285 SUPPRESSION OF MPF AND MAPK ACTIVITIES BY DIBUTYRYL CAMP DURING FIRST MEIOTIC MATURATION IMPROVES SUBSEQUENT DEVELOPMENT OF PORCINE OOCYTES
J.-S. Kim A , D.-B.n Koo A , J.-I. Chae A , Y.-K. Choo B , K.-K. Lee A and Y.-M. Han AA Laboratory of Development and Differentiation, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea
B Department of Life Science, Wonkwang University, Iksan, Korea. Email: leekk@kribb.re.kr
Reproduction, Fertility and Development 17(2) 293-293 https://doi.org/10.1071/RDv17n2Ab285
Submitted: 1 August 2004 Accepted: 1 October 2004 Published: 1 January 2005
Abstract
Maintaining the germinal vesicle (GV) stage in growing oocytes is essential for developmental competence of the eggs. In pig oocytes, MPF and MAPK activities are low during the GV stage, and their activities increase with progression of meiosis I. In general, cAMP that exists at high levels in GV oocytes inhibits germinal vesicle break down (GVBD). After gonadotrophin stimulation, the amount of cAMP in oocyte cytoplasm is decreased gradually for meiotic resumption. This study was conducted to examine the effect of dibutyryl cAMP (dbcAMP) on nuclear maturation, fertilization, and early embryo development of porcine oocytes. Oocytes were cultured in NCSU23 medium with or without dbcAMP for 22 h, and then cultured in fresh maturation medium for an additional 22 h prior to fertilization. The activities of MPF and MAPK were evaluated by Western blot analysis by using specific antibodies such as anti-cdc2 and anti-ERK1/2 in maturing pig oocytes at 22 h and 44 h. In vitro fertilization was performed with fresh ejaculated spermatozoa in the modified TRIS-buffered medium, and fertilized embryos were cultured in NCSU23 medium. When treated with dbcAMP for 22 h, most oocytes (204/224, 91.9%) were arrested in GV stage by blocking meiotic resumption. The activities of constituent proteins (cdc2 and ERK1/2) of MPF and MAPK were also suppressed in dbcAMP-treated oocytes. After completion of IVM, dbcAMP-treated oocytes showed a higher proportion of the metaphase II stage than non-treated ones (156/171, 91.3% vs. 121/167, 72.8%; P < 0.05). Furthermore, incubation of 44 h matured oocytes with dbcAMP for 22 h increased the MPF and MAPK activities. In the dbcAMP-treated group, penetration rate was increased (126/145, 80.0%) and polyspermy rate was reduced (26/126, 22.4%) as compared to the nontreated group (97/140, 69.3% and 46/97, 47.4%, respectively; P < 0.05). Furthermore, blastocyst formation of dbcAMP-treated eggs was also improved compared to the nontreated group (47/126, 37.0% vs. 28/146, 17.2%; P < 0.05). Our results suggest that the synchronization of meiotic resumption by dbcAMP may support meiotic maturation and in vitro development of pig eggs.
