263 DESCRIPTION OF CORTICAL GRANULE DISTRIBUTION IN OVINE MATURED OOCYTES: PRELIMINARY RESULTS

Abstract

In mammalian oocytes, the migration of cortical granules (CG) is an important step in cytoplasmic maturation and has been used as a criterion in the assessment of maturity and organelle organisation. With the use of fluorescent lectins, it is possible to identify specific sugars originating from the CG and to localise them to the oolemma, within the perivitelline space, or both. To our knowledge, no reports are available on fluorescence assessment of CG organisation in ovine oocytes. Therefore, the aim of this study was to describe the CG distribution of ovine oocytes after in vitro maturation (IVM). One hundred twenty-five oocytes from adults ewes were submitted to IVM in TCM-199 medium supplemented with 10% FBS for 24 h. The denuded oocytes were immersed in 0.1% pronase for 5 min to dissolve the zona pellucidae, fixed in 3% paraformaldehyde for 30 min, and incubated overnight in blocking solution at 4°C. Oocytes were placed into 0.1% Triton X-100 for 5 min for permeabilization and incubated in 10 µg mL–1 of fluorescent Lens culinaris agglutinin–fluorescein complex for 15 min at 37°C before being rinsed and mounted onto histological slides. The distribution of CG was evaluated with an Olympus fluorescence microscope (wavelength: 420 to 490 nm; Olympus, Tokyo, Japan), and oocytes were classified into three types according to the observed distributional pattern of the CG: (I) aggregates of CG distributed over the entire cytoplasm, (II) CG distributed in the cortex and forming a fluorescent halo around the plasma membrane, and (III) CG distributed uniformly over the entire cytoplasm without the presence of aggregates. Proportions of oocyte types were analysed by chi-square or, when appropriate, by Fisher’s exact test, using SAS version 8.2 (SAS Institute Inc., Cary, NC, USA). The proportion of type III oocytes (98/125, 78.4%) was significantly higher (P < 0.05) than those of types I (10/125, 8%) and II (17/125, 13.6%). No significant difference was observed between the proportions of type I and II oocytes. Analogous with the results described in the bovine, the type I distribution of CG in ovine oocytes has been considered a sign of a lack of maturation, even if the aggregates of CG are not as evident as in the bovine; types II and III were considered mature oocytes based on the cortical migration of CG. The cytoplasmic maturation is an essential process in directing and supporting the events of fertilization and early embryonic development. Therefore, describing the species-specific pattern of CG distribution is important for the assessment of oocyte maturation. More studies in the ovine are necessary to confirm the correlation between CG distribution and maturation status, as in bovine species.