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Low dose carbendazim disrupts mouse spermatogenesis might Be through estrogen receptor related histone and DNA methylation
Ecotoxicology and Environmental Safety 176 (2019) 242–249
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Low dose carbendazim disrupts mouse spermatogenesis might Be through estrogen receptor related histone and DNA methylation
T
Jing Liua, Pengfei Zhangb,c, Yong Zhaob, Hongfu Zhangd,∗ a
University Research Core, Qingdao Agricultural University, Qingdao, 266109, PR China College of Life Sciences, Qingdao Agricultural University, Qingdao, 266109, PR China c College of Animal Sciences and Technology, Qingdao Agricultural University, Qingdao, 266109, PR China d State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100193, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Carbendazim Spermatogenesis Estrogen receptor signaling Histone methylation DNA methylation
Pesticides, fungicides are reportedly involved in a decline in spermatozoa quality, especially motility, and a consequent increase in the rate of infertility. Fungicide carbendazim (CBZ) is widely used in agriculture and other aspects. Although CBZ is known to disrupt spermatogenesis, causing a decrease in spermatozoa concentration and motility, the mechanisms are not fully understood. We aimed to further explore the underlying mechanisms of CBZ disruption of spermatogenesis. Pubertal mice were exposed to low doses (0.1, 1 and 10 mg/ kg body weight) of CBZ for 5 weeks, then many factors related to spermatogenesis have been explored. It was found that 0.1–10 mg/kg body weight of CBZ exposure decreased mouse sperm motility and concentration, diminished the important protein factors (VASA, PGK2, B-Amy and CREM) for spermatogenesis, reduced sperm protein acrosin level, disrupted very vital epigenetic factors H3K27, 5 mC and 5 hmC. Furthermore, CBZ exposure damaged estrogen receptor alpha (ERα) pathway by decreased the protein levels of ERα and its targets PI3K and AKT. In summary low doses of CBZ exposure disrupted mouse spermatogenesis through estrogen receptor signaling; and that histone methylation and DNA methylation might play vital roles in CBZ disturbance of spermatogenesis through intertwining with estrogen signaling pathways. CBZ from the contamination in environment or food chain poses a serious threat to the normal development of spermatozoa. Therefore we strongly recommend to minimise the use of CBZ since it causes the severe issues on spermatogenesis.
1. Introduction Recent epidemiological studies report that infertility rates have been dramatically elevated from 7% to 8% in the 1960s to their current levels of 20%–35%, mostly due to a significant decrease in sperm motility (Levine et al., 2017; Centola et al., 2016). Semen quality is an essential biomarker of male overall reproductive function (Eisenberg et al., 2014). Among environmental chemicals, pesticides have been suggested to play a vital role in this deterioration in male fertility (Chiu et al., 2015). As a broad spectrum benzimidazole fungicide, carbendazim (methyl 2-benzimidazole carbamate, CBZ) can be a breakdown product of other fungicides, for example: benomyl and thiphanate-methyl. CBZ is widely used in agriculture, and is also used during the manufacturing of products such as paint, paper, and leather. In agriculture, it is used to treat foliar diseases on crops, and due to its widespread application, CBZ can be isolated from soil, ground water, and agricultural goods. Moreover,
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it has been found in indoor environments. In animals, CBZ has been found to impair liver and kidney function, to disrupt hematopoiesis and metabolism, and to induce chromosome aberrations by harming the spindle during mitosis and meiosis (Zubrod et al., 2014; Muthuviveganandavel et al., 2008; Akbarsha et al., 2000; Zuelke and Perreault, 1995; Sarrif et al., 1994). Epidemiologist have identified CBZ contamination worldwide and have determined that it negatively affects the reproductive ability of animals and human beings (Sakr and Shalaby, 2014). Humans can be directly exposed to CBZ through oral consumption (for example contaminated fruits, vegetables, and water), and inhalation and dermal exposure at home and in the work place (Bakirci et al., 2014; Boobis et al., 2008). CBZ has been found to possess endocrine-disrupting (androgen-like) actions (Durand et al., 2017) that interfere with spermatogenesis. Furthermore, exposure results in a decline in spermatozoa concentration and motility, an elevation in sperm abnormalities, an increase in oxidative stress and apoptosis, and a reduction in the rate and stability of microtubule assembly (Rama et al.,
Corresponding author. E-mail address: [email protected] (H. Zhang).
https://doi.org/10.1016/j.ecoenv.2019.03.103 Received 7 January 2019; Received in revised form 20 March 2019; Accepted 25 March 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
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procedure (Zhang et al., 2018).
2014; Adedara et al., 2013; Pacheco et al., 2012). DNA methylation, histone modifications, and noncoding RNAs are common epigenetic modifications which have been implicated in spermatogenesis (Gannon et al., 2014; Carrell, 2012; Ge et al., 2017). Furthermore, spermatogenesis is very sensitive to environmental contamination, which induces dramatic epigenetic alterations (Strazzullo and Matarazzo, 2017; Jenkins and Carrell, 2011). Such aberrations may lead to a decrease in male fertility, disruption of early embryo development, or diseases in offspring because they can be passed through generations to impact on future health and development (Wu et al., 2015; Aston et al., 2012). Many investigations have found that CBZ has endocrine disrupting effects that negatively influence testicular steroidogenesis to disrupt the production of testosterone, LH, FSH, GnRH, and to decrease the gene expression of estrogen receptor alpha (ERα) and ERβ (Rama et al., 2014). However, the mechanisms by which this takes place are unknown. During puberty, the testes undergo dramatic growth due to spermatogonial proliferation, the expansion of meiotic and haploid germ cells, and the increase in somatic cells such as Sertoli and Leydig cells. The pubertal period is therefore critical during male reproductive system development, and any disturbance in spermatogenesis at this stage will have a confounding impact on later fertility. Accordingly, the purpose of current study was to investigate the adverse effects of low concentrations of CBZ on spermatogenesis during the peripubertal stage in mice and the role of epigenetic modifications in this process.
2.5. Measurement of plasma steroid hormones ELISA kits (Nanjing Jiancheng Bioengineering Institute, China) for plasma testosterone (T) and estrogen (E) levels were used, following the manufacturer's instructions (Zhang et al., 2018; Wang et al., 2016). 2.6. Measurement of plasma AST and ALT Plasma levels of aspartate aminotransferase (AST/GOT) and alanine aminotransferase (ALT/GPT) were determined directly using appropriate kits (Nanjing Jiancheng Bioengineering Institute) and following the manufacturer's instructions. Six samples from each treatment were determined (Wang et al., 2016). 2.7. Western blotting Western blotting analysis took place as previously reported (Zhang et al., 2018; Wang et al., 2016; Zhao et al., 2016). The bands were quantified using Image-J software. The intensity of specific protein bands were initially normalized to actin, and subsequently to the control. More than six replicates were performed. 2.8. Detection of protein levels and location in the testes using immunofluorescence staining
2. Materials and methods (further details are provided in supplemental information)
Immunofluorescence staining of testicular samples is reported in our recent publication (Zhang et al., 2018; Wang et al., 2016). For each sample in each experiment, over 1000 cells were counted; data were then normalized to the control.
2.1. Study design (mouse exposure to CBZ) This investigation was performed in strict accordance with the recommendations provided in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Qingdao Agricultural University IACUC (Institutional Animal Care and Use Committee) (Zhang et al., 2018). This study set out to explore the impact of low doses of CBZ on spermatogenesis in mice and to investigate the underlying mechanisms. Male ICR mice were exposed to CBZ through oral gavage. The doses of CBZ used in this study in vivo were very low (0.1–10 mg/kg). However, CBZ has been used for animal studies from 100 to 600 mg/kg (Rama et al., 2014; Sakr and Shalaby, 2014; Farag et al., 2011). The CBA dosing solution was freshly prepared on a daily basis in corn oil. Four treatments were used with 30 mice per treatment as follows: (1) vehicle control (corn oil); (2) CBZ-0.1 mg/kg BW; (3) CBZ-1 mg/kg BW; (4) CBZ-10 mg/kg BW. Each mouse was administered 0.1 ml of the appropriate gavage each morning for five weeks starting at 25 d old.
2.9. Statistical analysis Quantitative data were presented as the mean ± SEM. ANOVA and the LSD multiple comparison test were used for statistical analyses. The statistical software SPSS (IBM Co., NY) was used for all analyses. All treatment groups were compared with each other for every parameter. Differences were considered statistically significant at p < 0.05. 3. Results 3.1. CBZ inhibited mouse spermatogenesis in vivo After a 5 week exposure to low doses of CBZ, mouse spermatozoa motility (grade A + B) was significantly decreased by CBZ 0.1, 1, and 10 mg/kg body weight (BW; Fig. 1A); spermatozoa concentration was significantly reduced by the CBZ 10 mg/kg exposure (Fig. 1B). Spermatogenesis is regulated by protein factors including Synaptonemal complex protein 3 (SYCP3), Deleted In Azoospermia Like (DAZL), DEAD-Box Helicase 4 (DDX4/VASA), KIT Proto-Oncogene Receptor Tyrosine Kinase (KIT), Glial Cell Derived Neurotrophic Factor (GDNF), CAMP Responsive Element Modulator (CREM), Zinc Finger Protein 37 Homolog (ZFP37), MYB Proto-Oncogene A (A-Myb), MYB Proto-Oncogene B (B-Myb), MutS Homolog 4 (MSH4), Phosphoglycerate Kinase 2 (PGK2), MutS Homolog 5 (MSH5), Snail Family Transcriptional Repressor 3 (SNAI 3), KIT ligand (KITLG). In current study VASA, B-myb, PGK2, and CREM were decreased by the CBZ 1 mg/kg BW, and 10 mg/kg BW exposures (Fig. 1C and D). Western blotting data for VASA was confirmed by IHF (Fig. 1E and F). At the same time the sperm markers acrosin and PNA were decreased by the CBZ 1 mg/kg BW and 10 mg/kg BW exposures in a dose dependent manner (Fig. 2A, B, and 2C). All the data suggested that CBZ inhibited mouse spermatogenesis in vivo, similar to findings in previous reports. However, low doses of CBZ exposure had just a slight effect on body parameters (Table 1).
2.2. Evaluation of spermatozoa motility using a computer-assisted sperm analysis system The motility of spermatozoa was assessed using a computer-assisted sperm assay (CASA) method according to World Health Organization guidelines (Zhang et al., 2018; Zhao et al., 2016; WHO, 2010). Spermatozoa motility data represented only grade A + grade B spermatozoa, since only these are considered functional. 2.3. Morphological observations of spermatozoa Abnormalities in spermatozoa were analysed according to a previously reported procedure (Zhang et al., 2018). 2.4. Assessment of acrosome integrity Acrosomal integrity was assessed following a previously reported 243
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Fig. 1. (A) Mouse spermatozoa motility (grade A + grade B). Y-axis = % of total cells, X-axis = the exposure doses (mg/kg body weight). (B) Mouse spermatozoa concentration. Y-axis = spermatozoa concentration, X-axis = the exposure doses (mg/kg body weight). (C) Protein levels of VASA, PGK2, B-Myb and CREM in mouse testis tissues detected by Western blotting. n > 3. (D) Quantitative data for Western blotting analysis of VASA, PGK2, B-Myb and CREM. (E) VASA positive cells in mouse testis tissues detected by immunofluorescence staining. Red: VASA; Blue: DAPI (for nuclei). n > 3. (F) Quantitative data for immunofluorescent staining analysis of VASA. a, b, c indicate a significant difference among different treatments (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
3.3. CBZ disrupted histone and DNA methylation in mouse testes
3.2. CBZ disturbed the estrogen receptor alpha (ERα) pathway in mouse testes
Histone methylation marker tri-methylation of H3 at lysine 27 (H3K27me3) and DNA methylation markers 5-methylcytosine (5 mC) and 5-hydroxymethylcytosine (5 hmC) are important for spermatogenesis and they were analysed in mouse testes in the current study. It was found that H3K27 was mainly expressed in spermatogonia stem cells (SSCs) and the number of H3K27 positive SSCs was significantly elevated by the CBZ 1 mg/kg BW and 10 mg/kg BW exposures in a dose dependent manner (Fig. 5A and B). The marker 5mc was identified in Leydig cells (Fig. 6A) and was decreased by the CBZ 1 mg/kg BW and 10 mg/kg BW exposures (Fig. 6B). The marker 5hmc was mainly expressed in SSCs and the number of 5hmc positive SSCs was reduced by the CBZ 10 mg/kg BW exposure (Fig. 6C and D). This data suggested that CBZ exposure adversely affected epigenetic markers with a resulting disturbance of spermatogenesis.
Estrogen receptor alpha (ERα) is reported to be a target of CBZ (Durand et al., 2017), thus ERα and its target genes Phosphoinositide 3kinase (PI3K) and AKT Serine/Threonine Kinase (AKT) were determined in the current investigation. It was found that the number of ERα positive leydig cells in mouse testes was significantly reduced by the CBZ 1 mg/kg BW, and 10 mg/kg BW exposures (Fig. 3A and B). Furthermore, the hormone production proteins Hydroxysteroid Dehydrogenase 3β1 (HSD3β1) and Hydroxysteroid Dehydrogenase 17β1 (HSD17β1) were reduced by the CBZ1 mg/kg BW, and 10 mg/kg BW exposures (Fig. 3C, D, and 3E). This suggested that CBZ disrupted the endocrine system in the testes. The number of PI3K positive cells in mouse testes was significantly decreased by the CBZ 1 mg/kg BW and 10 mg/kg BW exposures in a dose dependent manner (Fig. 4A and B). At the same time, AKT protein levels in the testes was decreased by the CBZ 1 mg/kg BW and 10 mg/kg BW exposures (Fig. 4C). This data suggested that CBZ inhibited spermatogenesis through the ERα pathway.
4. Discussion Male reproductive toxicology is attracting increasing concern 244
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Fig. 2. (A) Acrosin and PNA positive cells in mouse testis tissues detected by immunofluorescence staining. Red: Acrosin; Blue: DAPI (for nuclei). n > 3. (B) Quantitative data for immunofluorescent staining analysis of Acrosin. (C) Quantitative data for immunofluorescent staining analysis of PNA. a, b indicate a significant difference among different treatments (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
foodstuffs (Durand et al., 2017). Meanwhile, lots of insecticides, herbicides, and fungicides have been identified as endocrine disrupting chemicals (EDCs). EDCs mimic endogenous hormones to modify endocrine system function, even to interfere with an animal's survival, development, sexual differentiation, and reproduction (Zoeller et al., 2012). CBZ has been widely used in the control of various pathogens in agriculture, and it is now included in the priority list of EDCs by the
because human sperm quality has significantly deteriorated over the past few decades (Le Moal et al., 2014). Pesticides and other environmental chemicals have been implicated in this deterioration (Chiu et al., 2015). The extensive production and widespread use of pesticides has led to contamination of the environment, soil, groundwater sources, and even agricultural products. Because most pesticides are highly persistent, even in low amounts, their accumulation is magnified in
Table 1 Mouse body and plasma parameters. Data present as Average ± SEM. a, b, c indicate a significant difference among different treatments (n > 6; p < 0.05).
Body weight (g) Testis index (% of body weight) Kidney index (% of body weight) Spleen index (% of body weight) Liver index (% of body weight) E2 (ng/L) Testosterone (ng/L) Acrosome integrity (% of total cells) ALT AST
Control
CBZ 0.1
CBZ 1
CBZ 10
40.25 ± 1.50a 0.83 ± 0.04 1.78 ± 0.05 0.43 ± 0.05 5.85 ± 0.19 a 394.20 ± 73.59 1.33 ± 0.01 4.44 ± 0.45 117.5 ± 36.0 100.9 ± 20.4
39.07 ± 0.51 a 0.79 ± 0.031 1.70 ± 0.05 0.52 ± 0.09 5.64 ± 0.15 a 244.10 ± 52.90 1.30 ± 0.02 3.88 ± 0.71 117.1 ± 44.7 97.8 ± 33.4
38.87 ± 1.39 a 0.81 ± 0.024 1.91 ± 0.06 0.36 ± 0.02 5.60 ± 0.21 a 173.30 ± 10.40 1.29 ± 0.01 3.46 ± 0.33 115.9 ± 39.6 89.6 ± 26.6
36.26 ± 1.01 b 0.89 ± 0.021 1.70 ± 0.1 0.41 ± 0.04 5.13 ± 0.14 b 318.80 ± 120.50 1.29 ± 0.02 5.03 ± 0.58 56.5 ± 14.3 72.1 ± 34.0
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Fig. 3. (A) ERα positive cells in mouse testis tissues detected by immunofluorescence staining. Red: ERα; Blue: DAPI (for nuclei). n > 3. (B) Quantitative data for immunofluorescent staining analysis of ERα. (C) Protein levels of HSD17β1 and HSD3β1 in mouse testis tissues detected by Western blotting. n > 3. (D) Quantitative data for Western blotting analysis of HSD17β1. (E) Quantitative data for Western blotting analysis of HSD3β1. a, b, c indicate a significant difference among different treatments (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4. (A) PI3K positive cells in mouse testis tissues detected by immunofluorescence staining. Red: PI3K; Blue: DAPI (for nuclei). n > 3. (B) Quantitative data for immunofluorescent staining analysis of PI3K. (C) Protein levels of AKT in mouse testis tissues detected by Western blotting. n > 3. a, b, c indicate a significant difference among different treatments (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Many investigations have tried to explore the mechanisms underlying the CBZ disruption of spermatogenesis; they have found that CBZ influences testicular steroidogenesis with an alteration in the levels of various hormones (testosterone, LH, FSH, GnRH) and in gene expression levels of estrogen receptor alpha (ERα) and ERβ (Rama et al., 2014). However, the mechanisms by which CBZ disrupts spermatogenesis are not fully understood. In the current study, it was found that steroid hormone production proteins (HSD3β1 and HSD17β1) were reduced by CBZ exposure in mouse testes. These results are in line with earlier reports. Epigenetic modifications (DNA methylations, histone modifications, and non-coding RNAs) play vital roles in gametogenesis by regulating gene expression (Stewart et al., 2016). It has been found that histone methylation on lysine residues could activate or repress gene expression depending on the position of the residue modified (Dumasia et al., 2017; Bannister and Kouzarides, 2011). Di- or tri-methylation of H3 at lysine 4 (H3K4me2/3) and H3 acetylated at lysine 9 can activate gene expression; however, di- and tri-methylation of H3 at lysine 27 (H3K27me2/3) can repress gene expression (Bannister and Kouzarides, 2011). H3K27me3 is expressed in most male germ cells transcriptionally to repress the expression of somatic-specific genes to maintain their homeostasis and differentiation. The ERα signaling pathway is very important for spermatogenesis and it regulates histone methylation during this process (Dumasia et al., 2017). In the current investigation, we found that ERα levels were decreased by CBZ in mouse testes, and at the same time the ERα target genes PI3K and AKT were diminished by CBZ, which suggests that the ERα pathway was disrupted by CBZ. Moreover, our results showed that CBZ exposure promoted H3K27me3 expression in mouse spermatogonia cells. It has been reported that ERα agonist (4,4′,4’’-(4-Propyl-[1H] pyrazole-1,3,5triyl; PPT) promotes H3K27me3 expression (Dumasia et al., 2017). Our data indicate that the ERα pathway and histone modification may play an important role in CBZ disrupted spermatogenesis. In male gamete development, epigenome and epigenetic modifications play important roles throughout the establishment of primordial germ cells to the development of mature gametes (Stewart et al., 2016). DNA methylation is a major mechanism of genome reprogramming. 5Methylcytosine (5 mC) is important for cell fate determination and it modifies DNA-protein interactions and activates or represses gene expression. 5 mC and 5-hydroxymethylcytosine (5 hmC) are important during spermatogenesis (Stewart et al., 2016). DNA methylation modifies estrogen receptor (ER) gene expression, at the same time estrogen signaling regulates DNA methylation (Vrtačnik et al., 2014; Marques et al., 2013). Estrogen signaling and DNA methylation tightly interact together to regulate spermatogenesis (Vrtačnik et al., 2014; Marques et al., 2013). In our current investigation, we found that both 5 mC and 5 hmC were reduced by CBZ exposure which further suggests that the intertwined ER pathways and DNA methylation (5 mC and 5 hmC) were involved in the alteration of spermatogenesis by CBZ. In conclusion, the purpose of the current study was to further investigate the underlying mechanisms of CBZ disruption of spermatogenesis; we demonstrated that: i) CBZ disrupted mouse spermatogenesis with damage to sperm production proteins and sperm proteins; ii) CBZ disrupted spermatogenesis through estrogen receptor signaling; iii) histone methylation and DNA methylation might play vital roles in CBZ disturbance of spermatogenesis through intertwining with estrogen signaling pathways. Due to its over use or incorrect use, CBZ poses a serious threat to healthy sperm development. Therefore, greater attention should be paid to the use of CBZ to minimise its influence on human spermatogenesis.
Fig. 5. (A) H3K27me3 positive cells in mouse testis tissues detected by immunofluorescence staining. Red: H3K27me3; Blue: DAPI (for nuclei). n > 3. (B) Quantitative data for immunofluorescent staining analysis of H3K27me3. n > 3. a, b, c indicate a significant difference among different treatments (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
European Commission (Ferreira et al., 2008). Early investigations report that CBZ disrupts reproductive systems, induces germ cell apoptosis, and causes embryotoxicity or teratogenesis in mice, rats, hamsters, and even humans (Adedara et al., 2013; Farag et al., 2011; Yu et al., 2009; Akbarsha et al., 2001). Acting as an EDC, CBZ has been found to decrease spermatozoa concentration and motility, to increase sperm abnormalities, to elevate oxidative stress and apoptosis, to reduce the rate and stability of microtubule assembly, and to disrupt spermatogenesis (Durand et al., 2017; Rama et al., 2014; Adedara et al., 2013; Pacheco et al., 2012). In the current study, we found that very low doses of CBZ decreased mouse spermatozoa motility and concentration. Furthermore, it disrupted spermatogenesis with damage to sperm production proteins and alterations in sperm proteins. Low doses of CBZ exposure also had a slight influence on body weight and other body parameters.
Conflicts of interest The authors declare no competing financial interest.
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Fig. 6. (A) 5 mC positive cells in mouse testis tissues detected by immunofluorescence staining. Red: 5 mC; Blue: DAPI (for nuclei). n > 3. (B) Quantitative data for immunofluorescent staining analysis of 5 mC. (C) 5 hmC positive cells in mouse testis tissues detected by immunofluorescence staining. Red: 5 hmC; Blue: DAPI (for nuclei). n > 3. (D) Quantitative data for immunofluorescent staining analysis of 5 hmC. n > 3. a, b indicate a significant difference among different treatments (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Author contributions
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Low dose carbendazim disrupts mouse spermatogenesis might Be through estrogen receptor related histone and DNA methylation
T
Jing Liua, Pengfei Zhangb,c, Yong Zhaob, Hongfu Zhangd,∗ a
University Research Core, Qingdao Agricultural University, Qingdao, 266109, PR China College of Life Sciences, Qingdao Agricultural University, Qingdao, 266109, PR China c College of Animal Sciences and Technology, Qingdao Agricultural University, Qingdao, 266109, PR China d State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100193, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Carbendazim Spermatogenesis Estrogen receptor signaling Histone methylation DNA methylation
Pesticides, fungicides are reportedly involved in a decline in spermatozoa quality, especially motility, and a consequent increase in the rate of infertility. Fungicide carbendazim (CBZ) is widely used in agriculture and other aspects. Although CBZ is known to disrupt spermatogenesis, causing a decrease in spermatozoa concentration and motility, the mechanisms are not fully understood. We aimed to further explore the underlying mechanisms of CBZ disruption of spermatogenesis. Pubertal mice were exposed to low doses (0.1, 1 and 10 mg/ kg body weight) of CBZ for 5 weeks, then many factors related to spermatogenesis have been explored. It was found that 0.1–10 mg/kg body weight of CBZ exposure decreased mouse sperm motility and concentration, diminished the important protein factors (VASA, PGK2, B-Amy and CREM) for spermatogenesis, reduced sperm protein acrosin level, disrupted very vital epigenetic factors H3K27, 5 mC and 5 hmC. Furthermore, CBZ exposure damaged estrogen receptor alpha (ERα) pathway by decreased the protein levels of ERα and its targets PI3K and AKT. In summary low doses of CBZ exposure disrupted mouse spermatogenesis through estrogen receptor signaling; and that histone methylation and DNA methylation might play vital roles in CBZ disturbance of spermatogenesis through intertwining with estrogen signaling pathways. CBZ from the contamination in environment or food chain poses a serious threat to the normal development of spermatozoa. Therefore we strongly recommend to minimise the use of CBZ since it causes the severe issues on spermatogenesis.
1. Introduction Recent epidemiological studies report that infertility rates have been dramatically elevated from 7% to 8% in the 1960s to their current levels of 20%–35%, mostly due to a significant decrease in sperm motility (Levine et al., 2017; Centola et al., 2016). Semen quality is an essential biomarker of male overall reproductive function (Eisenberg et al., 2014). Among environmental chemicals, pesticides have been suggested to play a vital role in this deterioration in male fertility (Chiu et al., 2015). As a broad spectrum benzimidazole fungicide, carbendazim (methyl 2-benzimidazole carbamate, CBZ) can be a breakdown product of other fungicides, for example: benomyl and thiphanate-methyl. CBZ is widely used in agriculture, and is also used during the manufacturing of products such as paint, paper, and leather. In agriculture, it is used to treat foliar diseases on crops, and due to its widespread application, CBZ can be isolated from soil, ground water, and agricultural goods. Moreover,
∗
it has been found in indoor environments. In animals, CBZ has been found to impair liver and kidney function, to disrupt hematopoiesis and metabolism, and to induce chromosome aberrations by harming the spindle during mitosis and meiosis (Zubrod et al., 2014; Muthuviveganandavel et al., 2008; Akbarsha et al., 2000; Zuelke and Perreault, 1995; Sarrif et al., 1994). Epidemiologist have identified CBZ contamination worldwide and have determined that it negatively affects the reproductive ability of animals and human beings (Sakr and Shalaby, 2014). Humans can be directly exposed to CBZ through oral consumption (for example contaminated fruits, vegetables, and water), and inhalation and dermal exposure at home and in the work place (Bakirci et al., 2014; Boobis et al., 2008). CBZ has been found to possess endocrine-disrupting (androgen-like) actions (Durand et al., 2017) that interfere with spermatogenesis. Furthermore, exposure results in a decline in spermatozoa concentration and motility, an elevation in sperm abnormalities, an increase in oxidative stress and apoptosis, and a reduction in the rate and stability of microtubule assembly (Rama et al.,
Corresponding author. E-mail address: [email protected] (H. Zhang).
https://doi.org/10.1016/j.ecoenv.2019.03.103 Received 7 January 2019; Received in revised form 20 March 2019; Accepted 25 March 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
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procedure (Zhang et al., 2018).
2014; Adedara et al., 2013; Pacheco et al., 2012). DNA methylation, histone modifications, and noncoding RNAs are common epigenetic modifications which have been implicated in spermatogenesis (Gannon et al., 2014; Carrell, 2012; Ge et al., 2017). Furthermore, spermatogenesis is very sensitive to environmental contamination, which induces dramatic epigenetic alterations (Strazzullo and Matarazzo, 2017; Jenkins and Carrell, 2011). Such aberrations may lead to a decrease in male fertility, disruption of early embryo development, or diseases in offspring because they can be passed through generations to impact on future health and development (Wu et al., 2015; Aston et al., 2012). Many investigations have found that CBZ has endocrine disrupting effects that negatively influence testicular steroidogenesis to disrupt the production of testosterone, LH, FSH, GnRH, and to decrease the gene expression of estrogen receptor alpha (ERα) and ERβ (Rama et al., 2014). However, the mechanisms by which this takes place are unknown. During puberty, the testes undergo dramatic growth due to spermatogonial proliferation, the expansion of meiotic and haploid germ cells, and the increase in somatic cells such as Sertoli and Leydig cells. The pubertal period is therefore critical during male reproductive system development, and any disturbance in spermatogenesis at this stage will have a confounding impact on later fertility. Accordingly, the purpose of current study was to investigate the adverse effects of low concentrations of CBZ on spermatogenesis during the peripubertal stage in mice and the role of epigenetic modifications in this process.
2.5. Measurement of plasma steroid hormones ELISA kits (Nanjing Jiancheng Bioengineering Institute, China) for plasma testosterone (T) and estrogen (E) levels were used, following the manufacturer's instructions (Zhang et al., 2018; Wang et al., 2016). 2.6. Measurement of plasma AST and ALT Plasma levels of aspartate aminotransferase (AST/GOT) and alanine aminotransferase (ALT/GPT) were determined directly using appropriate kits (Nanjing Jiancheng Bioengineering Institute) and following the manufacturer's instructions. Six samples from each treatment were determined (Wang et al., 2016). 2.7. Western blotting Western blotting analysis took place as previously reported (Zhang et al., 2018; Wang et al., 2016; Zhao et al., 2016). The bands were quantified using Image-J software. The intensity of specific protein bands were initially normalized to actin, and subsequently to the control. More than six replicates were performed. 2.8. Detection of protein levels and location in the testes using immunofluorescence staining
2. Materials and methods (further details are provided in supplemental information)
Immunofluorescence staining of testicular samples is reported in our recent publication (Zhang et al., 2018; Wang et al., 2016). For each sample in each experiment, over 1000 cells were counted; data were then normalized to the control.
2.1. Study design (mouse exposure to CBZ) This investigation was performed in strict accordance with the recommendations provided in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Qingdao Agricultural University IACUC (Institutional Animal Care and Use Committee) (Zhang et al., 2018). This study set out to explore the impact of low doses of CBZ on spermatogenesis in mice and to investigate the underlying mechanisms. Male ICR mice were exposed to CBZ through oral gavage. The doses of CBZ used in this study in vivo were very low (0.1–10 mg/kg). However, CBZ has been used for animal studies from 100 to 600 mg/kg (Rama et al., 2014; Sakr and Shalaby, 2014; Farag et al., 2011). The CBA dosing solution was freshly prepared on a daily basis in corn oil. Four treatments were used with 30 mice per treatment as follows: (1) vehicle control (corn oil); (2) CBZ-0.1 mg/kg BW; (3) CBZ-1 mg/kg BW; (4) CBZ-10 mg/kg BW. Each mouse was administered 0.1 ml of the appropriate gavage each morning for five weeks starting at 25 d old.
2.9. Statistical analysis Quantitative data were presented as the mean ± SEM. ANOVA and the LSD multiple comparison test were used for statistical analyses. The statistical software SPSS (IBM Co., NY) was used for all analyses. All treatment groups were compared with each other for every parameter. Differences were considered statistically significant at p < 0.05. 3. Results 3.1. CBZ inhibited mouse spermatogenesis in vivo After a 5 week exposure to low doses of CBZ, mouse spermatozoa motility (grade A + B) was significantly decreased by CBZ 0.1, 1, and 10 mg/kg body weight (BW; Fig. 1A); spermatozoa concentration was significantly reduced by the CBZ 10 mg/kg exposure (Fig. 1B). Spermatogenesis is regulated by protein factors including Synaptonemal complex protein 3 (SYCP3), Deleted In Azoospermia Like (DAZL), DEAD-Box Helicase 4 (DDX4/VASA), KIT Proto-Oncogene Receptor Tyrosine Kinase (KIT), Glial Cell Derived Neurotrophic Factor (GDNF), CAMP Responsive Element Modulator (CREM), Zinc Finger Protein 37 Homolog (ZFP37), MYB Proto-Oncogene A (A-Myb), MYB Proto-Oncogene B (B-Myb), MutS Homolog 4 (MSH4), Phosphoglycerate Kinase 2 (PGK2), MutS Homolog 5 (MSH5), Snail Family Transcriptional Repressor 3 (SNAI 3), KIT ligand (KITLG). In current study VASA, B-myb, PGK2, and CREM were decreased by the CBZ 1 mg/kg BW, and 10 mg/kg BW exposures (Fig. 1C and D). Western blotting data for VASA was confirmed by IHF (Fig. 1E and F). At the same time the sperm markers acrosin and PNA were decreased by the CBZ 1 mg/kg BW and 10 mg/kg BW exposures in a dose dependent manner (Fig. 2A, B, and 2C). All the data suggested that CBZ inhibited mouse spermatogenesis in vivo, similar to findings in previous reports. However, low doses of CBZ exposure had just a slight effect on body parameters (Table 1).
2.2. Evaluation of spermatozoa motility using a computer-assisted sperm analysis system The motility of spermatozoa was assessed using a computer-assisted sperm assay (CASA) method according to World Health Organization guidelines (Zhang et al., 2018; Zhao et al., 2016; WHO, 2010). Spermatozoa motility data represented only grade A + grade B spermatozoa, since only these are considered functional. 2.3. Morphological observations of spermatozoa Abnormalities in spermatozoa were analysed according to a previously reported procedure (Zhang et al., 2018). 2.4. Assessment of acrosome integrity Acrosomal integrity was assessed following a previously reported 243
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Fig. 1. (A) Mouse spermatozoa motility (grade A + grade B). Y-axis = % of total cells, X-axis = the exposure doses (mg/kg body weight). (B) Mouse spermatozoa concentration. Y-axis = spermatozoa concentration, X-axis = the exposure doses (mg/kg body weight). (C) Protein levels of VASA, PGK2, B-Myb and CREM in mouse testis tissues detected by Western blotting. n > 3. (D) Quantitative data for Western blotting analysis of VASA, PGK2, B-Myb and CREM. (E) VASA positive cells in mouse testis tissues detected by immunofluorescence staining. Red: VASA; Blue: DAPI (for nuclei). n > 3. (F) Quantitative data for immunofluorescent staining analysis of VASA. a, b, c indicate a significant difference among different treatments (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
3.3. CBZ disrupted histone and DNA methylation in mouse testes
3.2. CBZ disturbed the estrogen receptor alpha (ERα) pathway in mouse testes
Histone methylation marker tri-methylation of H3 at lysine 27 (H3K27me3) and DNA methylation markers 5-methylcytosine (5 mC) and 5-hydroxymethylcytosine (5 hmC) are important for spermatogenesis and they were analysed in mouse testes in the current study. It was found that H3K27 was mainly expressed in spermatogonia stem cells (SSCs) and the number of H3K27 positive SSCs was significantly elevated by the CBZ 1 mg/kg BW and 10 mg/kg BW exposures in a dose dependent manner (Fig. 5A and B). The marker 5mc was identified in Leydig cells (Fig. 6A) and was decreased by the CBZ 1 mg/kg BW and 10 mg/kg BW exposures (Fig. 6B). The marker 5hmc was mainly expressed in SSCs and the number of 5hmc positive SSCs was reduced by the CBZ 10 mg/kg BW exposure (Fig. 6C and D). This data suggested that CBZ exposure adversely affected epigenetic markers with a resulting disturbance of spermatogenesis.
Estrogen receptor alpha (ERα) is reported to be a target of CBZ (Durand et al., 2017), thus ERα and its target genes Phosphoinositide 3kinase (PI3K) and AKT Serine/Threonine Kinase (AKT) were determined in the current investigation. It was found that the number of ERα positive leydig cells in mouse testes was significantly reduced by the CBZ 1 mg/kg BW, and 10 mg/kg BW exposures (Fig. 3A and B). Furthermore, the hormone production proteins Hydroxysteroid Dehydrogenase 3β1 (HSD3β1) and Hydroxysteroid Dehydrogenase 17β1 (HSD17β1) were reduced by the CBZ1 mg/kg BW, and 10 mg/kg BW exposures (Fig. 3C, D, and 3E). This suggested that CBZ disrupted the endocrine system in the testes. The number of PI3K positive cells in mouse testes was significantly decreased by the CBZ 1 mg/kg BW and 10 mg/kg BW exposures in a dose dependent manner (Fig. 4A and B). At the same time, AKT protein levels in the testes was decreased by the CBZ 1 mg/kg BW and 10 mg/kg BW exposures (Fig. 4C). This data suggested that CBZ inhibited spermatogenesis through the ERα pathway.
4. Discussion Male reproductive toxicology is attracting increasing concern 244
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Fig. 2. (A) Acrosin and PNA positive cells in mouse testis tissues detected by immunofluorescence staining. Red: Acrosin; Blue: DAPI (for nuclei). n > 3. (B) Quantitative data for immunofluorescent staining analysis of Acrosin. (C) Quantitative data for immunofluorescent staining analysis of PNA. a, b indicate a significant difference among different treatments (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
foodstuffs (Durand et al., 2017). Meanwhile, lots of insecticides, herbicides, and fungicides have been identified as endocrine disrupting chemicals (EDCs). EDCs mimic endogenous hormones to modify endocrine system function, even to interfere with an animal's survival, development, sexual differentiation, and reproduction (Zoeller et al., 2012). CBZ has been widely used in the control of various pathogens in agriculture, and it is now included in the priority list of EDCs by the
because human sperm quality has significantly deteriorated over the past few decades (Le Moal et al., 2014). Pesticides and other environmental chemicals have been implicated in this deterioration (Chiu et al., 2015). The extensive production and widespread use of pesticides has led to contamination of the environment, soil, groundwater sources, and even agricultural products. Because most pesticides are highly persistent, even in low amounts, their accumulation is magnified in
Table 1 Mouse body and plasma parameters. Data present as Average ± SEM. a, b, c indicate a significant difference among different treatments (n > 6; p < 0.05).
Body weight (g) Testis index (% of body weight) Kidney index (% of body weight) Spleen index (% of body weight) Liver index (% of body weight) E2 (ng/L) Testosterone (ng/L) Acrosome integrity (% of total cells) ALT AST
Control
CBZ 0.1
CBZ 1
CBZ 10
40.25 ± 1.50a 0.83 ± 0.04 1.78 ± 0.05 0.43 ± 0.05 5.85 ± 0.19 a 394.20 ± 73.59 1.33 ± 0.01 4.44 ± 0.45 117.5 ± 36.0 100.9 ± 20.4
39.07 ± 0.51 a 0.79 ± 0.031 1.70 ± 0.05 0.52 ± 0.09 5.64 ± 0.15 a 244.10 ± 52.90 1.30 ± 0.02 3.88 ± 0.71 117.1 ± 44.7 97.8 ± 33.4
38.87 ± 1.39 a 0.81 ± 0.024 1.91 ± 0.06 0.36 ± 0.02 5.60 ± 0.21 a 173.30 ± 10.40 1.29 ± 0.01 3.46 ± 0.33 115.9 ± 39.6 89.6 ± 26.6
36.26 ± 1.01 b 0.89 ± 0.021 1.70 ± 0.1 0.41 ± 0.04 5.13 ± 0.14 b 318.80 ± 120.50 1.29 ± 0.02 5.03 ± 0.58 56.5 ± 14.3 72.1 ± 34.0
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Fig. 3. (A) ERα positive cells in mouse testis tissues detected by immunofluorescence staining. Red: ERα; Blue: DAPI (for nuclei). n > 3. (B) Quantitative data for immunofluorescent staining analysis of ERα. (C) Protein levels of HSD17β1 and HSD3β1 in mouse testis tissues detected by Western blotting. n > 3. (D) Quantitative data for Western blotting analysis of HSD17β1. (E) Quantitative data for Western blotting analysis of HSD3β1. a, b, c indicate a significant difference among different treatments (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4. (A) PI3K positive cells in mouse testis tissues detected by immunofluorescence staining. Red: PI3K; Blue: DAPI (for nuclei). n > 3. (B) Quantitative data for immunofluorescent staining analysis of PI3K. (C) Protein levels of AKT in mouse testis tissues detected by Western blotting. n > 3. a, b, c indicate a significant difference among different treatments (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Many investigations have tried to explore the mechanisms underlying the CBZ disruption of spermatogenesis; they have found that CBZ influences testicular steroidogenesis with an alteration in the levels of various hormones (testosterone, LH, FSH, GnRH) and in gene expression levels of estrogen receptor alpha (ERα) and ERβ (Rama et al., 2014). However, the mechanisms by which CBZ disrupts spermatogenesis are not fully understood. In the current study, it was found that steroid hormone production proteins (HSD3β1 and HSD17β1) were reduced by CBZ exposure in mouse testes. These results are in line with earlier reports. Epigenetic modifications (DNA methylations, histone modifications, and non-coding RNAs) play vital roles in gametogenesis by regulating gene expression (Stewart et al., 2016). It has been found that histone methylation on lysine residues could activate or repress gene expression depending on the position of the residue modified (Dumasia et al., 2017; Bannister and Kouzarides, 2011). Di- or tri-methylation of H3 at lysine 4 (H3K4me2/3) and H3 acetylated at lysine 9 can activate gene expression; however, di- and tri-methylation of H3 at lysine 27 (H3K27me2/3) can repress gene expression (Bannister and Kouzarides, 2011). H3K27me3 is expressed in most male germ cells transcriptionally to repress the expression of somatic-specific genes to maintain their homeostasis and differentiation. The ERα signaling pathway is very important for spermatogenesis and it regulates histone methylation during this process (Dumasia et al., 2017). In the current investigation, we found that ERα levels were decreased by CBZ in mouse testes, and at the same time the ERα target genes PI3K and AKT were diminished by CBZ, which suggests that the ERα pathway was disrupted by CBZ. Moreover, our results showed that CBZ exposure promoted H3K27me3 expression in mouse spermatogonia cells. It has been reported that ERα agonist (4,4′,4’’-(4-Propyl-[1H] pyrazole-1,3,5triyl; PPT) promotes H3K27me3 expression (Dumasia et al., 2017). Our data indicate that the ERα pathway and histone modification may play an important role in CBZ disrupted spermatogenesis. In male gamete development, epigenome and epigenetic modifications play important roles throughout the establishment of primordial germ cells to the development of mature gametes (Stewart et al., 2016). DNA methylation is a major mechanism of genome reprogramming. 5Methylcytosine (5 mC) is important for cell fate determination and it modifies DNA-protein interactions and activates or represses gene expression. 5 mC and 5-hydroxymethylcytosine (5 hmC) are important during spermatogenesis (Stewart et al., 2016). DNA methylation modifies estrogen receptor (ER) gene expression, at the same time estrogen signaling regulates DNA methylation (Vrtačnik et al., 2014; Marques et al., 2013). Estrogen signaling and DNA methylation tightly interact together to regulate spermatogenesis (Vrtačnik et al., 2014; Marques et al., 2013). In our current investigation, we found that both 5 mC and 5 hmC were reduced by CBZ exposure which further suggests that the intertwined ER pathways and DNA methylation (5 mC and 5 hmC) were involved in the alteration of spermatogenesis by CBZ. In conclusion, the purpose of the current study was to further investigate the underlying mechanisms of CBZ disruption of spermatogenesis; we demonstrated that: i) CBZ disrupted mouse spermatogenesis with damage to sperm production proteins and sperm proteins; ii) CBZ disrupted spermatogenesis through estrogen receptor signaling; iii) histone methylation and DNA methylation might play vital roles in CBZ disturbance of spermatogenesis through intertwining with estrogen signaling pathways. Due to its over use or incorrect use, CBZ poses a serious threat to healthy sperm development. Therefore, greater attention should be paid to the use of CBZ to minimise its influence on human spermatogenesis.
Fig. 5. (A) H3K27me3 positive cells in mouse testis tissues detected by immunofluorescence staining. Red: H3K27me3; Blue: DAPI (for nuclei). n > 3. (B) Quantitative data for immunofluorescent staining analysis of H3K27me3. n > 3. a, b, c indicate a significant difference among different treatments (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
European Commission (Ferreira et al., 2008). Early investigations report that CBZ disrupts reproductive systems, induces germ cell apoptosis, and causes embryotoxicity or teratogenesis in mice, rats, hamsters, and even humans (Adedara et al., 2013; Farag et al., 2011; Yu et al., 2009; Akbarsha et al., 2001). Acting as an EDC, CBZ has been found to decrease spermatozoa concentration and motility, to increase sperm abnormalities, to elevate oxidative stress and apoptosis, to reduce the rate and stability of microtubule assembly, and to disrupt spermatogenesis (Durand et al., 2017; Rama et al., 2014; Adedara et al., 2013; Pacheco et al., 2012). In the current study, we found that very low doses of CBZ decreased mouse spermatozoa motility and concentration. Furthermore, it disrupted spermatogenesis with damage to sperm production proteins and alterations in sperm proteins. Low doses of CBZ exposure also had a slight influence on body weight and other body parameters.
Conflicts of interest The authors declare no competing financial interest.
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Fig. 6. (A) 5 mC positive cells in mouse testis tissues detected by immunofluorescence staining. Red: 5 mC; Blue: DAPI (for nuclei). n > 3. (B) Quantitative data for immunofluorescent staining analysis of 5 mC. (C) 5 hmC positive cells in mouse testis tissues detected by immunofluorescence staining. Red: 5 hmC; Blue: DAPI (for nuclei). n > 3. (D) Quantitative data for immunofluorescent staining analysis of 5 hmC. n > 3. a, b indicate a significant difference among different treatments (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Author contributions
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