ABSTRACT
DNA topoisomerase II (topo II) is an essential enzyme that mediates a variety of chromosome activities including DNA replication, transcription, recombination, and chromosome condensation and segregation. Isoform-specific anti-topo II antibodies were used to determine the distribution of topo II alpha and beta in mouse gametes and embryos. Immunoblot analysis with two anti-topo IIalpha antibodies revealed that a 170 kDa topo IIalpha band was present in ovary and testis. Mature sperm exhibited an 89 kDa band only, which may be a degradation product of topo IIalpha. Immunoblots probed with a monoclonal antibody that recognizes both isoforms, showed bands at 170 and 180 kDa, which correspond to topo IIalpha and beta, respectively. An additional 100 kDa band was also present in ovary and testis. Mature sperm did not exhibit staining with this antibody. We also localized topo II in mouse gametes and embryos up to the blastocyst stage using immunofluorescence microscopy. While both isoforms were found in nuclei and nucleoli of germinal vesicle oocytes, topo IIalpha localized to metaphase chromosomes during meiosis, and only to nucleoli during embryonic interphase. Topo IIbeta was absent from chromosomes of metaphase II oocytes, but localized to embryonic interphase nuclei. Both full-length isoforms were absent from sperm, indicating topo II is stored maternally. These results identify topo II as an important component of mouse oocyte and embryonic chromatin, and suggest its involvement in oocyte maturation and preimplantation embryonic development. The different immunofluorescent staining patterns indicate topo IIalpha and beta may serve different roles during the embryonic cell cycle.
Subject(s)
Blastocyst/cytology , Blastocyst/enzymology , DNA Topoisomerases, Type II/metabolism , Animals , Antibody Specificity , Blotting, Western , Cell Nucleolus/enzymology , Cell Nucleus/enzymology , DNA Topoisomerases, Type II/immunology , Embryonic and Fetal Development , Germ Cells/enzymology , Isoenzymes/immunology , Isoenzymes/metabolism , Meiosis , Mice , Microscopy, FluorescenceABSTRACT
Topoisomerase II (topo II) is an essential enzyme that alters DNA topology. This activity is important for a variety of chromosome functions including DNA replication, transcription, recombination, and chromosome condensation and segregation. Previously we localized topo II in mouse gametes and preimplantation embryos using isoform-specific antibodies demonstrating the presence of the enzyme in oocytes and embryos, but not sperm. To probe functions of topo II during preimplantation development, we treated mouse zygotes with 100 nM teniposide, and assessed embryo morphology and DNA replication. Teniposide blocked cleavage in 69% embryos; the remainder cleaved once but had abnormal nuclei. Teniposide-treated embryos were devoid of topo II immunofluorescence. Teniposide also prevented DNA replication, implicating topo II in this process. Embryos treated with a 2 hr pulse of teniposide recovered and developed to the blastocyst stage, indicating 100 nM teniposide did not induce apoptosis. To more specifically analyze topo IIalpha function, we treated zygotes with topo IIalpha-targeted antisense oligodeoxynucleotides. Most zygotes arrested at the 2-cell stage while controls developed into blastocysts indicating topo IIalpha is essential for preimplantation development. The absence of topo IIalpha, but not beta immunofluorescence in antisense-treated embryos confirms the specificity and impact of the treatment. In addition, topo IIalpha is newly synthesized at the 2-cell stage. These results establish an essential function for topo II in mouse preimplantation embryonic development.
