Total RNA and protein content, Cyclin B 1 expression and developmental competence of prepubertal goat oocytes

The aim of this study was to examine the relationship between the developmental competence of oocytes and their total RNA and protein contents, and the level of Cyclin B1 transcription. Ovaries from prepubertal goats were collected from a slaughterhouse. Oocytes were recovered by slicing and those with two or more layers of cumulus cells and homogenous cytoplasm were matured in vitro (20-25 oocytes per drop) for 27 h. Both before and after IVM, samples of oocytes were denuded and categorised into four group treatments by diameter (< 110 mu m, 110-125 mu m, 125-135 mu m; > 135 mu m), separated into sub-groups of 10 oocytes per treatment-replicate and stored in liquid nitrogen until total RNA content analysis by spectophotometry, total protein content analysis by a colorimetric assay and Cyclin B1 transcription analysis by RT-PCR. For the study of developmental competence, the rest of the matured oocytes were fertilised in vitro in groups of 20-25 for 24 h. Presumptive zygotes were denuded, sorted into the four categories of diameter noted above, and placed into culture drops in groups of 18-25 for in vitro culture. Cleavage rate was evaluated at 48 hpi and embryo development at 8 d post-insemination. There were four replicates of each treatment for each assay or evaluation point of the experiment. There were no significant differences between the size categories of oocytes at collection in total RNA content, total protein content and Cyclin B1 mRNA. There were significant differences (P < 0.05) in the expression of Cyclin B1 before IVM with oocytes in the > 135mm diameter category having the highest value for this variant. There were no significant differences in these characteristics between the categories of oocyte diameter after lVM except in respect of total RNA content, which was lower for the largest size of oocytes (> 135 mu m; mean +/- S.D. = 12.3 +/- 1.84 ng/oocyte) than the other three size groups (19.2 +/- 1.38-22.1 +/- 4.44 ng/oocyte; P < 0.05). Significant differences (P < 0.05) in cleavage rate were observed between the different oocyte size categories (< I 10 mu m, 3.0%; 110-125 vLm, 32%; 125-135 mu m, 50%; > 135 mu m, 73 %). Only oocytes > 125 mu m diameter developed to the blastocyst stage (125-135 mu m, 7%; > 135 mu m, 10%). This study showed that the RNA content and the Cyclin B1 RNA expression of prepubertal goat oocytes, and their development to embryos varied between the different size categories of the oocytes. (c) 2007 Elsevier B.V. All rights reserved.