Ultrastructural Pathology, Early Online, 1–5, 2015 ! Informa Healthcare USA, Inc. ISSN: 0191-3123 print / 1521-0758 online DOI: 10.3109/01913123.2015.1027436

ORIGINAL ARTICLE

Effect of Cadmium on Cellular Ultrastructure in Mouse Ovary Ying Wang, BS1, Xuejuan Wang, BS1, Yanwu Wang, PhD1, Rong Fan, BS1, Chao Qiu, PhD1, Shan Zhong, PhD1,2, Lei Wei, MD, PhD1,2, and Daji Luo, PhD1,2

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School of Basic Medical Sciences, Wuhan University, Wuhan, Hubei, P.R. China and 2Hubei Provincial Key Laboratory of Developmentally Originated Disease, School of Basic Medical Sciences, Wuhan University, Wuhan, Hubei, P.R. China

ABSTRACT This study aimed at analyzing the cytotoxicity and pathological effects of cadmium on the ovary. Our studies revealed that cadmium was deposited in the mouse ovary after 8 d cadmium injection in vivo. Also, the increase in the rate of body weight was slowed, while the gonadosomatic index was reduced in the CdCl2 group, compared with the control group. Meanwhile, cadmium affected the maturation of follicles, the degradation of corpus luteum, the arrangement of follicles and corpus luteum, and increased the number of atresia follicles. Besides, under the electron microscope, chromatin margination, karopyknosis, swelling of mature cisternae of Golgi apparatus, mitochondrial cristae disappearance, and swelling of the rough endoplasmic reticulum can be observed in the CdCl2 group mice. Collectively, our findings elucidated the morphological mechanism that the exposure of cadmium changed the ultrastructure of cells in ovary tissues. Keywords: Cadmium, mouse ovary, ultrastructure changes

although its toxic mechanism still remains largely unknown, and research on its prevention and cure still lacks sufficient understanding [3,8]. It is a vital way to understand the toxicity of Cd to elucidate the morphological changes of ovary cells after the exposure of Cd. Unfortunately, the morphological mechanism responsible for the toxic effects of Cd in the ovary is far from being completely understood. The present study was conducted to investigate the effect of Cd2+ on cellular structure and ultrastructure in mouse ovary.

Cadmium (Cd) is an industrial, environmental pollutant and a long biological half-life metal, arising primarily from battery, plastic, fertilizer industries, and cigarette smoke [1]. It has no known biological function and regulation pathway, and prolonged exposure causes long-term toxic effects to humans and animals. More and more studies show that Cd is a toxic metal, targeting the lung, liver, and kidney and causing nephrotoxicity, hepatotoxicity, and immunotoxicity after prolonged exposure [2–4]. In fact, a wide spectrum of deleterious effects on the reproductive tissues and the developing embryo has also been described [5]. In this way, abnormal concentrations of Cd were found not only in the follicular fluid but also in the placentas of smokers [6,7]. These observations support the concept that the reproductive organs of smokers are at higher risk of exposure to toxic levels of Cd. Thus, the reproductive toxicity of Cd has become of great concern in recent dozen years,

MATERIALS AND METHODS Animals and chemicals Female Kunming mice of 7–8 weeks old were purchased from the ABSL-III Laboratory of Wuhan University. All animal work was performed in

Received 18 February 2015; Accepted 5 March 2015; Published online 24 June 2015 Correspondence: Daji Luo, Department of Genetics, School of Basic Medical Sciences, Wuhan University, Wuhan, Hubei, China. E-mail: [email protected]

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accordance with protocols and guidelines approved by the Institutional Animal Care and Use Committee. All mice were housed under the identical conditions in an aseptic facility and given free access to water and food. CdCl2 was obtained from Sigma (St. Louis, MO).

After that, it was followed by postfixation with 1% osmium tetroxide. Samples were dehydrated in graded ethanol, embedded in Epon 812. The sections were examined under a TEM (Hitachi HT7700-SS, Tokyo, Japan) [12].

Animal treatment strategy

Statistical analysis

The animals were randomly divided into two groups – control group and CdCl2 group. There are six mice in each group, the control group and CdCl2 group animals were given vehicle solutions (NS, 20 mL/kg/d) and the CdCl2 group animals were intraperitoneal injected with a single dose of CdCl2 dissolved in NS (6 mg/kg/body weight) [9]. The treatment was continued for 8 d at the rate of 1 dose/d. The body weight of all mice was detected to evaluate the effects of the treatment on animals. At the end of time period, mice were fasted for 15 h. After killing, small pieces of ovary tissues from mice were obtained and processed for atomic absorption analysis of Cd, histopathology analysis, and transmission electron microscopy (TEM) examination.

The data are expressed as the mean ± SEM. Differences among means were analyzed by oneway ANOVA. p5.05 value was considered statistically significant with SPSS version 19.0 (SPSS Inc., Chicago, IL).

Atomic absorption analysis Six ovary tissues from control group and Cd group were pooled and mixed up completely, individually. Then 0.2 g (wet weight) of samples was digested with nitric acid (0.5 mol/L), evaporated, and diluted with HNO3 (10 mmol/L) [10]. The prepared samples were mineralized in a microwave oven (Mars Xpress 5, CEM, Princeton, NJ) [11]. Trace element concentrations in the mineralized samples were determined using the contrAA 700 atomic absorption spectrophotometer (Analytik Jena AG, Jena, Germany) with graphite furnace atomic absorption spectrometry (GF-AAS) for Cd (228 nm). All analyses were run in three replicates. The percent RSD did not exceed 5% for GF-AAS analysis.

RESULTS Effect of the treatment on body weight and gonadosomatic index The body weight and the gonadosomatic index (GSI) were applied to detect the effects of Cd in whole animal and tissues level. Weight of each mouse was at the range of 24–26 g before the treatment. After the treatment, there was a noticeable increase in weight in both groups. The average body weights of control group and CdCl2 group increased by 23.2% and 14.0%, respectively, compared with the weights before the treatment (Figure 1). The increase in the rate of body weight was slower in the CdCl2 group, compared with the control group. The GSI is commonly used to evaluate the gonad and gonad development [13]. At 8 d after the treatment, the GSI of control group was 3.1 (N = 6) and that of the CdCl2 group was 2.6 (N = 6), respectively (Figure 2). GSI of the CdCl2 group was lower than that of the control group.

Inverted microscope for ovary cells morphology Ovary sections were collected and fixed in 4% paraformaldehyde. Paraffin-embedded tissue sections (5 mm) were stained with hematoxylin and eosin (H&E), according to standard techniques [12]. Images were taken using a Nikon ECLIPSE Ci (Nikon Instruments, Inc., Tokyo, Japan).

TEM for cell ultrastructure Ovary was cut into 1 mm, three fragments, and fixed by immersion in 4% prechilled glutaraldehyde.

FIGURE 1. The effect of the treatment on body weights. Solid square and round represent the body weight in control group and CdCl2 group, respectively (six mice per group). Ultrastructural Pathology

Cadmium Effect on Ovary Cellular Ultrastructure

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FIGURE 2. The effect of the treatment on the GSI; *p5.05 (six mice per group).

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Cd (Figure 4B1–3). From the section view, the ovary of the CdCl2 group is larger than that of the control group (Figure 4A1 and B1). It was found that the distribution of follicles and corpus luteum were seriously affected and became highly confused in ovary tissues in the Cd group (Figure 4B1). The ovary contained all developmental stages of follicles in the control group. Also, follicles and ovary cortex cell developed and grew normally (Figure 4A2). However, few primordial follicles were found, and more atresia follicles were found in the ovary of the CdCl2 group. Meanwhile, the average size of corpus luteum in the CdCl2 group is larger than that in the control group, as the degradation of corpus luteum had not occurred (Figure 4B2). Although there was no significant difference in the size of follicles between the control and the CdCl2 groups, the division and growth of the ovary cortex cells were seriously affected and became highly confused in ovary tissues in the Cd group (Figure 4B2 and B3). The oocyte nucleus damage and the morphological changes of granulosa cells, and the cells of the internal and external theca layers of the ovarian follicle caused by Cd were found in the ovary of the CdCl2 group (Figure 4B3).

Cellular ultrastructure changes in mouse ovary cells

FIGURE 3. The graphite furnace atomization (GF-AAS) for Cd (228 nm); ***p5.001.

Atomic absorption analysis of Cd in mouse ovary To demonstrate the deposition of Cd in the tissue in vivo, the graphite furnace atomization (GF-AAS) was applied to detect the concentration of Cd (228 nm) in mice ovary. Standard Cd solution is diluted into 2.0 mg/L and the instrument automatically converts into (0, 0.5, 1.0, 1.5, and 1.0 mg/L) the standard series. Using software, the standard curve is illustrated for analyzing Cd concentrations of samples. After 8 d treatment, the concentration of Cd was 4.79 mg/kg in the ovaries of CdCl2 group animals, which is significantly different from that of Cd in the ovaries of control group animals (Figure 3).

In order to explore the ultrastructural changes of follicles, we used TEM to observe the morphology of mice follicles in both the control group and the CdCl2 group (Figure 5). Compared with cells in the control group (Figure 5A1–3), the cellular ultrastructure changes of granulosa cells and follicles in CdCl2 groups were more visible (Figure 5B1–3). The chromatins were accumulated under the nuclear membrane, but mild convolution or breakdown of the nuclear membrane and decondensation of chromatin was visible in the CdCl2 group (Figure 5B1). Furthermore, severe karyopyknosis was also visible (Figure 5B1). The mitochondria in the normal cells were oval and regular in shape and mitochondrial cristae can also be clearly observed (Figure 5A2). On the contrary, in CdCl2 groups, the shape of mitochondria was swelled to round, while the structure of mitochondrial cristae became blurry and even disappeared (Figure 5B2). Besides, swelling of mature cisternae of Golgi apparatus and swelling of the rough endoplasmic reticulum were observed (Figure 5B2 and 3).

Histopathological changes of mouse ovary DISCUSSION The control group demonstrated the normal basic structure of mouse ovary (Figure 4A1–3). The CdCl2 group showed an evident ovary damage caused by !

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Cd is a long biological half-life environmental pollutant, targeting the immuno-organ, and causing

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FIGURE 4. The morphological changes of ovary tissues of mice by H&E. A1–3 and B1–3 represent the changes of ovary tissues in control group and CdCl2 group, respectively. A1,B1, original magnification 20, A2,B2, original magnification 100, A3,B3, original magnification 400.

FIGURE 5. The morphological changes of ovary tissues of mice by electron microscope. A1–3 and B1–3 represent the changes of ovary tissues in control group and CdCl2 group, respectively. Original magnification: A1  3000, A2  7000, A3  20000, B1  4000, B2  20000, B3  20000.

immuno-toxicity after prolonged exposure [1–4]. Recently, the reproductive toxicity of Cd has become of great concern. However, its toxic mechanism still remains largely unknown, and also its molecular

mechanism and the morphological mechanism in gonads. The present study was carried out to evaluate the cytotoxicity and pathological effects of Cd on the ovary. We chose CdCl2 injections to deliver ions to Ultrastructural Pathology

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Cadmium Effect on Ovary Cellular Ultrastructure organisms of experimental mice because of two reasons: (1) well-controlled dosage; (2) in comparison to oral Cd administration, more pronounced effect [4,14]. It has been shown that Cd blood levels in injected rats were higher than in orally treated ones [14]. Using GF-AAS, Cd were detected and successfully deposited in the ovary of the CdCl2 group after 8 d treatment (Figure 3). It is obvious that Cd is harmful to gonads of human and animals, based on the data of body weight and GSI after the treatment (Figures 1 and 2). Compared to the control group, the increase in the rate of body weight and the GSI were reduced in the CdCl2 group. Interestingly, the size of the ovary in the CdCl2 group is large than that in the control group (Figure 4A1 and B1). According to our data, the distribution of follicles and corpus luteum became highly confused in ovary tissues in the Cd group (Figure 4B1). Folliculogenesis is controlled by the endocrine system. Gonadotropinreleasing hormone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), estrogen, and progesterone participate in an intricate process of positive and negative feedbacks to regulate folliculogenesis. It has been reported that there are significant differences in the expression level of FSH, LH, and testosterone between Cd exposure and non-Cd exposure [15,16]. It implied that Cd induced disorders of these hormones to result in the ectopic maturation of follicle (Figure 4B1), follicles atresia (Figure 4B2), and the abnormal division and growth of the ovary cortex cells (Figure 4B3). Cd induces alterations in the protein synthesis in the liver in vivo [4,17]. Ribosomes, endoplasmic reticulum, and Golgi apparatus were the most vital organelles on intracellular proteins synthesis. According to our data, swelling of mature cisternae of Golgi apparatus and swelling of the rough endoplasmic reticulum were observed in the CdCl2 group (Figure 5B2 and B3). It is suggested that Cd2+ brought about the most remarkable alterations of protein synthesis activity in the ovary. Collectively, our findings elucidated the morphological mechanism that the exposure of Cd changed the ultrastructure of cells in ovary tissues. Our results reminded that the reproductive toxicity of Cd should be concerned.

DECLARATION OF INTEREST The authors report no conflicts of interest.

FUNDING This work was supported by ‘‘the National Natural Science Foundation of China’’ (No. 31472263), the !

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Foundation of Health and Family Planning Commission of Hubei province (No. JS-2011009), and ‘‘the Fundamental Research Funds for the Central Universities’’ (No. 014301020204).

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Effect of Cadmium on Cellular Ultrastructure in Mouse Ovary.

This study aimed at analyzing the cytotoxicity and pathological effects of cadmium on the ovary. Our studies revealed that cadmium was deposited in th...
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