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Impairment of ovaries by 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD) exposure in utero associated with BMP15 and GDF9 in the female offspring rat
Accepted Manuscript Title: Impairment of ovaries by 2,3,7,8-Tetrachlorobenzo-p-dioxin (TCDD) exposure in utero associated with BMP15 and GDF9 in the female offspring rat Authors: Xiuli Zhang, Mengmeng Ji, Xuemei Tan, Kailun Yu, Xiaozhuan Liu, Ning Li, Zengli Yu PII: DOI: Reference:
S0300-483X(18)30299-3 https://doi.org/10.1016/j.tox.2018.08.015 TOX 52088
To appear in:
Toxicology
Received date: Revised date: Accepted date:
11-3-2018 8-8-2018 27-8-2018
Please cite this article as: Zhang X, Ji M, Tan X, Yu K, Liu X, Li N, Yu Z, Impairment of ovaries by 2,3,7,8-Tetrachlorobenzo-p-dioxin (TCDD) exposure in utero associated with BMP15 and GDF9 in the female offspring rat, Toxicology (2018), https://doi.org/10.1016/j.tox.2018.08.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title Impairment of ovaries by 2,3,7,8-Tetrachlorobenzo-p-dioxin (TCDD) exposure in utero associated with BMP15 and GDF9 in the female offspring rat
Xiuli Zhanga, b, Mengmeng Jia, Xuemei Tana, Kailun Yua, Xiaozhuan Liuc, Ning Lid, and Zengli
Health College of Zhengzhou University, No. 100 of Science Road, Zhengzhou, 450001,
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aPublic
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Yua*
China.
First Affiliated Hospital of Zhengzhou University, No. 1 of Jianshe East Road, Zhengzhou,
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bThe
University, No. 63 of Agricultural Road, Zhengzhou, 450002, China.
*Corresponding authors
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Email: [email protected]
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dHenan Agricultural
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Provincial Peoples Hospital, No. 7 of Weiwu Road, Zhengzhou, 450001, China.
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cHenan
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450052, China.
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AB STRACT
2,3,7,8-Tetrachlorobenzo-p-dioxin (TCDD) exposure in utero had been shown
to affect ovarian development and functions. However, its mechanism remained unknown. In this study, to investigate the effect of maternal exposure to TCDD on ovaries, the pregnant Sprague Dawley (SD) rats were treated with TCDD (100ng/kg 1
or 500ng/kg) or only vehicle and corn oil on the day 8 to 14 of gestation through the gavage with a stainless-steel feeding needle (once a day). The vaginal opening and estrous cycle of female offspring rats (F1) were monitored twice a day. The ovarian histology, follicle counts, real-time PCR, western blotting and DNA methylation
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analysis about Gdf9 and Bmp15 were carried out in F1 rats. The results showed that exposure to TCDD (especially the dose of 500ng/kg) in utero on GD8-14 might
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change the ovary weight, the concentration of E2 and FSH, the estrous cycles and
the numbers of primordial and secondary follicles of the offspring rats. In addition,
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the mRNA and protein expression of GDF9 and BMP15 was down-regulated, while
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the methylation patterns of Gdf9 and Bmp15 were not affected. In conclusion,
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maternal exposure to TCDD could affect the ovary development and functions which
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were possibly associated with down-regulation of mRNA and protein expression of
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GDF9 and BMP15. However, the down-regulation was not related to the pattern of
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methylation of Gdf9 and Bmp15.
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Key words: TCDD; Follicular development; GDF9; BMP15; Epigenetics
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1. Introduction
The fetus is highly vulnerable to various adverse environmental conditions
during the critical period of development when perturbation can lead to adult-onset diseases (Barker 2003, Crain et al. 2008, Langley-Evans and McMullen 2010, Zama and Uzumcu 2013). Its mechanism involves epigenetics that includes DNA 2
methylation, histone modification and chromatin remodeling, non-coding RNA regulation. Any disruption of the epigenome during the period of fetal development may result in altered gene expression in adulthood (Zama and Uzumcu 2009, Gore et al. 2011, Luense et al. 2011, Schwarz et al. 2010). 2,3,7,8-Tetrachlorobenzo-p-dioxin
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(TCDD), a byproduct of numerous industrial processes such as waste incineration, enters the body mainly through the digestive tract, respiratory tract and skin which
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can disturb the reproductive and endocrine systems. In addition, TCDD can enter the fetus through the blood-vessel barrier (Hurst et al. 2000), and interfere with ovarian
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follicular development of female offspring. The sensitivity of fetal rats to TCDD is
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100 times higher than that of adult rats, and even TCDD-insensitive species in
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adulthood are sensitive to TCDD during the fetal period (Wolf et al. 1999). Previous
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studies had demonstrated that TCDD exposure in utero was associated with reduced
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ovarian weight, decreased number of primordial, primary and antral follicles and corpus lutea, and even primary ovarian insufficiency (POI) (also known as
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premature ovarian failure (POF) in the adult female rat offspring (Wolf et al. 1999,
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Heimler et al. 1998, Nilsson et al. 2012). However, the mechanisms through which the maternal TCDD exposure affected the ovarian development and even resulted in
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POF in the female offspring rats are relatively unknown. The ovarian folliculogenesis is an orderly process which depends on complex
bidirectional communication between the oocyte and its surrounding somatic cells and involves steroid hormones, cytokines and growth factors (Kezele and Skinner 2003, Skinner 2005, Chen et al. 2007). Growth differentiation factor-9 (GDF 9) and 3
bone morphogenetic protein-15 (BMP 15), belonging to the transforming growth factor-β (TGF-β) superfamily, are secreted by oocytes and play crucial roles on ovarian follicular development (Gilchrist et al. 2008, Persani et al. 2014). BMP15 and GDF9 can form biologically active non-covalent homodimers (Persani et al.
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2014, Liao et al. 2003, Liao et al. 2004). BMP15 forms a complex with type I activin receptor-like kinase-6 (ALK6, also known as BMPRⅠB) and the BMP receptor type
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Ⅱ(BMPR2) on the membrane surface of granulosa cells (GCs) (Moore et al. 2003, Pulkki et al. 2012), and GDF9 interacts with the receptors of BMPR2 and ALK5 in
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GCs (Vitt UA et al. 2002, Mazerbourg et al. 2004, Kaivo-Oja et al. 2005). BMP15
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and GDF9 play pivotal roles on stimulating early follicular growth through
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promoting the proliferation of GCs (Otsuka and Shimasaki 2002, Fenwick et al.
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2013, Vitt et al. 2000, McNatty et al. 2005). The two growth factors can also
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modulate the differentiative effect of follicle-stimulating hormone (FSH) and control follicular maturation (Otsuka et al. 2000, Vitt et al. 2000, McNatty et al. 2005,
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Persani et al. 2014). In addition, BMP15 and GDF9 act on cumulus expansion and
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oocyte competence (Hussein et al. 2006, Elvin et al. 1999, Gui and Joyce 2005, Yeo et al. 2008). Importantly, previous studies have demonstrated that Bmp15 and Gdf9 gene mutations were associated with abnormal follicular development and POI/POF
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(Persani et al. 2014, Kumar et al. 2017). We hypothesized that low dose TCDD exposure in utero might impair ovarian folliculogenesis and even result in POI in female rat offspring. It was possibly related to the effect of TCDD exposure on the epigenetic regulation at the stage of 4
embryonic development which lead to the disorder of the expression of GDF9 and BMP15. To verify the hypothesis, the pregnant Sprague Dawley (SD) rats (n=42) were treated with TCDD (100ng/kg or 500ng/kg) or only vehicle and corn oil on the day 8 to 14 of gestation. The vaginal opening and estrous cycle of female offspring
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rat (F1) were monitored daily. On PND70, F1 rats were euthanized and the ovarian histology, follicle counts and apoptosis of GCs were conducted. Furthermore,
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real-time polymerase chain reaction (RT-PCR), Western blotting and DNA
methylation analysis of GDF9 and BMP15 were carried out in F1 rats of three
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groups.
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2. Materials and methods
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2.1 Animals and treatments
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The experimental protocols were designed in accordance with the Guide for the
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Care and Use of Laboratory Animals issued by the U.S. National Academy of Sciences and approved by the Animal Use and Care Committee at Public Health
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College of Zhengzhou University. Animals were treated humanely and with regard to
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alleviation of suffering.
The healthy female and male Sprague Dawley (SD) rats (8-10 weeks old)
purchased from experimental animal center of Zhengzhou university were housed in
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a temperature- and humidity-controlled room under a 12-hour light-dark cycle and fed an ad libitum diet. After one week adaptive feed, female and male rats were mated in 2:1 pattern. On the following morning, a sperm-positive vaginal smear was assigned as day 1 of gestation (GD1). The pregnant female rats (F0, n=42) were 5
randomly divided into three groups: control group (vehicle and corn oil, n=15), 100 ng/kg/day (low-dose TCDD, n=15), and 500 ng/kg/day (high-dose TCDD, n=12). TCDD was dissolved in dimethylsulfoxide (DMSO) and then diluted with corn oil. On the day 8 to 14 of gestation (Nilsson et al. 2008), the pregnant rats (F0) were
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administered daily by gavage with a stainless steel feeding needle. Each pregnant rat was weighted before receiving the treatment. The offspring of the F0 generation rats
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were the F1 generation.
2.2 The timing of vaginal opening (VO) and estrous cycle monitoring
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One rat was randomly picked from each litter and used to observe the timing of
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vaginal opening and estrous cycle monitoring. The vaginal opening of F1 was
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monitored at 8:00 AM daily, starting on the day 21 of postpartum, until the vagina
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was open. Thereafter, vaginal smears were obtained daily at 8:00 and 20:00 from the
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female rats to evaluate the estrous cycle over a 40-day-period. The estrous cycle of female rats is a repetitive but dynamic process. Each estrous cycle lasts average 4-5
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days and is generally divided into 4 stages (proestrus, estrus, metestrus and diestrus)
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identified by the analysis of the cell types presented in the vagina (Cora et al. 2015). The cell smears were stained with Methylene blue and estimated to determine the four stages of the estrous cycle as described previously (Cora et al. 2015).
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2.3 Tissue collection and processing 70-day-old F1 females were euthanized by CO2 inhalation for tissue collection at diestrus stage. Body, uterus and ovary weights were measured at dissection time. Left or right ovary of each female was randomly fixed in 4% Paraformaldehyde 6
Solution in PBS for histology and TUNEL analysis, and the other ovary was placed in tissue preservation solution and stored at -80℃ for subsequent molecular biological experiments. Blood samples were collected from abdominal aorta, allowed to clot for 30 min, centrifuged at 3500r/min for 15min and serum samples
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were stored at -80℃ for steroid hormone assays. 2.4 Serum estradiol (E2) and FSH levels
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Serum E2 and FSH levels (n=6, respectively in each group) were determined by commercial rat specific ELISA kits using Roche E170 MODULAR Immunoassay
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Analyzer following the manufacturer’s instructions. The standard curve was plotted
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according to the concentration of standard substance and optical density (OD) value
equation.
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2.5 Histology and Follicle counts
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and then the E2 and FSH concentration was calculated using standard curve
We randomly selected 5 litters per group and one rat per litter. Left or right
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ovaries were randomly assigned to be fixed in 4% Paraformaldehyde Solution in
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PBS and embedded in paraffin. The whole ovary was serially sectioned (5μm thick) and every 19th and 20th sections of the ovary were taken. One section was stained with hematoxylin and eosin for follicle counting, and the other for apoptosis assay. A
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total of 20 sections were taken from each ovary. Sections were visualized using a light microscopy (NIKON Eclipse Ci) and imaged using a digital sight DS-FI2 (NIKON, Japan). According to the classification by Pedersen and Peters (Pedersen and Peters 1968), follicles were classified into three categories: primordial follicles 7
(type 1 and 2), primary follicles (type 3a and 3b), secondary follicles (other growing follicles, type 4-8). Furthermore, the number of corpus luteum (CL) were also recorded. 2.6 Apoptosis assay
Mediated Nick End Labeling) Kit (Roche 11684817910) according to the
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manufacturer’s instructions. The nuclei were counterstained by DAPI (4’,
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Apoptosis was detected using TUNEL (Terminal-deoxynucleotidyl Transferase
6-diamidino-2-phenylindole). Images were drawn under 3D HISTECH Panoramic
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MIDI (Hungary) and the TUNEL assay was analyzed using Image-pro plus 6.0
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(Media Cybernetics, Inc., Rockville, MD, USA).The number of TUNEL-positive
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cells in the ovary was determined by counting at least three random microscopic
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fields from each sample, and the positive cells showed green fluorescence. The
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results were expressed by the percentages of apoptotic cells in each section. 2.7 RNA isolation and real-time RT-PCR
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Three litters were randomly selected from each group and subsequently one
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ovary was taken from each litter. Total RNA was extracted from ovary tissues of control (n=3) and TCDD (n=3, respectively) groups using Trizol (SK1321, Sangon Biotech, China). The quality and quantity of the isolated RNAs was determined
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using SMA4000 Spectrophotometer (Merinton). The total RNA was reverse transcribed with Revertaid Premium Reverse Transcriptase (Thermo ScientificTM EP0733) following the manufacturer’s instructions. Quantitative RT-PCR was performed using SG Fast qPCR Master Mix (High Rox) and Applied Biosystems 8
StepOnePlus Real-Time PCR Detection System. The reactions were incubated for 3 min at 95℃ followed by 45 cycles of 7 sec at 95℃, 10 sec at 57℃ and 15 sec at 72℃. All reactions were run in triplicate and relative mRNA expression was analyzed using the comparative cycle threshold method (2-∆∆CT) according to the
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manufacturer’s instructions (Applied Biosystems). The primers (Table 1) were designed using the Primer Premier 5.0 software according to the species-specific
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sequences from GenBank. 2.8 Western Blot Analysis
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Protein was extracted from the ovaries of either TCDD-exposed (n=3,
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respectively) and control (n=3) rats using RIPA lysis buffer (50Mm Tris-HCl,
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150mM NaCl, 1mM EDTA-Na2, 1% Triton X-100, 1% sodium deoxycholate, 0.1%
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SDS). The protein levels were determined by BCA solution (Thermo Scientific
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Pierce). For western blots, denatured protein was loaded into the wells of the SDS-PAGE in a 12% separating gel, and then electro-transferred to the
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polyvinylidene fluoride (PVDF) blotting membrane (Millipore, USA). The
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Membrane was blocked with 5% skim milk in TBS-T (TBS, 0.5% v/v Tween-20) for 1 h at room temperature and incubated overnight at 4℃ in primary antibody in blocking buffer. The following day, the membrane was incubated in a recommended
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dilution (1:3000) of conjugated secondary antibody in blocking buffer for 30min at room temperature. After three washes with TBS-T, the membrane was detected by chemiluminescence method. Densitometric analyses were performed using Alpha processing system (Alpha Innotech, USA ). 9
2.9 DNA methylation analysis The Methprimer software was used to predict the CpG island located in the promoter region (2000bp in front of the first exon). The result showed that there was one CpG island in the promoter region of Gdf9 and two pairs of primers were used
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to amplify the fragment. There was no CpG island in the promoter region of Bmp15, thence the fragment rich in CpG sites was amplified.
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DNA was extracted using DNA Extraction Kit (Sangon Biotech, China) and
quantified using SMA4000 Spectrophotometer (Merinton). 2ug DNA was diluted
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with DDW to 50ul and denatured with 3 M NaOH (5.5ul, freshly prepared) for 30
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min at 42℃. Freshly prepared 10mM hydroquinone (30ul) and 3.6 M sodium
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bisulfite (520ul) were added and then the mixture was incubated at 50℃ for 16h.
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The bisulfite-treated DNA was amplified by PCR using Taq DNA polymerase
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(TaKaRa). The sequences of primer were shown in Table X. PCR products were subcloned into pUC18-T vector (Sangon Biotech Co., Ltd, Shanghai, China) to
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analyze the methylation pattern of each clone using Sequencing. Ten clones were
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selected for each PCR product. Sequencing was used to find the methylation pattern of each CpG site of each clone. In this case, there were three samples in each group (from different litters) that included data of 10 clones.
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2.10 Statistical analysis All analysis was performed with SPSS version 21, and a P <0.05 was considered significant. The data were subjected to test for normality and homogeneity of variance. Statistically significant difference was determined by 10
one-way analysis of variance (ANOVA) followed by a least-significant difference (LSD) post hoc test. The nonparametric Kruskal-Wallis test was performed if the data were not normally distributed. The abnormal rate of estrous cyclicity was analyzed by Fisher exact test.
3.1 Effect of TCDD on the ovary weight and ovary coefficient
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3. Result
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Animals were assessed at necropsy for changes in general health. Treatment had no apparent effect on the general health of the rat. But, compared with the control
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group, there were differences in the weight of body and ovary. The body weight
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increased in the low-dose TCDD rats, while the ovary weight decreased in the
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high-dose TCDD group compared with those of the control group. Moreover, the
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ovary coefficient was decreases in the two treatment groups (Figure 1).
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3.2 Effect of TCDD on the timing of vaginal opening (VO) and estrous cyclicity The vaginal opening time was slightly delayed only in the low-dose TCDD
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group, while it was not distinct between the high-lose TCDD and the control rats
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(Figure 2). The estrous cyclicity of most rats was normal and the abnormal rate was 21% in the control group. Half of the rats in the low-dose TCDD group had abnormal estrous cycle (50%). In addition, the estrous cycle of most rats was
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disturbed (75%) in the high-lose TCDD group, and it varies in length, estrus and metestrus stage being longer (Figure 2). Although the abnormal rate appeared an increasing trend with the increase of TCDD dose, the statistical results showed that the abnormal rate of estrous cycle in high-does TCDD group was significantly high 11
compared with that of the control group, while there was no difference in abnormal rate of estrous cycle between the low dose TCDD and the control rats. 3.3 Effect of TCDD on the concentration of E2 and FSH Steroid hormone test revealed difference in the concentration of E2 and FSH in
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the treatment rats compared with those from the control group (Figure 2). The E2 levels decreased in the treatment groups, while the FSH concentration increased only
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in the high-dose TCDD rats.
3.4 Effect of TCDD on the morphology of ovaries, follicle counts and follicle cells
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apoptosis
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Microscopic evaluation of ovarian sections revealed significant differences in
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the number of follicles in different stages of development in ovaries of rats exposed
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to TCDD compared with the control rats (Figure. 3. A). Especially, the
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TCDD-exposed ovaries had significantly fewer primordial follicles than ovaries from the control group, while the number of secondary, preovulation follicles and
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corpus luteum were obviously elevated in the TCDD-exposed ovaries compared with
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those from control rats. It was verified by the result of follicle count in the present study (Figure. 3. B). The TUNEL staining suggested that treatment with TCDD could result in an increase in apoptosis (Figure. 3. C and D). There were differences
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in the number of positively staining follicles between the TCDD-exposed rats and control rats. However, the positively stained follicles appeared more in the developing follicles, and the apoptosis rates were 10% greater in the TCDD-exposed groups than those in the control group. The apoptotic granulosa cells surrounding the 12
corpus luteum were small in all ovaries of rats, although the result of TUNEL staining showed a statistical difference between the TCDD-exposure groups and the control group. 3.5 Effect of TCDD on the expression of mRNA and protein of Gdf9 and Bmp15
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The GDF9/BMP15 signal pathway, plays a pivotal role in follicle development. Alk5 and Alk6 are type I receptor of GDF9 and BMP15, respectively, and BMPRII
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is the common type II receptor of those. The result of RT-PCR showed that the
mRNA expression of Gdf9, Bmp15, Alk6, Bmpr2 significantly decreased in both
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low and high dose TCDD groups compared with the control group, while mRNA
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expression of Alk5 was reduced only in low-dose TCDD rats (Figure 4). To further
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explore whether TCDD exposure in utero affects GDF9/BMP15 signal pathway in
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ovary of offspring rats, we performed Western blotting assays and found that both
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GDF9 and BMP15 expression significantly decreased in the TCDD-exposure groups compared with the control group (Figure 4).
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3.6 Effect of TCDD on the promoter region methylation of Gdf9 and Bmp15
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To investigate the relationship between Gdf9 and Bmp15 gene promoter region methylation and their gene expression in TCDD-exposure rats, the DNA methylation analysis was carried out in the present study. However, we found that there were no
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differences in methylation patterns between the treatment and control group, either in Gdf9 or in Bmp15. Furthermore, we also observed that the degree of methylation in the promoter region of Gdf9 was very low (Figure 5), whereas it was very high in Bmp15 (Figure 6). We also analyzed the methylation rate of each CpG site. There 13
were 44 CpG sites in promoter region of Gdf9 of which 9 CpG sites in control group, 8 CpG sites in 100ng/kg TCDD group, and 6 CpG sites in 500ng/kg TCDD group were methylated, but the methylation level at each CpG site was much lower than 50% (Figure 7). There were 8 CpG sites in promoter region of Bmp15 of which the
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methylation level of each CpG site was higher than 50% and even methylation rate of some CpG sites reached 100%, but the methylation rate of each CpG site did not
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differ among the three groups (Figure 7). 4. Discussion
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The results of the present study showed that exposure to TCDD in utero on
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GD8-14 might change the ovary weight, the time of vaginal opening, the
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concentration of E2 and FSH, the estrous cycles and the numbers of primordial and
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secondary follicles of the offspring rats. Especially in 500ng/kg TCDD group, the
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rats had the characteristics of lower E2 level, higher FSH concentration and reduced the number of primordial follicles which might indicate a possibility of POI/POF.
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Because the number of primordial follicles is nonrenewable, if it is produced too
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little or exhausted too quickly, POI/POF would occur. The Seveso incident in Italy also proved that the increased risk of premature ovarian failure is associated with
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TCDD exposure (Eskenazi et al. 2005). There were no differences in the visual characteristics of ovaries among the
three groups, and the ovarian weight decreased only in the high-dose group. Previous studies had shown that a single oral dose of TCDD (1.0 ug/kg) on GD 8 caused a reduction in ovary weight in 30% of offspring rats (Gray and Ostby 1995), whereas 14
the same dose to pregnant Holtzman rats on GD15, there was no significant change in ovary weight and cross-sectional size of ovary (Chaffin et al. 1996, Heimler et al. 1998). This might be due to the fact that the maximum dose of TCDD was 500ng/kg in this study that was much lower than the lowest teratogenic dose (1.25ug/kg)
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(National Toxicology Program, 1991), whereas the LD50 (lethal dose, 50%) was 20ug/kg.
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The vaginal opening time was slightly delayed in low-dose TCDD group
compared with that in the control group. Approximately half of the rats in 100ng/kg
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TCDD group had disordered estrous cyclicity, and the abnormal rate of estrous
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cyclicity was 75% in 500ng/kg TCDD group. The disorders of estrous cyclicity
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disorders were mainly as extend or shorten the period of estrous cycle, prolongation
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of estrus and metestrus stage and continuous estrus. This was consistent with some
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previous studies. Exposure of adult rats to TCDD altered estrous cyclicity (Cummings et al. 1996, Li et al. 1995). One oral dose of TCDD (1ug/kg) was given
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to dams on GD8 which resulted in 47% of one-year-old offspring rats showing
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continuous estrus (Gray and Ostby 1995), and one oral dose of TCDD (1 or 2.5 ug/kg) to dams on GD15 could make the offspring rats possess abnormal estrous cyclicity and shortened estrus stage (Salisbury and Marcinkiewicz 2002). In the
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present study, serum E2 level decreased in both TCDD-treated groups, while serum FSH concentration only increased in higher-dose TCDD group. The possible mechanism was that TCDD is a high-affinity ligand for the aryl hydrocarbon receptro (AhR) which could significantly alter steroid hormone synthesis and the 15
transcriptional pathway through ligand binding receptor in the cells of ovarian (Heimler et al. 1998), or because that TCDD exposure induced the metabolism of estrogen which resulted in a decrease in estrogen levels binding and activating estrogen receptors. In addition, TCDD exposure could directly damage the ovary
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which led to a reduction of secretion of E2 by granulosa cells. Myllymaki et al. (Myllymaki et al. 2005) found that, after administration of TCDD (single oral dose,
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0, 0.04, 0.2 or 1.0ug/kg) to pregnant rats on GD13, the E2 level decreased and the
FSH level was elevated in 14 or 16-day old offspring rats in 1.0ug/kg TCDD group
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that was consistent with the results of the 500 ng/kg TCDD group in this study.
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However, Pesonen et al. (Pesonen et al. 2006) also gave a single dose TCDD (0.04,
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0.2 or 1.0ug/kg) to pregnant dams on GD13 and found that there were no significant
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differences in E2 and FSH levels compared with control group. Additionally,
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pregnant rats drank Canal waters contaminated with TCDD and their offspring rats of the first generation possess a reduction of E2 level and normal FSH concentration
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(Huang et al. 2011). Thence, it was controversial whether intrauterine exposure of
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TCDD affected E2 and FSH levels in offspring rats, or the effect of TCDD exposure on E2 and FSH levels differed by dose, exposure periods and species. The observation of ovarian tissue sections stained H&E was consistent with the
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result of follicle counting. The data showed that the primordial follicle number decreased and the number of secondary follicle and corpus luteum increased. However, there was controversy over the number of follicles at some stages affected by TCDD intrauterine exposure. Heimler et al (Heimler et al. 1998) found that 16
exposure to TCDD in utero and via lactation hindered maturation of follicles and caused a reduction of number of preantral and antral follicles. Although this was demonstrated by other studies that one oral dose TCDD (1ug/kg) exposure to pregnant rats on GD15 might significantly reduce the number of preantral and antral
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follicles (Chaffin et al. 1996, Heimler et al. 1998), but not affect the primordial follicle number (Heimler et al. 1998), Salisbury and Marcinkiewicz’s (Salisbury and
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Marcinkiewicz 2002) report suggested that one oral dose TCDD on GD15 did not
affect the number of primordial and preantral follicles. Furthermore, there was not a
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reduction of primordial follicle number in two-day-old offspring born in dams
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exposed to a single dose of TCDD (1.0ug/kg) on GD11, 15 and 18 which perhaps
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suggested that exposure to TCDD in utero did not affect the primordial follicle pool
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of offspring. Gray et al. (Gray et al. 1997) reported that when one single dose of
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TCDD (0.2 and 0.8ug/kg) to pregnant rats on GD15, the cystic follicles and corpus luteum increased and obvious ovarian interstitial hyperplasia was reported in their
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70-day-old offspring rats that was in line with our results. The process of follicular
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development is complex and requires not only the bidirectional communication between oocytes, granulosa and follicle cells, but also the correct-levels of sex hormones in the hypothalamus-pituitary-ovarian. Therefore, this might be the reason
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for the controversy, which required further extensive research to clarify. The TUNEL result showed that the apoptosis rate of granulosa cells surrounding the growing follicles increased in TCDD-treated groups compared with that in the control group. Although there were differences in the apoptosis rate of follicular cells of corpus 17
luteum, the rate was all very low in the three groups. Granulosa cells play an important role in follicular development and can express FSH receptors (Edson et al. 2009, Eppig et al. 2002, Liu et al. 2015, Liu et al. 2006). So the increased apoptosis rate of granulosa cells would affect follicles growth. Overall, TCDD exposure in
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utero affected the follicle development and steroid hormone secretion which interfered with estrous cyclicality and follicle growth.
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The GDF9/BMP15 signaling pathway plays an important role in ovarian follicle development. It regulates primordial follicle recruitment (Martins et al. 2008, Peng
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et al. 2010), boosts proliferation of granulosa cells (Gilchrist et al. 2006), inhibits
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granulosa cell apoptosis and luteinization, promotes maturation of oocyte, ovulation
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and steroid hormone synthesis (Su et al. 2008, Orisaka et al. 2009). GDF9 and
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BMP15 can form homodimers or heterodimers (Liao et al. 2003, Juengel et al. 2004,
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McIntosh et al. 2008, Mottershead et al. 2012). ALK5 and ALK6 are the type I receptors of GDF9 and BMP15 respectively. They share BMPRII as the type II
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receptor. Our results showed that TCDD exposure in utero resulted in a
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down-regulation of mRNA expression of Gdf9, Alk5, Bmp15, Alk6 and Bmpr2. The protein expression of GDF9 and BMP15 was also down-regulated. Previous studies proved that premature ovarian failure was associated with down-regulation of
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GDF9/BMP15 or mutation of Gdf9/Bmp15 (Liu et al. 2011, Mehdizadeh et al. 2016, Wei et al. 2014, Cui et al. 2017, Ma et al. 2015, Tiotiu et al. 2010, Pu et al. 2014, Fonseca et al. 2014, Chand et al. 2006). In this study, especially in the high-dose TCDD group, decreased number of primordial follicles and E2 level and increased 18
FSH concentration were similar to the phenotype of premature ovarian failure. In addition, prenatal exposure to endocrine disruptors (EDCs), including bisphenol A, dioxins, insecticides and cigarette smoke, caused down-regulation of expression of GDF9 and BMP15 in ovaries and increased apoptosis of germ cell/oocyte, even lead
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to the occurrence of premature ovarian failure (Patel et al. 2015). Therefore, we speculated that TCDD exposure in utero interfered with ovarian follicle development,
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and even resulted in premature ovarian failure. Its mechanism was associated with the down-regulation of GDF9/BMP15 (Stanley et al. 2015). However,
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down-regulation of GDF9 and BMP15 accompanied increased number of secondary
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follicle and corpus luteum. The reason was unknown and needed to be elucidated.
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Epigenetic reprogramming in the primordial germ cell undergoes a DNA
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demethylation and remethylation during early embryo development (Skinner et al.
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2010). TCDD exposure during this period may alter epigenome and transcriptome which affect expression of critical genes related to follicular development and then
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interfere with follicle development. There were no differences in methylation rates of
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promoter regions of gene Gdf9 and Bmp15 among the three groups in the present study. Therefore, we thought that down-regulation of mRNA and protein expression of related genes in GDF9/BMP15 signaling pathway by TCDD exposure was not
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related to the methylation patterns of Gdf9 and Bmp15. At present, although there were no related reports on the relationship between TCDD exposure and the methylation patterns of Gdf9 and Bmp15, Liu et al. (Liu et al. 2018) intragastrically administrated diethylhexyl phthalate (DEHP) to 21-day-old mouse and found that 19
the reduction of mRNA level of Gdf9 was not associated with the DNA methylation level in promoter region of Gdf9. This was consistent with our result. Histone modification is also one of the epigenetic mechanisms, and histone acetylation is the most studied in histone modification. Histone acetylation mainly
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occurs on the lysine residues of histone (H) 3 and 4 and plays an important role in transcriptional regulation of genes. Under the action of histone acetyltransferases
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(HATs) and histone deacetylases (HDACs), histone acetylation and deacetylation in
the nucleus maintains histone acetylation homeostasis. Histone acetylation promotes
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the transcription of genes, whereas histone deacetylation represses transcription of
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genes (Lin and Dent 2006). Lukas et al. (Vrba et al. 2008) found that H3 and H4
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acetylation of promoter region of Gdf9 was associated with mRNA expression level
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of Gdf9 (Vrba et al. 2008). Moreover, in the study of reproductive toxicity induced
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by soluble fluoride salt in mice, it was found that decreased mRNA expression level
2015).
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of Gdf9 was related to histone acetylation, but not DNA methylation (Yin et al.
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In summary, the present results propose that maternal exposure to TCDD (especially the dose of 500ng/kg) not only significantly affected E2 and FSH levels, but also resulted in decreased primordial follicles and increased the numbers of
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secondary follicles and corpus lutea. This might be associated with down-regulation of mRNA and protein expression of GDF9/BMP15. However, the down-regulation was not related to the pattern of methylation of promoter regions in Gdf9 and Bmp15. 20
Conflicts of interest statement The authors declare that there are no conflicts of interest. Acknowledgements This research was supported by grants from the National Nature Science
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Foundation of China (21577119 and U1604183)
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exposure in rats. Biology of reproduction 88: 52.
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Figure. 1. The effects of exposure to TCDD in utero on body weight, ovary weight and ovary
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coefficient of female offspring rats. The ovary coefficient is ovary weight/body weight ratio.
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Data were analyzed using one-way ANOVA, **P <0.05, compared with the control group.
Figure. 2. The effect of exposure to TCDD in utero on the day of vaginal opening, abnormal rate of estrous cyclicity, serum E2 and FSH concentration in female offspring rats. The data of vaginal opening, E2 and FSH concentration were analyzed using one-way ANOVA, while the abnormal rate of estrous cyclicity was analyzed using Fisher exact test, **P <0.05, compared with the control group. 28
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Figure. 3. The effect of TCDD exposure in utero on the morphology of ovary, follicle counts and
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follicle cells apoptosis. (A) Morphology of ovaries by H&E staining. The number of primordial follicle was decreased, and the number of secondary follicle and corpus luteum were increased in
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TCDD-treated groups; (B) The result of follicle counting, **P <0.05, compared with the control group; (C) The apoptosis assay was detected using TUNEL and the positive cells showed green
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fluorescence; (D) The rate of follicle cells apoptosis. Data were analyzed using one-way ANOVA,
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**P <0.05, compared with the control group.
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Figure. 4. The effect of TCDD exposure in utero on the mRNA and protein expression of GDF9/BMP15. (A) The mRNA expression of GDF9/BMP15 signaling pathway related to genes in female offspring rats was analyzed using real time-PCR. **P <0.05, compared with the control group. (B and C) The protein expression of GDF9 and BMP15 was detected using western blotting. Data were analyzed using one-way ANOVA,**P <0.05, compared with the control group. 30
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Figure. 5. The effect of TCDD exposure in utero on methylation pattern of promoter region of
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Gdf9. (A and B) The two fragment PCR used to detect the methylation rate of CpG island in promoter region of Gdf9. Ten clones were selected for each PCR product. Sequencing was used
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to find the methylaiton pattern of each CpG site of each clone. The white cycles meant unmethlated CpG sites and the black cycles meant methylated CpG sites. There were no differences in methylation pattern of Gdf9 among three groups. Data were analyzed using
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nonparametric Kruskal-Wallis test.
31
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Figure. 6. The effect of TCDD exposure in utero on methylation pattern of promoter region of
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Bmp15. Ten clones were selected for each PCR product. Sequencing was used to find the
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methylaiton pattern of each CpG site of each clone. The white cycles meant unmethlated CpG
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sites and the black cycles meant methylated CpG sites. There were no differences in methylation pattern of Bmp15 among three groups. Data were analyzed using nonparametric Kruskal-Wallis
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test.
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Figure. 7. The methylation rate of each CpG site in promoter regions of Gdf9 and Bmp15. (A) There were 44 CpG sites in Gdf9 of which 9 CpG sites in control group, 8 CpG sites in 100ng/kg
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TCDD group and 6 CpG sites in 500ng/kg TCDD group were methylated. (B) There were 8 CpG sites in promoter region of Bmp15 of which the methylation level of each CpG site was higher
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than 50% and even methylation rate of some CpG sites reached 100%. Data were analyzed using
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nonparametric Kruskal-Wallis test.
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Table. 1.Primer sequences used for the real-time PCR of GDF9/BMP15 signaling pathway related genes. Gene
Primer
Sequence 5'→3'
Product size (bp)
Annealing/extension temperature (℃)
GAPDH
Forward
CAAGTTCAACGGCACAGTCAA
140
59.4
Reverse
CGCCAGTAGACTCCACGACA
Forward
CCAGCAACCAGATGACAGGA
Reverse
TCACAGTGGAGGAGGAAGCA
Forward
GAGAACCGCACGATTGGAG
Reverse
CACAGTGGCTCTGATTAGTTGGT
Forward
TTGACATTCCACCCAACACC
Reverse
CATCTCCTTGCAATCTCCCA
Forward
AAGCCTGGAAAGAAAATAGCCT
Reverse
ACATTGGGTTGACCGTTGG
Forward
AACCGCACTGTCATTCACCA
Reverse
TTTGCCGATGCTTTCTTGTAG
Alk6 Bmpr2
58.5 150 151
58.4 58.1
174
58.9 58.4
192
59.2 58.5
N A M ED PT CC E A
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58.5 58.7
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Alk5
58.8
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Bmp15
138
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Gdf9
59.2
S0300-483X(18)30299-3 https://doi.org/10.1016/j.tox.2018.08.015 TOX 52088
To appear in:
Toxicology
Received date: Revised date: Accepted date:
11-3-2018 8-8-2018 27-8-2018
Please cite this article as: Zhang X, Ji M, Tan X, Yu K, Liu X, Li N, Yu Z, Impairment of ovaries by 2,3,7,8-Tetrachlorobenzo-p-dioxin (TCDD) exposure in utero associated with BMP15 and GDF9 in the female offspring rat, Toxicology (2018), https://doi.org/10.1016/j.tox.2018.08.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title Impairment of ovaries by 2,3,7,8-Tetrachlorobenzo-p-dioxin (TCDD) exposure in utero associated with BMP15 and GDF9 in the female offspring rat
Xiuli Zhanga, b, Mengmeng Jia, Xuemei Tana, Kailun Yua, Xiaozhuan Liuc, Ning Lid, and Zengli
Health College of Zhengzhou University, No. 100 of Science Road, Zhengzhou, 450001,
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aPublic
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Yua*
China.
First Affiliated Hospital of Zhengzhou University, No. 1 of Jianshe East Road, Zhengzhou,
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bThe
University, No. 63 of Agricultural Road, Zhengzhou, 450002, China.
*Corresponding authors
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Email: [email protected]
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dHenan Agricultural
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Provincial Peoples Hospital, No. 7 of Weiwu Road, Zhengzhou, 450001, China.
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cHenan
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450052, China.
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AB STRACT
2,3,7,8-Tetrachlorobenzo-p-dioxin (TCDD) exposure in utero had been shown
to affect ovarian development and functions. However, its mechanism remained unknown. In this study, to investigate the effect of maternal exposure to TCDD on ovaries, the pregnant Sprague Dawley (SD) rats were treated with TCDD (100ng/kg 1
or 500ng/kg) or only vehicle and corn oil on the day 8 to 14 of gestation through the gavage with a stainless-steel feeding needle (once a day). The vaginal opening and estrous cycle of female offspring rats (F1) were monitored twice a day. The ovarian histology, follicle counts, real-time PCR, western blotting and DNA methylation
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analysis about Gdf9 and Bmp15 were carried out in F1 rats. The results showed that exposure to TCDD (especially the dose of 500ng/kg) in utero on GD8-14 might
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change the ovary weight, the concentration of E2 and FSH, the estrous cycles and
the numbers of primordial and secondary follicles of the offspring rats. In addition,
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the mRNA and protein expression of GDF9 and BMP15 was down-regulated, while
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the methylation patterns of Gdf9 and Bmp15 were not affected. In conclusion,
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maternal exposure to TCDD could affect the ovary development and functions which
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were possibly associated with down-regulation of mRNA and protein expression of
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GDF9 and BMP15. However, the down-regulation was not related to the pattern of
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methylation of Gdf9 and Bmp15.
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Key words: TCDD; Follicular development; GDF9; BMP15; Epigenetics
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1. Introduction
The fetus is highly vulnerable to various adverse environmental conditions
during the critical period of development when perturbation can lead to adult-onset diseases (Barker 2003, Crain et al. 2008, Langley-Evans and McMullen 2010, Zama and Uzumcu 2013). Its mechanism involves epigenetics that includes DNA 2
methylation, histone modification and chromatin remodeling, non-coding RNA regulation. Any disruption of the epigenome during the period of fetal development may result in altered gene expression in adulthood (Zama and Uzumcu 2009, Gore et al. 2011, Luense et al. 2011, Schwarz et al. 2010). 2,3,7,8-Tetrachlorobenzo-p-dioxin
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(TCDD), a byproduct of numerous industrial processes such as waste incineration, enters the body mainly through the digestive tract, respiratory tract and skin which
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can disturb the reproductive and endocrine systems. In addition, TCDD can enter the fetus through the blood-vessel barrier (Hurst et al. 2000), and interfere with ovarian
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follicular development of female offspring. The sensitivity of fetal rats to TCDD is
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100 times higher than that of adult rats, and even TCDD-insensitive species in
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adulthood are sensitive to TCDD during the fetal period (Wolf et al. 1999). Previous
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studies had demonstrated that TCDD exposure in utero was associated with reduced
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ovarian weight, decreased number of primordial, primary and antral follicles and corpus lutea, and even primary ovarian insufficiency (POI) (also known as
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premature ovarian failure (POF) in the adult female rat offspring (Wolf et al. 1999,
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Heimler et al. 1998, Nilsson et al. 2012). However, the mechanisms through which the maternal TCDD exposure affected the ovarian development and even resulted in
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POF in the female offspring rats are relatively unknown. The ovarian folliculogenesis is an orderly process which depends on complex
bidirectional communication between the oocyte and its surrounding somatic cells and involves steroid hormones, cytokines and growth factors (Kezele and Skinner 2003, Skinner 2005, Chen et al. 2007). Growth differentiation factor-9 (GDF 9) and 3
bone morphogenetic protein-15 (BMP 15), belonging to the transforming growth factor-β (TGF-β) superfamily, are secreted by oocytes and play crucial roles on ovarian follicular development (Gilchrist et al. 2008, Persani et al. 2014). BMP15 and GDF9 can form biologically active non-covalent homodimers (Persani et al.
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2014, Liao et al. 2003, Liao et al. 2004). BMP15 forms a complex with type I activin receptor-like kinase-6 (ALK6, also known as BMPRⅠB) and the BMP receptor type
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Ⅱ(BMPR2) on the membrane surface of granulosa cells (GCs) (Moore et al. 2003, Pulkki et al. 2012), and GDF9 interacts with the receptors of BMPR2 and ALK5 in
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GCs (Vitt UA et al. 2002, Mazerbourg et al. 2004, Kaivo-Oja et al. 2005). BMP15
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and GDF9 play pivotal roles on stimulating early follicular growth through
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promoting the proliferation of GCs (Otsuka and Shimasaki 2002, Fenwick et al.
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2013, Vitt et al. 2000, McNatty et al. 2005). The two growth factors can also
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modulate the differentiative effect of follicle-stimulating hormone (FSH) and control follicular maturation (Otsuka et al. 2000, Vitt et al. 2000, McNatty et al. 2005,
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Persani et al. 2014). In addition, BMP15 and GDF9 act on cumulus expansion and
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oocyte competence (Hussein et al. 2006, Elvin et al. 1999, Gui and Joyce 2005, Yeo et al. 2008). Importantly, previous studies have demonstrated that Bmp15 and Gdf9 gene mutations were associated with abnormal follicular development and POI/POF
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(Persani et al. 2014, Kumar et al. 2017). We hypothesized that low dose TCDD exposure in utero might impair ovarian folliculogenesis and even result in POI in female rat offspring. It was possibly related to the effect of TCDD exposure on the epigenetic regulation at the stage of 4
embryonic development which lead to the disorder of the expression of GDF9 and BMP15. To verify the hypothesis, the pregnant Sprague Dawley (SD) rats (n=42) were treated with TCDD (100ng/kg or 500ng/kg) or only vehicle and corn oil on the day 8 to 14 of gestation. The vaginal opening and estrous cycle of female offspring
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rat (F1) were monitored daily. On PND70, F1 rats were euthanized and the ovarian histology, follicle counts and apoptosis of GCs were conducted. Furthermore,
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real-time polymerase chain reaction (RT-PCR), Western blotting and DNA
methylation analysis of GDF9 and BMP15 were carried out in F1 rats of three
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groups.
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2. Materials and methods
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2.1 Animals and treatments
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The experimental protocols were designed in accordance with the Guide for the
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Care and Use of Laboratory Animals issued by the U.S. National Academy of Sciences and approved by the Animal Use and Care Committee at Public Health
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College of Zhengzhou University. Animals were treated humanely and with regard to
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alleviation of suffering.
The healthy female and male Sprague Dawley (SD) rats (8-10 weeks old)
purchased from experimental animal center of Zhengzhou university were housed in
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a temperature- and humidity-controlled room under a 12-hour light-dark cycle and fed an ad libitum diet. After one week adaptive feed, female and male rats were mated in 2:1 pattern. On the following morning, a sperm-positive vaginal smear was assigned as day 1 of gestation (GD1). The pregnant female rats (F0, n=42) were 5
randomly divided into three groups: control group (vehicle and corn oil, n=15), 100 ng/kg/day (low-dose TCDD, n=15), and 500 ng/kg/day (high-dose TCDD, n=12). TCDD was dissolved in dimethylsulfoxide (DMSO) and then diluted with corn oil. On the day 8 to 14 of gestation (Nilsson et al. 2008), the pregnant rats (F0) were
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administered daily by gavage with a stainless steel feeding needle. Each pregnant rat was weighted before receiving the treatment. The offspring of the F0 generation rats
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were the F1 generation.
2.2 The timing of vaginal opening (VO) and estrous cycle monitoring
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One rat was randomly picked from each litter and used to observe the timing of
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vaginal opening and estrous cycle monitoring. The vaginal opening of F1 was
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monitored at 8:00 AM daily, starting on the day 21 of postpartum, until the vagina
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was open. Thereafter, vaginal smears were obtained daily at 8:00 and 20:00 from the
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female rats to evaluate the estrous cycle over a 40-day-period. The estrous cycle of female rats is a repetitive but dynamic process. Each estrous cycle lasts average 4-5
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days and is generally divided into 4 stages (proestrus, estrus, metestrus and diestrus)
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identified by the analysis of the cell types presented in the vagina (Cora et al. 2015). The cell smears were stained with Methylene blue and estimated to determine the four stages of the estrous cycle as described previously (Cora et al. 2015).
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2.3 Tissue collection and processing 70-day-old F1 females were euthanized by CO2 inhalation for tissue collection at diestrus stage. Body, uterus and ovary weights were measured at dissection time. Left or right ovary of each female was randomly fixed in 4% Paraformaldehyde 6
Solution in PBS for histology and TUNEL analysis, and the other ovary was placed in tissue preservation solution and stored at -80℃ for subsequent molecular biological experiments. Blood samples were collected from abdominal aorta, allowed to clot for 30 min, centrifuged at 3500r/min for 15min and serum samples
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were stored at -80℃ for steroid hormone assays. 2.4 Serum estradiol (E2) and FSH levels
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Serum E2 and FSH levels (n=6, respectively in each group) were determined by commercial rat specific ELISA kits using Roche E170 MODULAR Immunoassay
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Analyzer following the manufacturer’s instructions. The standard curve was plotted
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according to the concentration of standard substance and optical density (OD) value
equation.
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2.5 Histology and Follicle counts
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and then the E2 and FSH concentration was calculated using standard curve
We randomly selected 5 litters per group and one rat per litter. Left or right
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ovaries were randomly assigned to be fixed in 4% Paraformaldehyde Solution in
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PBS and embedded in paraffin. The whole ovary was serially sectioned (5μm thick) and every 19th and 20th sections of the ovary were taken. One section was stained with hematoxylin and eosin for follicle counting, and the other for apoptosis assay. A
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total of 20 sections were taken from each ovary. Sections were visualized using a light microscopy (NIKON Eclipse Ci) and imaged using a digital sight DS-FI2 (NIKON, Japan). According to the classification by Pedersen and Peters (Pedersen and Peters 1968), follicles were classified into three categories: primordial follicles 7
(type 1 and 2), primary follicles (type 3a and 3b), secondary follicles (other growing follicles, type 4-8). Furthermore, the number of corpus luteum (CL) were also recorded. 2.6 Apoptosis assay
Mediated Nick End Labeling) Kit (Roche 11684817910) according to the
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manufacturer’s instructions. The nuclei were counterstained by DAPI (4’,
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Apoptosis was detected using TUNEL (Terminal-deoxynucleotidyl Transferase
6-diamidino-2-phenylindole). Images were drawn under 3D HISTECH Panoramic
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MIDI (Hungary) and the TUNEL assay was analyzed using Image-pro plus 6.0
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(Media Cybernetics, Inc., Rockville, MD, USA).The number of TUNEL-positive
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cells in the ovary was determined by counting at least three random microscopic
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fields from each sample, and the positive cells showed green fluorescence. The
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results were expressed by the percentages of apoptotic cells in each section. 2.7 RNA isolation and real-time RT-PCR
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Three litters were randomly selected from each group and subsequently one
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ovary was taken from each litter. Total RNA was extracted from ovary tissues of control (n=3) and TCDD (n=3, respectively) groups using Trizol (SK1321, Sangon Biotech, China). The quality and quantity of the isolated RNAs was determined
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using SMA4000 Spectrophotometer (Merinton). The total RNA was reverse transcribed with Revertaid Premium Reverse Transcriptase (Thermo ScientificTM EP0733) following the manufacturer’s instructions. Quantitative RT-PCR was performed using SG Fast qPCR Master Mix (High Rox) and Applied Biosystems 8
StepOnePlus Real-Time PCR Detection System. The reactions were incubated for 3 min at 95℃ followed by 45 cycles of 7 sec at 95℃, 10 sec at 57℃ and 15 sec at 72℃. All reactions were run in triplicate and relative mRNA expression was analyzed using the comparative cycle threshold method (2-∆∆CT) according to the
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manufacturer’s instructions (Applied Biosystems). The primers (Table 1) were designed using the Primer Premier 5.0 software according to the species-specific
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sequences from GenBank. 2.8 Western Blot Analysis
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Protein was extracted from the ovaries of either TCDD-exposed (n=3,
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respectively) and control (n=3) rats using RIPA lysis buffer (50Mm Tris-HCl,
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150mM NaCl, 1mM EDTA-Na2, 1% Triton X-100, 1% sodium deoxycholate, 0.1%
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SDS). The protein levels were determined by BCA solution (Thermo Scientific
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Pierce). For western blots, denatured protein was loaded into the wells of the SDS-PAGE in a 12% separating gel, and then electro-transferred to the
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polyvinylidene fluoride (PVDF) blotting membrane (Millipore, USA). The
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Membrane was blocked with 5% skim milk in TBS-T (TBS, 0.5% v/v Tween-20) for 1 h at room temperature and incubated overnight at 4℃ in primary antibody in blocking buffer. The following day, the membrane was incubated in a recommended
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dilution (1:3000) of conjugated secondary antibody in blocking buffer for 30min at room temperature. After three washes with TBS-T, the membrane was detected by chemiluminescence method. Densitometric analyses were performed using Alpha processing system (Alpha Innotech, USA ). 9
2.9 DNA methylation analysis The Methprimer software was used to predict the CpG island located in the promoter region (2000bp in front of the first exon). The result showed that there was one CpG island in the promoter region of Gdf9 and two pairs of primers were used
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to amplify the fragment. There was no CpG island in the promoter region of Bmp15, thence the fragment rich in CpG sites was amplified.
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DNA was extracted using DNA Extraction Kit (Sangon Biotech, China) and
quantified using SMA4000 Spectrophotometer (Merinton). 2ug DNA was diluted
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with DDW to 50ul and denatured with 3 M NaOH (5.5ul, freshly prepared) for 30
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min at 42℃. Freshly prepared 10mM hydroquinone (30ul) and 3.6 M sodium
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bisulfite (520ul) were added and then the mixture was incubated at 50℃ for 16h.
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The bisulfite-treated DNA was amplified by PCR using Taq DNA polymerase
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(TaKaRa). The sequences of primer were shown in Table X. PCR products were subcloned into pUC18-T vector (Sangon Biotech Co., Ltd, Shanghai, China) to
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analyze the methylation pattern of each clone using Sequencing. Ten clones were
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selected for each PCR product. Sequencing was used to find the methylation pattern of each CpG site of each clone. In this case, there were three samples in each group (from different litters) that included data of 10 clones.
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2.10 Statistical analysis All analysis was performed with SPSS version 21, and a P <0.05 was considered significant. The data were subjected to test for normality and homogeneity of variance. Statistically significant difference was determined by 10
one-way analysis of variance (ANOVA) followed by a least-significant difference (LSD) post hoc test. The nonparametric Kruskal-Wallis test was performed if the data were not normally distributed. The abnormal rate of estrous cyclicity was analyzed by Fisher exact test.
3.1 Effect of TCDD on the ovary weight and ovary coefficient
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3. Result
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Animals were assessed at necropsy for changes in general health. Treatment had no apparent effect on the general health of the rat. But, compared with the control
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group, there were differences in the weight of body and ovary. The body weight
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increased in the low-dose TCDD rats, while the ovary weight decreased in the
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high-dose TCDD group compared with those of the control group. Moreover, the
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ovary coefficient was decreases in the two treatment groups (Figure 1).
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3.2 Effect of TCDD on the timing of vaginal opening (VO) and estrous cyclicity The vaginal opening time was slightly delayed only in the low-dose TCDD
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group, while it was not distinct between the high-lose TCDD and the control rats
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(Figure 2). The estrous cyclicity of most rats was normal and the abnormal rate was 21% in the control group. Half of the rats in the low-dose TCDD group had abnormal estrous cycle (50%). In addition, the estrous cycle of most rats was
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disturbed (75%) in the high-lose TCDD group, and it varies in length, estrus and metestrus stage being longer (Figure 2). Although the abnormal rate appeared an increasing trend with the increase of TCDD dose, the statistical results showed that the abnormal rate of estrous cycle in high-does TCDD group was significantly high 11
compared with that of the control group, while there was no difference in abnormal rate of estrous cycle between the low dose TCDD and the control rats. 3.3 Effect of TCDD on the concentration of E2 and FSH Steroid hormone test revealed difference in the concentration of E2 and FSH in
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the treatment rats compared with those from the control group (Figure 2). The E2 levels decreased in the treatment groups, while the FSH concentration increased only
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in the high-dose TCDD rats.
3.4 Effect of TCDD on the morphology of ovaries, follicle counts and follicle cells
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apoptosis
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Microscopic evaluation of ovarian sections revealed significant differences in
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the number of follicles in different stages of development in ovaries of rats exposed
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to TCDD compared with the control rats (Figure. 3. A). Especially, the
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TCDD-exposed ovaries had significantly fewer primordial follicles than ovaries from the control group, while the number of secondary, preovulation follicles and
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corpus luteum were obviously elevated in the TCDD-exposed ovaries compared with
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those from control rats. It was verified by the result of follicle count in the present study (Figure. 3. B). The TUNEL staining suggested that treatment with TCDD could result in an increase in apoptosis (Figure. 3. C and D). There were differences
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in the number of positively staining follicles between the TCDD-exposed rats and control rats. However, the positively stained follicles appeared more in the developing follicles, and the apoptosis rates were 10% greater in the TCDD-exposed groups than those in the control group. The apoptotic granulosa cells surrounding the 12
corpus luteum were small in all ovaries of rats, although the result of TUNEL staining showed a statistical difference between the TCDD-exposure groups and the control group. 3.5 Effect of TCDD on the expression of mRNA and protein of Gdf9 and Bmp15
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The GDF9/BMP15 signal pathway, plays a pivotal role in follicle development. Alk5 and Alk6 are type I receptor of GDF9 and BMP15, respectively, and BMPRII
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is the common type II receptor of those. The result of RT-PCR showed that the
mRNA expression of Gdf9, Bmp15, Alk6, Bmpr2 significantly decreased in both
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low and high dose TCDD groups compared with the control group, while mRNA
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expression of Alk5 was reduced only in low-dose TCDD rats (Figure 4). To further
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explore whether TCDD exposure in utero affects GDF9/BMP15 signal pathway in
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ovary of offspring rats, we performed Western blotting assays and found that both
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GDF9 and BMP15 expression significantly decreased in the TCDD-exposure groups compared with the control group (Figure 4).
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3.6 Effect of TCDD on the promoter region methylation of Gdf9 and Bmp15
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To investigate the relationship between Gdf9 and Bmp15 gene promoter region methylation and their gene expression in TCDD-exposure rats, the DNA methylation analysis was carried out in the present study. However, we found that there were no
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differences in methylation patterns between the treatment and control group, either in Gdf9 or in Bmp15. Furthermore, we also observed that the degree of methylation in the promoter region of Gdf9 was very low (Figure 5), whereas it was very high in Bmp15 (Figure 6). We also analyzed the methylation rate of each CpG site. There 13
were 44 CpG sites in promoter region of Gdf9 of which 9 CpG sites in control group, 8 CpG sites in 100ng/kg TCDD group, and 6 CpG sites in 500ng/kg TCDD group were methylated, but the methylation level at each CpG site was much lower than 50% (Figure 7). There were 8 CpG sites in promoter region of Bmp15 of which the
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methylation level of each CpG site was higher than 50% and even methylation rate of some CpG sites reached 100%, but the methylation rate of each CpG site did not
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differ among the three groups (Figure 7). 4. Discussion
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The results of the present study showed that exposure to TCDD in utero on
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GD8-14 might change the ovary weight, the time of vaginal opening, the
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concentration of E2 and FSH, the estrous cycles and the numbers of primordial and
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secondary follicles of the offspring rats. Especially in 500ng/kg TCDD group, the
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rats had the characteristics of lower E2 level, higher FSH concentration and reduced the number of primordial follicles which might indicate a possibility of POI/POF.
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Because the number of primordial follicles is nonrenewable, if it is produced too
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little or exhausted too quickly, POI/POF would occur. The Seveso incident in Italy also proved that the increased risk of premature ovarian failure is associated with
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TCDD exposure (Eskenazi et al. 2005). There were no differences in the visual characteristics of ovaries among the
three groups, and the ovarian weight decreased only in the high-dose group. Previous studies had shown that a single oral dose of TCDD (1.0 ug/kg) on GD 8 caused a reduction in ovary weight in 30% of offspring rats (Gray and Ostby 1995), whereas 14
the same dose to pregnant Holtzman rats on GD15, there was no significant change in ovary weight and cross-sectional size of ovary (Chaffin et al. 1996, Heimler et al. 1998). This might be due to the fact that the maximum dose of TCDD was 500ng/kg in this study that was much lower than the lowest teratogenic dose (1.25ug/kg)
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(National Toxicology Program, 1991), whereas the LD50 (lethal dose, 50%) was 20ug/kg.
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The vaginal opening time was slightly delayed in low-dose TCDD group
compared with that in the control group. Approximately half of the rats in 100ng/kg
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TCDD group had disordered estrous cyclicity, and the abnormal rate of estrous
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cyclicity was 75% in 500ng/kg TCDD group. The disorders of estrous cyclicity
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disorders were mainly as extend or shorten the period of estrous cycle, prolongation
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of estrus and metestrus stage and continuous estrus. This was consistent with some
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previous studies. Exposure of adult rats to TCDD altered estrous cyclicity (Cummings et al. 1996, Li et al. 1995). One oral dose of TCDD (1ug/kg) was given
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to dams on GD8 which resulted in 47% of one-year-old offspring rats showing
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continuous estrus (Gray and Ostby 1995), and one oral dose of TCDD (1 or 2.5 ug/kg) to dams on GD15 could make the offspring rats possess abnormal estrous cyclicity and shortened estrus stage (Salisbury and Marcinkiewicz 2002). In the
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present study, serum E2 level decreased in both TCDD-treated groups, while serum FSH concentration only increased in higher-dose TCDD group. The possible mechanism was that TCDD is a high-affinity ligand for the aryl hydrocarbon receptro (AhR) which could significantly alter steroid hormone synthesis and the 15
transcriptional pathway through ligand binding receptor in the cells of ovarian (Heimler et al. 1998), or because that TCDD exposure induced the metabolism of estrogen which resulted in a decrease in estrogen levels binding and activating estrogen receptors. In addition, TCDD exposure could directly damage the ovary
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which led to a reduction of secretion of E2 by granulosa cells. Myllymaki et al. (Myllymaki et al. 2005) found that, after administration of TCDD (single oral dose,
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0, 0.04, 0.2 or 1.0ug/kg) to pregnant rats on GD13, the E2 level decreased and the
FSH level was elevated in 14 or 16-day old offspring rats in 1.0ug/kg TCDD group
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that was consistent with the results of the 500 ng/kg TCDD group in this study.
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However, Pesonen et al. (Pesonen et al. 2006) also gave a single dose TCDD (0.04,
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0.2 or 1.0ug/kg) to pregnant dams on GD13 and found that there were no significant
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differences in E2 and FSH levels compared with control group. Additionally,
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pregnant rats drank Canal waters contaminated with TCDD and their offspring rats of the first generation possess a reduction of E2 level and normal FSH concentration
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(Huang et al. 2011). Thence, it was controversial whether intrauterine exposure of
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TCDD affected E2 and FSH levels in offspring rats, or the effect of TCDD exposure on E2 and FSH levels differed by dose, exposure periods and species. The observation of ovarian tissue sections stained H&E was consistent with the
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result of follicle counting. The data showed that the primordial follicle number decreased and the number of secondary follicle and corpus luteum increased. However, there was controversy over the number of follicles at some stages affected by TCDD intrauterine exposure. Heimler et al (Heimler et al. 1998) found that 16
exposure to TCDD in utero and via lactation hindered maturation of follicles and caused a reduction of number of preantral and antral follicles. Although this was demonstrated by other studies that one oral dose TCDD (1ug/kg) exposure to pregnant rats on GD15 might significantly reduce the number of preantral and antral
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follicles (Chaffin et al. 1996, Heimler et al. 1998), but not affect the primordial follicle number (Heimler et al. 1998), Salisbury and Marcinkiewicz’s (Salisbury and
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Marcinkiewicz 2002) report suggested that one oral dose TCDD on GD15 did not
affect the number of primordial and preantral follicles. Furthermore, there was not a
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reduction of primordial follicle number in two-day-old offspring born in dams
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exposed to a single dose of TCDD (1.0ug/kg) on GD11, 15 and 18 which perhaps
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suggested that exposure to TCDD in utero did not affect the primordial follicle pool
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of offspring. Gray et al. (Gray et al. 1997) reported that when one single dose of
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TCDD (0.2 and 0.8ug/kg) to pregnant rats on GD15, the cystic follicles and corpus luteum increased and obvious ovarian interstitial hyperplasia was reported in their
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70-day-old offspring rats that was in line with our results. The process of follicular
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development is complex and requires not only the bidirectional communication between oocytes, granulosa and follicle cells, but also the correct-levels of sex hormones in the hypothalamus-pituitary-ovarian. Therefore, this might be the reason
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for the controversy, which required further extensive research to clarify. The TUNEL result showed that the apoptosis rate of granulosa cells surrounding the growing follicles increased in TCDD-treated groups compared with that in the control group. Although there were differences in the apoptosis rate of follicular cells of corpus 17
luteum, the rate was all very low in the three groups. Granulosa cells play an important role in follicular development and can express FSH receptors (Edson et al. 2009, Eppig et al. 2002, Liu et al. 2015, Liu et al. 2006). So the increased apoptosis rate of granulosa cells would affect follicles growth. Overall, TCDD exposure in
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utero affected the follicle development and steroid hormone secretion which interfered with estrous cyclicality and follicle growth.
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The GDF9/BMP15 signaling pathway plays an important role in ovarian follicle development. It regulates primordial follicle recruitment (Martins et al. 2008, Peng
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et al. 2010), boosts proliferation of granulosa cells (Gilchrist et al. 2006), inhibits
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granulosa cell apoptosis and luteinization, promotes maturation of oocyte, ovulation
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and steroid hormone synthesis (Su et al. 2008, Orisaka et al. 2009). GDF9 and
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BMP15 can form homodimers or heterodimers (Liao et al. 2003, Juengel et al. 2004,
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McIntosh et al. 2008, Mottershead et al. 2012). ALK5 and ALK6 are the type I receptors of GDF9 and BMP15 respectively. They share BMPRII as the type II
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receptor. Our results showed that TCDD exposure in utero resulted in a
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down-regulation of mRNA expression of Gdf9, Alk5, Bmp15, Alk6 and Bmpr2. The protein expression of GDF9 and BMP15 was also down-regulated. Previous studies proved that premature ovarian failure was associated with down-regulation of
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GDF9/BMP15 or mutation of Gdf9/Bmp15 (Liu et al. 2011, Mehdizadeh et al. 2016, Wei et al. 2014, Cui et al. 2017, Ma et al. 2015, Tiotiu et al. 2010, Pu et al. 2014, Fonseca et al. 2014, Chand et al. 2006). In this study, especially in the high-dose TCDD group, decreased number of primordial follicles and E2 level and increased 18
FSH concentration were similar to the phenotype of premature ovarian failure. In addition, prenatal exposure to endocrine disruptors (EDCs), including bisphenol A, dioxins, insecticides and cigarette smoke, caused down-regulation of expression of GDF9 and BMP15 in ovaries and increased apoptosis of germ cell/oocyte, even lead
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to the occurrence of premature ovarian failure (Patel et al. 2015). Therefore, we speculated that TCDD exposure in utero interfered with ovarian follicle development,
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and even resulted in premature ovarian failure. Its mechanism was associated with the down-regulation of GDF9/BMP15 (Stanley et al. 2015). However,
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down-regulation of GDF9 and BMP15 accompanied increased number of secondary
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follicle and corpus luteum. The reason was unknown and needed to be elucidated.
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Epigenetic reprogramming in the primordial germ cell undergoes a DNA
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demethylation and remethylation during early embryo development (Skinner et al.
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2010). TCDD exposure during this period may alter epigenome and transcriptome which affect expression of critical genes related to follicular development and then
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interfere with follicle development. There were no differences in methylation rates of
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promoter regions of gene Gdf9 and Bmp15 among the three groups in the present study. Therefore, we thought that down-regulation of mRNA and protein expression of related genes in GDF9/BMP15 signaling pathway by TCDD exposure was not
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related to the methylation patterns of Gdf9 and Bmp15. At present, although there were no related reports on the relationship between TCDD exposure and the methylation patterns of Gdf9 and Bmp15, Liu et al. (Liu et al. 2018) intragastrically administrated diethylhexyl phthalate (DEHP) to 21-day-old mouse and found that 19
the reduction of mRNA level of Gdf9 was not associated with the DNA methylation level in promoter region of Gdf9. This was consistent with our result. Histone modification is also one of the epigenetic mechanisms, and histone acetylation is the most studied in histone modification. Histone acetylation mainly
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occurs on the lysine residues of histone (H) 3 and 4 and plays an important role in transcriptional regulation of genes. Under the action of histone acetyltransferases
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(HATs) and histone deacetylases (HDACs), histone acetylation and deacetylation in
the nucleus maintains histone acetylation homeostasis. Histone acetylation promotes
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the transcription of genes, whereas histone deacetylation represses transcription of
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genes (Lin and Dent 2006). Lukas et al. (Vrba et al. 2008) found that H3 and H4
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acetylation of promoter region of Gdf9 was associated with mRNA expression level
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of Gdf9 (Vrba et al. 2008). Moreover, in the study of reproductive toxicity induced
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by soluble fluoride salt in mice, it was found that decreased mRNA expression level
2015).
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of Gdf9 was related to histone acetylation, but not DNA methylation (Yin et al.
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In summary, the present results propose that maternal exposure to TCDD (especially the dose of 500ng/kg) not only significantly affected E2 and FSH levels, but also resulted in decreased primordial follicles and increased the numbers of
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secondary follicles and corpus lutea. This might be associated with down-regulation of mRNA and protein expression of GDF9/BMP15. However, the down-regulation was not related to the pattern of methylation of promoter regions in Gdf9 and Bmp15. 20
Conflicts of interest statement The authors declare that there are no conflicts of interest. Acknowledgements This research was supported by grants from the National Nature Science
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Foundation of China (21577119 and U1604183)
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exposure in rats. Biology of reproduction 88: 52.
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Figure. 1. The effects of exposure to TCDD in utero on body weight, ovary weight and ovary
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coefficient of female offspring rats. The ovary coefficient is ovary weight/body weight ratio.
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Data were analyzed using one-way ANOVA, **P <0.05, compared with the control group.
Figure. 2. The effect of exposure to TCDD in utero on the day of vaginal opening, abnormal rate of estrous cyclicity, serum E2 and FSH concentration in female offspring rats. The data of vaginal opening, E2 and FSH concentration were analyzed using one-way ANOVA, while the abnormal rate of estrous cyclicity was analyzed using Fisher exact test, **P <0.05, compared with the control group. 28
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Figure. 3. The effect of TCDD exposure in utero on the morphology of ovary, follicle counts and
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follicle cells apoptosis. (A) Morphology of ovaries by H&E staining. The number of primordial follicle was decreased, and the number of secondary follicle and corpus luteum were increased in
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TCDD-treated groups; (B) The result of follicle counting, **P <0.05, compared with the control group; (C) The apoptosis assay was detected using TUNEL and the positive cells showed green
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fluorescence; (D) The rate of follicle cells apoptosis. Data were analyzed using one-way ANOVA,
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**P <0.05, compared with the control group.
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Figure. 4. The effect of TCDD exposure in utero on the mRNA and protein expression of GDF9/BMP15. (A) The mRNA expression of GDF9/BMP15 signaling pathway related to genes in female offspring rats was analyzed using real time-PCR. **P <0.05, compared with the control group. (B and C) The protein expression of GDF9 and BMP15 was detected using western blotting. Data were analyzed using one-way ANOVA,**P <0.05, compared with the control group. 30
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Figure. 5. The effect of TCDD exposure in utero on methylation pattern of promoter region of
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Gdf9. (A and B) The two fragment PCR used to detect the methylation rate of CpG island in promoter region of Gdf9. Ten clones were selected for each PCR product. Sequencing was used
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to find the methylaiton pattern of each CpG site of each clone. The white cycles meant unmethlated CpG sites and the black cycles meant methylated CpG sites. There were no differences in methylation pattern of Gdf9 among three groups. Data were analyzed using
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nonparametric Kruskal-Wallis test.
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Figure. 6. The effect of TCDD exposure in utero on methylation pattern of promoter region of
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Bmp15. Ten clones were selected for each PCR product. Sequencing was used to find the
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methylaiton pattern of each CpG site of each clone. The white cycles meant unmethlated CpG
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sites and the black cycles meant methylated CpG sites. There were no differences in methylation pattern of Bmp15 among three groups. Data were analyzed using nonparametric Kruskal-Wallis
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test.
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Figure. 7. The methylation rate of each CpG site in promoter regions of Gdf9 and Bmp15. (A) There were 44 CpG sites in Gdf9 of which 9 CpG sites in control group, 8 CpG sites in 100ng/kg
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TCDD group and 6 CpG sites in 500ng/kg TCDD group were methylated. (B) There were 8 CpG sites in promoter region of Bmp15 of which the methylation level of each CpG site was higher
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than 50% and even methylation rate of some CpG sites reached 100%. Data were analyzed using
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nonparametric Kruskal-Wallis test.
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Table. 1.Primer sequences used for the real-time PCR of GDF9/BMP15 signaling pathway related genes. Gene
Primer
Sequence 5'→3'
Product size (bp)
Annealing/extension temperature (℃)
GAPDH
Forward
CAAGTTCAACGGCACAGTCAA
140
59.4
Reverse
CGCCAGTAGACTCCACGACA
Forward
CCAGCAACCAGATGACAGGA
Reverse
TCACAGTGGAGGAGGAAGCA
Forward
GAGAACCGCACGATTGGAG
Reverse
CACAGTGGCTCTGATTAGTTGGT
Forward
TTGACATTCCACCCAACACC
Reverse
CATCTCCTTGCAATCTCCCA
Forward
AAGCCTGGAAAGAAAATAGCCT
Reverse
ACATTGGGTTGACCGTTGG
Forward
AACCGCACTGTCATTCACCA
Reverse
TTTGCCGATGCTTTCTTGTAG
Alk6 Bmpr2
58.5 150 151
58.4 58.1
174
58.9 58.4
192
59.2 58.5
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58.5 58.7
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Alk5
58.8
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Bmp15
138
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Gdf9
59.2