Nuclear Status and Cytoplasmic Ultrastructure of Canine Oocyte in Relation to in vitro Spontaneous Denudation

Introduction - In contrast with most mammals, the canine oocyte is released at the germinal vesicle (GV, immature) stage and nuclear maturation is completed after 60-72h spent in the oviduct. In vitro maturation (IVM) rate is really low (10-30%), which represents the main limiting factor and a key step for further development of assisted reproductive technologies in canine species. In other mammals, oocyte meiotic resumption is controlled by factors secreted by the surrounding granulosa cells and transmitted to the oocyte via transzonal projections. In the canine oocyte, the exact role of granulosa cells is poorly documented. Some authors reported that removal of granulosa cells before IVM may improve meiotic resumption rate (Reynaud et al., 2006). Moreover, Otoi et al. (2007) showed that spontaneous denudation may lead to higher meiosis resumption rates.

Objectives - In this study, we aimed to analyze the effect of granulosa cells (absence/presence) on meiosis resumption and oocyte quality. Chromatin configuration was evaluated by confocal microscopy, ensuring a low rate of undetermined nuclear status (Saint- Dizier et al., 2004). Furthermore, we evaluated the cytoplasmic and nuclear qualities of the oocytes by an ultrastructural approach by Electron Transmission Microscopy.

Materials and methods - A total of 3440 oocytes from 34 anoestrus and metoestrus pubertal bitches were collected and in vitro matured for 72h in M199/20% Fetal Calf Serum. At the end of IVM, 2 oocytes populations were fixed for evaluation: Denuded oocytes (DO) with at least 75% of their surface spontaneously denuded were distinguished from Cumulus- Enclosed-Oocytes (CEO), with 100% of their surface still surrounded by granulosa cells.

Results - Spontaneous denudation was observed in 12.9% of the oocytes. The degeneration rates were not significantly different between groups: 33.4% for DO and 24.8% for CEO. Considering the non-degenerated oocytes, the meiosis resumption rate was significantly higher for the DO than the CEO (respectively 10 % versus 2.8 %). However, despite this higher maturation rate, ultrastructural analysis revealed that DO at metaphase I and II stages still presented an immature cytoplasm with few organelles. On the contrary, CEO at MI and MII stages were shown closer characteristics to in vivo oocytes but the spatial distribution of mitochondria was heterogeneous.

Conclusions - even if spontaneous denudation during culture may lead to higher MI/MII rates, the quality of these oocytes seems to be deficient to support fertilization and early embryo development. Spontaneous denudation during IVM may therefore not be a good criteria to sort oocytes before in vitro fertilization.