Abstract
Meiotic maturation of mammalian oocytes is a complex process during which microfilaments and microtubules provide the framework for chromosomal reorganisation and cell division. The aim of this study was to use fluorescence and confocal laser scanning microscopy to examine changes in the distribution of these important cytoskeletal elements and their relationship to chromatin configuration during the maturation of horse oocytes in vitro. Oocytes were cultured in M199 supplemented with pFSH and eLH and, at 0, 12, 24, and 36 hr after the onset of culture, they were fixed for immunocytochemistry and stained with markers for microtubules (a monoclonal antiโฮฑโtubulin antibody), microfilaments (AlexaFluor 488 Phalloidin) and DNA (TOโPRO~3~). At the germinal vesicle stage, oocyte chromatin was amorphous and poorly condensed and the microfilaments and microtubules were distributed relatively evenly throughout the ooplasm. After germinal vesicle breakdown, the microtubules were aggregated around the now condensed chromosomes and the microfilaments had become concentrated within the oocyte cortex. During metaphase I, microtubules were detected only in the meiotic spindle, as elongated asters encompassing the aligned chromosomes, and, as maturation progressed through anaphaseโI and telophaseโI, the spindle assumed a more eccentric position and gradually rotated to assist in the separation of the homologous chromosomes and in the subsequent formation of the first polar body. During metaphase II, the meiotic spindle was a symmetrical, barrelโshaped structure with two poles and with the chromosomes aligned along its midline. At this stage, microtubules were found intermingled with chromatin within the polar body and, although, the bulk of the microfilaments remained within the oocyte cortex, a rich domain was found overlying the spindle. Thus, during the in vitro maturation of horse oocytes both the microfilament and microtubular elements of the cytoskeleton were seen to reorganise dramatically in a fashion that appeared to enable chromosomal alignment and segregation. Mol. Reprod. Dev. 60: 260โ269, 2001. ยฉ 2001 WileyโLiss, Inc.