PARTHENOGENETIC DEVELOPMENT AND PROTEIN-PATTERNS OF NEWLY MATURED BOVINE OOCYTES AFTER CHEMICAL ACTIVATION

Development of an effective activation protocol is of great importance for studying oocyte competence and embryo cloning. Experiments were d esigned to examine effects of intracellular calcium elevating agents s uch as calcium ionophore A23187 (CaA) and ethanol, or protein synthesi s and phosphorylation inhibitors such as cycloheximide (CH) and 6-dime thylaminopurine (6-DMAP), or a sequential combination of these agents on both parthenogenetic development and protein patterns of newly matu red bovine oocytes. Oocytes were matured for 24 hr in M-199 supplement ed with follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol at 39 degrees C in humidified air. They were then activa ted by various treatments and cultured in KSOM. Protein patterns at 15 hr after treatment were determined on 8-15% gradient SDS-PAGE and sil ver stained. Results demonstrated that none of the chemical agents-CaA , ethanol, 6-DMAP, or cycloheximide-could effectively induce parthenog enetic development of young bovine oocytes. When compared with the sin gle treatments, sequentially combined treatments of CaA with 6-DMAP or with cycloheximide plus cytochalasin D (CD) significantly increased t he rates of cleavage (78-82% versus 3-13%) and blastocyst development (31-40% versus 0%), which were comparable with those of IVF group (80% and 35%, respectively; P> 0.05). Supplementation with CD to the combi ned CaA and CH treatment improved rates of cleavage and blastocyst dev elopment versus without CD supplementation (31% versus 7%; P< 0.05). F luorescent microscopy revealed that 95% (n = 40) of oocytes treated wi th CaA plus 6-DMAP had one pronucleus (PN) and one polar body (PB), wh ile 88% (n = 40) in the CaA plus cycloheximide-treated group had one P N and two PBs and 85% (n = 40) in CaA plus cycloheximide and CD group had two PNs and one PB. Treatment by CaA alone resulted in 73% of oocy tes (n = 40) arrested at a metaphase stage with two PBs (named as meta phase III or MIII). Protein patterns were similar for chemically activ ated and in vitro-fertilized (IVF) oocytes in that the 138- and 133-kD a proteins, whose functions are not yet known, were present in the met aphase-stage (MII 24 hr, MII 40 hr, and MIII) oocytes but were absent in PN-stage oocytes regardless of treatment. Therefore, these proteins seem to be metaphase-associated proteins. Taken together, we conclude that optimal parthenogenetic development of newly matured bovine oocy tes can be obtained by calcium ionophore treatment followed by incubat ion in either 6-DMAP or cycloheximide plus cytochalasin D and that the reduction of the 138- and 133-kDa proteins might be necessary for the full activation of bovine oocytes. (C) 1998 Wiley-Liss, Inc.