242 DETECTION OF MICROTUBULES BY POLARIZED LIGHT MICROSCOPY IN BOVINE OOCYTES

Abstract

Polarized light microscopy (PLM) is being used as a tool in assisted reproductive technologies in humans and, more recently, in farm animals. Polarized light microscopy may allow for a quick localization or removal of nuclear structures, or both in the oocyte without fluorochrome staining and ultraviolet exposure, and may reduce nuclear damage when performing intra-cytoplasmic sperm injection. The aim of this study was to assess the efficiency of PLM to detect microtubule-polymerized protein within in vitro-matured bovine oocytes. Cumulus–oocyte complexes from slaughterhouse ovaries were matured in vitro for 23 h in bicarbonate-buffered TCM-199, FSHp (1 μg mL^–1), LH (5 μg mL^–1), 17β-estradiol (1 μg mL^–1), and 10% fetal calf serum at 38.7°C in 5% CO₂ in air with high humidity. After in vitro maturation, oocytes (n = 98) were denuded with 0.5% hyaluronidase and were placed individually in 10-μL drops of TCM-199-HEPES-BSA in a glass Petri dish. Polarized light microscopy was used to detect the presence of polymerized protein that could be forming a meiotic spindle. To confirm the presence of the polymerized protein and the meiotic spindle, each individual oocyte was subjected to immunostaining and chromatin detection as described by Morató et al. 2008 Mol. Reprod. Dev. 75, 191–201. The experiment was replicated 6 times. There was an absolute positive correlation (r = 1; P < 0.0001) between the signal obtained by PLM and the presence of microtubule-polymerized protein as confirmed by immunostaining. A barrel-shaped spindle was observed in 40% of the individual samples, whereas disorganized microtubule structures were obtained in 55% of the samples. The biological implication of these findings needs to be explored. However, PLM seems to be an efficient system to detect polymerized protein in in vitro-matured bovine oocytes.