Microtubule patterning during meiotic maturation in mouse oocytes is determined by cell cycle-specific sorting and redistribution of gamma-tubulin.

The topography of microtubule assembly events during meiotic maturation of animal oocytes demands tight spatial control and temporal precision. To better understand what regulates the timing and location of microtubule assembly, synchronously maturing mouse oocytes were evaluated with respect to gamma-tubulin, pericentrin, and total tubulin polymer fractions at specific stages of meiotic progression. gamma-Tubulin remained associated with cytoplasmic centrosomes through diakinesis of meiosis-1. Following chromatin condensation and perinuclear centrosome aggregation, gamma-tubulin relocated to a nuclear lamina-bounded compartment in which meiosis-1 spindle assembly occurred. gamma-Tubulin was stably associated with the meiotic spindle from prometaphase-1 through to anaphase-2, but also exhibited cell cycle-specific relocalization to cytoplasmic centrosomes. Specifically, anaphase onset of both meiosis-1 and -2 was characterized by the concomitant appearance of gamma-tubulin and microtubule nucleation in subcortical centrosomes. Brief pulses of taxol applied at specific cell cycle stages enhanced detection of gamma-tubulin compartmentalization, consistent with a gamma-tubulin localization-dependent spatial restriction of microtubule assembly during meiotic progression. In addition, a taxol pulse during meiotic resumption impaired subsequent gamma-tubulin sorting, resulting in monopolar spindle formation and cell cycle arrest in meiosis-1; despite cell cycle arrest, polar body extrusion occurred roughly on schedule. Therefore, sorting of gamma-tubulin is involved in both the timing of location of meiotic spindle assembly as well as the coordination of karyokinesis and cytokinesis in mouse oocytes.

Combelles CM ，Albertini DF 《DEVELOPMENTAL BIOLOGY》

Sorting and reorganization of centrosomes during oocyte maturation in the mouse.

:In animal oocytes, the centrosome exists as an acentriolar aggregate of centrosomal material that is regulated in a dynamic manner throughout the process of meiotic maturation. Recently, it has been demonstrated that in female meiotic systems spindle assembly is likely regulated by chromosomal and microtubule/microtubule-associated influences. The purpose of this study was to analyze the distribution of the integral centrosomal protein, pericentrin, during the course of meiotic maturation. The function of the centrosome during meiotic progression was evaluated by exposing oocytes to pharmacological agents that perturb cytoplasmic homeostasis (cycloheximide, nocodazole, cytochalasin D, taxol, and vanadate). Pericentrin was localized to the spindle poles during metaphase of meiosis-I as O- and C-shaped structures. At anaphase, these structures fragment, become displaced from the spindle poles, and associate with the lateral spindle margin. The metaphase spindle at meiosis-II had incomplete pericentrin rings at both spindle poles. Vanadate treatment, a known inhibitor of dynein-ATPase, resulted in meiotic arrest, constriction of the spindle pole, and an aggregation of pericentrin at the spindle poles. After taxol exposure, pericentrin incorporation into both spindle poles and cytoplasmic centrosomes was increased. Treatment of oocytes with cycloheximide, nocodazole, and cytochalasin D, influenced early events associated with chromosome capture and spindle assembly and altered the number and distribution of cytoplasmic centrosomes. Thus, although pericentrin incorporation is not required for meiotic spindle formation, the dynamic reorganization of pericentrin and changes in centrosome microtubule nucleating capacity are involved in critical cell cycle transitions during meiotic maturation.

Carabatsos MJ ，Combelles CM ，Messinger SM ，Albertini DF ... -  《MICROSCOPY RESEARCH AND TECHNIQUE》

Microtubule assembly after treatment of pig oocytes with taxol: correlation with chromosomes, gamma-tubulin, and MAP kinase.

In this study, taxol was used as a tool to study the correlation of microtubule assembly with chromosomes, gamma-tubulin and phosphorylated mitogen-activated protein (MAP) kinase in pig oocytes at different maturational stages. Taxol treatment did not affect meiotic resumption and chromosome condensation but inhibited/disrupted chromosome alignment at the metaphase plate and bipolar spindle formation and thus meiotic progression. Microtubules were co-localized with chromosomes and were found to emanate from the chromosomes in taxol-treated oocytes, suggesting that chromosomes may serve as a source of microtubule organization. In addition, the concentric emanation of microtubules within the chromosome-surrounded area in taxol-treated oocytes suggests that microtubule emanation from the chromosomes may be directed by other microtubule-organizing material. The formation of one large spindle or >/=2 spindles in oocytes after taxol removal shows that minus end microtubule-organizing material can be normally located on both sides of chromosomes only when the chromosomes are aligned on the metaphase plate. The co-localization of gamma-tubulin and phosphorylated MAP kinase with microtubule assembly in both control and taxol-treated oocytes suggests that these two proteins are associated microtubule-nucleating material in pig oocytes. However, Western blot analysis showed that neither cytoplasmic microtubule aster formation nor extensive microtubule assembly in the chromosome region induced by taxol was caused by super-activation of MAP kinase. Taxol also induced microtubule assembly depending on chromosome distribution in the first polar body. The results suggest that chromosomes are always co-localized with microtubules and that emanation of microtubules from the chromosomes may be regulated/directed by microtubule-organizing material including gamma-tubulin and phosphorylated MAP kinase in pig oocytes.

Sun QY ，Lai L ，Wu GM ，Park KW ，Day BN ，Prather RS ，Schatten H ... -  《MOLECULAR REPRODUCTION AND DEVELOPMENT》

Allocation of gamma-tubulin between oocyte cortex and meiotic spindle influences asymmetric cytokinesis in the mouse oocyte.

Barrett SL ，Albertini DF 《BIOLOGY OF REPRODUCTION》

Distinctions in meiotic spindle structure and assembly during in vitro and in vivo maturation of mouse oocytes.

Sanfins A ，Lee GY ，Plancha CE ，Overstrom EW ，Albertini DF ... -  《BIOLOGY OF REPRODUCTION》