Mechanisms Underlying The Abnormality Of Sexual Development And Stability In Mouse Induced By Representative Endocrine Disrupting Chemicals

Endocrine disrupting chemicals (EDCs) are widely distributed in air, soil and water, affecting the sexual development and sex stability maintenance, leading to developmental deficiencies including imbalance in sex ratio, sexual precocity and sex reversal. Recent studies have found that besides the key genes on sex chromosome, a series of autosomal transcriptional factors and signaling molecules play an important role in the regulation of biopotential gonad differentiation during the sexual development of mammals. Sex determination genes also regulate sex stability after sexual maturity. Thus, it is of significance to investigate the effects of EDCs on gene regulation pathway in sexual development and stability in mammals, which contributes to illustrate the mechanism of their reproductive and developmental toxicity. The present study selected the representive anti-estrogen EDC, tamoxifen (TAM), which is as the model chemical, evaluating its effects on some key autosomal genes expression regulating sexual development and stability. Furthermore, molecular mechanisms of two typical environmental EDCs, bisphenol A (BPA) and tributyltin (TBT), were studied underlying their interference with sexual development in mice. The main contents and results of the present study are as follows,1. Effects of TAM exposure during embryonic period on sex determination genes in mice. Pregnant Institute of Cancer Research (ICR) mice were intraperitoneal injected with 1 mg/kg bw-TAM at 10.5 days post coitem (dpc). The mice were sacrificed and the gonads were collected at 13.5 dpc. Realtime reverse transcription polymerase chain reaction (Realtime RT-PCR) and whole-mount in situ hybridization (WISH) were used to detect the sex determination genes expression. The results showed that TAM induced abnormal up-regulation of platelet-derived growth factor alpha (Pdgfra) in XX gonad and the expression pattern is similar to that observed in control XY gonad, implying that TAM induceed coelomic blood vessel in the XX gonad. Moreover, TAM up-regulated Sty-related high-mobility group box 9 (Sox9). Accordingly, the ovary-specific marker, forkhead transcription factor gene (Foxl2) was depressed. The changes in the SOX9 and FOXL2 protein were consiternt with those of the gene expression. The in vitro assay with organotypic cultures further confirmed that TAM induced the over-expression of Pdgfra and fibroblast growth factor 9 (Fg/9) in XX gonad and depressed the Fgf9 expression in the XY gonad. In addition, the ratio of 17?-estradiol and testosterone (E2/T) was significantly decreased. The results implicated that TAM could up-regulate testis determination genes and inhibite the ovary-specific genes expression in XX gonad, which initiate the testis differentiation and finally leads to sexual development abnormalities.2. Effects of TAM exposure during sex maturity period on key genes involved in signaling pathway regulating the sex stability in female adult mice. ICR mice were intraperitoneal injected with 75 or 225 mg/kg-bw TAM for 7 d beginning on postnatal day (PND) 49. Five days after exposure, the serum and ovaries were collected. Realtime RT-PCR, immunohistochemistry and Western blot were performed to detect the expression levels and pattern of genes and proteins regulating sex determination. The results showed that TAM induced the up-regulation of platelet/endothelial cell adhesion molecule 1 (PECAM-1), implying that vascular endothelial cells were induced in TAM-treated ovaries. Moreover, TAM induced the over-expression of Sox9 and Fgf9. Accordingly, expressions of Foxl2, wingless-related MMTV integration site 4(Wnt4) and its downstream gene, follistatin (Fst), were depressed. The levels of 17?-estradiol, progesterone and luteinizing hormone decreased. Meanwhile, the levels of testosterone increased. The results indicated that the ovary-specific factors, Foxl2, Wnt4 and its downstream genes were downregulated, while testis-specific factors, Sox9 and Fgf9, were induced by TAM, which leads to the interference of the maintenance of ovary and sex stability.3. Effects of BPA exposure during embryonic and lactational period onkey genes involved in sexual development in male mice. Pregnant ICR mice were gavaged from 0.5 dpc to PND21 with 0.1 mg/kg-bw BPA. The testis and serum were collected from the male pups were on PNDO,7,21, and 49. Realtime RT-PCR, immunohistochemistry and Western blot were performed to detect the expression of key genes and proteins involved in sexual development. Results showed that the expression of SOX9 and FGF9 protein were not changed after sexual maturation by BPA exposure. However, BPA caused inhibition in the expressions of anti-Mullerian hormone (Amh), Sox9 and Fgf9 during neonatal and sexual maturation periods. In addition, BPA down-regulated the expression of zinc finger transcription factors termed GATA-binding protein 4 (Gata4) in male pups, and especially inhibited its normal up-regulation at puberty. Besides, BPA activated Foxl2, DSS-AHC critical region on the X chromosome gene 1(Daxl), Wnt4 and Fst. Results indicated that developmental exposure to BPA in male pups initiated the ovary regulation and depressed the testis regulation in mice, which may account for the feminization caused by BPA.4. Effects of TBT exposure during embryonic and lactational period on key genes involved in sexual development in male mice. Pregnant ICR mice were gavaged from 0.5 dpc to PND21 with 20?g/kg-bwTBT. The testis and serum were collected from the male pupswere on PNDO,7,21 and 49. Realtime RT-PCR, immunohistochemistry and Western blot were performed to detect the expression of key genes and proteins in sexual development. Results showed that E2/T was increased in adult male pups, with the absence of change in adult female pups. On PNDO, Sox9?Fgf9 and Amh were significantly depressed. Gata4 expressed earlier and the expression temporal profiles were interrupted after treatment with TBT. Besides, the levels of Gata4 expression were significantly increased. The expression of Foxl2 and other ovary regulation genes were not significantly changed after treatment with TBT. Results indicated that key sex determination genes detected in the present study were not significantly affected in male mice after TBT exposure during embryonic and lactational period. However, the sexual development and the balance of hormone levels were interrupted until sexual maturity in male pups.In summary, the present study illustrated that effects of three representive EDCs on autosomal sex determination pathway caused by these chemicals. A new potential mechanism was identified on the reproductive and developmental toxicity of EDCs. The present findings contribute to a better understanding of the imbalance in sex ratio in human and animals, as well as the reproduction dysgenesis after exposure to environmental EDCs, and effects of early-life exposure to EDCs on adult health and disease, which provids a scientific basis for evaluating the health risk of EDCs.