Department of Anatomy, Biochemistry & Physiology, Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI, USA.
Department of Obstetrics, Gynecology & Women's Health, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI, USA.
Biol Reprod. 2022 Apr 26;106(4):730-740. doi: 10.1093/biolre/ioab237.
Origin recognition complex subunit 4 (ORC4) is a DNA-binding protein required for DNA replication. During oocyte maturation, after the last oocyte DNA replication step and before zygotic DNA replication, the oocyte undergoes two meiotic cell divisions in which half the DNA is ejected in much smaller polar bodies. We previously demonstrated that ORC4 forms a cytoplasmic cage around the DNA that is ejected in both polar body extrusion (PBE) events. Here, we used ZP3 activated Cre to delete exon 7 of Orc4 during oogenesis to test how it affected both predicted functions of ORC4: its recently discovered role in PBE and its well-known role in DNA synthesis. Orc4 deletion severely reduced PBE. Almost half of Orc4-depleted germinal vesicle (GV) oocytes cultured in vitro were arrested before anaphase I (48%), and only 25% produced normal first polar bodies. This supports the role of ORC4 in PBE and suggests that transcription of the full-length Orc4 during oogenesis is required for efficient PBE. Orc4 deletion also abolished zygotic DNA synthesis. Fewer Orc4-depleted oocytes developed to the metaphase II (MII) stage, and after activation these oocytes were arrested at the two-cell stage without undergoing DNA synthesis. This confirms that transcription of full-length Orc4 after the primary follicle stage is required for zygotic DNA replication. The data also suggest that MII oocytes do not have a replication licensing checkpoint as cytokinesis progressed without DNA synthesis. Together, the data confirm that oocyte ORC4 is important for both PBE and zygotic DNA synthesis.
卵母细胞 ORC4 是 DNA 复制所必需的 DNA 结合蛋白。在卵母细胞成熟过程中，在最后一次卵母细胞 DNA 复制步骤之后，在合子 DNA 复制之前，卵母细胞经历两次减数分裂，其中一半的 DNA 被排出到更小的极体中。我们之前证明 ORC4 在两个极体挤出（PBE）事件中形成围绕被排出的 DNA 的细胞质笼。在这里，我们使用 ZP3 激活 Cre 在卵发生过程中删除 Orc4 的外显子 7，以测试它如何影响 ORC4 的两个预测功能：它最近在 PBE 中的作用及其在 DNA 合成中的众所周知的作用。Orc4 的缺失严重降低了 PBE。在体外培养的近一半 Orc4 耗尽的生发泡（GV）卵母细胞在第一次有丝分裂后期（48%）之前停滞不前，只有 25%产生正常的第一极体。这支持了 ORC4 在 PBE 中的作用，并表明在卵发生过程中全长 Orc4 的转录是 PBE 所必需的。Orc4 的缺失也消除了合子 DNA 合成。更少的 Orc4 耗尽的卵母细胞发育到中期 II（MII）阶段，并且在激活后，这些卵母细胞在没有进行 DNA 合成的情况下停滞在二细胞阶段。这证实了初级卵泡阶段后全长 Orc4 的转录是合子 DNA 复制所必需的。数据还表明，MII 卵母细胞没有复制许可检查点，因为有丝分裂没有进行而没有 DNA 合成。总之，数据证实卵母细胞 ORC4 对 PBE 和合子 DNA 合成都很重要。
