Influence of different stages of the oestrous cycle on cumulus-oocyte communications in canine oocytes : effects on the efficiency of in vitro maturation

The aim of this study was to investigate the effects of different stages of the oestrous cycle on the communications between dog oocytes and cumulus cells, as well as their possible relationships with the meiotic competence of oocytes matured in vitro. Ovaries were harvested from bitches undergoing anoestrus or late pro-oestrus. Maturation of cumulus-oocyte complexes (COCs) was performed in TCM-199 with 0.3% (w/v) BSA, 0.5 iu FSH ml-1 and 0.5 iu LH ml-1 for 72 h. At the time of collection (0 h), and after 24, 48 or 72 h of culture, some oocytes were stained with Lacmoid to evaluate the nuclear stage of maturation. At 0, 24 or 48 h of in vitro maturation (IVM), oocytes were microinjected (n = 84) with a 3% (w/v) Lucifer yellow solution to evaluate the presence of cumulus-oocyte communications through gap junctions. At t = 0, communications were open in 89% of COCs collected during late pro-oestrus, whereas such communications were not evident in any of the oocytes collected during anoestrus. Communications remained permeable for the first 24 h of maturation in many late pro-oestrous oocytes (67%), whereas no fluorescence diffusion was observed at 48 h of maturation. The presence of cumulus-oocyte communications in late pro-oestrous oocytes was accompanied by their ability to resume meiosis and reach telophase I or metaphase II at higher rates than in oocytes collected during anoestrus (11.1% versus 0%, respectively; P < 0.01). These results indicate that, during anoestrus, there are no permeable gap junctions between oocytes and surrounding cumulus cells at the time of isolation from the ovary. This finding indicates that such oocytes are unsuitable for IVM, as confirmed by their inability to reach telophase I or metaphase II in culture. In contrast, a positive correlation was observed between the presence of permeable gap junctions and meiotic competence in COCs isolated at late pro-oestrus. However, the small proportion of oocytes reaching telophase I and metaphase II indicates that further efforts to define better culture conditions are still required.