Spindle Formation and Dynamics of γ-Tubulin and Nuclear Mitotic Apparatus Protein Distribution During Meiosis in Pig and Mouse Oocytes1

Abstract

This work focuses on the assembly and transformation of the spindle during the progression through the meiotic cell cycle. For this purpose, immunofluorescent confocal microscopy was used in comparative studies to determine the spatial distribution of alpha- and gamma-tubulin and nuclear mitotic apparatus protein (NuMA) from late G2 to the end of M phase in both meiosis and mitosis. In pig endothelial cells, consistent with previous reports, gamma-tubulin was localized at the centrosomes in both interphase and M phase, and NuMA was localized in the interphase nucleus and at mitotic spindle poles. During meiotic progression in pig oocytes, gamma-tubulin and NuMA were initially detected in a uniform distribution across the nucleus. In early diakinesis and just before germinal vesicle breakdown, microtubules were first detected around the periphery of the germinal vesicle and cell cortex. At late diakinesis, a mass of multi-arrayed microtubules was formed around chromosomes. In parallel, NuMA localization changed from an amorphous to a highly aggregated form in the vicinity of the chromosomes, but gamma-tubulin localization remained in an amorphous form surrounding the chromosomes. Then the NuMA foci moved away from the condensed chromosomes and aligned at both poles of a barrel-shaped metaphase I spindle while gamma-tubulin was localized 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 inhibitor of microtubule polymerization, induced disappearance of the pole staining of NuMA in pig metaphase II oocytes, whereas the mouse meiotic spindle pole has been reported to be resistant to the treatment. These results suggest that the nature of the meiotic spindle differs between species. The axis of the pig meiotic spindle rotated from a perpendicular to a parallel position 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 midzone during anaphase I and telophase I in pig oocytes. We postulate that in the centrosome-free meiotic spindle, NuMA aggregates the spindle microtubules at the midzone during anaphase and telophase and that the polarity of meiotic spindle microtubules might become inverted during spindle elongation.

Pins homolog LGN regulates meiotic spindle organization in mouse oocytes

“…In mouse oocytes, LGN-N-GFP localized to the spindle apparatus, but was not restricted to the spindle poles. This localization pattern is similar to that of NuMA, which is localized along the meiotic spindle microtubules in mouse oocytes [26]. We speculate that the localization difference between LGN (and LGN-N) on mitotic spindles in MDCK cells and LGN (and LGN-N) on meiotic spindles in mouse oocytes may stem from the different localization of NuMA.…”

“…Previously, researchers have reported that LGN regulates mitotic spindle organization in somatic cells by interacting with and inhibiting the microtubule binding and stabilizing activity of NuMA [16,18]. In mouse oocytes, instead of concentrating at the spindle poles like γ-tubulin, NuMA is localized along spindle microtubules [26], similar to the spindle localization of LGN. Therefore, excessive LGN might inhibit NuMA and consequently disturb the targeting of Kif2a and/or other microtubule depolymerizing factors to the spindle poles and ultimately result in elongated meiotic spindles in mouse oocytes.…”

NuMA distribution and microtubule configuration in rabbit oocytes and cloned embryos

“…The NuMA foci were translocated from the condensed chromosomes and became aligned at both poles of the meiotic spindle during the metaphase I and II stages. Comparing with previous studies, we found that NuMA distribution in rabbit species is similar to pig (Lee et al 2000) but different from mouse (Lee et al 2000, Tang et al 2004. In mouse oocytes, NuMA was localized along spindle microtubules (Lee et al 2000) and at cytasters (Tang et al 2004).…”

“…As the role in meiosis, NuMA appeared to localize to spindle poles of the meiotic pig and mouse oocytes and treatment with nocodazole disrupted the localization of NuMA (Lee et al 2000). In maturating Xenopus oocytes, the microinjection of NuMA antibodies perturbed the organization of the transient MT array and the meiotic spindle (Becker et al 2003).…”

“…In previous studies, the researchers analyzed the expression pattern and function of NuMA in mouse and pig oocytes (Lee et al 2000) and early embryos (Tang et al 2004) as well as mouse (Zhong et al 2005, Van Thuan et al 2006 and pig cloned embryos. But the dynamic changes of NuMA and microtubules during meiosis I-II transition in rabbit oocytes have not been reported.…”