N OCIETY' - the Society for Reproductive Biology

SUCCESSFUL DEVELOPMENT OF A 6·DMAP REPLACEMENT FOR BOVINE OOCYTEACTIVATION RESEARCH.W.G. James, O.La~ham-Kaplan and A.a. TrounsonInstitute ofReproduction and Development, Monash University, Melbourne, Australia.IntroductionAn efficient method of activation following nucleartransfer and sperm injection in bovine is necessary toinduce successful preimplantation development. Theactivation treatment currently considered to be themost successful is the Ca++-ionophore A23187followed by 6-dimethylaminopurine (6-DMAP) (1).A similar success with oocyte activation has beenobserved in the sheep with ethanol exposure followedby 6-DMAP (2). However, concerns have been raisedto the effects the non-specific protein kinase inhibitor6-DMAP might have on embryo development (3,4).6-DMAP has been shown to accelerate thedisappearance of phosphorylated forms of proteins.Development of 6-DMAP treated mouse embryosresulted in severely compromised post-implantationdevelopment with no live pups born (3).The agentcycloheximide can be used to replace 6-DMAP,targetting the synthesis of proteins rather than theirphosphorylation. Cycloheximide treatment is capableof activating both young and aging oocytes at highrates. The majority of other artificial stimuli areunable to activate young oocytes adequately (5). Thishas often limited nuclear transfer procedures to theuse of aging oocytes, in which cytoskeletaldeterioration has been observed (5). The presentstudy investigated the effect of cycloheximidetreatment following ethanol exposure on IVM bovineoocytes.Materials & MethodsImmature oocytes were aspirated from abattoircollected ovaries and matured in-vitro for 24hr (6).Prior to activation, oocytes were denuded of allcumulus by vortexing for 3min in 50111 hyaluronidasesolution (lmg/ml). Mature oocytes that had extrudeda ~rst. polar body were used for the study. TheactIvatIOn treatments which were examined are: [1]A23187 (38IlM; 10min) followed by 6-DMAP (2JlM;5hr); [2] ethanol (7%;5min) followed by 6-DMAP(2IlM ; 5hr); [3] ethanol (7%;5min) followed bycycloheximide (IOllg/ml) and cytochalasin-B(5Ilg/ml) (5hr); [4] ethanol alone (7%;5min). 6­DMA~ treatment results in diploid parthogenomes,so to SImulate this cytochalasin-B was combined withthe cycloheximide treatment. Treated oocytes werewashed through drops of equilibratedSOF120AAIBSA medium, and cultured for 3 days inSOF120AAIBSA at 39°C at 7% O 2, 5% CO , 288% N rOn day 3 (day 0 = day of activation) parthenoteswere transferred to fresh medium and cultured for afurther 4 days, after which blastocyst formation wasdetermined.ResultsNo significant difference in cleavage rates wasobserved between the activation treatments thatproduce diploid parthogenomes (Table 1). However,the cleavage rate of ethanol treated eggs, whichproduces haploid parthogenomes, was significantlylower than the diploid treatments. No significantdifference in blastocyst formation rates was observedbetween the A23187/6-DMAP and Eth/6-DMAPtreatments, however, a significantly higher rate wasobserved between Eth/cyclo/cytoB and Eth/6-DMAP(P