We previously reported that paternal preconception chronic ethanol exposure in mice imparts adult male offspring with reduced ethanol drinking preference and consumption, increased ethanol sensitivity, and attenuated stress responsivity. That same chronic ethanol exposure paradigm was later revealed to affect the sperm epigenome by altering the abundance of several small noncoding RNAs, a mechanism that mediates the intergenerational effects of numerous paternal environmental exposures. Although recent studies have revealed that the unique RNA signature of sperm is shaped during maturation in the epididymis via extracellular vesicles (EVs), formal demonstration that EVs mediate the effects of paternal preconception perturbations is lacking. Therefore, in the current study we tested the hypothesis that epididymal EV preparations are sufficient to induce intergenerational effects of paternal preconception ethanol exposure on offspring. To test this hypothesis, sperm from ethanol-naïve donors were incubated with epididymal EV preparations from chronic ethanol (Ethanol EV-donor) or control-treated (Control EV-donor) mice prior to in vitro fertilization (IVF) and embryo transfer. Progeny were examined for ethanol- and stress-related behaviors in adulthood. Ethanol EV-donors imparted reduced body weight at weaning and imparted modestly increased limited access ethanol intake to male offspring. Ethanol-EV donors also imparted increased basal anxiety-like behavior and reduced sensitivity to ethanol-induced anxiolysis to female offspring. Although Ethanol EV-donor treatment did not recapitulate the ethanol- or stress-related intergenerational effects of paternal ethanol following natural mating, these results demonstrate that coincubation of sperm with epididymal EV preparations is sufficient to impart intergenerational effects of ethanol through the male germline. This mechanism may generalize to the intergenerational effects of a wide variety of paternal preconception perturbations.

我们之前曾报道，小鼠的父系先入之见慢性乙醇暴露使成年雄性后代乙醇饮用偏好和消耗减少，乙醇敏感性增加，应激反应减弱。同样的慢性乙醇暴露范例后来被发现通过改变几个小的非编码 RNA 的丰度来影响精子表观基因组，这是一种介导许多父系环境暴露的代际效应的机制。尽管最近的研究表明，精子的独特 RNA 特征是在附睾成熟过程中通过细胞外囊泡 (EV) 形成的，但缺乏 EV 介导父亲先入为主扰动影响的正式证据。所以，在当前的研究中，我们测试了附睾 EV 制剂足以诱导父系先入为主乙醇暴露对后代的代际影响的假设。为了验证这一假设，在实验前，将来自未接触过乙醇的供体的精子与来自慢性乙醇（乙醇 EV 供体）或对照处理（对照 EV 供体）小鼠的附睾 EV 制剂一起孵育体外受精 (IVF) 和胚胎移植。检查后代在成年期的乙醇和压力相关行为。乙醇 EV 捐赠者在断奶时体重减轻，并适度增加雄性后代有限的乙醇摄入量。乙醇-EV 供体还增加了雌性后代的基础焦虑样行为，并降低了对乙醇诱导的抗焦虑作用的敏感性。尽管乙醇 EV 供体治疗没有概括自然交配后父本乙醇的乙醇或压力相关的代际效应，但这些结果表明，精子与附睾 EV 制剂的共孵育足以通过雄性种系传递乙醇的代际效应。这种机制可以推广到各种父系先入之见扰动的代际效应。