320 MPF ACTIVITY AND DEVELOPMENTAL COMPETENCE IN DIFFERENT SIZES OF PREPUBERTAL GOAT OOCYTES

Abstract

Developmental competence of prepubertal goat oocytes recovered from a slaughterhouse is low, probably due to an incomplete cytoplasmic maturation. Regulation of cytoplasmic maturation is still unknown, although maturation-promoting factor (MPF) is suggested to play an important role in this process. To better understand the role of MPF in cytoplasmic maturation, we have studied MPF kinase activity in oocytes with different developmental competence. Ovaries were obtained from a slaughterhouse, and oocytes were recovered by slicing and were selected according to morphological criteria. Some oocytes were denuded and classified in diameter groups (<110 μm, 110–125 μm, 125–135 μm, and >135 μm), placed in lysis buffer (50 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 5 mM EDTA, 0.01% Brij35, 1 mM phenyl methyl sultonyl fluoride (PMSF), 0.05 mg/mL leupeptin, 50 mM 2-mercaptoethanol, 25 mM α-glycerophosphate, 1 mM Na-orthovanadate) and frozen in liquid N₂. Cell extracts were stored at −80°C until use. The rest of oocytes were matured in vitro in medium TCM199 supplemented with hormones, 10% (DBS), and 400 μM cysteamine, for 27 h in 5% CO₂ in air and 38.5°C. After IVM, a sample of oocytes were also denuded, classified by diameters, and frozen as described above. The rest of oocytes were used for IVF in mDM with spermatozoa capacitated with heparin and ionomicin. After 24 h, presumptive zygotes were cultured for 7 days in medium SOF in 5% CO₂, 5% O₂, and 90% N₂ at 38.5°C. At 48 h post-insemination, we added 0.1 μL FBS per embryo. Embryonic development was evaluated with Hoechst staining after IVC. MPF kinase activity was detected using the MESACUP cdc2 kinase assay kit (MBL Woburn, MA, USA). Briefly, the oocyte extract corresponding to 10 oocytes was mixed with 10× reaction buffer (25 mM HEPES buffer (pH 7.5), 10 mM MgCl₂) and 10% biotinylated MV peptide (SLYSSPGGAYC). We added 0.1 mM ATP to start the reaction. The mixture was incubated at 30°C for 30 min. The reaction was finished by adding 200 μL of PBS containing 50 mM EGTA. The phosphorylated MV peptide was detected by specific antibody using an ELISA procedure, and the OD was measured at 492 nm. Fisher's exact test was used to analyze IVC results, and ANOVA to analyze cdc2 kinase activity results. We considered differences statistically significant when P < 0.05. Results are shown in Table 1. We observed that embryonic cleavage and blastocyst rate increased with oocyte diameter. The MPF activity detected was higher in the largest oocytes after IVM. As a consequence, we could establish that oocytes with a higher MPF activity are more capable of maintaining embryonic development until the blastocyst stage, which may indicate the important role that MPF plays in cytoplasmic maturation.

Table 1. Cleavage, blastocyst rate, and MPF kinase activity in different sizes of prepubertal goat oocytes