Potabattula Ramya, Dittrich Marcus, Hahn Thomas, Schorsch Martin, Ptak Grazyna Ewa, Haaf Thomas
Institute of Human Genetics, Julius Maximilians University, 97074 Würzburg, Germany.
Department of Bioinformatics, Julius Maximilians University, 97074 Würzburg, Germany.
Int J Mol Sci. 2025 Apr 28;26(9):4197. doi: 10.3390/ijms26094197.
In this study, droplet digital PCR and deep bisulfite sequencing were used to study the absolute and active rDNA copy number (CN) and the effect of paternal age on human and mouse sperm. The absolute CN ranged from 98 to 404 (219 ± 47) in human and from 98 to 177 (133 ± 14) in mouse sperm. Methylation of the human upstream control element/core promoter (UCE/CP) region and the 5' external transcribed spacer, as well as that of the mouse CP, the spacer promoter, and 28S rDNA, significantly increased with donor age and absolute CN. Overall, rDNA hypomethylation was much more pronounced in mouse sperm, with 101.7 ± 11.4 copies showing a completely (0%) unmethylated and 11.3 ± 2.8 (8.5%) a slightly methylated (1-10%) CP region, compared to humans with 25.7 ± 9.5 (12%) completely unmethylated and 83.0 ± 19.8 slightly methylated UCE/CP regions. Although the absolute CN was much higher in human sperm, the number of copies with a hypomethylated (0-10%) promoter was comparable in humans (108.7 ± 28.3) and mice (113.0 ± 12.2). However, in mice, the majority (77%) of all copies were completely unmethylated, whereas in humans a high percentage (38%) showed one or two single CpG methylation errors. These different germline methylation dynamics may be due to species differences in reproductive strategies and lifespan. Complete demethylation of the sperm rDNA promoter in mice may be essential for embryonic genome activation, which already occurs at the 2-cell stage in mice and at the 4-8-cell stage in humans. The paternal age effect has been conserved between humans and mice with some notable differences. In humans, the number of hypomethylated (0-10%) copies decreased with age, whereas in mice only the completely unmethylated copies decreased with age. The number of methylated rDNA copies (>1% in mice and >10% in humans) significantly increased with age.
在本研究中，采用液滴数字PCR和深度亚硫酸氢盐测序技术，研究人类和小鼠精子中核糖体DNA（rDNA）的绝对拷贝数和活性拷贝数（CN）以及父本年龄的影响。人类精子中rDNA的绝对拷贝数范围为98至404（219±47），小鼠精子中为98至177（133±14）。人类上游控制元件/核心启动子（UCE/CP）区域和5'外部转录间隔区以及小鼠CP、间隔区启动子和28S rDNA的甲基化程度均随供体年龄和绝对拷贝数显著增加。总体而言，rDNA低甲基化在小鼠精子中更为明显，101.7±11.4个拷贝的CP区域完全未甲基化（0%），11.3±2.8个拷贝（8.5%）的CP区域轻度甲基化（1%-10%）；相比之下，人类精子中有25.7±9.5个拷贝（12%）完全未甲基化，83.0±19.8个拷贝轻度甲基化。虽然人类精子中的绝对拷贝数要高得多，但启动子低甲基化（0%-10%）的拷贝数在人类（108.7±28.3）和小鼠（113.0±12.2）中相当。然而，在小鼠中，所有拷贝的大多数（77%）完全未甲基化，而在人类中，高比例（38%）的拷贝显示出一两个单个CpG甲基化错误。这些不同的生殖系甲基化动态可能是由于生殖策略和寿命的物种差异。小鼠精子rDNA启动子的完全去甲基化可能对胚胎基因组激活至关重要，这在小鼠的2细胞阶段就已发生，而在人类中则发生在4-8细胞阶段。父本年龄效应在人类和小鼠之间具有保守性，但也存在一些显著差异。在人类中，低甲基化（0%-10%）拷贝数随年龄下降，而在小鼠中，只有完全未甲基化的拷贝数随年龄下降。甲基化rDNA拷贝数（小鼠中>1%，人类中>10%）随年龄显著增加。
