Ochratoxin A exposure impairs porcine granulosa cell growth via the

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Food Safety and Toxicology

Ochratoxin A exposure impairs porcine granulosa cell growth via the PI3K-AKT signaling pathway Tian-Yu Zhang, Xiao-Feng Sun, Lan Li, Jin-Mei Ma, RuiQian Zhang, Xue-Lian Liu, Na Li, Paul W.Dyce, and Wei Shen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06361 • Publication Date (Web): 16 Jan 2019 Downloaded from http://pubs.acs.org on January 17, 2019

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Journal of Agricultural and Food Chemistry

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Ochratoxin A exposure impairs porcine granulosa cell growth

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via the PI3K-AKT signaling pathway

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Tian-Yu Zhang 1, Xiao-Feng Sun 2, Lan Li 2, Jin-Mei Ma 3, Rui-Qian Zhang 1, Na Li 1, Xue-

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Lian Liu 1, Paul W. Dyce 4, Wei Shen 2,*

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1 College of Animal Science and Technology, Qingdao Agricultural University, Qingdao

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266109, China;

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2 College of Life Sciences, Institute of Reproductive Sciences, Qingdao Agricultural

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University, Qingdao 266109, China;

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3 Animal Husbandry and Veterinary Station of Penglai City, Yantai 265600, China;

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4 Department of Animal Sciences, Auburn University, Auburn, AL 36849, USA

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* Correspondence and reprint requests to:

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Prof. Wei Shen, E-mail: [email protected]; [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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The mycotoxin ochratoxin A (OTA), a naturally occurring food contaminant, has a toxic

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effect on the growth and development of follicles in pigs. However, little is known

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regarding the specific toxic effects of OTA exposure on oocytes and granulosa cells (GCs).

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In this study, we cultured porcine ovarian GCs and exposed them to OTA in vitro in order

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to explore the mechanism causing the negative effects. Initially it was found that OTA

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exposure inhibited cell viability in a time and dose dependent manner. We also showed that

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OTA exposure increased oxidative stress, and decreased proliferation ratio and increased

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apoptosis ratio in GCs. We revealed an important role for the PI3K/AKT signal pathway in

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GC proliferation and apoptosis by RNA-seq analysis. The results not only showed that OTA

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treatment significantly affected the expression of genes within the PI3K/AKT pathway, but

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also demonstrated a concrete relationship between the PI3K/AKT pathway and GC cell

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proliferation and apoptosis. In a conclusion, the results demonstrated that OTA exposure

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impaired porcine GC growth via PI3K-AKT signaling pathway.

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Keywords: Ochratoxin A; Granulosa cells; RNA-seq; PI3K/AKT signaling pathway

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Introduction

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Follicular growth and atresia are two major events involved during mammalian

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folliculogenesis. Follicular growth is largely the results of granulosa cell (GC) proliferation

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and differentiation. Particularly, GCs have been demonstrated a critical role in determining

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the follicular development through providing signaling molecules and nutrients essential

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for follicular development and maturation 1. However, mammalian follicular atresia is a

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common physiological process during follicular development and is characterized by GC

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apoptosis 2. Apoptosis of GCs occurs through mitochondria-mediated events, and the

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expression of apoptotic factors including Bcl-2 and Caspase family members 3.

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Ochratoxin A (OTA) is produced by the fungi Aspergillus and Penicilliu. In addition,

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OTA is a very high contaminant in food and animal feed and can be detected not only in

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all types of grains and cereal products, but also frequently in coffee, cacao, spices, soy,

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nuts, beer, grapes and their products 4,5. Thus, OTA has been considered to be an important

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factor causing the pollution in agricultural products, water resources and animal diseases.

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OTA probably enters the body of human through skin exposure or airborne transmission,

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while the main way is through the food contaminated by OTA 6. OTA was first described

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in 1965 7, and now more than half a century later, many animal studies have demonstrated

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that OTA has nephrotoxicity, hepatotoxicity, neurotoxicity, teratogenicity and

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immunotoxicity. Toxicity from OTA has been shown to be caused by mechanisms related

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to oxidative stress, cell proliferation and effects on some important signaling pathways 8.

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OTA nephropathy and carcinogenicity are well known, and resulted in the Endemic

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Nephropathy (EN) crisis in Europe 9. It belongs to group of well described toxins found in



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the environment that are suspected of perturbing the reproductive systems of males and

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females. We and others have demonstrated that OTA exposure affects the animal testis

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development and sperm motility

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affect the synthesis of steroids hormones 8, but the causative mechanism has not been well

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described.

10.

Furthermore, OTA, as an endocrine disruptor, can

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Mycotoxin exposure has been shown to reduce fertility in livestock animals, notably

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in pigs 11. Recently, HT-2 toxin, fumonisin B1, deoxynivalenol and zearalenone have been

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shown to affect the oxidative stress levels, along with the proliferation, apoptosis and

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steroidogenesis production rates of GCs

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mammals, many reports have focused on the toxic mechanisms of OTA, but few reports

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have investigated

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activated by OTA exposure in porcine GCs, including the TNFα/THFR2, FasL/Fas, and

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TRAIL/TRAILRs receptors 16. In addition, focal adhesion functions including regulate the

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gene expression, cell growth and proliferation17,18, but few reports involve the relationship

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between its’ receptors with GCs by OTA exposure.

15.

12,13,14.

Because of its serious carcinogenicity in

Interestingly, some surface receptors have been suggested to be

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In previous research, we found that exposure to OTA could affect the quality of boar

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sperm via the PTEN/AKT signaling pathways in vitro 10. It is well known that the PI3K-

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AKT pathway plays a crucial role in the regulation of GC growth and apoptosis during

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follicular development 19. Oxidative stress can promote the apoptosis of GCs 20 and AKT

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can block apoptosis by phosphorylation of several downstream signaling molecules,

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including the inhibition of Caspase-9 and the inactivation of Bcl-2 family members.

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Therefore, activation of AKT not only protects GCs from apoptosis but also regulates


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cellular proliferation 21. Whether OTA acts through the AKT pathway remains to be fully

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elucidated 22.

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In order to investigate whether OTA exposure affects GC growth and apoptosis, an in

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vitro GC culture model was used to study the effects of OTA exposure on GCs. RNA-seq

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was utilized to identify genes whose expression was altered by OTA exposure.

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Bioinformatics was used to further characterize genetic pathways in the GCs that were

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altered following OTA exposure.

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Materials and Methods

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Reagents and antibodies

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OTA was purchased from Pribolab Pte. Ltd (IAC-040-3, Singapore). Cell culture media

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was supplemented with OTA (dissolved in DMSO) at a concentration ranging from 10 μM

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to 160 μM and maintained for 24 h. The granulosa cells cultured with 0.1 % DMSO were

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used as OTA-0 μM as a control group.

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Primary antibodies used in this study appear in Table S1. Secondary antibodies were

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CY3/FITC-labeled goat anti-rabbit (A0516/A0562), FITC-labeled goat anti-mouse

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(A0568), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (A0216), and

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HRP-conjugated goat anti-rabbit IgG (A0208). All secondary antibodies were purchased

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from Beyotime Biotechnology Co., Ltd. (Nantong, China).

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High glucose DMEM (SH30022.01), purchased from HyClone Co., Ltd. (Beijing,

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China), epidermal growth factor (EGF, SRP3196), follicle stimulating hormone (FSH,

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2293), luteinizing hormone (LH, 5269) and L-cysteine (L-Cys, C5360) were purchased



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from Sigma Co., Ltd. (USA). Penicillin-streptomycin (PS, 15140122), fetal bovine serum

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(FBS, 10099-141) and M199 (11150-059) were purchased from Gibco Co., Ltd. (Australia).

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Furthermore, bovine serum albumin (BSA, A8020), Tween-20 (T8220) and Trition X 100

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(T8200) were purchased from Solarbio Co., Ltd. (Beijing, China).

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Porcine GCs and cumulus-oocyte complexes (COCs) cultured in vitro

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The porcine ovaries were collected from the slaughterhouse of Qingdao Wanfu Group Co.,

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Ltd. and preserved in physiological saline solution at 37 °C until arrival at the laboratory

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within 2 h from collection. GCs from follicles on the ovaries with diameters of 3-5 mm

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were collected using a 10 ml syringe connected to a 12 gauge needle. The collected GCs

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were pelleted by centrifuging at 1,500 rpm for 3 min and washed with phosphate-buffered

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saline (PBS) 3 times. Then, these cells were cultured in High glucose DMEM medium

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supplemented with 10 % FBS, 1 % penicillin/streptomycin, 0.5 % gentamycin sulfate at 37

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°C in an atmosphere of 5 % CO2 in air 14. Primary GCs were passaged into a 6 cm petri

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dish at a density of 1×10 6 cells/well after 36 h of culture.

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Ovarian follicles with diameters greater than 5 mm were aspirated using a 20 ml

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syringe with a 16 gauge needle connected in order to obtain COCs. Oocytes with 2-4 layers

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of cumulus cells (CCs) and a uniform cytoplasm were selected for culture. After washing

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three times, the collected COCs were then cultured in maturation media containing 500 μl

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M199 medium supplemented with 0.5 μg/ml LH, 0.5 μg/ml FSH, 10 ng/ml EGF, 0.57 mΜ

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L-Cys, 1 % PS and 10 % porcine follicular fluid in a 24-well dish with at a density of 30-

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40 COCs/well at 38.5 °C in an atmosphere of 5 % CO2 in air. Following 12 h and 42 h of


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culture, the CCs were removed from the COCs using hyaluronidase, and then the denuded

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oocytes were picked up. The rate of porcine oocytes at the germinal vesicle (GV) stage to

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the germinal vesicle breaking down (GVBD) and polar body extrusion (PBE) stage were

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analyzed according to our previous research 23.

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Granulosa cell viability assays

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A Cell Counting Kit-8 (CCK-8, Sangon, E606335, Shanghai, China) was utilized to

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determine the GC viability and proliferation rates. Briefly, following exposure to

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concentrations of OTA for various treatment times, 100 μl of cell suspension (about 5,000

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cells / well) was removed and 10 μl of CCK-8 Solution was added in one well of a 96-well

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plate. The plate was then incubated 4 h in a cell culture incubator. Finally, the optical

144

density (OD value) was calculated with a Microplate Reader (Bio-Rad, iMarkTM, USA),

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and the absorbances of each experimental group were measured at 450 nm.

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RNA extraction from GCs and quantitative real-time PCR (qRT-PCR)

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According to the manufacturer's instructions, porcine GCs’s total RNA was extracted by

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using an RNAprep pure MicroKit (Aidlab, RN28, Beijing, China) and using reverse

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transcription using a cDNA Synthesis Kit (TransGen, AT311-03, Beijing, China) into

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cDNA. The resulting cDNA was then subjected to qRT-PCR by using a Light-Cycler 480

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Real-Time PCR System (Roche, Germany) with the Light-Cycler

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Master Mix Kit (Roche) following the manufacturers’ protocol. Relative mRNA expression

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levels were analyzed using the 2(−ΔΔCt) method

1

®

480 SYBR Green I

and normalized against the mRNA



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expression of the housekeeping gene Gapdh. The information of the primer sequences are

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summarized in Table S2.

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RNA-seq and bioinformatics analysis

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The RNA-seq data was obtained by using the Illumina Hiseq 2000 platform from Novogene

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Co., Ltd. (Beijing, China). A total of three independent biological samples were analyzed

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in each group. Resulting RNA-seq data were uploaded to the SRA repository under

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accession number: PRJNA490225. Differentially expressed genes (DEGs) were identified

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using the R Bioconductor/DESeq2 package. Data of differential expression analysis was

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normalized previously to avoid possible bias

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considered statistically significant.

24,25,

and adjusted P-value < 0.01 was

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We used the R Bioconductor/clusterProfiler package for analysis of functional profiles,

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Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) of DEGs 26.

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The software used was edgeR (3.12.1) following a previous publication

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enrichment analysis (GSEA) shows concordant differences between two groups’ statistical

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significance. GSEAs with normalized enrichment score (NES) > 1 and false discovery rate

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(FDR) adjusted P-value < 0.05, were considered statistically significant 28. Search Tool for

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the Retrieval of Interacting Genes/Proteins (STRING) contains the related interactions

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between known and predicted proteins and genes, and Cytoscape software was used to

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visualize the protein–protein interaction (PPI) results 29.

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Apoptosis analysis by flow cytometry


27.

Gene set

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Porcine GCs were exposed to OTA for 24 h, and were washed and collected three times

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with phosphate-buffered saline (PBS). Cells were then analyzed following the protocol of

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an Annexin V-FITC/PI Cell Apoptosis kit (TransGen, FA101) with a FACSCaliburTM Flow

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Cytometer (BD Bioscience, Mississauga, USA). Early apoptotic cells would only be stained

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by Annexin V, necrotic cells and late apoptotic cells would be stained by Annexin V and

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PI, respectively. At least 10,000 cells were collected from all groups for each time. Data

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were analyzed using Flowjo 10.2 software.

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GSH Assay

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The levels of glutathione (GSH) in the GCs was determined using a GSH Assay Kit

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(Beyotime, S0053). After OTA exposure for 24 h, the GCs were collected and washed in

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PBS three times. A total of 1 x 10 6 cells from each group were collected for testing. Briefly,

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samples were pelleted at 10,000 g by centrifuging at 4 °C for 10 min, and then DNTB was

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added to each sample. Finally, a microplate reader was used to measure the absorbance at

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412 nm. The measured results were expressed as pmol /106 cells.

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Immunocytochemistry (ICC)

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The GCs were collected and fixed with 4 % paraformaldehyde (PFA) for 4 h at 4 °C, and

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then incubated with 1 % PBST (1% Trition X 100 dissolved in PBS) for 0.5 h at room

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temperature. . The samples were then blocked with sealing fluid, and then incubated with

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the primary antibodies (Table S1) in blocking solution overnight at 4 °C. The next day,

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after washing with PBS containing 1 % BSA (Solarbio, A8020), the samples were



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incubated with secondary antibodies at 37 °C for 2 h. The negative controls were incubated

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with secondary antibodies only (primary omitted). Finally, the nucleus of GCs were stained

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with Hoechst33342 (Beyotime, C1022). After staining, fluorescent images were collected

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using a Fluorescent Microscope (Olympus, BX51, Japan). The relative fluorescence

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intensity per unit area was determined using ImageJ software 30.

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TdT-mediated dUTP nick end labeling (TUNEL) and 5-Ethynyl-2'-deoxyuridine

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(EdU) staining

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A TUNEL BrightRed Apoptosis Detection Kit (Vazyme, A11302, Nanjing, China) was

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utilized to determine the apoptotic status of GCs. Briefly, GCs were washed with PBS and

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collected three times, followed by fixation with 4 % PFA for 2 h. After nuclei visualization

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by Hoechst33342, images were taken under fluorescence microscopy, the TUNEL positive

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cells were counted and analyzed 31. Greater than 2,000 positive cells were used each time

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and at least three biological replicates were performed.

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The percentage of proliferative GCs were evaluated using the Cell-Light EdU DNA

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cell proliferation kit (RiboBio, C10371-1, Guangzhou, China). Briefly, EdU was added to

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the culture medium 2 h before fixed with 4 % PFA, then GCs were incubated in the staining

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reaction mix for 30 min. Finally, nucleus of GCs were stained with Hoechst33342. The

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images were taken under fluorescence microscopy. EdU positive cells were counted and

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analyzed 32. At least 2,000 positive GCs were counted each time and at least three biological

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replicates were analyzed.

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Western blotting

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Western blotting (WB) was performed using standard protocols. Firstly, total protein was

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isolated from GC samples using RIPA lysis buffer (Beyotime, P0013C) according to the

224

manufacturer’s instructions. Then the protein samples were analyzed by SDS-PAGE on

225

different concentrations of stacking gel and separating gel depending on the molecular

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weight of the target protein. Separated proteins were then transferred onto polyvinylidene

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fluoride (PVDF) membranes by electrophoresis. After blocked in TBST (Tris-buffered

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saline with Tween-20) containing 10 % BSA at 4 °C for at least 2 h, the membranes were

229

then incubated with primary antibodies (Table S1) at different dilutions in TBST buffer

230

containing 10 % BSA at 4 °C overnight. The next day following washing, the membranes

231

were incubated with secondary antibodies for 2 h at room temperature. The BeyoECL plus

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kit (Beyotime, P0018) was used for signal detection, and the AlphaView SA software was

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used to analyze the relative expression levels of the detected proteins.

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Statistical analysis

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All statistical analysis were performed with at least three independent replicates for each

237

experiment, and data were statistically analyzed by one-way analysis of variance (ANOVA)

238

followed by the Tukey’s test to analysis the mean. Data were represented as mean ± SEM

239

(Standard Error of Mean) and differences were considered significant at P < 0.05 (*) and

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extremely significant at P < 0.01 (**).

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Results



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OTA exposure inhibited the growth of porcine GCs in vitro

244

To investigate whether OTA exposure affects GC growth and apoptosis, we utilized an in

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vitro GC culture model combined with RNA-seq. The technical schematic of our

246

experimental design is presented in Fig. 1A.

247

In order to investigate the effects of OTA exposure on porcine GCs in vitro, primary

248

GCs were exposed for various time at various concentrations. The effects of OTA exposure

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on cell viability and growth were determined using a CCK-8 assay at 4 exposure time points

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(12, 24, 36, 48 h) with concentration gradients ranging from 10 µM to 160 µM. The results

251

suggested that the cell viability was significantly reduced in the OTA treated groups when

252

compared with that of control groups after culturing for 24 h and 48 h. We found 20 µM or

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greater OTA exposure for 24 h significantly decreased cell viability (Fig. 1B; P < 0.05 or

254

P < 0.01). Furthermore, we found that treatment of GCs for 48 h with 10 µM or greater of

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OTA was significantly reduced compared to the control group (Figs. 1B and 1C). Then we

256

using WB to analysis the expression of proliferating cell nuclear antigen (PCNA) at the

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protein level 33. We found that the expression of PCNA was downregulated in OTA treated

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groups (Fig. 1D). The data suggested that 20 µM or greater OTA exposure could inhibit the

259

growth of porcine GCs in vitro.

260 261

RNA-seq analysis of the effect of OTA exposure on the gene expression of porcine GCs

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To study the mechanism of OTA exposure, RNA-seq was performed after GC OTA

263

exposure. Total three RNA-seq samples of each groups (0 µM (Control), 20 µM, and 40

264

µM OTA treated GCs) were been collected. Firstly, the variations of the samples from the


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control and OTA treatment groups were analyzed using PCA (Fig. S1A). The venn diagram

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demonstrated a total of 2,323 genes significantly different in terms of expression levels

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between the control and OTA treated GCs (Fig. 2A). Then the volcano map demonstrated

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the changes in down-regulated and up-regulated DEGs in the OTA treatments compared to

269

the control group with fold-changes greater than or equal to 2 (Fig. S1B). A heatmap

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determined that most DEGs were consistently affected by OTA exposure (Fig. S1C).

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In order to know the function and relationship in these DEGs, GO term and KEGG

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pathway analyses were performed. The DEGs between control and OTA treated groups

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were shown in GO enrichment results (Fig. 2B, Fig. S1D and Table S3). The enriched GO

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terms of biological process included cellular response to stress (P adjust = 2.21E-07; Counts

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= 269), cell cycle (P

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1.63E-03; Counts = 155) and apoptotic processes (P adjust = 7.75E-03; Counts = 227); the

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enriched GO terms of cellular components included the catalytic complex (P adjust = 1.07E-

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06; Counts = 264), focal adhesion (P adjust = 8.66E-27; Counts = 171), adherens junctions

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(P adjust = 1.15E-02; Counts = 138); the enriched terms of molecular function included RNA

280

binding (P adjust = 4.21E-12; Counts = 244) and kinase activity (P adjust = 4.91E-03; Counts

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= 166). Collectively, the GO enrichment results indicated that many DEGs had been

282

affected by OTA, especially the analysis of biological process remind us that OTA exposure

283

could affect GC apoptotic processes generally and significantly.

adjust

= 3.98E-03; Counts = 207), regulation of cell cycle (P

adjust

=

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The DEGs in OTA treatment groups compared to control group were shown in KEGG

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enrichment results (Fig. S2A and Tables S4-5). KEGG enrichment results indicated that

286

PI3K-Akt signaling pathway (P adjust = 0.012804549; Counts = 162) were activated, other



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major pathways included the cell cycle (P

288

(P adjust = 1.16E-03; Counts = 76), progesterone-mediated oocyte maturation (P adjust = 1.1E-

289

02; Counts = 51) and apoptosis (P adjust = 2.56E-02; Counts = 71) (Fig. 2C and Table S6).

290

Then we chose three important signaling pathways including focal adhesion, cell cycle and

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apoptosis of OTA treated on porcine GCs. Venn diagram demonstrated that the intersection

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of DEGs in three groups were 31 (24.03 %), 23 (22.54 %) and 27 (27.27 %), respectively

293

(Fig. S2B).

adjust

= 1.16E-03; Counts = 76), focal adhesion

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It was worth noting that most DEGs (162) were in the PI3K-AKT signaling pathway,

295

and Venn diagram demonstrated that there was a relationship between that and other major

296

pathways including focal adhesion, cell cycle, apoptosis and progesterone-mediated oocyte

297

maturation with 61, 13, 15 and 10 DEGs, respectively (Fig. 2D). Thus, we inferred that the

298

PI3K-AKT signaling pathway may play an important role in the effects of OTA exposure.

299

The RNA-seq data were also analyzed by GSEA. Coincidently, the GSEA analysis

300

showed that the PI3K_AKT_MTOR_SIGNATURE gene set was enriched in the 40 μM

301

OTA treated group compared to the control group. The results showed |NES| >1, FDR P -

302

value < 0.1 (Fig. S2C), which was the further evidence that the PI3K/AKT signaling

303

pathway was significant influenced by OTA exposure. So a series of analysis of

304

bioinformatics suggested that OTA played an important role in the regulation of the

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PI3K/AKT signaling pathway and had key connections with cellular apoptosis and

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proliferation.

307 308

OTA exposure activated the surface receptors and induced the oxidative stress of GCs


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In order to verify the RNA-seq results and further elucidate the potential regulatory

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mechanism, we investigated if OTA could activate GC surface receptors. The RNA-seq

311

data suggested that focal adhesion was activated by OTA. Integrin beta subunits (ITGB) 1,

312

a heterodimeric cell-surface receptor, an Integrin family member involved in so many

313

important cell functions 34, and coagulation factor II (F2R) receptor, a G-protein coupled

314

receptor (GPCR) family member

315

Fluorescence intensity and WB results showed that the expression of these two major

316

proteins, ITGB1 and F2R, was significantly increased in the OTA exposed groups

317

compared to that of the control group, respectively (Figs. 3A and 3B; P < 0.05 or P < 0.01).

318

In addition, GSH is the main source of sulfhydryl in most living cells which serves as

319

a key antioxidant. It was shown in Fig. 3C that the intracellular GSH content in GCs

320

exposed to OTA was significantly reduced compared to control group (P < 0.01).

321

Furthermore, WB analysis indicated that the protein levels of the oxidant enzymes

322

Superoxide dismutase 1 (SOD1), Glutathione peroxidase 1 (GPX-1) and Catalase (CAT)

323

In the OTA treated groups, they were significantly increased compared to the control group

324

(Fig. 3D; P < 0.05 or P < 0.01).

35,

were involved in the regulation of GC response.

325 326

OTA exposure affected the proliferation and apoptosis of porcine GCs

327

GC proliferation is complex and multifaceted during follicular development and is

328

important for growth of the oocyte and maintaining ovarian functions. EdU analysis

329

showed that GC proliferation significantly decreased following OTA exposure compared

330

to that of the untreated control group (Fig. 4A; P < 0.01). Progression of cell proliferation



331

is intricately regulated by the cyclin dependent kinase (CDK) complexes, and the two most

332

important CDK inhibitors CDKN1A and CDKN1B. WB results demonstrated that the

333

protein expressions of both CDKN1A and CDKN1B were significantly induced, and CDK2,

334

CDK4, CCND1 and c-MYC were significantly decreased in OTA treated groups compared

335

to that of the control group (Figs. 4B and 4C; P < 0.05 or P < 0.01). Furthermore, qRT-

336

PCR results were consistent with the WB results showing increased Cdkn1a and Cdkn1b

337

transcripts and decreased Cdk2, Cdk4, Ccnd1, and c-Myc transcripts following OTA

338

exposure (Fig. 4D; P < 0.05 or P < 0.01).

339

Previous analysis of GO term and KEGG pathways indicated that OTA not only

340

regulated the process of proliferation but also affected apoptosis of porcine GCs. In order

341

to investigate the mechanism by which OTA affected apoptosis of porcine GCs, the mRNA

342

and protein expression of apoptosis related genes such as Bax, Bcl2l1 and Casp9 were

343

analyzed using qRT-PCR and WB. The results showed that the mRNA and protein

344

expressions of these genes were significantly increased in the OTA treated groups

345

compared with control group (Figs. 5A-5C; P < 0.05 or P < 0.01). Furthermore, TUNEL

346

analysis revealed that OTA treatment significantly induced an increase in the percentage of

347

TUNEL positive porcine GCs in (Fig. 5D; P < 0.01). GCs were stained simultaneously with

348

Annexin V/ PI in order to categorize them as early apoptotic, late apoptotic, or necrotic

349

using flow cytometry. Percentages of necrosis/late apoptotic GCs were increased by OTA

350

exposure, and live cells significantly decreased when compared to the control group (Fig.

351

5E; P < 0.05 or P < 0.01).

352


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OTA exposure affected the cell fate of porcine GCs via the PI3K-AKT pathway

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KEGG pathway analysis indicated that OTA exposure affected the cell fate decisions

355

between growth and apoptosis of porcine GCs via the PI3K-AKT signaling pathway. To

356

investigate the regulation mechanism of the PI3K-AKT signaling pathway in porcine GCs

357

exposed to OTA, these related genes were detected using qRT-PCR and WB. The qRT-

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PCR results for Pik3r1, Pik3r5, Pten, Akt1, Akt2 and Akt3 all showed consistent expression

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levels with the RNA-seq data (Fig. 6A; P < 0.05 or P < 0.01). Analysis using WB

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determined that PIK3R1, PIK3R5 and PTEN were activated and that the protein expression

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was significantly increased, and the p-AKT (Ser473)/AKT ratio was significantly decreased

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in the OTA treated groups compare with that of the control group (Figs. 6B and 6C; P

Ochratoxin A exposure impairs porcine granulosa cell growth via the

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