Dual effects of molybdenum on mouse oocyte quality and ovarian oxidative stress

Abstract

A sub-acute toxicity test was performed to investigate the effects of molybdenum (Mo) on ovarian function. ICR adult female mice were exposed to Mo by free access to distilled water containing the Mo at 5, 10, 20, and 40 mg/L for 14 days. Compared to the control group, M II oocyte morphology, ovary index, and ovulation improved within the 5 mg/L Mo group, but were negatively affected by Mo at 40 mg/L. Morphologically abnormal ovarian mitochondria were observed at ≥ 20 mg/L. These alterations accompanied the changes in superoxide dismutase (SOD), glutathione peroxidise (GPx), and malondialdehyde (MDA) levels in ovaries. In conclusion, Mo affects oocyte quality possibly through regulating ovarian oxidative stress in a dose-dependent manner. It appears that Mo may improve ovarian function at a suitable concentration, which might be a candidate for the treatment of female infertility.

Keywords:

• granulosa cells
• molybdenum
• mouse
• oocyte
• ovary
• oxidative stress

Introduction

There is growing interest in the possible health threat posed by endocrine-disrupting chemicals, which have effects on male and female reproduction [Diamanti-Kandarakis et al. 2009]. Molybdenum (Mo) is one of the endocrine disruptors widely present in food, water [Underwood 1981; Yu et al. 2011], and industrial products [Braydich-Stolle et al. 2005; Ema et al. 2010; Pandey and Singh 2002]. Molybdenum is also an environmental pollutant discharged from combustion and is present in contact lens solutions and as a color additive in cosmetics [ACGIH 1995]. Its wide distribution greatly increases the risk of animal and human environmental exposure to Mo.

It has been established that excessive ingestion of Mo can cause male reproductive toxicity in animals and humans [Bersenyi et al. 2008; Lyubimov et al. 2004; Meeker et al. 2008; Pandey and Singh 2002; Wirth and Mijal 2010]. However, fewer studies and conflicting information has been presented with respect to its effect on female reproductive health. Fungwe et al. [1990] indicated that a high level of Mo caused prolonged estrous cycle and abnormal embryogenesis in rats. Similarly, Huang et al. [2011] suggested that at a high-dose (8.51 µg/mg average content levels in soil) exposure during human pregnancy was related to the increased risk of neural tube defects. In contrast, other studies showed no effects on the estrous cycle, conception rates, and reproductive indices in rats or guinea pigs [Howell et al. 1993; Lyubimov et al. 2004]. To our knowledge, the mechanism of Mo affecting female reproductive health is far from clear.

Oxidative stress is a possible mechanism by which ovarian damage could accumulate presenting as oocyte deformity and eventually female infertility [Behrman et al. 2001; Gupta et al. 2006; Hussain et al. 2005; Lim and Luderer 2011; Tessaro et al. 2012; Wang et al. 2012]. It occurs when the formation of reactive oxygen species (ROS) exceeds the ability of the cells to defend themselves from increased ROS [Gupta et al. 2006]. A biochemical and molecular cascade (lipids, proteins, DNA, and/or RNA) of a whole organ can be affected with ensuing detriment [Pelicano et al. 2003]. Antioxidants, such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT), are important in protecting ovarian cells from oxidative stress [Matzuk et al. 1998; Tilly and Tilly 1995]. Malondialdehyde (MDA) reflects the level of peroxidation and the degree of cell injury [Sharma 2004]. Thus, ovarian oxidative stress can be evaluated by detecting SOD and GPx, as well as the level of MDA [Bozkurt et al. 2012].

Mitochondrial integrity and function also reflect the degree of oxidative stress [Hagen et al. 2004; Lenaz 2001; Pelicano et al. 2003; Schuh et al. 2005]. This reflects the ROS by-product during the metabolic processes involving the mitochondrial electron transfer chain [Hagen et al. 2004; Lenaz 2001; Pelicano et al. 2003]. Reduced mitochondrial membrane potential, defective activity, and impaired functions damaged ovaries, germinal vesicle (GV), or metaphase  II (M II) oocytes [Gupta et al. 2006; Hussain et al. 2005; Ou et al. 2012; Tessaro et al. 2012].

Thus, this study was designed to investigate the possible toxicity of Mo on the mouse ovary and to explore the possible mechanism affecting female reproductive health. At high doses alterations in M II oocyte morphology, ovarian index, ovarian oxidative stress, and ultrastructure of mitochondria in ovarian cells were detected after mice were exposed to Mo for 14 days. This may be associated with ovarian oxidative stress.

Results and Discussion

Effects of Mo exposure on ovary parameters and M II oocyte morphology

Distinct hyperemia was observed in the ovaries exposed to ≥ 20 mg/L Mo (Fig.1). This suggested that ovarian varicocele can follow Mo exposure. However, at 5 mg/L, Mo significantly improved the ovulation number (Table 1) and M II oocyte morphology, but did not affect the ovary index. In contrast, at 40 mg/L the ovary index significantly decreased and reduced M II oocyte morphology (P < 0.01) (Fig. 2) was observed but it did not significantly change the ovulation number.

Figure 1.  The ovaries exposed to molybdenum (Mo) at 20 mg/L (B) and 40 mg/L (C) exhibited the obvious hyperemia (black arrows), compared to the control group (A) (white arrows).

Figure 2.  Morphological images of M II oocytes (×400). Compared to the control (A), the morphological abnormalities of M II oocytes were observed in mice exposed to molybdenum at ≥20 mg/L, such as no first polar body (B), distorted cytoplasm (C), transformative zona pellucida and excessive vacuolization (D), shrunken cytoplasm (E), and centrally severe granulation (F).

Table 1. Effects of molybdenum exposure on ovary parameters and M II oocyte morphology.

Mo (mg/L)	Ovary index (×10^−3)*	No. ovulation	Abnormal rate/%**
0 (Control)	0.9985 ± 0.0643	31.5000 ± 2.7773	31.8542 ± 2.3361
5	0.9807 ± 0.0511	54.3500 ± 2.8316^a	18.6753 ± 0.8782^a
10	0.9854 ± 0.0527	45.4500 ± 2.6526^a	28.4928 ± 1.9862
20	0.9533 ± 0.0596	41.6000 ± 3.2577^b	34.4304 ± 3.0439
40	0.8394 ± 0.0538^a	35.7368 ± 3.6714	45.4962 ± 3.3147^a

Mo: molybdenum; Data represent means ± SD.

Compared with the control group, ^aP < 0.01, ^bP < 0.05.

*Ovary index = wet ovary weight / total wet body weight; **Abnormal rate = (no. total ovulation - no. normal M II oocytes) / No. total ovulation.

Effects of Mo on the levels of SOD, GPx, and MDA of ovaries in mice

As shown in Table 2, compared to control, Mo at 5 mg/L stimulated SOD and GPx activities, but did not obviously change the content of MDA. At 40 mg/L Mo significantly decreased the activity of SOD and GPx as the level of MDA significantly increased. In comparison, the effect of Mo was tempered on these oxidative stress parameters at 10 mg/L or 20 mg/.

Table 2. Effects of molybdenum exposure on the levels of SOD, GPx, and MDA in ovaries.

Mo (mg/L)	SOD activities (U/mg prot)	GPx activities (U/mg prot)	MDA contents (nM/mg prot)
0 (Control)	115.22 ± 14.32	32.68 ± 1.32	1.0394 ± 0.1282
5	118.56 ± 10.45	36.48 ± 2.03^b	0.9913 ± 0.0113
10	131.38 ± 11.52^b	39.57 ± 3.19^a	0.9756 ± 0.1428
20	100.71 ± 8.78^b	31.39 ± 2.44	1.6071 ± 0.1087^a
40	76.62 ± 6.40^a	22.61 ± 1.73^a	1.6808 ± 0.1886^a

Mo: molybdenum; SOD: superoxide dismutase; GPx: glutathione peroxidise; MDA: malondialdehyde. Data represent means ± SD. Compared with the control group, ^aP < 0.01; ^bP < 0.05.

Ultra-structural changes of ovary tissue after mice exposed to Mo

Transmission electron microscopy (TEM) images (Fig. 3) showed that the ovaries were damaged to some extent by exposure to ≥ 20 mg/L Mo. As a whole, the cytoplasm of ovarian cells contained relatively few organelles, and smaller, morphologically abnormal, swollen mitochondria with distorted cristae in the 20 mg/L and 40 mg/L group compared to the control group (Fig. 3C and F). The vacuolization, mild swelling, disappearance of cristae, and incomplete membrane were observed in damaged intra-ovarian oocytes (Fig. 3A and B) and granulosa cells (Fig. 3D and E).

Figure 3.  Transmission electron microscopic images of the intra-ovarian oocytes (A-C) and the granulosa cells (D-F) in ovaries. Compared to the control (C, F, ×40,000), the mitochondrial vacuolization (A; ×5,000) and the swollen mitochondria with distorted cristae (B; ×40,000) in ovarian oocytes, and the mitochondrial vacuolization in granulosa cells (D, E; ×10,000) were observed in the 20 mg/L (A, D) and 40 mg/L (B, E) group. The arrow indicated the zona pellucida in A, and the arrows or cycle indicated the mitochondria in B-F.

The results presented in this study suggest that Mo dually influences ovarian function in a dose-dependent manner. M II oocyte quality was significantly improved when oxidative stress was decreased with 5 mg/L Mo. On the contrary, oxidative damage increased at the 40 mg/L and M IIoocyte quality was significantly decreased.

To our knowledge, this is the first report concerning the improvement of in vivo oocyte quality by oral administration of Mo at a low dose (but not at the physiological dose). Molybdenum at 5 mg/L significantly increased ovulation as well as decreased the rate of abnormal oocytes (Table 1) and ovarian oxidative stress (Table 2). This indicated that at low dose, Mo could improve ovarian function. A similar improvement in reproductive capacity was observed in our previous study. At 5 mg/L Mo improved the development of mouse embryos cultured in vitro [Bi et al. 2013], and at 12.5 mg/L enhanced the mouse sperm quality and testicular function in vivo [Zhai et al. 2013]. Additionally, in vitro plasma membrane leakage was promoted by 5 mg/L and 10 mg/L Mo [Braydich-Stolle et al. 2005]. These beneficial effects of Mo on the reproductive system were probably due to the biological functions of Mo at low concentrations. The results in this study are in line with the above studies.

Molybdenum at high levels significantly increased ovarian oxidative damage (Table 2), which might be due to the imbalance of trace metals. Guo et al. [2012] showed that the imbalance of trace metals is associated with oxidative stress. At ≥ 40 mg/L Mo significantly decreased the level of ovarian Cu, Zn, and Mn (unpublished results). This would likely have decreased the activities of Cu-Zn SOD and Mn SOD because that Cu and Zn and Mn are prosthetic groups of Cu-Zn SOD and Mn SOD, respectively [Sevilla et al. 1982]. A decrease in Mn SOD activity has been shown to promote lipid, protein, and DNA damage [Adachi et al. 1991].

The ovary index (weight) significantly decreased when the dose of Mo was increased to the 40 mg/L. This likely reflects ovarian apoptotic cell death that is regulated by sex steroids [Billig et al. 1993]. Molybdate interacts directly with steroid (androgens and estrogens) receptors, stabilizes receptors for androgens and estrogens, preventing receptor inactivation or transformation [Dahmer et al. 1984; Van Niekerk and Van Niekerk 1989]. The high dietary intake of Mo could disturb the metabolic fate of the major estrogens [Beattie and Weersink 1992; Murayama and Fukai 1985], which can affect ovary weight [Hegele-Hartung et al. 2004]. The association between ovarian weight and estrogen was also reported in adult female rats exposed to neonatal prazosin [Fitch and Feder 1992]. The results in the present study are consistent with a report by du Plessis et al. [1999] where a Mo (38 mg/kg feed) and sulphur (0.34%) supplemented diet of adult South African Mutton Merino ewes yielded reduced ovarian weight.

The M II oocyte morphology was affected by Mo at 40 mg/L in a significantly negative way (Table 1). A similar phenomenon has been observed in other types of cells. Our previous study showed that at ≥ 40 mg/L, Mo significantly decreased morphology and development of in vitro cultured embryos [Bi et al. 2013]. Ammonium tetrathiomolybdate at 10 mg/L resulted in a marked reduction in cellular clumping, while at 100 mg/L and higher resulted in an absence of clumping [Kendall et al. 2003]. In addition, ammonium molybdate at 330 mg/L inhibited cell division and differentiation in vitro [Kolesarova 2009]. The morphological changes are similar to previous observations.

Ovarian hyperemia was observed in the group of animals exposed to ≥ 20 mg/L. The exact mechanism for this is unclear and requires attention. However, our previous study and others have shown that Mo could provoke a varicocele in male mice [Zhai et al. 2013] or men [Makker et al. 2009]. Studies have suggested that the long-lasting ovarian varicocele can cause hypofunction of the ovaries, similar to testicular varicocele, and could be a cause of infertility [Galkin et al. 1991; Tarazov et al. 2011]. Ovarian hyperemia was accompanied with more morphologically abnormal M II oocytes, lighter ovaries, damaged oocytes, and granulosa cells in ovaries. These results support the above hypothesis and were consistent with our previous study on the testis toxicity of Mo [Zhai et al. 2013].

The decreased ovary index observed and the increased abnormal M II oocyte morphology might be mediated by the increased oxidative damage, which might cause the ovarian dysfunctions. Studies have shown that oxidative damage is a common mediator of apoptosis [Buttke and Sandstrom 1994] in ovarian endothelial cells and luteal cells [Faucher et al. 2005; Nakamura et al. 2001]. Further, it can arrest the ovarian cell-cycle [Clopton and Saltman 1995] and inhibit antral follicle growth [Wang et al. 2012]. In this study, the mitochondria of the intra-ovarian oocytes and granulosa cells were injured to some extent and exhibited the ultra-structural pathological changes in ≥ 20 mg/L groups (Fig. 2), consistent with increased oxidative damage.

In conclusion, the present study suggests that Mo has both the nutritional and toxic function on the ovarian function and M II oocyte quality in a dose-dependent manner. At a low dose, Mo improved overall quality but at a high dose it damaged the ovarian function and M II oocyte quality. The damage likely reflects the action of ovarian oxidative stress. To our knowledge, this is the first report concerning the improvement of oocyte quality in vivo by dose dependent oral administration of Mo.

Methods and Materials

Chemicals

Unless otherwise stated, all components used in this study were purchased from Sigma–Aldrich Corp. (St. Louis, MO, USA).

Animals

The study design was approved by the Animal Care and Use Committee at the Henan University of Science and Technology. Adult ICR strain female mice, aged 4 to 8 w, were used for toxicity experiments. All mice used in this study were maintained under Good Laboratory Practice (GLP) conditions. The mice had free access to a commercial standard pellet diet and distilled water ad libitum.

Exposure of mice to Mo

One hundred and twenty five healthy ICR strain mice, of proven fertility, were divided randomly into five groups of 25 animals each. The mice in different groups were respectively allowed free access to the distilled water containing the Mo (sodium molybdate dihydrate) at concentrations of 0, 5, 10, 20, or 40 mg/L for 14 d.

Transmission electron microscopy of ovaries

The ovaries were quickly removed and cut into blocks. Blocks were retrimmed into 1 mm³ and further prefixed with 2.5% glutaraldehyde in a 0.2 M phosphate buffer (pH 7.4) at 4°C for 3 h. The tissues were rinsed three times in the same buffer, postfixed with 1% OsO₄ at 4°C for 2 h, and dehydrated in ethanol. The specimens for transmission electron microscopy were embedded in Epon. Ultra-thin sections cut horizontally were prepared and double stained with uranyl acetate and lead citrate. The ovarian ultrastructure was observed under a transmission electron microscope (JEM-2000EX; JEOL, Tokyo, Japan), and images were recorded. The ultrastructure position of the oocytes was identified according to the position of the zona pellucida, as shown by the arrow in Figure 3A, the oocyte ultrastructure was situated on the inside of the zona pellucida.

Collection of ovaries and oocytes

The mice in the Mo-exposed groups and control were superovulated by intraperitoneal injection of 10 IU PMSG followed 48 h later by 10 IU of hCG. At 20 h post-hCG, followed by body weighing, the ovaries were quickly removed, photographed, and weighed, then stored at −20°C for measurement of biochemical indicators. At the same time, cumulus–oocyte complexes (COCs) were collected from oviducts, and the cumulus cells were dispersed in M2 medium containing 0.1% hyaluronidase, then the oocytes were washed and collected in M2 medium, and counted under a stereomicroscope.

Evaluation of oocyte quality

Oocytes were morphologically classified as normal and abnormal according to the Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology [2011] report under an inverted phase contrast microscope at ×400 magnification (TE-2000, Nikon, Tokyo, Japan). Briefly, the normal oocyte morphology (Fig. 2 A) was that of a spherical structure enclosed by a uniform zona pellucida, with a uniform translucent cytoplasm free of inclusions, a size-appropriate polar body, and a size-appropriate perivitelline space. The oocytes in other morphologies (Fig. 2B-F) were considered abnormal. The percentage of abnormal oocytes was calculated relative to the total number of ovulations.

Measurement of biochemical indicators

Ovaries were added into precooled 0.9% saline with a ratio of 1:9 (g:mL) and made into 10% homogenate using a Ultraschallprozessor (UP200S, Dr. Hielscher Gmbh, Germany), for the measurement of biochemical indicators. The activities of SOD and GPx, and the level of MDA in ovaries were determined by using the corresponding assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions.

Statistical analysis

Except for the abnormality rate of oocytes, all of the data were expressed as the mean ± standard error of the mean and statistically analyzed by single variable of the general linear model using SPSS software (SPSS 17. 0). The number of ovulations and the abnormality rate of oocytes were analyzed by the Chi-Square Test. Differences between experimental groups were considered significant at P < 0.05 or extreme at P < 0.01.

Abbreviations

Mo:	=	molybdenum
ROS:	=	reactive oxygen species
SOD:	=	superoxide dismutase
GPx:	=	glutathione peroxidise
MDA:	=	malondialdehyde
CAT:	=	catalase
GV:	=	germinal vesicle
M II:	=	metaphase II
TEM:	=	transmission electron microscopy

Acknowledgments

Chunjie Zhang Ph.D, Yinju Li Ph.D, Yumei Liu Ph.D, and Lifang Han laboratory assistant are thanked for assistance in the usage of instruments, Long Ta Ph.D for analysis of pathology slices, and Ms. Kerry Alicia Webber for the improvement of language for the writing of this paper.

Declaration of interest: This work was supported by the National Natural Science Foundation of China (Project No. 30901026) and the Higher Education Finance Scheme for Young Backbone Teachers in Henan Province (Project No. 2010GGJS- 071), China. The authors report no declarations of interest.

Author contributions: Conceived and designed the experiments: F-JL, Y-LZ, X-LY, X-LC; Performed the experiments: X-LC, X-WZ, L-JJ, QQ; Analyzed the data: F-JL, Z-JL, X-GM; Contributed reagents/ materials/ analysis tools: R-ZS; Wrote the manuscript: F-JL, X-LC, Y-LZ, R-ZS.