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Allocation of Gamma-Tubulin Between Oocyte Cortex and Meiotic Spindle Influences Asymmetric Cytokinesis in the Mouse Oocyte¹

Program in Cellular, Molecular and Developmental Biology,³ Tufts University School of Medicine, Boston, Massachusetts 02111 Department of Molecular and Integrative Physiology,⁴ The Center for Reproductive Sciences,⁵ University of Kansas, Kansas City, Kansas 66160 Marine Biological Laboratory,⁶ Woods Hole, Massachusetts 02543

ABSTRACT

In oocytes, asymmetric cytokinesis represents a conserved strategy for karyokinesis during meiosis to retain ooplasmic maternal factors needed after fertilization. Given the role of gamma-tubulin in cell cycle progression and microtubule dynamics, this study focused on gamma-tubulin as a key regulator of asymmetric cytokinesis in mouse oocytes. Gamma-tubulin properties were studied using multiple-label digital imaging, Western blots, quantitative RT-PCR, and microinjection strategies in mouse oocytes matured in vivo (IVO) or in vitro (IVM). Quantitative image analysis established that IVO oocytes extrude smaller first polar bodies (PBs), contain smaller spindles, and have more cytoplasmic microtubule organizing centers (MTOCs) relative to IVM oocytes. Maturation in culture was shown to alter gamma-tubulin distribution, as evidenced by incorporation throughout the meiotic spindle and within the first PB. Western blot analysis confirmed that total gamma-tubulin content remained elevated in IVM oocytes compared with IVO oocytes. Analysis of gamma-tubulin mRNA during maturation revealed fluctuations in IVO oocytes, whereas IVM oocytes maintained relatively stable at lower levels for the time points examined (0–16 h). Selective reduction of gamma-tubulin mRNA by injection of siRNA diminished both spindle and PB size, whereas overexpression of enhanced green fluorescent protein gamma-tubulin had the opposite effect. Together, these studies reinforce the notion that limiting gamma-tubulin availability during meiotic maturation ensures coordination of karyokinesis and cytokinesis and conservation of gamma-tubulin as an embryonic reserve.

cumulus cells,, follicle-stimulating hormone, meiosis, oocyte development, ovum

¹Supported by National Institutes of Health grant RO1-HD42076, the Eshe Fund, Serono Reproductive Biology Institute, and The Hall Family Foundation.

Correspondence: ²David F. Albertini, 3901 Rainbow Blvd., University of Kansas Medical Center, Kansas City, KS 66160. FAX: 913 588 0456; e-mail: dalbertini{at}kumc.edu