Institute for Biogenesis Research, Department of Anatomy, Biochemistry and Physiology, University of Hawaii at Manoa, John A Burns School of Medicine, Honolulu, Hawaii, United States of America.
PLoS One. 2013;8(2):e56385. doi: 10.1371/journal.pone.0056385. Epub 2013 Feb 19.
Mouse zygotes do not activate apoptosis in response to DNA damage. We previously reported a unique form of inducible sperm DNA damage termed sperm chromatin fragmentation (SCF). SCF mirrors some aspects of somatic cell apoptosis in that the DNA degradation is mediated by reversible double strand breaks caused by topoisomerase 2B (TOP2B) followed by irreversible DNA degradation by a nuclease(s). Here, we created zygotes using spermatozoa induced to undergo SCF (SCF zygotes) and tested how they responded to moderate and severe paternal DNA damage during the first cell cycle. We found that the TUNEL assay was not sensitive enough to identify the breaks caused by SCF in zygotes in either case. However, paternal pronuclei in both groups stained positively for γH2AX, a marker for DNA damage, at 5 hrs after fertilization, just before DNA synthesis, while the maternal pronuclei were negative. We also found that both pronuclei in SCF zygotes with moderate DNA damage replicated normally, but paternal pronuclei in the SCF zygotes with severe DNA damage delayed the initiation of DNA replication by up to 12 hrs even though the maternal pronuclei had no discernable delay. Chromosomal analysis of both groups confirmed that the paternal DNA was degraded after S-phase while the maternal pronuclei formed normal chromosomes. The DNA replication delay caused a marked retardation in progression to the 2-cell stage, and a large portion of the embryos arrested at the G2/M border, suggesting that this is an important checkpoint in zygotic development. Those embryos that progressed through the G2/M border died at later stages and none developed to the blastocyst stage. Our data demonstrate that the zygote responds to sperm DNA damage through a non-apoptotic mechanism that acts by slowing paternal DNA replication and ultimately leads to arrest in embryonic development.
鼠受精卵不会因 DNA 损伤而引发细胞凋亡。我们之前曾报道过一种独特的可诱导精子 DNA 损伤，称为精子染色质碎片化（SCF）。SCF 在某些方面与体细胞凋亡相似，即 DNA 降解是由拓扑异构酶 2B（TOP2B）介导的可逆双链断裂引起的，随后由核酶（s）引起不可逆的 DNA 降解。在这里，我们使用诱导发生 SCF 的精子创建了受精卵（SCF 受精卵），并测试了它们在第一个细胞周期中对中度和重度父系 DNA 损伤的反应。我们发现，在任何情况下，TUNEL 检测都不够敏感，无法识别 SCF 在受精卵中引起的断裂。然而，在受精后 5 小时，即 DNA 合成前，两组的父系原核均对 γH2AX 呈阳性染色，γH2AX 是 DNA 损伤的标志物，而母系原核则呈阴性。我们还发现，中度 DNA 损伤的 SCF 受精卵的两个原核均正常复制，但严重 DNA 损伤的 SCF 受精卵的父系原核延迟了 DNA 复制的启动，最多延迟 12 小时，而母系原核则没有明显延迟。两组的染色体分析均证实，父系 DNA 在 S 期后降解，而母系原核形成正常染色体。DNA 复制延迟导致向 2 细胞期的进展明显延迟，并且大部分胚胎在 G2/M 边界处停滞，表明这是胚胎发育中的一个重要检查点。那些通过 G2/M 边界的胚胎在后期死亡，没有一个发育到囊胚期。我们的数据表明，受精卵通过一种非凋亡机制对精子 DNA 损伤做出反应，该机制通过减缓父系 DNA 复制起作用，最终导致胚胎发育停滞。
