Bowman Christopher J, Turner Katie J, Sar Madhabananda, Barlow Norman J, Gaido Kevin W, Foster Paul M D
CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709, USA.
Toxicol Sci. 2005 Jul;86(1):161-74. doi: 10.1093/toxsci/kfi172. Epub 2005 Apr 13.
Di(n-butyl) phthalate (DBP) is a common plasticizer and solvent that disrupts androgen-dependent male reproductive development in rats. In utero exposure to 500 mg/kg/day DBP on gestation days (GD) 12 to 21 decreases androgen biosynthetic enzymes, resulting in decreased fetal testicular testosterone levels. One consequence of prenatal DBP exposure is malformed epididymides in adult rats. Reduced fetal testosterone levels may be responsible for the malformation, since testosterone is required for Wolffian duct stabilization and their development into epididymides. Currently, little is understood about the molecular mechanisms of Wolffian duct differentiation. The objective of this study was to identify changes in gene expression associated with altered morphology of the proximal Wolffian duct following in utero exposure to DBP. Pregnant Crl:CD(R) (SD) rats were gavaged with corn oil vehicle or 500 mg/kg/day DBP from GD 12 to GD 19 or 21. There were only small morphological differences between control and DBP-exposed Wolffian ducts on GD 19. On GD 21, 89% of male fetuses in the DBP dose group showed marked underdevelopment of Wolffian ducts, characterized by decreased coiling. RNA was isolated from Wolffian ducts on GD 19 and 21. Together with empirical information, cDNA microarrays were used to help identify candidate genes that could be associated with the morphological changes observed on GD 21. These candidate genes were analyzed by real-time RT-PCR. Changes in mRNA expression were observed in genes within the insulin-like growth factor (IGF) pathway, the matrix metalloproteinase (MMP) family, the extracellular matrix, and in other developmentally conserved signaling pathways. On GD 19, immunolocalization of IGF-1 receptor protein demonstrated an increase in cytoplasmic expression in the mesenchymal and epithelial cells. There was also a variable decrease in androgen receptor protein in ductal epithelial cells on GD 19. This study provides insight into the effects of antiandrogens on the molecular mechanisms involved in Wolffian duct development. The altered morphology and changes in gene expression following DBP exposure are suggestive of altered paracrine interactions between ductal epithelial cells and the surrounding mesenchyme during Wolffian duct differentiation due to lowered testosterone production.
邻苯二甲酸二丁酯（DBP）是一种常见的增塑剂和溶剂，会干扰大鼠雄激素依赖的雄性生殖发育。在妊娠第12至21天子宫内暴露于500毫克/千克/天的DBP会降低雄激素生物合成酶，导致胎儿睾丸睾酮水平下降。产前暴露于DBP的一个后果是成年大鼠附睾畸形。胎儿睾酮水平降低可能是畸形的原因，因为睾酮是中肾管稳定及其发育成附睾所必需的。目前，对于中肾管分化的分子机制了解甚少。本研究的目的是确定子宫内暴露于DBP后与近端中肾管形态改变相关的基因表达变化。将怀孕的Crl:CD(R)（SD）大鼠从妊娠第12天至第19天或第21天用玉米油载体或500毫克/千克/天的DBP灌胃。在妊娠第19天，对照和暴露于DBP的中肾管之间只有微小的形态差异。在妊娠第21天，DBP剂量组中89%的雄性胎儿显示中肾管明显发育不全，其特征是盘绕减少。在妊娠第19天和第21天从大鼠中肾管分离RNA。结合经验信息，使用cDNA微阵列来帮助鉴定可能与妊娠第21天观察到的形态变化相关的候选基因。通过实时RT-PCR分析这些候选基因。在胰岛素样生长因子（IGF）途径、基质金属蛋白酶（MMP）家族、细胞外基质以及其他发育保守的信号通路中的基因中观察到mRNA表达变化。在妊娠第19天，IGF-1受体蛋白的免疫定位显示间充质和上皮细胞的细胞质表达增加。在妊娠第19天，导管上皮细胞中的雄激素受体蛋白也有不同程度的减少。本研究深入了解了抗雄激素对中肾管发育相关分子机制的影响。DBP暴露后形态改变和基因表达变化表明，由于睾酮产生降低，在中肾管分化过程中导管上皮细胞与周围间充质之间的旁分泌相互作用发生了改变。
