Dynamics of the spindle pole matrix protein NuMA and dynactin during human oocyte and zygote development

Abstract

OBJECTIVE: The nature of the meiotic spindle differs between species. In particular, its transformation during the progression through the meiotic cell cycle up to the zygote stage after fertilization is poorly understood in humans. This work focuses on the study of the assembly of the nuclear mitotic apparatus protein (NuMA), α/β tubulins and dynactin during human female meiosis and fertilization. DESIGN: Immunofluorescence microscopy and pharmacological inhibition studies. MATERIALS AND METHODS: Confocal microscopy was used to determine the spatial distribution of NuMA and α/β tubulins from GV oocytes to pronuclear stage zygotes after fertilization. A total of 86 oocytes and discarded supernumerary pronuclear stage zygotes were analyzed. The material were fixed and permeabilized with a combination of 2% formaldehyde and 1%Triton X-100 previously described. Incubation with primary and secondary antibodies was done over night at room temperature and for one hour at 37°C respectively. DNA was labeled using TOTO-3. Inhibition of phosphatases and ATPases activity (dynein dependent transport) was carried out during 1 and 2 hs in 400 μM Sodium-Orthovanadate (SOV). After treatment, the distribution of NuMA and dynactin was analyzed. RESULTS: At GV stage, NuMA was evenly spread across the nucleoplasm. Before GVBD, some discrete NuMA foci were detected close to chromosomes but most of the NuMA protein was still diffusely localized in the nucleus. After GVBD, NuMA became tightly associated with the chromosome cluster. Metaphase I and II oocytes show NuMA localized at the spindle poles as discrete spots. After fertilization, all normal (2PNs) and abnormal (≥3PNs) pronuclear stage zygotes show NuMA translocated to the interphase nuclei. SOV treatment during 2hs of human oocyte maturation induces the formation of DNA spread, ectopic localization of NuMA and lower maturation rate compared to controls. CONCLUSIONS: The study of NuMA in germ cells and zygotes is relatively new and largely unexplored in humans. The identification of NuMA in different stages of matured oocytes and zygotes suggests its importance during bipolar spindle assembly. Further investigation of NuMA in abnormal oocytes and embryos, will enhance our understanding of its contribution during the assembly of microtubule spindle and the segregation of chromosomes during meiosis and mitosis.