Chang Ching-Chien, Nagy Zsolt Peter, Abdelmassih Roger, Yang Xiangzhong, Tian X Cindy
Center for Regenerative Biology/Department of Animal Science, University of Connecticut, Storrs, 06269, USA.
Biol Reprod. 2004 Mar;70(3):752-8. doi: 10.1095/biolreprod.103.024497. Epub 2003 Nov 12.
During the haploidization process, it is expected that diploid chromosomes of somatic cells will be reduced to haploid for the generation of artificial gametes. In the present study, we aimed to use enucleated mouse oocytes at the germinal vesicle-stage (G2/M) as recipients for somatic cells that are also synchronized to the G2/M stage for haploidization. The reconstructed oocytes were then induced to undergo meiosis in vitro and observed for their nuclear morphology and microtubule network formation at various expected stages of the meiotic division. Following in vitro maturation, more than half (62/119, 52.1%) of the reconstructed oocytes completed the first round of meiosis-like division, as evidenced by the extrusion of pseudopolar bodies (PBs). However, accelerated PB extrusion, approximately 3-4 h earlier than that by control oocytes occurred. Furthermore, abnormally large pseudo-PBs, as large as four times the normal PB sizes, were observed. During the process of in vitro maturation at both the expected stages of metaphase I (MI) and metaphase II (MII), condensed chromosomes were observed in 38.7% and 55.2% of oocytes, respectively. However, two other types of nuclear configurations were also observed: 1) uneven distribution of chromatin and 2) an interphase-like nucleus, indicating deficiencies in chromosome condensation. Following oocyte activation, more than half (21/33, 63.6%) of the reconstructed oocytes with pseudo-PBs formed separated pseudopronuclei (PN), suggesting formation of functional spindles. The formation of bipolar spindle-like microtubule network at both the expected MI and MII stages during in vitro maturation was confirmed by immunohistochemistry. In summary, this study demonstrated that a high proportion of G2/M somatic nuclei appear to undergo meiosis-like division, in two successive steps, forming a pseudo-PB and two separate pseudo-PN upon in vitro maturation and activation treatment. Moreover, the enucleated geminal vesicle cytoplast retained its capacity for meiotic division following the introduction of a somatic G2/M nucleus.
在单倍体化过程中，预期体细胞的二倍体染色体将减少为单倍体，以产生人工配子。在本研究中，我们旨在使用处于生发泡期（G2/M期）的去核小鼠卵母细胞作为体细胞的受体，这些体细胞也同步至G2/M期以进行单倍体化。然后诱导重构卵母细胞在体外进行减数分裂，并在减数分裂的各个预期阶段观察其核形态和微管网络形成。体外成熟后，超过一半（62/119，52.1%）的重构卵母细胞完成了第一轮减数分裂样分裂，假极体（PBs）的排出证明了这一点。然而，假极体排出加速，比对照卵母细胞早约3 - 4小时。此外，观察到异常大的假极体，其大小高达正常极体的四倍。在体外成熟的预期中期I（MI）和中期II（MII）阶段，分别在38.7%和55.2%的卵母细胞中观察到浓缩染色体。然而，还观察到另外两种核构型：1）染色质分布不均和2）间期样核，表明染色体浓缩存在缺陷。卵母细胞激活后，超过一半（21/33，63.6%）带有假极体的重构卵母细胞形成了分离的假原核（PN），表明形成了功能性纺锤体。通过免疫组织化学证实了体外成熟过程中预期的MI和MII阶段均形成了双极纺锤体样微管网络。总之，本研究表明，高比例的G2/M体细胞核似乎经历了减数分裂样分裂，分两个连续步骤，在体外成熟和激活处理后形成假极体和两个分离的假原核。此外，引入体细胞G2/M核后，去核的生发泡细胞质体保留了其减数分裂能力。
