Epigenetic Transgenerational Inheritance of Altered Sperm Histone Retention Sites

Female and male rats of an outbred female Sprague Dawley rats were administered daily intraperitoneal injections of vinclozolin (100 mg/kg BW/day), or DDT (25 mg/kg BW/day) or dimethyl sulfoxide (DMSO) in oil (1 µl/kg BW/day vehicle). Treatment lineages are designated “control” or “vinclozolin” or “DDT” lineages. The gestating female rats treated were designated as the F0 generation. The offspring of the F0 generation rats were the F1. Non-littermate females and males aged 70-90 days from F1 generation of control, vinclozolin or DDT were bred to obtain F2 generation offspring. The F2 generation rats were similarly bred to obtain the F3 generation offspring. Individuals were maintained for 120 days and euthanized for sperm collection. Sperm from multiple individual rats was pooled into single samples. For the control samples, 2 pools were made using 6 animals and 1 pool with 5 animals. For the DDT samples, 3 pools were mady using 4 animals. For the Vinclozolin samples, 3 pools were made using 4 animals. Genomic DNA was isolated and histone chromatin immunoprecipitation with genomic DNA was performed. Each pooled sample received a separate index primer. NGS was performed at the WSU Spokane Genomics Core using Ilumina HiSeq 2500 with a PE50 application, with a read size of approximately 50 bp and approximately 35 million reads per pool.本实验选取远交系Sprague Dawley大鼠雌雄个体，每日通过腹腔注射分别给予烯菌酮（vinclozolin，100 mg/kg体重/天）、滴滴涕（DDT，25 mg/kg体重/天），或以油为溶剂的二甲基亚砜（DMSO，1 µl/kg体重/天，作为载体对照）。根据处理方式将实验谱系划分为对照组、烯菌酮组与DDT组。经上述处理的孕鼠为F0代，其繁育的后代为F1代。选取对照组、烯菌酮组及DDT组F1代中70~90日龄的非同窝雌雄个体进行交配，获得F2代子代；将F2代大鼠按相同流程交配，得到F3代子代。所有个体饲养至120日龄后实施安乐死以采集精子，将多只个体的精子混合为单一样本：对照组设置2个混合池，分别由6只大鼠的精子混合而成，另设1个混合池由5只大鼠的精子混合而成；DDT组设置3个混合池，每个混合池由4只大鼠的精子混合而成；烯菌酮组同样设置3个混合池，每个混合池由4只大鼠的精子混合而成。提取基因组DNA（genomic DNA）后，以基因组DNA为底物开展组蛋白染色质免疫共沉淀（chromatin immunoprecipitation, ChIP）实验。每个混合样本使用独立的索引引物（index primer）进行标记。随后在华盛顿州立大学斯波坎分校基因组学核心实验室（WSU Spokane Genomics Core），采用Illumina HiSeq 2500测序平台以PE50双端测序模式开展下一代测序（next-generation sequencing, NGS），单端读长约为50 bp，每个混合池的测序读数约为3500万条。