Microarray analysis of rat testis following combined fetal and postnatal DBP exposure

Provided later Pregnant Fisher 344 rats will be purchased from Charles River Laboratories, Inc. and delivered to CIIT on gestational day (GD) 7 (GD0 = day first vaginal plug positive). At gestational day 12 (GD12), the dams will be exposed once/day until GD20 to 50 mg/kg dibutyl phthalate (DBP) in corn oil vehicle via oral gavage. Each dose group will contain 4-6 vehicle control or phthalate treated dams. Groups of animals will be sacrificed at GD20, postnatal day (PND) 35, and PND90 for endpoint analysis. At GD20, treated and control animals will be examined for various endpoints including body weight, testicular histopathology, gene expression profile via microarray analysis, and anogenital distance (AGD). AGD (at parturition; PND1) and nipple number/location (at PND14 and day of sacrifice) will be determined on animals in the postnatal groups. At PND35 or 90, one male from each in utero corn oil vehicle or DBP exposed group will receive a second gavage of either corn oil or 500 mg/kg DBP. 6 hours after the second gavage, the following endpoints will be examined: 1) testis histopathology; 2) spermatid head quantification (PND90 only); 3) testis and body weights; 5) genome-wide gene expression (via microarray); and 6) germ cell apoptosis (TUNEL assay).

本研究将从查尔斯河实验室有限公司（Charles River Laboratories, Inc.）购入怀孕的Fisher 344大鼠，并于妊娠第7天（GD7，GD0指首次检测到阴道栓阳性的当日）运送至CIIT。于妊娠第12天（GD12）起，每日经口灌胃给予玉米油溶剂中的50mg/kg邻苯二甲酸二丁酯（dibutyl phthalate, DBP），直至妊娠第20天（GD20）。每个剂量组包含4-6只溶剂对照组或DBP处理组孕鼠。分别在GD20、出生后第35天（PND35）和出生后第90天（PND90）处死各组动物以开展终点检测。在GD20时，对处理组与对照组动物检测多项终点指标，包括体重、睾丸组织病理学、通过微阵列分析检测基因表达谱以及肛殖距（anogenital distance, AGD）。出生后组的动物需检测肛殖距（分娩时即PND1）以及乳头数量与位置（PND14及处死当日）。在PND35或PND90时，从每个宫内暴露于玉米油溶剂或DBP的组中选取1只雄性动物，再次经口灌胃给予玉米油或500mg/kg的DBP。第二次灌胃6小时后，检测以下终点指标：1）睾丸组织病理学；2）精子头计数（仅PND90组）；3）睾丸与体重；5）全基因组基因表达（通过微阵列分析）；6）生殖细胞凋亡（TUNEL测定法）。