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Reproductive Toxicology 46 (2014) 121–128

Contents lists available at ScienceDirect

Reproductive Toxicology

j ourna l h o mepa ge: www.elsev ier .com/ locate / reprotox

eproductive toxicity in acrylamide-treated female mice

uanwei Weia, Jian Lib, Xingmei Lia, Lei Zhanga, Fangxiong Shia,∗

Laboratory of Animal Reproduction, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, ChinaInstitute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China

r t i c l e i n f o

rticle history:eceived 30 December 2013eceived in revised form 6 March 2014ccepted 15 March 2014vailable online 18 April 2014

eywords:

a b s t r a c t

We investigated the reproductive toxicity of acrylamide in female mice. The results from immunohisto-chemistry provided evidence that nitric oxide synthase (NOS) signaling was involved in the process offollicular development and atresia. Oral administration of acrylamide to female mice led to significantlyreduced body weights, organ weights and the number of corpora lutea (P < 0.05). Serum progesteroneconcentrations were significantly reduced (P < 0.05) concomitant with the increasing doses of acryla-mide; however, 17�-estradiol (E2) concentrations were unchanged with treatment. Measurement of

crylamideeproductive toxicityvaryitric oxide synthase

NOS activities indicated that total NOS (TNOS), iNOS and eNOS activities were significantly increased(P < 0.05) with increasing doses of acrylamide. The results from in vitro study indicated that acrylamidereduced the viability of mouse granulosa cells in a dose-dependent manner. In summary, acrylamideaffected bodily growth and development, as well as reproductive organs, the number of corpora luteaand progesterone production in female mice, possibly acting through the NOS signaling pathway.

© 2014 Elsevier Inc. All rights reserved.

. Introduction

Acrylamide (AA, CAS Reg. No. 79-06-1), an industrially produced,�-unsaturated (conjugated), highly water-soluble and chemi-ally reactive molecule, is widely used in several industries, suchs polymers, cosmetics, paper and textiles, wastewater treatmentnd laboratory gels [1–3]. Besides utilization in industry and theaboratory, the presence of acrylamide in certain food cooked atigh temperature was reported by Swedish researchers in 20004] and 2002 [5]. Furthermore, earlier toxicological studies foundhat acrylamide is carcinogenic and causes tumors [6,7]. It has beenemonstrated that acrylamide can be readily absorbed across thekin and move into the bloodstream following dermal exposure8–10]. These findings increased concerns about the effect of acryl-mide on human health, and acrylamide has been classified as aancer risk [11] by the International Agency for Research on Can-er (IARC), with many studies reporting a correlation with cancer.crylamide is formed at high temperatures above 120 ◦C during theooking of carbohydrate-rich foodstuffs [12,13], especially whenlucose reacts with asparagine [12–15]. The highest concentrations

f acrylamide were found in French fries and potato chips, and evenn diverse foods such as breads, cereals, cakes, coffee and cocoa16]. It is also reported that acrylamide is present in tobacco smoke

∗ Corresponding author. Tel.: +86 25 84399112; fax: +86 25 84399112.E-mail address: fxshi@njau.edu.cn (F. Shi).

ttp://dx.doi.org/10.1016/j.reprotox.2014.03.007890-6238/© 2014 Elsevier Inc. All rights reserved.

[17,18]. Thus, acrylamide can be absorbed through contaminatedfoods or water, and via dermal contact and respiration. Recently,several studies have been focused on acrylamide as a cytotoxicmaterial. It was reported that exposure to monomeric acrylamideresulted in peripheral neuropathy that was accompanied by weak-ness of the limbs [19]. It is also believed that acrylamide is clearlygenotoxic both in somatic [20–23] and germ cells [8,23–25]. Ourprevious study revealed the effects of acrylamide on male repro-duction [26], however, the exact mechanisms for acrylamide’seffects on female ovarian follicular development and atresia remainto be elucidated.

Studies have shown that an acrylamide analog synergisticallyactivates soluble guanylyl cyclase (sGC) in the presence of sub-maximal concentrations of nitric oxide (NO), and it may serveas a novel activator of sGC for the treatment of a variety ofdisorders associated with reduced NO signaling [27]. In fact,acrylamide-treated rats displayed differential expression of sGC inthe seminiferous epithelium [26].

It is believed that NO plays important roles in folliculogene-sis, oocyte growth and maturation, atresia, ovulation, luteolysis,steroidogenesis and prostaglandin biosynthesis [28]. NO is synthe-sized from l-arginine by NO synthase (NOS), and is designated asneuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS

(eNOS) [29,30]. It is reported that iNOS and eNOS are localizedin oocytes, granulosa cells, theca cells [31,32] and luteal tissues[33,34]. Studies also suggested that nNOS is found in non-neuronalcells [35,36]. However, little is known about the involvement of the

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hree types of NOS during the process of ovarian follicular devel-pment and atresia.

To expand our understanding of acrylamide toxicity on femaleeproduction, and to further assess the involvement of acryla-ide in NOS signaling, NOSs cellular localization and expression

atterns, we evaluated NOS activities, body weights, reproductivergan weights, number of corpora lutea, serum estradiol and pro-esterone levels and cell viability in prepubertal mice treated dailyith acrylamide for 30 days.

. Materials and methods

.1. Ethics statement

Animal procedures were conducted in accordance with theuidelines of the Institutional Animal Care and Use Committee ofanjing Agricultural University. All mice were sacrificed in a state ofnconsciousness after the mice were anesthetized with halothanend the ovaries were removed.

.2. Reagents

Acrylamide monomer dry crystals (C3H5NO, >99.9% purity, CASo. 000079-06-1) were purchased from Sigma–Aldrich. Coomassierilliant blue protein assay kits, NO kits and NOS kits were pur-hased from Nanjing Jiancheng Biological Engineering Co., Ltd.,hina. The nNOS, iNOS and eNOS antibodies were purchased fromuhan Boshide Engineering Co. Ltd., China. RIA kits for 17�-

stradiol (E2) and progesterone (P4) detection were purchasedrom Beijing North Biotechnology Institute, China. All other chem-cals were purchased reagent grade.

.3. Drug treatment

Acrylamide was dissolved in sterile distilled water, stored underefrigeration (approximately 4 ◦C), and replenished twice per day toetain stability of the dosing solutions. According to the publishedeports, the doses of acrylamide ranged from 5 to 50 mg/kg d, last-ng for several days to several weeks of treatment [7,26,37–39].n the current investigation, mice were administered water orallyn the control group, and the experimental groups were given 20r 40 mg/kg d of acrylamide in water for 30 consecutive days. Theethod of acrylamide administration used was the same as we

eported previously [26].

.4. Animals and conditions

Thirty-six female Kunming mice at 35 days of age were weightanked into three groups of 12 animals each, and acrylamide wasdministered to each group at a dose of 0, 20 or 40 mg/kg d for0 consecutive days. Additionally, 24 female Kunming mice of theame age were injected intraperitoneally with 10 units of PMSG andacrificed 48 h later. The ovaries were obtained and transferred intoetri dishes (35 mm × 15 mm) filled with PBS and then pricked with

syringe under a surgical dissecting microscope to release gran-losa cells (GCs). Another five female 35-day-old Kunming miceere sacrificed, and the ovaries used for immunohistochemistry.ll mice were purchased from Qinglongshan Laboratory Animalompany (Nanjing, China). Animals were maintained under a 14-

light, 10-h dark schedule with food and sterile distilled watervailable ad libitum. The experimental protocol was approved in

ccordance with the Guide for the Care and Use of Laboratory Ani-als prepared by the Institutional Animal Care and Use Committee,anjing Agricultural University. After the mice were anesthetizedith halothane, ovaries were collected and ovarian protein extracts

ology 46 (2014) 121–128

collected for NOS activity measurement. After the mice were anes-thetized with halothane, samples of blood were collected from allanimals. Serum was then separated and stored at −70 ◦C until use.

2.5. RIA for E2 and P4 concentrations

Blood was collected from different groups of mice and cen-trifuged at 5000 × g for 10 min to separate the serum. The serumwas used to quantify the concentrations of E2 and P4 using E2and P4 RIA kits respectively, in the General Hospital of the NanjingMilitary Command.

2.6. Measurement of NOS activity

The total NOS, eNOS and iNOS activities were measured using acommercial reagent (Jiancheng Bioengineering Institute, Nanjing,China). Briefly, the NOS activity was determined by measuring therelease of local NO generated via a five-electron oxidation of ter-minal guanidinium nitrogen on l-arginine by NOS. NO then boundto the nucleophilic materials and generated a colored compound.Afterwards, the reaction was terminated with citric acid. The opti-cal density at 530 nm (OD530) was detected using a microplatereader (Bio-TEK Instruments, Winooski, VT, USA). In the presentstudy, one of the ovaries from each mouse was collected and twoof these ovaries were then pooled. Thus, each group contained sixreplicates. The concentration of ovarian protein homogenates was800 �g/ml, and the procedures were performed strictly accordingto the manufacturer’s protocols.

2.7. Immunohistochemistry of NOS

To examine the localization of nNOS, iNOS and eNOS inmouse ovaries, continuous ovarian sections from female micewere used. Immunohistochemical staining was performed usingSABC method, which was a three-step method to form thestreptavidin–biotin complex, with polyclonal antibodies to nNOS(diluted 1:200), iNOS (1:200), or eNOS (1:200). A few sectionswere picked randomly from the serial sections, mounted onslides coated with APES (3-aminopropyl-triethoxysilane) and driedfor 24 h at 37 ◦C. The antibodies were diluted in PBS contain-ing 1% (w/v) bovine serum albumin (BSA), and the sectionswere incubated overnight at 4 ◦C with primary antibodies. Thespecific protein immunoreactivity was visualized with 0.05% 3,3′-diaminobenzidine (DAB) in 10 mM PBS containing 0.01% (v/v) H2O2for 2 min, and counterstained with hematoxylin. The negative con-trol was normal rabbit serum (NRS) instead of primary antibody.The images were captured under the microscope. To assign theintensity of staining for nNOS, iNOS and eNOS, three independentobservers blinded to the experimental design were asked to exam-ine the photomicrographs, using a method previously described[40]: −, no staining detected; +, weak; ++, moderate; +++, strongstaining. Relative levels of immunostaining were evaluated andrepeated at least four times.

2.8. Histopathological studies

After fixation in paraformaldehyde solution (4%), mouse ovarieswere embedded in paraffin, and sectioned serially at 5 �m. Thesesections were analyzed for morphologic changes of the ovaries.Slides at every time-point were stained with hematoxylin and eosin(H&E) and observed under a light microscope (Nikon, Tokyo, Japan).

2.9. Mouse GCs culture

GCs were collected from mouse ovaries using a syringe; washedwith PBS and incubated in DMEM/F12(1:1) with 10% fetal bovine

Q. Wei et al. / Reproductive Toxic

Table 1Classification criteria of premature mouse ovarian follicles.a

Follicle Morphologic characteristics of cells

Primordial follicles Oocyte partially or completely encapsulated bysquamous pregranulosa cells.

Primary follicles Granulosa cells (with single layer) showenlargement.

Secondary follicles Oocyte encapsulated by more than two layers ofgranulosa cells; no antrum formation.

Atretic follicles Follicles with apoptotic granulosa cells in thefollicular antrum, and apoptosis of granulosa cellswere accompanied by the presence of nuclei withcondensed chromatin stained with hematoxylin.

a Referenced mainly from our laboratory’s previous study (Ding et al., 2010, WeiesT

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t al., 2012, Wei & Shi, 2013). The stages of follicle development (primordial, primary,econdary and atretic follicle) were divided based on the classification criteria inable 1.

erum in an incubator with 5% CO2 in air at 37 ◦C. Penicillin100 units/ml) and streptomycin (100 mg/ml) were included in theell cultures. It has been reported that a broad spectrum of acryl-mide concentrations have been used in in vitro experiments onarious types of cell lines, as well as using different lengths of expo-ure [41–44]. Based upon this information we therefore selectedhe effective acrylamide concentration for mouse granulosa cellsn 64-h cultures. The final concentrations applied were 0.5 mM and

mM of acrylamide in cell culture medium and GCs were grownn coverslips in 6-well culture plates, or in 96-well culture platesCostar; Corning) at a density of 3–5 × 104 cells per well.

.10. In vitro cell viability assays

Mouse GCs were counted in a hemocytometer, and GCs werehen seeded in 96-well culture plates (Costar; Corning) at a densityf 3–5 × 104 cells per well. The cell viability assay at the end of theulture period was performed using a MTT kit (KeyGEN, Nanjing,hina) according to the manufacturer’s instructions. The opticalensity at 550 nm (OD550) was detected using an ELISA readerBio-TEK Instruments, Winooski, VT, USA).

.11. Statistical analysis

All results are expressed as means ± SEM. The statistical anal-sis was performed with a t-test to compare two groups, and annalysis of variance followed by Tukey’s range test for multipleomparisons. P < 0.05 was considered to be statistically significant.

. Results

.1. Different expression patterns of nNOS, iNOS and eNOS in thevaries of premature mice

In order to provide evidence that NOS was involved in the pro-ess of follicular development and atresia, we collected the ovariesf 35-day-old mice, and investigated the cellular localizationatterns of nNOS, iNOS and eNOS in the ovary by immunohisto-hemistry. Mouse ovarian follicles were classified based on thelassification criteria in Table 1. In the ovary, a positive reaction wasbserved as brown staining (marked with red arrows). No specifictaining was observed in the negative control sections (Fig. 1D1, D2nd D3). The results showed that the immunostaining pattern ofNOS, iNOS and eNOS varied among different follicle stages duringhe process of follicular development. Relative expression levels of

mmunostaining of nNOS, iNOS and eNOS in the ovaries are shownn Table 2.

Immunohistochemical results showed that nNOS, iNOS andNOS were localized to the cytoplasm of oocytes in secondary

ology 46 (2014) 121–128 123

follicles (Fig. 1A1, A2, C1, C2), however, expression of iNOS wasmore intense than either nNOS or eNOS. The expression of nNOS,iNOS and eNOS were decreased in atretic follicles compared withhealthy follicles.

In granulosa cells, nNOS and iNOS staining increased with follic-ular development, while there was almost no staining of eNOS. Inpregranulosa cells, the staining with iNOS was weak, while nNOSand eNOS were negligible (Fig. 1A3, B3 and C3). In addition, nNOS,iNOS and eNOS were not detected in the apoptotic granulosa cells(marked with black triangle, Fig. 1A1, B1 and C1).

3.2. Body weights and organ weights are decreased afteracrylamide treatment

After administration of acrylamide, the body weights and repro-ductive organ weights were measured and compared (Table 3).

At the beginning of the experiment, the body weights showedno significant difference (P > 0.05) among different groups, but afteradministration of acrylamide, the body weights of mice in the con-trol group were significantly greater than those of the mice treatedwith 20 mg/kg d or 40 mg/kg d acrylamide-treated group (P < 0.01).The absolute and relative organ weights (of uteri and ovaries) wereall reduced significantly (P < 0.01) in the 20 mg/kg d acrylamide-treated group and in the 40 mg/kg d acrylamide-treated group,which suggested that acrylamide manifested toxicity related toboth growth and development of the body and its reproductiveorgans.

3.3. Acrylamide reduces the number of the corpora lutea

The Corpus luteum is very important for the maintenance ofovarian function [45]. In order to detect the effect of acrylamideon the state of mouse ovaries, ovaries were sectioned serially at5 �m and stained with H&E (Fig. 2), and then the number of cor-pus luteum were counted and statistical analyzed (Fig. 3). Theresult showed that the number of corpus luteum of control groupwas significantly higher than 20 mg/kg d acrylamide-treated group,what’s more, there was almost no corpus luteum observed in the40 mg/kg d acrylamide-treated group (Figs. 2 and 3).

There was also a decrease in the proportion of primordial fol-licles and an increase in the proportion of antral follicles in theovaries of mice treated with acrylamide. The dynamic changes offollicular development in mice were characterized by the propor-tion of follicles at each stage. The proportions of primordial, primaryand antral follicles is shown in Fig. 4.

Compared with the control group, with increasing doses ofacrylamide, the proportion of primordial follicles was decreased,whereas the proportion of primary follicles was increased at thelow dose of acrylamide, but there was no difference between thehigh dose and the control group.

3.4. Acrylamide leads to a decrease in P4 but not in E2concentrations in the serum of mice

The concentrations of estradiol (E2) and progesterone (P4) in theserum from different groups of mice are shown in Fig. 5. Comparedwith control group, with the increasing doses of acrylamide, serumP4 concentrations were significantly decreased (P < 0.05) (Fig. 5A).

In addition, there was a significant difference between the 40 mgand 20 mg groups (P < 0.05). With increasing doses of acrylamide,serum E2 concentrations showed no significant difference betweenthe acrylamide-treated and control groups (P > 0.05) (Fig. 5B).

124 Q. Wei et al. / Reproductive Toxicology 46 (2014) 121–128

Fig. 1. Immunostaining of nNOS (A), iNOS (B) and eNOS (C) in the ovary of prepubertal mice. (Photographs in differently colored square boxes are enlarged below and markedwith A1, 2, 3, B1, 2, 3, C1, 2, 3 and D1, 2, 3.) Staining of nNOS (A), iNOS (B) and eNOS (C) were qualitatively different in the granulosa cells at various stages of folliculardevelopment.

Table 2Relative levels of immunostaining of nNOS, iNOS and eNOS in the non-apoptotic granulosa cells of mice ovaries.a

Protein Stages Primordial follicle Primary follicle Secondary follicles Atretic follicles

nNOSOocyte + + + +Granulosa cells − ++ +++ +++

iNOSOocyte ++ +++ +++ +Granulosa cells ++ +++ ++ +++

eNOSOocyte − − + −Granulosa cells − − + −

a −, no staining detected; +, weak; ++, moderate; +++, strong staining; N/A, not available.

Q. Wei et al. / Reproductive Toxicology 46 (2014) 121–128 125

Table 3Effects of acrylamide on the absolute and relative organ weight (n = 12, mean ± SEM).a

Weights Control 20 mg 40 mg

Body weight (BW)/g 30.44 ± 0.40 26.79 ± 0.52** 18.68 ± 0.61**Uterus weight/g 0.28 ± 0.01 0.19 ± 0.01** 0.03 ± 0.01**Organ coefficient of uterus (g/100 g) 0.91 ± 0.03 0.71 ± 0.02** 0.16 ± 0.01**Ovary weight (mg) 16.78 ± 0.98 8.72 ± 0.47** 3.9 ± 0.44**Organ coefficient of ovary (mg/100 g) 55.34 ± 3.47 32.46 ± 1.50** 20.60 ± 1.88**

a The statistical differences among groups were analyzed by one-way ANOVA. P < 0.05 was considered to be statistically significant and designated “*”; and P < 0.01 wasdesignated “**”.

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of acrylamide on male reproductive function [26], the effects ofacrylamide on ovarian follicular development and atresia in the

Fig. 2. Histologic observations on the acrylamide-

.5. NOS activities increased after acrylamide treatment

In order to study the involvement of NOS in acrylamide-treatedice, we measured tNOS, iNOS and eNOS activities in the ovary.The results showed that tNOS, iNOS and eNOS activities were

ignificantly increased (P < 0.05) with increasing doses of acryla-ide (Fig. 6A and B). However, at each acrylamide dose, there is no

ifference between iNOS and eNOS (P > 0.05) (Fig. 6B).

.6. Cell viability decreases after acrylamide treatment in vitro

To further study the toxic effects on mouse GCs in vitro, GCsere cultured in the presence (0.5 mM and 5.0 mM) or absence of

crylamide for 48 h, and we observed the cellular growth state on

overslips in 6-well culture plates under an inverted microscope. Toonfirm the observations, cell viability was also determined using

MTT kit. Cell culture for MTT in 96 wells was under the same con-itions. The results showed that acrylamide inhibited GC growth

ig. 3. Effects of acrylamide on the number of corpora lutea in ovaries (n = 12,ean ± SEM), compared to the control group. The statistical differences among

roups were analyzed using ANOVA and Tukey’s range test. Values with differentuperscripts are significantly different (P < 0.05).

d mouse ovary. (A) Control; (B) 20 mg; (C) 40 mg.

commensurate with the increasing dose of acrylamide (Fig. 7A–C).Cell viability was significantly decreased in the acrylamide-treatedgroup compared with the control group (Fig. 7D).

4. Discussion

It was reported that acrylamide exerts carcinogenic effectsthat can harm human health [7,6]. Previous studies have demon-strated that acrylamide induces central-peripheral neuropathythrough axon damage in the peripheral nervous system, and causesataxia, skeletal muscle weakness and body weight loss [46,47].Although our previously published data revealed the toxic effects

female remained to be elucidated. To enrich our understanding ofacrylamide toxicity on female reproduction, we examined the

Fig. 4. The proportion of follicles was determined as a percentage of total oocytenumbers counted per section. Each bar represents the mean ± SEM.

126 Q. Wei et al. / Reproductive Toxicology 46 (2014) 121–128

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ig. 5. Serum levels of P4 (A) and E2 (B). Data represent the mean ± SEM from 12

ange test. Values with different superscripts are significantly different (P < 0.05).

ffects of acrylamide on the mouse, using both in vivo and in vitroodels.Although blood concentrations of acrylamide in animals after

ral treatments was not clear, a broad spectrum of acrylamideoncentrations have been used in in vitro experiments on vari-us types of cell lines to study the toxic effect of acrylamide. Itas been reported that exposure of acrylamide at 0.5–5.0 mM for8 h could effectively induce elevated expressions of heat shockroteins (HSPs) (Hsp90, Hsp70 and Hsp27, respectively) in humaneuroblastoma cells [41]. Therefore, 0.5 mM and 5 mM acrylamideere used to confirm the in vitro toxic effect in GCs in our current

tudy.The results of immunohistochemistry provided evidence that

OS is involved during the process of follicular growth, develop-ent and atresia. Cellular expression and localization patterns of

NOS, iNOS and eNOS varied during these processes, indicating thatOS signaling is involved.

Our present study clearly demonstrated that oral administra-ion of acrylamide to female immature mice led to a significanteduction in body weight, organ weight and relative organ weight.hese results are consistent with acrylamide-manifested toxicity

riented principally toward growth and development [26,48]. Fur-her histological examination showed that the number of ovarianorpora lutea was reduced in the 20 mg/kg d acrylamide-treatedroup compared with controls, and that there was almost no

ig. 6. Effect of acrylamide on the level of tNOS (A), iNOS (B) and eNOS (B) in the ovaries osing ANOVA and Tukey’s range test. Values with different superscripts are significantly d

The statistical differences among groups were analyzed using ANOVA and Tukey’s

corpora lutea observed in the 40 mg/kg d acrylamide-treated group.In addition, there was a diminution in the proportion of primor-dial follicles commensurate with increasing doses of acrylamide,whereas the percentage of primary follicles was increased. Theseresults suggest that acrylamide affects follicular development andthe corpus luteum formation.

We also demonstrated that serum P4 concentrations were sig-nificantly decreased with increasing doses of acrylamide; however,for E2, there was no significant difference in peripheral E2 betweenthe acrylamide-treated and control groups. The P4 reduction mayexplain the decreased number of corpora lutea induced by acryla-mide, as the primary function of the corpus luteum is the secretionof the hormone P4, which is required for maintenance of normalovarian functions in mammals [49]. The attenuated number of cor-pora lutea caused by acrylamide could then affect overall femalereproductive function.

Measurement of NOS showed that tNOS, iNOS and eNOSactivities were significantly increased with increasing doses ofacrylamide administration. Our previous study demonstrated thatNOS was implicated in rat [50] and porcine [51] follicular devel-opment. This suggested to us that acrylamide may affect female

reproduction through NOS activities. However, these findings needto be corroborated by more evidences.

Previous reports showed that acrylamide or its analogs affectsGC which provides clues as to how acrylamide may affect NOS

f mice (n = 6, mean ± SEM). The statistical differences among groups were analyzedifferent (P < 0.05).

Q. Wei et al. / Reproductive Toxicology 46 (2014) 121–128 127

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ig. 7. Phase contrast images and cell viability of mGCs cultured for 64 h with two

tatistical differences among groups were analyzed by using ANOVA and Tukey’s ra

ignaling [26,27]. Our results provide further evidence that NOSignaling may be involved in acrylamide toxicity in the mouse.owever, the exact mechanisms underlying the toxic effects ofcrylamide are still unclear.

In conclusion, our investigation provide the evidence that NOSay be involved in follicular development and atresia, and acryl-

mide significantly affected growth of the body as well as theeproductive organs in the female mouse, possibly acting throughOS signaling pathway. However, the role of NOS in acrylamide-

nduced reproductive toxicity still need further investigation. Indition, in vitro study indicated that acrylamide affected the prolif-ration and viability of mouse granulosa cells in a dose-dependentanner. Moreover, this is the first study to show that acrylamideanifests reproductive toxicity in female mice. Thus, our study sug-

ests that exposure to acrylamide causes decreases corpus luteumumber, thereby affecting the female reproductive function. Thesendings may be helpful in the study of the toxic effect of acrylamiden animal reproduction overall.

onflict of interest statement

The authors declare that there are no conflicts of interest.

ransparency document

The Transparency document associated with this article can beound in the online version.

cknowledgments

This study was supported by the National Natural Scienceoundation of China (No. 31172206) and a Grant-in-Aid for Inno-ative Training of Doctoral Students in Jiangsu Province, ChinaNo. CXZZ12-0294). We also express our gratitude to Dr. Reinhold

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ntrations of acrylamide (0.5 mM or 5 mM). (A) Control; (B) 0.5 �M; (C) 5.0 �M. Thest. Values with different superscripts are significantly different (P < 0.05).

J. Hutz of the Department of Biological Sciences, University ofWisconsin-Milwaukee in USA for reading the original manuscriptand offering valuable suggestions.

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