Paternal over- and under-nutrition program fetal and placental development in a sex-specific manner

Under- and over-nutrition are directly linked to poor physiological, metabolic and reproductive health. While the association between paternal diet and offspring well-being is becoming established, the underlying mechanisms are yet to be fully defined. The aim of this study was to establish and compare the impact of over- and under-nutrition on male reproductive fitness and post-fertilisation development, with the additional exploration of the role that specific micronutrient supplementation may play in ameliorating any detrimental effects of poor paternal diet. Male C57/BL6J mice were fed either control diet (CD), isocaloric low protein diet (LPD), 'Western' diet (WD) or LPD or WD supplemented with methyl-donors and carriers (MD-LPD or MD-WD respectively) for 8 weeks before mating with virgin C57/BL6J females maintained on standard mouse chow. Placental tissue was collected at embryonic day (E)8.5 to assess early placental morphology and metabolism or E17.5 for sex-specific transcriptomic profiling. Post-mating male tissues were harvested for assessment of testicular morphology, gene expression and metabolic profiling and fecal microbiome was analysed. This study found significant increases in adiposity, hepatic cholesterol and FFAs in WD and MDWD fed males, alongside an increase in the abundance of Defferibacteres, Proteobacteria and reduction TM7 abundance in fecal samples compared to CD fed males. Analysis of testicular gene expression revealed 402 (267 down-regulated, 135 upregulated) genes and 285 (187 down-regulated, 98 up-regulated) genes differentically expressed in WD and MD-WD-fed males, respectively, with minimal changes observed in LPD and MD-LPD groups compared to CD-fed males. Placenta from LPD fed fathers had a significant reduction in lactate production at E8.5 and significant sex-specific influences of paternal diet on late gestation fetal and placental growth were observed in both LPD and MD-LPD groups, which had a significantly higher proportion of fetuses with weights below the 10th centile when compared to CD fetuses. We also identified a significant reduction in sexually dimorphic gene expression in response to paternal diet, with 301, 13, 0, 14 and 15 placental genes to be differentially expressed between males and females in the CD, LPD, MD-LPD, WD and MD-WD. We observed that males consuming a high fat/sugar diet (WD and MD-WD), displayed a series of physiological changes in their metabolic health, gut bacterial profiles and testicular morphology and gene expression. While both over- and under-nutritional regimens had no impact on fundamental male fertility, we observed a significant loss in sexual-dimorphism in the late gestation placenta in response to our experimental diets. Such loss of sexual- dimorphism could provide one process through which poor paternal diet programs offspring ill-health in adulthood. Overall design: To investigate the impact sub-optimal diet fed males had on the placental RNA profiles of their offspring. We fed male mice either control diet (CD; 18% casein, 21% sucrose, 0% milk fat, 0% cholesterol), isocaloric low protein diet (LPD; 9% casein, 24% sucrose, 0% milk fat, 0% cholesterol), 'Western' diet (WD; 19% casein, 34% sucrose, 20% milk fat, 0.15% cholesterol) or LPD or WD supplemented with methyl-donors and carriers (5 g/kg diet choline chloride, 15 g/kg diet betaine, 7.5 g/kg diet methionine, 15 mg/kg diet folic acid, 1.5 mg/kg diet vitamin B12; termed MD-LPD or MD-WD respectively) for a minimum of 8 weeks before males were mated with standard chow fed females. At gestational day 17.5 females were euthanised and placentae were collected, fetal DNA was sexed using 3 primer sets specific for regions on the Y (Sry and Zfy) and X (Dxnds3) chromosomes and placental RNA was extracted from 1 placenta per litter (N=8, with 4 male and 4 female placenta in each diet group)

营养不足与营养过剩均与机体生理、代谢及生殖健康受损直接相关。尽管父代饮食与子代健康之间的关联已逐渐得到证实，但其潜在的分子机制仍有待完全阐明。本研究旨在明确并对比营养不足与营养过剩对雄性生殖健康及受精后发育的影响，并额外探索特定微量营养素补充在改善父代不良饮食所引发的损害中所发挥的作用。将雄性C57/BL6J小鼠分为四组，分别饲喂对照饲料（CD）、等热量低蛋白饲料（LPD）、西式饲料（WD），以及添加甲基供体与载体的LPD或WD（分别记为MD-LPD与MD-WD），饲喂8周后，与饲喂标准小鼠饲料的未交配C57/BL6J雌鼠进行交配。分别在胚胎发育第8.5天（E8.5）采集胎盘组织以评估早期胎盘形态与代谢状态，或在E17.5采集胎盘以进行性别特异性转录组分析。交配后采集雄性小鼠组织，用于评估睾丸形态、基因表达及代谢谱，并分析粪便微生物组。本研究发现，与饲喂CD的雄性小鼠相比，饲喂WD及MD-WD的小鼠体脂、肝脏胆固醇及游离脂肪酸（Free Fatty Acids, FFAs）水平显著升高；同时其粪便样本中脱铁杆菌门（Deferribacteres）、变形菌门（Proteobacteria）的丰度显著上升，而TM7门的丰度则显著下降。对睾丸基因表达的分析显示，与CD组雄性小鼠相比，WD组与MD-WD组分别有402个（267个下调、135个上调）及285个（187个下调、98个上调）基因存在差异表达，而LPD组与MD-LPD组的基因表达变化极小。父代饲喂LPD的胎盘在E8.5时乳酸生成量显著降低；同时，在LPD组与MD-LPD组中，父代饮食对妊娠晚期胎儿及胎盘生长存在显著的性别特异性影响，且这两组中体重低于第10百分位数的胎儿比例显著高于CD组。我们还发现，父代饮食会显著降低胎盘的性别二态性基因表达：在CD、LPD、MD-LPD、WD及MD-WD各组中，雌雄胎盘间存在差异表达的基因数分别为301、13、0、14及15个。我们观察到，饲喂高脂高糖饲料（WD及MD-WD）的雄性小鼠，其代谢健康、肠道菌群组成、睾丸形态及基因表达均出现了一系列生理改变。尽管营养不足与营养过剩两种膳食模式均未对雄性基础生育力造成影响，但我们观察到，实验性膳食会导致妊娠晚期胎盘的性别二态性显著丧失。这种性别二态性的丧失，可能是父代不良饮食程序化子代成年后健康受损的潜在机制之一。实验整体设计：本研究旨在探究父代饲喂次优膳食对子代胎盘RNA表达谱的影响。将雄性小鼠分为四组，分别饲喂对照饲料（CD：18%酪蛋白、21%蔗糖、0%乳脂、0%胆固醇）、等热量低蛋白饲料（LPD：9%酪蛋白、24%蔗糖、0%乳脂、0%胆固醇）、西式饲料（WD：19%酪蛋白、34%蔗糖、20%乳脂、0.15%胆固醇），以及添加甲基供体与载体的LPD或WD（添加量为：5g/kg饲料氯化胆碱、15g/kg饲料甜菜碱、7.5g/kg饲料蛋氨酸、15mg/kg饲料叶酸、1.5mg/kg饲料维生素B12，分别记为MD-LPD与MD-WD），每组饲喂至少8周后，将雄性小鼠与饲喂标准饲料的雌鼠进行交配。在妊娠第17.5天处死雌鼠并采集胎盘；利用针对Y染色体（Sry、Zfy）及X染色体（Dxnds3）特定区域的3对引物对胎儿DNA进行性别鉴定；每窝选取1个胎盘提取RNA（每组n=8，包含4个雄性胎盘与4个雌性胎盘）。