PARENT SESSION
GAMETE BIOLOGY AND GAMETOGENESIS - A

Monday, August 2, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(161) THE M-II MEIOTIC SPINDLE PHENOTYPE IN VIVO IS DETERMINED EARLY IN MEIOTIC PROGRESSION.

Sanfins, Alexandra^{1, 2}, Plancha, Carlos ², Overstrom, Eric ³, Albertini, David¹, ¹ Department of Anatomy and Cellular Biology, Boston, MA² Instituto de Medicina Molecular, Lisboa, Portugal³ Department of Biomedical Sciences, Grafton, MA

ABSTRACT- We have shown that naturally ovulated or superovulated mouse oocytes exhibit properties consistent with enhanced quality (e.g. reduced spindle size, increased number of MTOCs) relative to in vitro matured (IVM) oocytes (Sanfins et al., 2003; BOR 69:2059-2067). To better understand when in the course of meiotic progression these distinctions in oocyte phenotype arise, we have conducted two sets of experiments. First, oocytes were recovered hourly from 1 to 5 hours post-hCG to establish the rate of GVBD and spindle assembly. In vivo matured (IVO) oocytes retain the GV for a longer period of time when compared to their IVM counterparts (GVBD occurs at 3 hours in IVO oocytes versus 2 hours in IVM oocytes). In contrast, most IVO oocytes have completed spindle assembly by 5 hours post-hCG whereas this milestone is protracted in IVM oocytes (> 5 hours). To determine the effects of the intrafollicular residence time on achieving IVO properties, oocytes were collected 1.5 and 5 hours post-hCG and were cultured as intact cumulus complexes up to 16 hours. Oocytes removed 5 h post-hCG exhibited striking similarities to IVO oocytes whereas those removed at 1.5 h more closely resembled IVM oocytes, suggesting that delayed GVBD in vivo influences the mature oocyte phenotype. Since GVBD was delayed in IVO oocytes, we next analyzed the effects of delaying GVBD by roscovitine on the M-II phenotype. Overall reversibility of the drug was more effective after 3 hours when compared to 6 hours (74.8% M-IIs at 3 hours versus 48.8% at 6 hours) and under both conditions oocytes maintained a G2 cell cycle state. After either 3 or 6 hours of treatment and wash out, oocytes that progressed to M-II exhibited reduced spindle size and an increased number of MTOCs consistent with attainment of an IVO state. In conclusion, these studies have identified a critical window between 1.5-5 hours following hCG when events crucial to the final development of the oocyte are initiated. This work will aid in defining better conditions for IVM using markers that reflect key aspects of nuclear and cytoplasmic maturation.

KEY WORDS: in vitro, spindle, in vivo, maturation