Abstract:
Developmental competence of prepubertal (1- to 2-month old) goat oocytes is compromised, probably because of an incomplete cytoplasmic maturation. Oocyte selection for IVM-IVF is based on morphological criteria. The main regulator of oocyte nuclear maturation is maturation promoting factor (MPF), and it could also be involved in cytoplasmic maturation. The present study tried to determined p34cdc2 (the catalytic subunit of MPF) expression in oocytes of different sizes before and after IVM. Prepubertal goat oocytes were matured in a conventional IVM medium (TCM199 with serum, hormones, and cysteamine) for 27 h. At collection time, a sample of oocytes was classified into 4 groups according to their diameter (<110 ?m; 110?125 ?m; 125?135 ?m; and >135 ?m), and nuclear stage was evaluated. After IVM, oocytes were classified by diameter (as described before) and stained to analyze nuclear stage. Before and after IVM, a sample of 10 oocytes of each diameter group was frozen at ?80�C. These oocytes were used to perform the detection of p34cdc2-RNA by RT-PCR. Briefly, RNA of 10 oocytes was extracted with TriReagent (Sigma-Aldrich, Madrid, Spain), and used to perform RT using the ThermoScript kit (Invitrogen, Barcelona, Spain). The cDNA corresponding to two oocytes was amplified by PCR, and each amplification band was measured by densitometry using the Quantity One PC program. Rabbit globin mRNA was used as an extrinsic control of the whole process. Nuclear stage data were analyzed by Fisher's exact test, and RT-PCR data were analyzed by one-way ANOVA test. At collection time, a high percentage of prepubertal goat oocytes had resumed meiosis. After IVM, the percentage of MII-oocytes was higher in larger-sized oocytes. At collection time, significantly higher p34cdc2-RNA expression was found in 125?135 ?m oocytes. After IVM, no differences were found among oocyte groups. During maturation, a decrease of p34cdc2-RNA was found in 125?135 ?m oocytes. In contrast, an increase of p34cdc2-RNA was found in 110-125 ?m oocytes. The nuclear stage in the smallest oocytes show their reduced ability to resume meiosis. IVM-oocytes of different diameters showed no difference in p34cdc2-RNA expression. This study was supported by MCYT (Spain), with the grant number AGL2000-0353.