Regulação da maturação e expansão do cumulus pelas proteínas quinase A e C em oócitos bovinos

Abstract

The present study was conducted to determine the effect of protein kinase-C (PK-C) and the possible interaction with the protein kinase-A (PK-A) in the resumption of meiosis, nuclear maturation and cumulus expansion in bovine oocytes. Also, the effect of PK-C stimulation in cytoplasmic maturation and embryo development was investigated. In a pre-experiment, oocytes in germinal vesicle (GV), metaphase I (MI) and metaphase II (MH) stages were fixed in acetic acid: methanol (1:3) either on slides (FLL) or in petri dishes (FPL) and stained with 1% of lacmoid in phosphate buffered saline (PBS). Also, the oocytes were fixed on slides immersed in acetic acid: methanol (1:3) and stained with Giemsa (FLG). In the experiments that studies the role of second messengers on oocyte maturation, the oocytes were aspirated using a vacuum pump from 2-8mm follicles, from ovaries obtained in a slaughterhouse, and were cultured in modified TCM 199 medium containing 25mM hepes, 2.2mg/ml sodium bicarbonate and 3mg/ml bovine serum albumin (BSA) at 39°C under an atmosphere of 5% CO2 in air and saturated humidity. The following experiments were accomplished: l) the oocytes were stimulated by different second messengers during 7 or 18h to evaluate nuclear maturation (at 7h and 18h) and cumulus expansion (at 18h); 2) oocytes were stimulated in the presence of fetal bovine serum, and the effects of PK-A and PK-C in the regulation of nuclear maturation were investigated; 3) oocytes were cultured during a period of 24h in the presence of estrous cow serum suppiemented with LH (1.5mg/ml), FSH and LH (0.5mg/ml and 1.5mg/ml) or PMA (active phorbol ester, stimulator of PK-C; l00nM), for investigating cleavage and embryo development rates. In the pre-experiment, the GV stage was accurately identified only with the FLL technique. However, FPL and FLG procedures were more efficient for oocytes in the MI and MII stages. PMA and l .2-dioctanoyl-sn-glycerol (DiC8) stimulated meiotic resumption and nuclear maturation in a dose dependent manner (0-l00nM and 0-l0mM, respectively). The treatment with forskolin (FK) significantly inhibits resumption of meiosis and decreases the percentage of oocytes in the MH stage, but PK-C stimulation reversed this inhibitory effect. Forkolin stimulated cumulus expansion in the presence and absence of PMA; however, PMA had only a slight effect in cumulus expansion. There were no significant differences in cleavage and embryo development among treatment groups. The results suggest that PK-C is involved in the regulation of oocyte maturation in the bovine, stimulating both the resumption of meiosis and nuclear maturation. Also, PK-C suppresses the inhibitory effect of PK-A on oocyte nuclear maturation. In regarding to cumulus expansion, PK-C and PK-A are involved in the regulation of cumulus cells mucification.