Spindle formation and dynamics of gamma-tubulin and nuclear mitotic apparatus protein distribution during meiosis in pig and mouse oocytes

This work focuses on the assembly and transformation of the spindle during the progression through the meiotic cell cycle. For this purpose, immunoflu orescent confocal microscopy was used in comparative studies to determine t he spatial distribution of alpha- and gamma-tubulin and nuclear mitotic app aratus protein (NuMA) from late G2 to the end of M phase in both meiosis an d mitosis. In pig endothelial tells, consistent with previous reports, gamm a-tubulin was localized at the centrosomes in both interphase and M phase, and NuMA was localized in the interphase nucleus and at mitotic spindle pol es. During meiotic progression in pig oocytes, gamma-tubulin and NuMA were initially detected in a uniform distribution across the nucleus. In early d iakinesis and just before germinal vesicle breakdown, microtubules were fir st detected around the periphery of the germinal vesicle and cell cortex. A t rate diakinesis, a mass of multi-arrayed microtubules was formed around c hromosomes. In parallel, NuMA localization changed from an amorphous to a h ighly aggregated form in the vicinity of the chromosomes, but gamma-tubulin localization remained in an amorphous form surrounding the chromosomes. Th en the NuMA foci moved away from the condensed chromosomes and aligned at b oth poles of a barrel-shaped metaphase I spindle white gamma-tubulin was lo calized along the spindle microtubules, suggesting that pig meiotic spindle poles are formed by the bundling of microtubules at the minus ends by NuMA . interestingly, in mouse oocytes, the meiotic spindle pole was composed of several gamma-tubulin foci rather than NuMA. Further, nocodazole, an inhib itor of microtubule polymerization, induced disappearance of the pole stain ing of NuMA in pig metaphase ii oocytes, whereas the mouse meiotic spindle pole has been reported to be resistant to the treatment. These results sugg est that the nature of the meiotic spindle differs between species. The axi s of the pig meiotic spindle rotated from a perpendicular to a parallel pos ition relative to the cell surface during telophase I. Further, in contrast to the stable localization of NuMA and gamma-tubulin at the spindle poles in mitosis, NuMA and gamma-tubulin became relocalized to the spindle midzon e during anaphase I and telophase I in pig oocytes. We postulate that in th e centrosome-free meiotic spindle, NuMA aggregates the spindle microtubules at the midzone during anaphase and telophase and that the polarity of meio tic spindle microtubules might become inverted during spindle elongation.