Nuclear and cytoplasmic maturation of canine oocytes related to in vitro denudation

Abstract

In vitro maturation (IVM) of bitch oocytes is, to date, a very inefficient process, with common metaphase rates approximately 0-20% and mean degeneration rates approximately 20-30%. In other mammals, meiotic resumption is controlled in the cumulus-oocyte complex by the disappearance of the coupling between granulosa cells and the oocyte. The first aim of this study was to evaluate the influence on meiotic resumption of a mechanical denudation of the oocytes before maturation. The nuclear stage was determined by DNA staining with ethidium-homodimer-2 under confocal microscopy. Denuded (n = 318) and control (n = 378; no mechanical denudation) oocytes had similar degeneration rates (respectively 32.1 vs 28.6%). However, meiosis resumption rates were significantly higher for denuded oocytes (DO; 26.9 vs 17.8%). Secondly, we aimed to evaluate oocytes experiencing spontaneous denudation during the 72 h IVM period. Denuded oocytes, having lost cumulus cells on at least 75% of their perimeter (n = 440), were compared with surrounded oocytes (SO), with 100% of their perimeter surrounded by granulosa cells (n = 860). As above, the nuclear stage was determined by confocal microscopy, but cytoplasmic maturation was also evaluated through transmission electron microscopy. Degeneration rates but also meiosis resumption and metaphase rates were significantly higher for denuded than for SO (9.6 vs 2.8% for metaphase rate). Nevertheless, ultrastructurally, metaphase DO have scarcer organelles unevenly distributed, with smooth endoplasmic reticulum concentrated in aggregates in the cortical zone. Denudation, whether mechanical or spontaneous, is thus an inefficient mean to obtain metaphase II oocytes suitable for in vitro fertilization.