Intergenerational impact of paternal lifetime exposures to both folic acid deficiency and supplementation on reproductive outcomes and imprinted gene methylation
Intergenerational impact of paternal lifetime exposures to both folic acid deficiency and supplementation on reproductive outcomes and imprinted gene methylation
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DOI：
10.1093/molehr/gax029
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发表时间：
2017-07-01
影响因子：
4
通讯作者：
Trasler, Jacquetta
中科院分区：
文献类型：
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作者：
Ly, Lundi;Chan, Donovan;Trasler, Jacquetta
在整个生殖细胞发育过程中，父亲暴露于叶酸缺乏（FD）和/或叶酸补充（FS）的饮食是否会对男性生殖细胞产生不利影响，从而影响后代的健康结果？暴露于FD和FS饮食的雄性小鼠在其一生中表现出精子数量减少和印记基因甲基化改变，并有证据表明其对后代的不良影响，包括出生后断奶前死亡率增加和印记基因甲基化的变异性。胎儿期是男性生殖细胞发育过程中获得DNA甲基化模式的关键时期，需要足够的甲基供体。此外，DNA甲基化模式在出生后精子发生过程中继续重塑。以前的研究表明，叶酸缺乏可改变精子表观基因组并增加胎儿形态异常的发生率。雌性BALB/c小鼠（F0）在怀孕前4周以及整个怀孕和哺乳期被置于四种氨基酸限定的饮食之一：叶酸对照3 mg/kg）、10倍高FS（10 FS，20 mg/kg）或20倍高FS（20 FS，40 mg/kg）饮食。将F1代雄性断奶至其各自的产前饮食，以允许在生殖系表观遗传重编程的所有窗口期间暴露于饮食：擦除、重建和维持阶段。将F0代雌性与普通饲料喂养的雄性交配以产生F1窝，其生殖细胞在整个胚胎发育期间暴露于饮食。随后将F1代雄性小鼠与普通饲料喂养的雌性小鼠交配。从每只F1雄性动物中产生两窝未暴露于实验饲料的F2窝;在胚胎第18.5天（E）收集一窝，分娩一窝并在出生后进行随访。DNA甲基化在一个整体水平和在差异甲基化区域的印记基因（H19，印记母代表达转录本（非蛋白编码）-H19，小核核糖核蛋白多肽N-Snrpn，KCNQ 1相反链/反义转录本1（非蛋白质编码）-Kcnq 1 ot 1，在F1精子中分别通过发光甲基化分析和亚硫酸氢盐焦磷酸测序来评估父系表达基因1-Peg 1和父系表达基因3-Peg 3），F2 E18.5胎盘和F2 E18.5大脑皮质。F1雄性在终生暴露于叶酸缺乏和最高剂量叶酸补充（20 FS）后，精子数均降低（均P < 0.05）。20 FS暴露的F1雄性F2 E18.5日龄窝仔的着床后丢失率增加（P < 0.05）。来自7 FD和20 FS暴露的F1雄性的F2窝具有显著更高的出生后-断奶前幼仔死亡率（均P < 0.05）。10 FS暴露组男性精子印迹基因H19甲基化变异增加，P < 0.05; 7 FD和20 FS组也发现H19内少数位点的甲基化变异增加（P < 0.05）。虽然20 FS饮食导致F2 E18.5胎盘中印迹基因Snrpn和Peg 3的甲基化的个体间改变，但在7 FD和10 FS组中，Peg 1和/或Peg 3中检测的个体位点的千分之50%受到影响。F2 E18.5d10FS组脑皮质中Peg 1甲基化存在个体间差异（P < 0.05）。需要进一步的研究来了解叶酸缺乏和补充对发育中的男性生殖细胞的不良影响的机制。全基因组DNA和组蛋白甲基化组研究以及基因表达研究需要更好地了解叶酸暴露，改变生殖细胞表观基因组和后代outcomes.The研究结果之间的联系提供了进一步的支持父系传播的环境影响。研究结果表明，叶酸缺乏和高剂量补充叶酸都可能对生殖细胞发育和生殖健康有害，部分原因是改变了精子中的DNA甲基化。加拿大卫生研究院（CIHR #89944）。作者声明他们没有利益冲突。
Do paternal exposures to folic acid deficient (FD), and/or folic acid supplemented (FS) diets, throughout germ cell development adversely affect male germ cells and consequently offspring health outcomes?Male mice exposed over their lifetimes to both FD and FS diets showed decreased sperm counts and altered imprinted gene methylation with evidence of transmission of adverse effects to the offspring, including increased postnatal-preweaning mortality and variability in imprinted gene methylation.There is increasing evidence that disruptions in male germ cell epigenetic reprogramming are associated with offspring abnormalities and intergenerational disease. The fetal period is the critical time of DNA methylation pattern acquisition for developing male germ cells and an adequate supply of methyl donors is required. In addition, DNA methylation patterns continue to be remodeled during postnatal spermatogenesis. Previous studies have shown that lifetime (prenatal and postnatal) folic acid deficiency can alter the sperm epigenome and increase the incidence of fetal morphological abnormalities.Female BALB/c mice (F0) were placed on one of four amino-acid defined diets for 4 weeks before pregnancy and throughout pregnancy and lactation: folic acid control (Ctrl; 2 mg/kg), 7-fold folic acid deficient (7FD; 0.3 mg/kg), 10-fold high FS (10FS, 20 mg/kg) or 20-fold high FS (20FS, 40 mg/kg) diets. F1 males were weaned to their respective prenatal diets to allow for diet exposure during all windows of germline epigenetic reprogramming: the erasure, re-establishment and maintenance phases.F0 females were mated with chow-fed males to produce F1 litters whose germ cells were exposed to the diets throughout embryonic development. F1 males were subsequently mated with chow-fed female mice. Two F2 litters, unexposed to the experimental diets, were generated from each F1 male; one litter was collected at embryonic day (E)18.5 and one delivered and followed postnatally. DNA methylation at a global level and at the differentially methylated regions of imprinted genes (H19, Imprinted Maternally Expressed Transcript (Non-Protein Coding)-H19, Small Nuclear Ribonucleoprotein Polypeptide N-Snrpn, KCNQ1 Opposite Strand/Antisense Transcript 1 (Non-Protein Coding)-Kcnq1ot1, Paternally Expressed Gene 1-Peg1 and Paternally Expressed Gene 3-Peg3) was assessed by luminometric methylation analysis and bisulfite pyrosequencing, respectively, in F1 sperm, F2 E18.5 placenta and F2 E18.5 brain cortex.F1 males exhibited lower sperm counts following lifetime exposure to both folic acid deficiency and the highest dose of folic acid supplementation (20FS), (both P < 0.05). Post-implantation losses were increased amongst F2 E18.5 day litters from 20FS exposed F1 males (P < 0.05). F2 litters derived from both 7FD and 20FS exposed F1 males had significantly higher postnatal-preweaning pup death (both P < 0.05). Sperm from 10FS exposed males had increased variance in methylation across imprinted gene H19, P < 0.05; increased variance at a few sites within H19 was also found for the 7FD and 20FS groups (P < 0.05). While the 20FS diet resulted in inter-individual alterations in methylation across the imprinted genes Snrpn and Peg3 in F2 E18.5 placenta, a parts per thousand 50% of individual sites tested in Peg1 and/or Peg3 were affected in the 7FD and 10FS groups. Inter-individual alterations in Peg1 methylation were found in F2 E18.5 day 10FS group brain cortex (P < 0.05).Not applicable.The cause of the increase in postnatal-preweaning mortality was not investigated post-mortem. Further studies are required to understand the mechanisms underlying the adverse effects of folic acid deficiency and supplementation on developing male germ cells. Genome-wide DNA and histone methylome studies as well as gene expression studies are required to better understand the links between folic acid exposures, an altered germ cell epigenome and offspring outcomes.The findings of this study provide further support for paternally transmitted environmental effects. The results indicate that both folic acid deficiency and high dose supplementation can be detrimental to germ cell development and reproductive fitness, in part by altering DNA methylation in sperm.This study was supported by a grant to J.M.T. from the Canadian Institutes of Health Research (CIHR #89944). The authors declare they have no conflicts of interest.