262 MEIOTIC SPINDLE CONFORMATION ASSESSMENT BY POLARIZED LIGHT MICROSCOPY IN SHEEP AND GOAT OOCYTES

Abstract

Polarized light microscopy (PLM) allows detection of microtubule-polymerized protein in in vitro-matured sheep and goat oocytes (Caamaño et al. 2011 Reprod. Fertil. Dev. 23, 226–227). Spindle birefringence, measured as the mean retardance value, has been proposed as a marker of meiotic spindle conformation in humans and mice. Because the conformation of the meiotic spindle cannot be readily assessed by PLM, in this study we aimed to measure the mean retardance value of normal and abnormal meiotic spindles from in vitro-matured prepubertal sheep and goat oocytes. Oocytes were matured in vitro for 27 h and were then individually assessed by PLM (Oosight System, Research Instruments Ltd., Falmouth, Cornwall, UK), and the mean retardance value was analysed in meiotic spindles using specific software (Oosight). Meiotic spindle conformation was determined in individual oocytes by immunostaining and chromatin detection, as described by Morató et al. (2008 Mol. Reprod. Dev. 75, 191–201). A barrel-shaped spindle was considered a normal spindle configuration. Only oocytes with meiotic spindles identified by both PLM and immunostaining were used in this study. The experiment was replicated four times. Data were analysed by the general linear models procedure of SAS (SAS Institute Inc., Cary, NC, USA). A normal barrel-shaped spindle was observed in 80.8% of the sheep oocytes (n = 47) and in 83.9% of the goat oocytes (n = 62). In sheep, the mean retardance value in oocytes with a normal meiotic spindle conformation did not differ from that in oocytes with abnormal spindles (4.42 ± 0.26 nm v. 3.92 ± 0.54 nm). Similar results were obtained with goat oocytes with normal (2.94 ± 0.20 nm) v. abnormal spindle conformation (2.77 ± 0.08 nm). These results indicate that the mean retardance value does not distinguish between oocytes with normal and abnormal meiotic spindle conformation, as assessed by subsequent immunostaining.

Grant support from INIA-RZ2007-00013-00-00. M. Muñoz was sponsored by RYC08-03454.