title: Nuclear and microtubule dynamics of G2/M somatic nuclei during haploidization in germinal vesicle-stage mouse oocytes.
abstract: 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.
authors: Center for Regenerative Biology/Department of Animal Science, University of Connecticut, Storrs, 06269, USA.
authors: Ching-Chien
authors: CC
authors: Chang
authors: Zsolt Peter
authors: ZP
authors: Nagy
authors: Roger
authors: R
authors: Abdelmassih
authors: Xiangzhong
authors: X
authors: Yang
authors: X Cindy
authors: XC
authors: Tian
mesh_terms: D000818:Animals; D002455:Cell Division / Q000502:physiology; D002467:Cell Nucleus / Q000502:physiology*; D005260:Female; D016195:G2 Phase / Q000502:physiology; D006238:Haploidy; D066298:In Vitro Techniques; D008540:Meiosis / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008870:Microtubules / Q000502:physiology*; D009865:Oocytes / Q000166:cytology / Q000502:physiology*; D027724:Reproductive Techniques, Assisted; D008941:Spindle Apparatus / Q000502:physiology