| Zearalenone (ZEA), a nonsteroidal estrogenic mycotoxin found in various crops, is generated mainly by Fusarium genus. Recently, the crops of most countries are contaminated by ZEA. In most part of China, the temperature and humidity is suitable for the growth of molds. Therefore, the contamination of ZEA in crops and feed is seriously in our country. Many researches suggested that ZEA can bring about morphology changes,dysfunction and degradation of the reproductive organ. Pregnant animals with ZEA-contaminated feed intake cause miscarriage, stillbirth and monster. Recent investigations on reproductive toxicity of ZEA have been focused on the effects of ZEA on ovarian granulosa cells and oocytes. Few studies on the fuction of uterine, especially the effects of ZEA on endometrial stromal cells in vitro. In this paper, we firstly investigated the toxicity of ZEA on puberty female mice, and preliminary explore the toxicity and molecular mechanism of ZEA on mouse endometrial stromal cells. On these basis, we used the RNA-seq technology to analysis the transcriptome level of mouse endometrial stromal cells treated with ZEA to screen the key functional decline genes, and to further provide theory support for revealing the toxicity mechanism of ZEA on the mouse endometrial stromal cells. The main contents and results of this study are as follows:1. The toxicity of ZEA on the puberty female miceThe experiment was to explore the toxicity of different dose ZEA (Omg/kg (control),0.1mg/kg ZEA, 0.5mg/kg and 1mg/kg ZEA BW) on the prepubertal female mice with intraperitoneal injection. The results showed that the 0.5mg/kg ZEA BW and lmg/kg ZEA BW treatment could significantly reduce the body weight as compared with the control group continuous after intraperitoneal injection of ZEA for 3 weeks, (P<0.05). After 4 weeks intraperitoneal injection, the indice of spleen and uterus of lmg/kg ZEA BW treatment were significantly increased as compared with the control group (P<0.05). The mRNA expression of SOD and CAT gene of lmg/kg ZEA BW treatment were significantly reduced as compared with the control group (P<0.05) in liver. In kidney, the 0.5mg/kg ZEA BW and 1mg/kg ZEA BW treatment could significantly reduce the mRNA expression of SOD gene as compared with the control group (P<0.05). In uterus, the the ratio of Bax/Bcl-2 was significantly increased and the mRNA expression of ER? was significantly reduced of lmg/kg ZEA BW treatment as compared with the control group (P<0.05). The mRNA expression of Bax and Caspase-3 and the the ratio of Bax/Bcl-2 were significantly increased of lmg/kg ZEA BW treatment as compared with the control group (P<0.05). The estrous cycle of mice was normal in the control and 0.1mg/kg ZEA BW treatment, however,with increasing the dose of ZEA, the estrous cycle appeared disorder, the oestrus in 0.5mg/kg ZEA BW treatment was prolongation, while in 1mg/kg ZEA BW treatment, the estrus was longer, and most of the mice skipped the proestrus into estrus. There was no difference of the rate of seeing embolus in each group, the average was about 68.75 percent.There was relatively large difference of the pregnancy rate in each group, the 1mg/kg ZEA BW treatment had the lowest pregnancy rate (45.45 percent), which was reduced about 36.37 percent compared to the control (81.82 percent). The litter size of mice in 0.5mg/kg ZEA BW and lmg/kg ZEA BW was significantly reduced compared with the control group(P<0.01). The results suggested that ZEA treatment can markly affect the growth of mice and damage the main viscera and reproductive organ. It also can affect the expression level of key antioxidant enzyme genes, steroid hormone receptor genes and proapoptotic genes,resulting in disorder of estrous cycle appear and reduction of fertility.2. The toxicity of ZEA on mouse endometrial stromal cellsThe experiment was to study the toxicity of ZEA on mouse endometrial stromal cells by cell counting kit-8 (CCK-8), flow cytometry and TdT mediated dUTP Nick End Labeling (TUNEL) assay. The results showed that the total number and viability of mouse endometrial stromal cells reached a peak after 4-5d culture, followed by a decreasing. ZEA treatment could significantly reduce the cell viability of endometrial stromal cells in a dose-and time-dependent manner (P<0.05). ZEA treatment could significantly reduce the proportion of S phase and significantly increase the proportion of G2/M phase (P<0.05).Meanwhile, ZEA treatment copuld significantly increase the apoptosis rate of endometrial stromal cells (P<0.05). The results suggested that ZEA treatment could cause cell shrinkage and floating, reduce the cell viability, cause G2/M phase arrest and DNA replication block,as well as casuing apoptosis of mouse endometrial stromal cells.3. The toxicity mechanisms of ZEA on mouse endometrial stromal cellsThe experiment was to anslysis the toxicity mechanisms of ZEA on mouse endometrial stromal cells by qRT-PCR, Immunoblotting, caspase activity assay, CCK-8 assay and flow cytometry assay. The results showed that the mRNA expression of p53, Gadd45a, Bax,Caspase-3 and Caspase-9 were significantly increased as compared with the control group with increasing the dose of ZEA (P<0.05). The mRNA expression of CCNB1, cdc2 and Bcl-2 were significantly reduced as compared with the control group (P<0.05). The ratio of Bax/Bcl-2 was significantly increased as compared with the control group (P<0.01).Compared to the control group, the protein ratio of Bax/Bcl-2 and the activity of Caspase-3 and Caspase-9 were significantly increased (P<0.05). ZEA treatment led to significant loss of mitochondrial transmembrane potential and enhance reactive oxygen species (ROS)levels (P<0.01). Pretreated with caspase inhibitors (Z-VAD-FMK and Z-LEHD-FMK), theviability mouse endometrial stromal cells was significantly increased and the apoptosis rate was significantly reduced as compared with the 100?M ZEA treatment alone. Overall, the results suggested that ZEA treatment caused the G2/M arrest through changing the expression level of p53, Gadd45a, CCNB1 and cdc2 gene, and caused apoptosis throughchanging the expression level of the mitochondrial apoptosis pathway related genes Bax,Bcl-2, Caspase-3 and Caspase-9.4. The transcriptional characteristics of mouse endometrial stromal cells treated with ZEAIn this experiment, we used the RNA-seq technology to comprehensively analysis the transcriptome level of mouse endometrial stromal cells treated with ZEA. The sequencing results showed that there were 3846 differentially expressed unigenes (DEGs) in ZEA treatment as compared with the control, among those, 1810 genes were up-regulated and 2036 genes wrer down-regulated. GO functional analysis and KEGG Pathway functional analysis revealed that most of the GO terms and KEGG pathways were involved in the embryo implantation and pregnancy establishment. Meanwhile, by de novo assembly and mapping, there were 39527611 (81.04%) sequences can be matched with the mouse reference genes, 34264 alternative splicing events, 1591 genes were optimized and 275 novel transcripts were discovered in control group. In ZEA-treated group, there were 40768991 (83.27%) sequences can be matched with the mouse reference genes, 41584.alternative splicing events, 1646 genes were optimized and 319 novel transcripts were discovered. Further researches are needed to verify whether these differences are involved in the functional change of mouse endometrial stromal cells treated with ZEA. |
PDF Full Text Request