194 IN VITRO MATURATION AND RNA CONTENT AND DISTRIBUTION OF PORCINE OOCYTES DERIVED FROM SMALL AND MEDIUM FOLLICLES AND CLASSIFIED BY BRILLIANT CRESYL BLUE ASSAY

Abstract

Oocytes collected from the surface of slaughterhouse ovaries clearly have heterogeneous quality. The objective was to characterize a change or difference in RNA distribution and the content of porcine oocytes that were collected from small (SF; 1–2 mm in diameter) and medium follicles (MF; 3–6 mm in diameter) and assessed for glucose-6-phosphate dehydrogenase (G6PD) activity by brilliant cresyl blue (BCB) assay. Following BCB staining, porcine cumulus–oocyte complexes (COC) with dark blue (DB; G6PD-inactive, suggesting good quality) and light blue (LB; G6PD-active, suggesting immaturity) ooplasm were then separately cultured in vitro to evaluate nuclear maturation and analyze the characteristics of the RNA aspect. RNA distributions in cumulus cell mass and ooplasm were labeled with fluorescence, SYTO RNA select green and then examined at different periods of culture for in vitro maturation (IVM; with gonadotropins and dbcAMP for 20 h and then without those for 24 h). Total RNA content of oocytes and cumulus cell mass were measured by using a manufacture's kit (Invitrogen Quant-iT RNA assay kit). Statistical analyses of results from 3 to 5 replicated trials were performed by ANOVA with a Bonferroni/Dunn post-hoc test (significance, P < 0.05). When oocytes were classified by BCB assay, the percentage of oocytes with DB cytoplasm was much higher (P < 0.05) in oocytes from MF (72.5% of 208) compared with those of SF (53.6% of 352). Regardless of the origin of oocytes (SF vs MF), the incidence of mature oocytes following culture was higher in DB than LB (64.0% of 147 and 72.1% of 133 vs 50.9% of 108 and 52.8% of 54, respectively). Twenty hours after the start of IVM, the DB oocytes had a higher proportion of RNA-free zone inside the germinal vesicle compared to the LB oocytes (66.1% of 193 and 26.8% of 159 in MF; 47.9% of 185 and 17.9% of 184 in SF; P < 0.05). When the total content of RNA was examined during IVM, both oocytes from SF and MF contained higher levels of total RNA at the beginning of IVM as compared with 20 and 44 h after the start of IVM, whereas the content (40 denuded oocytes/sample) was higher in oocytes with DB cytoplasm from MF than those from SF (P < 0.05). Regardless of the origin of oocytes from SF and MF, the total content of RNA in oocytes with LB cytoplasm was significantly lower than in oocytes with DB cytoplasm (P < 0.05) before the start of IVM. The total RNA content of cumulus cells (40 COC/sample) before IVM culture was also higher in the cell mass surrounding the DB oocytes than the LB ones and in the cell mass surrounding the oocytes from MF rather than SF (P < 0.05). These results demonstrate a possibility that differences in RNA distribution and content in oocytes, as well as the content in cumulus cells, reflect in the ability of porcine oocytes to mature in vitro.