolar spindles were 19.72, 40.58, and 29.62%, respectively. The percentages of bipolar spindles in the experimental groups were lower than that in the control group (P < 0.01), and the percentage of multipolar spindles in 1 lg mL21 Aroclor-1254 treated group was higher than that in the control group (P < 0.05). The effect of Aroclor-1254 on the spindle area of mouse oocytes in metaphase meiosis II was shown in Figure 1(b). The spindle area of mouse oocytes from the control group (n 5 57), 1 lg mL21 Aroclor-1254 treated group (n 5 50) and 10 lg mL21 Aroclor-1254 treated group (n 5 50) were 404.0 6 4.198, 313.9 6 5.714, and 300.7 6 6.675 lm2, respectively. The spindle area of mouse oocytes from the experimental group was smaller than that from the control group (P < 0.01). The effect of Aroclor-1254 on the length of spindle of mouse oocytes in metaphase meiosis II was shown in Figure 1(c). The length of spindle in the control group (n 5 57), 1
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Fig. 1. Effect of Aroclor-1254 on the spindle shape of mouse oocytes. (a) The percentage of oocytes with bipolar, multipolar, nonpolar spindles by the treatment of Aroclor-1254. (b) The effect of Aroclor-1254 on the spindle area (Control, n 5 57; 1 lg mL21, n 5 50; 10 lg mL21, n 5 50). (c) The length of the spindle under the treatment of Aroclor-1254 with different concentrations (Control, n 5 57; 1 lg mL21, n 5 50; 10 lg mL21, n 5 50).*Compared to the control group, P < 0.05; **Compared to the control group, P < 0.01. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
lg mL21 Aroclor-1254 treated group (n 5 50) and 10 lg mL21 Aroclor-1254 treated group (n 5 50) were 25.67 6 0.4845, 25.21 6 0.5219, and 27.35 6 0.6180 lm, respectively. The length of spindle in 10 lg mL21 Aroclor1254 treated group was longer than that in the control group (P < 0.05). The localization of g-tubulin and a-tubulin was detected. In the control group, as shown in Figure 2(a), the oocytes developed to metaphase of the first meiotic division after 8 h cultivation in vitro, chromosome (Blue) aligned at the equatorial plate completely, g-tubulin (Red) was detected as two dots associated with the spindle poles, and a-tubulin (Green) under the traction of microtubule organization assembled as a bipolar spindle. The oocytes with PB I extrusion developed
Environmental Toxicology DOI 10.1002/tox
to metaphase of the second meiotic division after 14-h cultivation in vitro, chromosome (Blue) aligned on the equatorial plate, g-tubulin (Red) aggregated at the spindle poles regularly, and a-tubulin (Green) assembled as a normal bipolar spindle. Compared with the control group, oocytes developed to metaphase of the first meiotic division after 8-h cultivation and developed to metaphase of the second meiotic division after 14-h cultivation, chromosomes (Blue) revealed an obvious loss and no gathering at the equatorial plate, g-tubulin (Red) gathered into many spots at the spindle poles and a-tubulin (Green) assembled as a multipolar spindle in 1 lg mL21 Aroclor-1254 treated group, as shown in Figure 2(a,b); the localization of g-tubulin, chromosome and a-tubulin in
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Fig. 2. Effect of Aroclor-1254 on g-tubulin (red), a-tubulin (green) and chromosomes (blue) during the maturation of mouse oocytes after cultivation in vitro for 8 h (a) and 14 h (b). (A–D) The COCs cultured in M2 medium without treatment of Aroclor1254 (control), (E–H) the COCs treated by 1 lg mL21 Aroclor-1254, (I–L) the COCs treated by 10 lg mL21 Aroclor-1254. Scale bar represents 25 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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oocytes from 10 lg mL21 Aroclor-1254 treated group was similar as that from 1 lg mL21 Aroclor-1254 treated group. As shown in Figure 3(a,b), Aurora-A (Red) distributed at spindle and aligned at meiosis spindle poles, chromosome (Blue) aligned at the equatorial plate completely, and atubulin (Green) assembled as a bipolar spindle in the control group. In contrast, chromosome (Blue) exhibited an obvious loss and no gathering at the equatorial plate, Aurora-A (Red) gathered into many dots at the spindle poles and a-tubulin (Green) assembled as a multipolar spindle in the Aroclor1254 treated groups.
The Effect of Aroclor-1254 on the Arrangement of Chromosome in Oocytes The mouse oocytes developed to the metaphase of meiosis II after 14-h cultivation during in vitro maturation. The localization of chromosome was detected. Chromosomes (blue) arranged at the equatorial plate neatly in most oocytes from the control group were shown in Figure 4(a). A (87.74 6 2.15%). The abnormally arranged chromosomes (not arranged at the equatorial plate in oocytes) were shown in Figure 4(a). B to F. Furthermore, the population of oocytes with abnormally arranged chromosomes was evaluated and shown in Figure 4(b). The population of oocytes with abnormally arranged chromosome in the control group, 1 lg mL21 Aroclor-1254 treated group and 10 lg mL21 Aroclor-1254 treated group was 12.26% 6 2.15%, 63.33% 6 3.33%, and 83.31% 6 5.27%, respectively. The population of oocytes with abnormally arranged chromosomes in Aroclor-1254 treated groups was significantly higher than that in the control group (P < 0.01).
The Effect of Aroclor-1254 on DNA Damage in Cumulus Cells As shown in Figure 5, g-H2AX was stained in cumulus cells. The g-H2AX in Aroclor-1254 treated groups was more than that in the control group. The percentage of DNA damage in cumulus cells was significantly higher, when COCs were exposed to Aroclor-1254 for 14 h in vitro, which revealed a significant difference with that from the control group (P < 0.01).
DISCUSSION In the present study, although Aroclor-1254 did not affect the progression of mouse oocyte maturation, it could result in the increase of DNA damage in cumulus cells, the disruption of g-tubulin and Aurora-A localization and abnormal morphology of the spindles in the mesiosis, as well as impaired assembly of the spindles during mouse oocyte maturation. Many investigations have reported that the exposure to Aroclor-1254 can induce reproductive toxicity in mammals,
Environmental Toxicology DOI 10.1002/tox
and Aroclor-1254 can inhibit the maturation of oocytes and reduce developmental potential of oocytes in cattles, mice and pigs (Kholkute et al., 1994b; Pocar et al., 2001; Brevini et al., 2009). During oocyte maturation, a bipolar spindle is assembled for chromosome congression and segregation. Chromosome mis-alignment and mis-segregation can result in oocyte aneuploidy (Yin et al., 2008), suggesting that Aroclor-1254 can affect normal movement of g-tubulin and Aurora-A in mouse oocyte meiosis. In metaphase meiosis I and II, a-tubulin reveals the assembled multipolar spindle. g-tubulin is a component of g-tubulin ring complex (gTuRC) and plays an important role in microtubule nucleation (Stearns, 2004). It localizes in MI and MII spindles (Young et al., 2000). g-tubulin associates with microtubules and may be important for spindle formation (Schatten and Sun, 2011). Aurora-A plays a key role in mitosis and meiosis, and it associates with the function of centrosomes and spindles, and drives the maturation and segregation of centrosomes and spindle assembles (Crane et al., 2004; Hoar et al., 2007; Mori et al., 2007). In mouse oocytes, Aurora-A localizes to microtubules and microtubule-organized centers (Saskova et al., 2008). Spindle analysis was used to evaluate the quality of oocytes (Albertini et al., 2003; De Santis et al., 2005). A normal spindle is necessary for the regular chromosome alignment and segregation during the maturation of oocytes (Eichenlaub-Ritter et al., 2002; Vogt et al., 2008). In somatic cells, the errors in chromosome separation may cause aneuploid chromosome, thus inducing cancers (Kops et al., 2005; Malmanche et al., 2006). Aneuploid chromosome in Germ cells can result in aneuploid in embryo and lead spontaneous abortion in human (Bond and Chandley, 1983). Our present study suggests that oocytes exposed to Aroclor-1254 in vitro are characterized by abnormal chromosomal arrangements (Fig. 4) and multipolar spindle configuration (Figs. 2 and 3). The spindle area and the percentage of “Spindle” spindle in Aroclor-1254 treated groups are smaller, and the length of spindle in 10 lg mL21 Aroclor-1254 treated group is longer. Although the injury mechanism of spindles is not clear yet, mitochondria may play an important role in the regulation of microtubules and the assembly of spindles (Kr€uger and Tolic´-Nørrelykke, 2008). Studies on mouse oocytes suggest that errors in chromosome segregation during maturation is age-related (Eichenlaub-Ritter and Boll, 1989). Oocytes need sufficient ATP for transcription and translation during oocyte growth, and nuclear and cytoplasmic maturation (Eichenlaub-Ritter et al., 2004). Mitochondrial Ca21-sequestration is inhibited in rats exposed to PCBs (Kodavanti et al., 1996). We speculate that Aroclor-1254 may affect intracellular release or concentration change of Ca21 in the cytoplasm of oocytes so that mitochondria cannot provide enough energy. Once no sustainable energy is provided, gtubulin and Aurora-A cannot draw a-tubulin to assemble bipolar spindle. These data suggest that Aroclor-1254 exposure is likely to induce multipolar spindle. Although our
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Fig. 3. Effect of Aroclor-1254 on Aurora-A (red), a-tubulin (green) and chromosomes (blue) during the maturation of mouse oocytes after cultivation in vitro for 8 h (a) and 14 h (b). (A–D) The COCs cultured in M2 medium without treatment of Aroclor1254 (control), (E–H) the COCs treated by 1 lg mL21 Aroclor-1254, (I–L) the COCs treated by 10 lg mL21 Aroclor-1254. Scale bar represents 25 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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Fig. 4. Effect of Aroclor-1254 on the arrangement of chromosome in oocytes after cultivation for 14 h in vitro. (a) The localization of chromosome (blue) after oocytes treated by Aroclor-1254. (A) Chromosome (blue) arranged at the equatorial plate neatly. (B–F) The abnormally arranged chromosome. Scale bar represents 25 mm. (b) The population of oocytes with abnormally arranged chromosomes in the treatment groups (Control, n 5 50; 1 lg mL21, n 5 50; 10 lg mL21, n 5 50). **Compared to the control group, P < 0.01. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
results are the first to report the abnormal spindle configuration induced by the exposure to Aroclor-1254 in vitro, further evidence is needed to better elucidate this aspect. Previous study about spindle morphology and assembly focused on the function of spindle assembly checkpoint (SAC), such as Bub3, BubR1, Mad1, Mad2, Bub1, and Mps1. Touati et al. found that conditional knockout BubR1
Environmental Toxicology DOI 10.1002/tox
in mouse oocyte affected spindle assembly and chromosome segregation (Touati et al., 2015), but poly bodies can be extruded normally. Another published article in 2010 also verified that poly bodies could be extruded while spindle assembly is affected (Ou et al., 2010). Therefore, the phenomenon in our experiment that poly body I can be extruded while spindle was affected as well as other studies was
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Fig. 5. Effect of Aroclor-1254 on cumulus cells. (a) Confocal micrograph of cumulus oocyte complex treated for 14 h by Aroclor-1254 showing DNA (red) and g-H2AX (green). (A–C) The COCs cultured in M2 medium without treatment of Aroclor1254 (control), (D–F) the COCs treated by 1 lg mL21 Aroclor-1254, (G–I) the COCs treated by 10 lg mL21 Aroclor-1254. Scale bar represents 25 mm. (b) The percentage of DNA damage in cumulus cells after exposure to Aroclor-1254 (Control, n 5 40; 1 lg mL21, n 5 40; 10 lg mL21, n 5 40). **Compared with the control group, P < 0.01. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Environmental Toxicology DOI 10.1002/tox
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observed. However, the accurate and detail mechanisms should be further studied. g-H2AX is one of the earliest markers of double-strand break (DSB) in eukaryotes (Keogh et al., 2006). There is a close correlation between g-H2AX foci and DSB number. g-H2AX foci analysis can provide a sensitive monitor of DSB formation (L€obrich et al., 2010). The mammalian oocyte and its surrounding cumulus cells are dependent throughout the growth and development of the oocyte. The cumulus cells can protect the growing oocyte from the disadvantageous environment and supply nutrients to the oocyte through gap junctions between the cumulus mass and the oocyte (Mori et al., 2000). Granular cells play a critical role in the regulation of oocyte maturation and fertilization (Carabatsos et al., 2000). Our data have demonstrated the increased level of DNA damage in cumulus cells after exposure to Aroclor-1254 in vitro. Taken together, the presence of DNA damage in cumulus cells and the multipolar spindle in oocytes may also indicate a specific role of cumulus cells in the modulation of Aroclor-1254 toxicity during oocyte maturation. It is possible to conjecture that cumulus cells may be target cells for toxicological injury induced by Aroclor-1254 during oocyte maturation. In conclusion, Aroclor-1254 treatment in vitro can induce abnormal spindle configuration. The effect of Aroclor-1254 on oocyte fertilization and embryonic development from the zygote to blastocyst stage is not evaluated. The further investigations on Aroclor-1254 toxicity to reproductive function are highly desired. It is clear that Aroclor-1254 can affect oocyte outgrowth potential through a wide variety of biological processes and molecular mechanisms. These findings may be the rational explanation of reduced fecundity among women exposed to PCBs. The authors thank Rongbing Zheng, Rongshu Fu, and Fan Zhang for their technical assistance.
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Kholkute SD, Rodriguez J, Richard Dukelow W. 1994a. Reproductive toxicity of Aroclor-1254: Effects on oocyte, spermatozoa, in vitro fertilization, and embryo development in the mouse. Reprod Toxicol 8:487–493. Kholkute SD, Rodriguez J, Richard Dukelow W. 1994b. Effects of polychlorinated biphenyls (PCBs) on in vitro fertilization in the mouse. Reprod Toxicol 8:69–73. Kimura M, Matsuda Y, Yoshioka T, Okano Y. 1997. Cell cycledependent expression and spindle pole localization of a novel human protein kinase, Aik, related to Aurora of Drosophila and yeast Ipl1. J Biol Chem 272:13766–13771. Kodavanti PR, Ward TR, McKinney JD, Tilson HA. 1996. Inhibition of microsomal and mitochondrial Ca21-sequestration in rat cerebellum by polychlorinated biphenyl mixtures and congeners. Arch Toxicol 70:150–157. Kops GJ, Weaver BA, Cleveland DW. 2005. On the road to cancer: Aneuploidy and the mitotic checkpoint. Nat Rev Cancer 5: 773–785. Kr€uger N, Tolic´-Nørrelykke IM. 2008. Association of mitochondria with spindle poles facilitates spindle alignment. Curr Biol 18:R646–R647. L€obrich M, Shibata A, Beucher A, Fisher A, Ensminger M, Goodarzi AA, Barton O, Jeggo PA. 2010. gammaH2AX foci analysis for monitoring DNA double-strand break repair: Strengths, limitations and optimization. Cell Cycle 9:662–669. Malmanche N, Maia A, Sunkel CE. 2006. The spindle assembly checkpoint: Preventing chromosome mis-segregation during mitosis and meiosis. FEBS Lett 580:2888–2895. Marumoto T, Zhang D, Saya H. 2005. Aurora-A—A guardian of poles. Nat Rev Cancer 5:42–50. Mori D, Yano Y, Toyo-oka K, Yoshida N, Yamada M, Muramatsu M, Zhang D, Saya H, Toyoshima YY, Kinoshita K, Wynshaw-Boris A, Hirotsune S. 2007. NDEL1 phosphorylation by Aurora-A kinase is essential for centrosomal maturation, separation, and TACC3 recruitment. Mol Cell Biol 27:352– 367. Mori T, Amano T, Shimizu H. 2000. Roles of gap junctional communication of cumulus cells in cytoplasmic maturation of porcine oocytes cultured in vitro. Biol Reprod 62:913–919. Moritz M, Agard DA. 2001. g-Tubulin complexes and microtubule nucleation. Curr Opin Struct Biol 11:174–181. Oakley BR, Oakley CE, Yoon Y, Jung MK. 1990. g-Tubulin is a component of the spindle pole body that is essential for microtubule function in Aspergillus nidulans. Cell 61:1289–1301. Ou XH, Li S, Xu BZ, Wang ZB, Quan S, Li M, Zhang QH, Ouyang YC, Schatten H, Xing FQ, Sun QY. 2010. p38a MAPK is a MTOC-associated protein regulating spindle assembly, spindle length and accurate chromosome segregation during mouse oocyte meiotic maturation. Cell Cycle 9:4130–4143. Palacios MJ, Joshi HC, Simerly C, Schatten G. 1993. Gammatubulin reorganization during mouse fertilization and early development. J Cell Sci 104:383–389. Petro EM, Leroy JL, Covaci A, Fransen E, De Neubourg D, Dirtu AC, De Pauw I, Bols PE. 2012. Endocrine-disrupting chemicals
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in human follicular fluid impair in vitro oocyte developmental competence. Hum Reprod 27:1025–1033. Pocar P, Perazzoli F, Luciano AM, Gandolfi F. 2001. In vitro reproductive toxicity of polychlorinated biphenyls: Effects on oocyte maturation and developmental competence in cattle. Mol Reprod Dev 58:411–416. Raynaud-Messina B, Merdes A. 2007. g-Tubulin complexes and microtubule organization. Curr Opin Cell Biol 19:24–30. Roghi C, Giet R, Uzbekov R, Morin N, Chartrain I, Guellec Couturier LR, Doree A, Philippe M, Prigent MC. 1998. The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly. J Cell Sci 111:557–572. Saskova A, Solc P, Baran V, Kubelka M, Schultz RM, Motlik J. 2008. Aurora kinase A controls meiosis I progression in mouse oocytes. Cell Cycle 7:2368–2376. Schatten H, Sun QY. 2011. Centrosome dynamics during mammalian oocyte maturation with a focus on meiotic spindle formation. Mol Reprod Dev 78:757–768. Schiebel E. 2000. g-Tubulin complexes: Binding to the centrosome, regulation and microtubule nucleation. Curr Opin Cell Biol 12:113–118. Stearns T. 2004. The tubulin superfamily. In: Nigg EA, editor. Centrosomes in Development and Disease. Weinheim: WileyVCH Verlag GmbH & Co. KGaA. pp 17–25. Terada Y, Uetake Y, Kuriyama R. 2003. Interaction of Aurora-A and centrosomin at the microtubule-nucleating site in Drosophila and mammalian cells. J Cell Biol 162:757–764. Touati SA, Buffin E, Cladie`re D, Hached K, Rachez C, van Deursen JM, Wassmann K. 2015. Mouse oocytes depend on BubR1 for proper chromosome segregation but not for prophase I arrest. Nat Commun 6:6946. Trapp M, Baukloh V, Bohnet HG, Heeschen W. 1984. Pollutants in human follicular fluid. Fertil Steril 42:146. Vogt E, Kirsch-Volders M, Parry J, Eichenlaub-Ritter U. 2008. Spindle formation, chromosome segregation and the spindle checkpoint in mammalian oocytes and susceptibility to meiotic error. Mutat Res 651:14–29. Yao LJ, Zhong ZS, Zhang LS, Chen DY, Schatten H, Sun QY. 2004. Aurora-A is a critical regulator of microtubule assembly and nuclear activity in mouse oocytes, fertilized eggs, and early embryos. Biol Reprod 70:1392–1399. Yin S, Sun XF, Schatten H, Sun QY. 2008. Molecular insights into mechanisms regulating faithful chromosome separation in female meiosis. Cell Cycle 7:2997–3005. Young A, Dictenberg JB, Purohit A, Tuft R, Doxsey SJ. 2000. Cytoplasmic dynein-mediated assembly of pericentrin and g tubulin onto centrosomes. Mol Biol Cell 11:2047–2056. Younglai EV, Foster WG, Hughes EG, Trim K, Jarrell JF. 2002. Levels of environmental contaminants in human follicular fluid, serum, and seminal plasma of couples undergoing in vitro fertilization. Arch Environ Contam Toxicol 43:121–126.
Environmental Toxicology DOI 10.1002/tox
Key Laboratory of Animal Resistance Research, College of Life Science, Shandong Normal University, Jinan, China
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Affiliated Hospital of Jining Medical University, Jining, China
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Department of Toxicology, Shandong Center for Disease Control and Prevention, Jinan, China
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State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
Received 29 March 2015; revised 17 June 2015; accepted 27 June 2015 ABSTRACT: Polychlorinated biphenyls (PCBs), as typical environmental estrogen disruptors, are a structurally-related group of halogenated aromatic hydrocarbons that are composed of 209 isomers and present as a mixture in the environment. PCBs congener with different numbers and positions of chlorine atoms substituted on the biphenyl moiety. Aroclor-1254 is a mixture of more than 60 PCB congeners. Previous studies have provided the evidence that PCBs have severe negative effects on reproductive functions, but the effects of PCBs on spindle assembly during mouse oocyte maturation in vitro have not been reported. In the present study, female ICR mouse immature oocytes were cultured in M2 medium with 1 and 10 lg mL21 Aroclor-1254 separately in vitro. The percentage of germinal vesicle breakdown (GVBD) and the first polar body extrusion were recorded. The results showed no significant difference in the percentage of GVBD or the first polar body extrusion between control oocytes and Aroclor-1254treated oocytes. Further studies showed that the normal localization of g-tubulin and Aurora-A kinase was interfered and a-tubulin assembling into spindle was affected when mouse oocytes were exposed to Aroclor-1254. The length of spindle from 10 lg mL21 Aroclor-1254-treated oocytes was longer than that from control oocytes, and the spindle area in the Aroclor-1254-treated groups were decreased. Furthermore, the percentage of DNA damage in cumulus cells revealed an increase after exposed to Aroclor1254. These results will provide the important reference for the prevention of reproductive disorders C 2015 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2015. caused by PCBs. V Keywords: Aroclor-1254; mouse; oocyte; spindle; g-tubulin; Aurora-A
INTRODUCTION Correspondence to: S.-Z. Liu; e-mail: [email protected] Contract grant sponsor: National Nature Science Foundation of China. Contract grant numbers: 31101068, 31101034. Contract grant sponsor: Shandong Province Postdoctoral Innovation Project. Contract grant number: 201103102. Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22169
Polychlorinated biphenyls (PCBs), as typical environmental estrogen disruptors, are manufactured in the 1960s and widely used in industrial production for its chemical stability. Although PCBs are banned to be produced and used in the 1980s, PCBs are difficult to be degraded, stable, fatsoluble, and easy to assemble with food chains so that there is a lot of residual PCBs in the natural environment.
C 2015 Wiley Periodicals, Inc. V
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During oocyte maturation, oocytes develop to meiosis II after a series of biological events such as germinal vesicle breakdown (GVBD), chromosome condensation and spindle assembly. Because of going through cell division, the spindle plays an important role during oocyte maturation. g-tubulin is a very conserved protein, and it can be detected in almost eukaryotic cells. g-tubulin, one of pericentriolar materials (PCMs), plays a key role during the positioning of spindle (Brinkley, 1985; Joshi, 1994). Many studies on g-tubulin have demonstrated that it is usually gathered on the bipolar of spindle during mitosis (Schiebel, 2000; Moritz and Agard, 2001; Raynaud-Messina and Merdes, 2007). g-tubulin not only gathers on the bipolar of spindle, but also has a dot in cytoplasm during the meiosis of mouse oocytes (Gueth-Hallonet et al., 1993; Palacios et al., 1993; Combelles and Albertini, 2001). Spindle cannot assemble normally and chromosomes cannot aggregate adequately when the expression of g-tubulin is inhibited (Oakley et al., 1990). Many investigations on the function of g-tubulin in spindle assembly especially in meiosis have been conducted; however, the effects of PCBs on g-tubulin in spindle assembly during meiosis have not been reported. The Aurora family kinase is essential for the formation of bipolar spindle, the segregation of chromosomes and the completion of cytokinesis (Carmena and Earnshaw, 2003). Aurora-A kinase can help g-tubulin ring complex (g-TuRC) to target to centrin, and then influences the replication and maturity of centrosome (Terada et al., 2003). Aurora-A kinase also plays a key role in the segregation of centrosomes, and the overexpression of Aurora-A can lead to centrosome amplification (Dutertre et al., 2002; Barr and Gergely, 2007). Some reports have confirmed that the abnormal expression of Aurora-A kinase can lead to the abnormal segregation of centrosomes, thus resulting in the decrease in the length of the spindle or the formation of monopolar spindle (Glover et al., 1995). It has been proved that Aurora-A kinase can be co-localized with the dipolar of centrosome and spindle (Kimura et al., 1997). The abnormal expression of Aurora-A kinase also can lead to the formation of multipole or monopole spindle, because the instability of AuroraA kinase can result in the impact on the replication and segregation of centrosomes (Roghi et al., 1998). The studies on Aurora-A kinase in somatic cells suggests that it is essential for spindle assembly in mitosis, and it is necessary for the spindle assembly during meiosis (Castro et al., 2003; Crane et al., 2004; Yao et al., 2004). Therefore, the purpose of this study is to investigate the effect of Aroclor-1254 on g-tubulin, Aurora-A kinase and a-tubulin in mouse oocytes, and to explore the effect of Aroclor-1254 on the spindle assembly in mouse oocytes in vitro. Several reports have described that PCBs can be accumulated in human follicular fluid and the concentration of PCBs can be ranged from micrograms to milligrams per milliliter (Trapp et al., 1984; Jarrell et al., 1993; Petro et al.,
Environmental Toxicology DOI 10.1002/tox
2012). In in vitro study, low concentration of Aroclor-1254 exposure has been shown to disrupt oocyte maturation, fertilization, and developmental competence in pigs and cattles (Campagna et al., 2001; Pocar et al., 2001). In addition, Aroclor-1254 can affect in vitro fertilization and embryonic development of mice (Kholkute et al., 1994a). The concentrations of Aroclor-1254 in this study are 0.01, 0.1, 1.0, and 10.0 lg mL21, and the minimum effective dose (0.1 lg mL21) is the average level detected in human follicular fluid (Younglai et al., 2002). These studies have provided the evidence that PCBs have a severe negative effect on reproductive function, but the effects of PCBs on spindle assembly in mouse oocytes have not been reported. It is the first time to explore the effect of Aroclor-1254 on spindle assembly in mouse oocytes in vitro. Our study has found that Aroclor-1254 could disturb the normal movement of g-tubulin and Aurora-A kinase, and impair spindle assembly.
MATERIALS AND METHODS Animals Animal care and use were conducted in accordance with the Animal Research Institute Committee Guidelines of the Ethics Committee of Shandong Normal University, China. Mice were maintained on a strict 12 h light/dark cycle, temperature was maintained at 21–258C and the mice were given free accessibility to food and water. The food was provided by key laboratory for animals in Shandong Normal University. Female ICR mice with the age of 6- to 8-weeks old and body weight of 25–28 g were provided by the Beijing HFK Bio-Technology (Beijing, China). The mice were allowed to adapt for at least 7 days in experimental conditions before experiments.
Chemicals Aroclor-1254 was purchased from Accustandard (USA). Pregnant mare serum gonadotropin (PMSG) was purchased from Ningbo Second Hormone Factory (China). All other reagents were obtained from Sigma Chemical Co, unless indicated otherwise.
Collection of COCs and Treatment The healthy female mice (not in estrus period) were super-ovulated with an intraperitoneal injection of 10 IU PMSG. After PMSG injection for 46 h, the mice were killed by cervical dislocation. Ovarian was harvested from the reproductive system of mouse and put it in M2 medium. Under a dissecting microscope, follicles were punctured by sterile syringe. Cumulus oocyte complexes (COCs) were collected with homemade mouth straw, and then cleaned for follow-up study.
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TABLE I. Effect of Aroclor-1254 on the maturation of COCs in vitro Group(lg mL21)
No. of oocytes (n)
No. of GVBD(n)
No. of PBI (n)
GVBD(%)
PB I(%)
238 227 248
233 220 241
200 182 200
97.78 6 0.86 96.87 6 0.98 97.06 6 0.92
86.12 6 1.96 82.67 6 2.76 83.24 6 2.56
0 1 10
Aroclor-1254 was dissolved in absolute ethanol at the concentration of 500 mg mL21 and serially diluted in M2 medium to the desired concentrations. The COCs were divided into three groups, the experimental group with 1 and 10 lg mL21 of Aroclor-1254 in M2 medium, the control group with M2 medium directly culturing at 378C CO2 incubator for 8 or 14 h.
Immunofluorescence After culturing, oocytes were collected and fixed with 4% (w/v) paraformaldehyde in PBS (pH 7.4) for 30 min at room temperature. Fixed samples were permeabilized in incubation buffer (0.5% Triton X-100 in 20 mM HEPES, pH 7.4, 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose) for 30 min. After washing three times in PBS containing 0.01% TritonX100, samples were incubated in block solution (PBS containing 1% BSA) for 1 h at room temperature. Then, the samples were incubated with primary antibody for 1 h. After washing three times in PBS containing 0.01% Triton-X100, the samples were incubated with second antibody for 1 h. Nuclear status of oocytes was evaluated by staining with 1 lg mL21 propidium iodide (PI) or 4’,6-diamidino-2-phenylindole (DAPI) in PBS for 10 min. Finally, the samples were examined under a Zeiss confocal laser scanning microscope. The length and the area of the spindle were recorded using Image ProPlus software, the percentage of abnormal arranged chromosome is the ratio of the oocytes of abnomal arranged chromosome to the total oocytes. Moreover, the DNA damage in cumulus cells was evaluated, the percentage of DNA damage is the ratio of labeled cells to total cells.
Statistical Analysis Data were analyzed using SPSS 18.0 statistical software. At least three replicates were conducted for each treatment. Data were analyzed using chi-square test and ANOVA, and presented as Mean 6 SD. Statistical significance was determined when P value was 0.05).
The Effect of Aroclor-1254 on the Spindle Morphology of Mouse Oocytes Mouse cumulus oocyte complexes were cultured for 8 and 14 hs in M2 medium with different concentrations of Aroclor-1254 during in vitro maturation. The spindles in metaphase meiosis II were detected by the localization of atubulin (Green) and could be divided into three kinds (bipolar, multipolar, and nonpolar) of spindles depending on the shape. The effect of Aroclor-1254 on the spindle shape of mouse oocytes was shown in Figure 1(a). In the control group, the percentages of bipolar, multipolar, and nonpolar spindles were 63.9, 25.89, and 10.18%, respectively. In 1 lg mL21 Aroclor-1254 treated group, the percentages of bipolar, multipolar and nonpolar spindles were 17.78, 60.56, and 21.67%; and in 10 lg mL21 Aroclor-1254 treated group, percentages of bipolar, multipolar and nonpolar spindles were 19.72, 40.58, and 29.62%, respectively. The percentages of bipolar spindles in the experimental groups were lower than that in the control group (P < 0.01), and the percentage of multipolar spindles in 1 lg mL21 Aroclor-1254 treated group was higher than that in the control group (P < 0.05). The effect of Aroclor-1254 on the spindle area of mouse oocytes in metaphase meiosis II was shown in Figure 1(b). The spindle area of mouse oocytes from the control group (n 5 57), 1 lg mL21 Aroclor-1254 treated group (n 5 50) and 10 lg mL21 Aroclor-1254 treated group (n 5 50) were 404.0 6 4.198, 313.9 6 5.714, and 300.7 6 6.675 lm2, respectively. The spindle area of mouse oocytes from the experimental group was smaller than that from the control group (P < 0.01). The effect of Aroclor-1254 on the length of spindle of mouse oocytes in metaphase meiosis II was shown in Figure 1(c). The length of spindle in the control group (n 5 57), 1
Environmental Toxicology DOI 10.1002/tox
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Fig. 1. Effect of Aroclor-1254 on the spindle shape of mouse oocytes. (a) The percentage of oocytes with bipolar, multipolar, nonpolar spindles by the treatment of Aroclor-1254. (b) The effect of Aroclor-1254 on the spindle area (Control, n 5 57; 1 lg mL21, n 5 50; 10 lg mL21, n 5 50). (c) The length of the spindle under the treatment of Aroclor-1254 with different concentrations (Control, n 5 57; 1 lg mL21, n 5 50; 10 lg mL21, n 5 50).*Compared to the control group, P < 0.05; **Compared to the control group, P < 0.01. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
lg mL21 Aroclor-1254 treated group (n 5 50) and 10 lg mL21 Aroclor-1254 treated group (n 5 50) were 25.67 6 0.4845, 25.21 6 0.5219, and 27.35 6 0.6180 lm, respectively. The length of spindle in 10 lg mL21 Aroclor1254 treated group was longer than that in the control group (P < 0.05). The localization of g-tubulin and a-tubulin was detected. In the control group, as shown in Figure 2(a), the oocytes developed to metaphase of the first meiotic division after 8 h cultivation in vitro, chromosome (Blue) aligned at the equatorial plate completely, g-tubulin (Red) was detected as two dots associated with the spindle poles, and a-tubulin (Green) under the traction of microtubule organization assembled as a bipolar spindle. The oocytes with PB I extrusion developed
Environmental Toxicology DOI 10.1002/tox
to metaphase of the second meiotic division after 14-h cultivation in vitro, chromosome (Blue) aligned on the equatorial plate, g-tubulin (Red) aggregated at the spindle poles regularly, and a-tubulin (Green) assembled as a normal bipolar spindle. Compared with the control group, oocytes developed to metaphase of the first meiotic division after 8-h cultivation and developed to metaphase of the second meiotic division after 14-h cultivation, chromosomes (Blue) revealed an obvious loss and no gathering at the equatorial plate, g-tubulin (Red) gathered into many spots at the spindle poles and a-tubulin (Green) assembled as a multipolar spindle in 1 lg mL21 Aroclor-1254 treated group, as shown in Figure 2(a,b); the localization of g-tubulin, chromosome and a-tubulin in
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Fig. 2. Effect of Aroclor-1254 on g-tubulin (red), a-tubulin (green) and chromosomes (blue) during the maturation of mouse oocytes after cultivation in vitro for 8 h (a) and 14 h (b). (A–D) The COCs cultured in M2 medium without treatment of Aroclor1254 (control), (E–H) the COCs treated by 1 lg mL21 Aroclor-1254, (I–L) the COCs treated by 10 lg mL21 Aroclor-1254. Scale bar represents 25 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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oocytes from 10 lg mL21 Aroclor-1254 treated group was similar as that from 1 lg mL21 Aroclor-1254 treated group. As shown in Figure 3(a,b), Aurora-A (Red) distributed at spindle and aligned at meiosis spindle poles, chromosome (Blue) aligned at the equatorial plate completely, and atubulin (Green) assembled as a bipolar spindle in the control group. In contrast, chromosome (Blue) exhibited an obvious loss and no gathering at the equatorial plate, Aurora-A (Red) gathered into many dots at the spindle poles and a-tubulin (Green) assembled as a multipolar spindle in the Aroclor1254 treated groups.
The Effect of Aroclor-1254 on the Arrangement of Chromosome in Oocytes The mouse oocytes developed to the metaphase of meiosis II after 14-h cultivation during in vitro maturation. The localization of chromosome was detected. Chromosomes (blue) arranged at the equatorial plate neatly in most oocytes from the control group were shown in Figure 4(a). A (87.74 6 2.15%). The abnormally arranged chromosomes (not arranged at the equatorial plate in oocytes) were shown in Figure 4(a). B to F. Furthermore, the population of oocytes with abnormally arranged chromosomes was evaluated and shown in Figure 4(b). The population of oocytes with abnormally arranged chromosome in the control group, 1 lg mL21 Aroclor-1254 treated group and 10 lg mL21 Aroclor-1254 treated group was 12.26% 6 2.15%, 63.33% 6 3.33%, and 83.31% 6 5.27%, respectively. The population of oocytes with abnormally arranged chromosomes in Aroclor-1254 treated groups was significantly higher than that in the control group (P < 0.01).
The Effect of Aroclor-1254 on DNA Damage in Cumulus Cells As shown in Figure 5, g-H2AX was stained in cumulus cells. The g-H2AX in Aroclor-1254 treated groups was more than that in the control group. The percentage of DNA damage in cumulus cells was significantly higher, when COCs were exposed to Aroclor-1254 for 14 h in vitro, which revealed a significant difference with that from the control group (P < 0.01).
DISCUSSION In the present study, although Aroclor-1254 did not affect the progression of mouse oocyte maturation, it could result in the increase of DNA damage in cumulus cells, the disruption of g-tubulin and Aurora-A localization and abnormal morphology of the spindles in the mesiosis, as well as impaired assembly of the spindles during mouse oocyte maturation. Many investigations have reported that the exposure to Aroclor-1254 can induce reproductive toxicity in mammals,
Environmental Toxicology DOI 10.1002/tox
and Aroclor-1254 can inhibit the maturation of oocytes and reduce developmental potential of oocytes in cattles, mice and pigs (Kholkute et al., 1994b; Pocar et al., 2001; Brevini et al., 2009). During oocyte maturation, a bipolar spindle is assembled for chromosome congression and segregation. Chromosome mis-alignment and mis-segregation can result in oocyte aneuploidy (Yin et al., 2008), suggesting that Aroclor-1254 can affect normal movement of g-tubulin and Aurora-A in mouse oocyte meiosis. In metaphase meiosis I and II, a-tubulin reveals the assembled multipolar spindle. g-tubulin is a component of g-tubulin ring complex (gTuRC) and plays an important role in microtubule nucleation (Stearns, 2004). It localizes in MI and MII spindles (Young et al., 2000). g-tubulin associates with microtubules and may be important for spindle formation (Schatten and Sun, 2011). Aurora-A plays a key role in mitosis and meiosis, and it associates with the function of centrosomes and spindles, and drives the maturation and segregation of centrosomes and spindle assembles (Crane et al., 2004; Hoar et al., 2007; Mori et al., 2007). In mouse oocytes, Aurora-A localizes to microtubules and microtubule-organized centers (Saskova et al., 2008). Spindle analysis was used to evaluate the quality of oocytes (Albertini et al., 2003; De Santis et al., 2005). A normal spindle is necessary for the regular chromosome alignment and segregation during the maturation of oocytes (Eichenlaub-Ritter et al., 2002; Vogt et al., 2008). In somatic cells, the errors in chromosome separation may cause aneuploid chromosome, thus inducing cancers (Kops et al., 2005; Malmanche et al., 2006). Aneuploid chromosome in Germ cells can result in aneuploid in embryo and lead spontaneous abortion in human (Bond and Chandley, 1983). Our present study suggests that oocytes exposed to Aroclor-1254 in vitro are characterized by abnormal chromosomal arrangements (Fig. 4) and multipolar spindle configuration (Figs. 2 and 3). The spindle area and the percentage of “Spindle” spindle in Aroclor-1254 treated groups are smaller, and the length of spindle in 10 lg mL21 Aroclor-1254 treated group is longer. Although the injury mechanism of spindles is not clear yet, mitochondria may play an important role in the regulation of microtubules and the assembly of spindles (Kr€uger and Tolic´-Nørrelykke, 2008). Studies on mouse oocytes suggest that errors in chromosome segregation during maturation is age-related (Eichenlaub-Ritter and Boll, 1989). Oocytes need sufficient ATP for transcription and translation during oocyte growth, and nuclear and cytoplasmic maturation (Eichenlaub-Ritter et al., 2004). Mitochondrial Ca21-sequestration is inhibited in rats exposed to PCBs (Kodavanti et al., 1996). We speculate that Aroclor-1254 may affect intracellular release or concentration change of Ca21 in the cytoplasm of oocytes so that mitochondria cannot provide enough energy. Once no sustainable energy is provided, gtubulin and Aurora-A cannot draw a-tubulin to assemble bipolar spindle. These data suggest that Aroclor-1254 exposure is likely to induce multipolar spindle. Although our
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Fig. 3. Effect of Aroclor-1254 on Aurora-A (red), a-tubulin (green) and chromosomes (blue) during the maturation of mouse oocytes after cultivation in vitro for 8 h (a) and 14 h (b). (A–D) The COCs cultured in M2 medium without treatment of Aroclor1254 (control), (E–H) the COCs treated by 1 lg mL21 Aroclor-1254, (I–L) the COCs treated by 10 lg mL21 Aroclor-1254. Scale bar represents 25 mm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Environmental Toxicology DOI 10.1002/tox
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Fig. 4. Effect of Aroclor-1254 on the arrangement of chromosome in oocytes after cultivation for 14 h in vitro. (a) The localization of chromosome (blue) after oocytes treated by Aroclor-1254. (A) Chromosome (blue) arranged at the equatorial plate neatly. (B–F) The abnormally arranged chromosome. Scale bar represents 25 mm. (b) The population of oocytes with abnormally arranged chromosomes in the treatment groups (Control, n 5 50; 1 lg mL21, n 5 50; 10 lg mL21, n 5 50). **Compared to the control group, P < 0.01. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
results are the first to report the abnormal spindle configuration induced by the exposure to Aroclor-1254 in vitro, further evidence is needed to better elucidate this aspect. Previous study about spindle morphology and assembly focused on the function of spindle assembly checkpoint (SAC), such as Bub3, BubR1, Mad1, Mad2, Bub1, and Mps1. Touati et al. found that conditional knockout BubR1
Environmental Toxicology DOI 10.1002/tox
in mouse oocyte affected spindle assembly and chromosome segregation (Touati et al., 2015), but poly bodies can be extruded normally. Another published article in 2010 also verified that poly bodies could be extruded while spindle assembly is affected (Ou et al., 2010). Therefore, the phenomenon in our experiment that poly body I can be extruded while spindle was affected as well as other studies was
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Fig. 5. Effect of Aroclor-1254 on cumulus cells. (a) Confocal micrograph of cumulus oocyte complex treated for 14 h by Aroclor-1254 showing DNA (red) and g-H2AX (green). (A–C) The COCs cultured in M2 medium without treatment of Aroclor1254 (control), (D–F) the COCs treated by 1 lg mL21 Aroclor-1254, (G–I) the COCs treated by 10 lg mL21 Aroclor-1254. Scale bar represents 25 mm. (b) The percentage of DNA damage in cumulus cells after exposure to Aroclor-1254 (Control, n 5 40; 1 lg mL21, n 5 40; 10 lg mL21, n 5 40). **Compared with the control group, P < 0.01. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] Environmental Toxicology DOI 10.1002/tox
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observed. However, the accurate and detail mechanisms should be further studied. g-H2AX is one of the earliest markers of double-strand break (DSB) in eukaryotes (Keogh et al., 2006). There is a close correlation between g-H2AX foci and DSB number. g-H2AX foci analysis can provide a sensitive monitor of DSB formation (L€obrich et al., 2010). The mammalian oocyte and its surrounding cumulus cells are dependent throughout the growth and development of the oocyte. The cumulus cells can protect the growing oocyte from the disadvantageous environment and supply nutrients to the oocyte through gap junctions between the cumulus mass and the oocyte (Mori et al., 2000). Granular cells play a critical role in the regulation of oocyte maturation and fertilization (Carabatsos et al., 2000). Our data have demonstrated the increased level of DNA damage in cumulus cells after exposure to Aroclor-1254 in vitro. Taken together, the presence of DNA damage in cumulus cells and the multipolar spindle in oocytes may also indicate a specific role of cumulus cells in the modulation of Aroclor-1254 toxicity during oocyte maturation. It is possible to conjecture that cumulus cells may be target cells for toxicological injury induced by Aroclor-1254 during oocyte maturation. In conclusion, Aroclor-1254 treatment in vitro can induce abnormal spindle configuration. The effect of Aroclor-1254 on oocyte fertilization and embryonic development from the zygote to blastocyst stage is not evaluated. The further investigations on Aroclor-1254 toxicity to reproductive function are highly desired. It is clear that Aroclor-1254 can affect oocyte outgrowth potential through a wide variety of biological processes and molecular mechanisms. These findings may be the rational explanation of reduced fecundity among women exposed to PCBs. The authors thank Rongbing Zheng, Rongshu Fu, and Fan Zhang for their technical assistance.
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