Dynamic changes of gamma-tubulin in preimplantation development of parthenogenetic mouse embryos / 生理学报

ABSTRACT

Tubulin is the major protein of microtubule. alpha- and beta- tubulins form heterodimers, while gamma-tubulin regulates microtubule organization. The present study aimed to observe the dynamic changes of gamma-tubulin in preimplantation development of parthenogenetic mouse embryos. Immunofluorescence and laser confocal microscopy were used to detect the location of gamma-tubulin in preimplantation parthenogenetic embryos activated by SrCl2. The oocytes were collected at 13-14 h after hCG injection, and then activated with 10 mmol/L SrCl2 in Ca(2+)-free CZB medium with 5 mmol/L cytochalasin B (CB), fixed at 1 h intervals until 6 h after activation. The results showed that spindle was paralleled with the cell membrane all the time, when the meiosis of MII mouse oocytes resumed. The rotation of spindle was inhibited, but karyokinesis was not influenced. At 0 h after activation, i.e. at metaphase, gamma-tubulin was distributed mainly on the two poles of spindle. At 1 h after activation, i.e. at anaphase, following the separation of chromosomes, gamma-tubulin was transformed from dense to disperse. At 2 h after activation, gamma-tubulin was localized between the segregated sister chromatids at telophase. However, at 3-6 h after activation, gamma-tubulin concentrated around the two female pronuclei during their formation and juxtaposition. Moreover, another group of MII oocytes were activated for 6 h and cultured in droplets of KSOM medium under mineral oil in 5% CO2 in air at 37 degrees C to permit parthenogenetic development. The embryos were collected and fixed at 3 h, 14 h, 16 h, and 18 h of culture. At 3 h after culture, i.e. at mitotic interphase, it was shown that amorphous gamma-tubulin distributed around the nuclei of early parthenogenetic embryos. At 24 h after culture, i.e. at prometaphase, gamma-tubulin migrated along the spindle microtubule to the two poles. Our results showed that gamma-tubulin had similar location patterns at metaphase, anaphase and telophase in meiosis and mitosis. It was concluded that gamma-tubulin assembly in parthenogenetically activated oocytes facilitated the formation of negative pole cap and the stabilization of microtubule, thus promoting the spindle formation at meiosis and mitosis. The relocation of gamma-tubulin at anaphase and telophase might be induced by the event of segregation of homologous chromosome being pulled away by the spindle. gamma-tubulin might contribute to the migration and juxtaposition of the two female pronuclei as well.