Total RNA and protein content, Cyclin B1 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 microm, 110-125 microm, 125-135 microm; >135 microm), 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 >135 mm diameter category having the highest value for this variant. There were no significant differences in these characteristics between the categories of oocyte diameter after IVM except in respect of total RNA content, which was lower for the largest size of oocytes (>135 microm; 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 (<110 microm, 3.0%; 110-125 microm, 32%; 125-135 microm, 50%; >135 microm, 73%). Only oocytes >125 microm diameter developed to the blastocyst stage (125-135 microm, 7%; >135 microm, 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.
Anguita
Begoña
B
Departament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.
Paramio
Maria-Teresa
MT
Jiménez-Macedo
Ana R
AR
Morató
Roser
R
Mogas
Teresa
T
Izquierdo
Dolors
D
D000818:Animals; D002478:Cells, Cultured; D019926:Cyclin B / Q000235:genetics*; D056744:Cyclin B1; D047108:Embryonic Development / Q000502:physiology; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D005786:Gene Expression Regulation; D006041:Goats / Q000196:embryology / Q000502:physiology*; D008297:Male; D009865:Oocytes / Q000378:metabolism / Q000502:physiology*; D011506:Proteins / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D013997:Time Factors
D019926:Cyclin B; D056744:Cyclin B1; D011506:Proteins; D012313:RNA