Hybrid embryos produced ; a murine model to decrease generation interval in domestic species

Theriogeno/ogy41:313,

1994

HYBRID EMBRYOS PRODUCED IN VITRO; A MURINE M O D E L TO DECREASE GENERATION I N T E R V A L IN DOMESTIC SPECIES B. G. Tatham, A.T. Dowsing and A.O. Trounson. Centre for Early Human Development, Monash University, Melbourne, 3168 Australia. Decreased generation interval overcomes a limitation to genetic progress in breeding domestic animals. Hybrid embryo production uses drugs which control the cell cycle, combined with electrofusion to manipulate embryos. Preventing DNA synthesis of pre pronuclear zygotes with aphidicolin (APC), and stimulating with caffeine resulted in 2 cell embryos with each blastomere containing a haploid DNA content. After APC and caffeine treatment blastomeres were electrofused to reconstitute the diploid genome. In this murine model C57BIxCBA F1 mice were killed at 20 hours post hCG when pre pronuclear zygotes were extracted and allocated randomly into 5 groups. Group 1 was cultured in 0.05 lxg/ml APC and 0.5 mM caffeine in M16 for 20 hours, electrofused in Zimmerman fusion medium (ZFM) and cultured in M16 until the two cell stage when embryos were transferred opposite to controls into four pseudopregnant recipients or cultured to the blastocyst stage. Group 2 was cultured in 0.05 ixg/ml APC, Group 3 was cultured in 0.5 mM caffeine for 20 hours when cleavage was assessed and embryos cultured to the blastocyst stage. Group 4 was cultured in M16 for 20 hours, electrofused in ZFM and cultured to the tetraploid blastocyst stage. Group 5 was cultured in M16 for 20 hours and either transferred as above or cultured to the blastocyst stage.

Table 1: Effect of Drugs and Eiectrofusion on Murine Zygotes Blastocyst(%) Resorption(%) Foetus(%) Group(%) Cleavage(%) Fusion(%) 1 APC+Caff 262/295 (89) 134/149 (94) 61/109 (56) 8/24 (33) 0/24 (0) 2 APC 37/202 (18) ND 27/146 (19) ND ND 3 Caffeine 140/154 (90) ND 74/101 (73) ND ND 4 Electrofusion 81/90 (90) 74/81 (91) 34/45 (76) ND ND 5 Control 215/237 (91) ND 73/93 (79) 2/20 (10) 14/20 (70) After APC and caffeine treatment diploid pronuclear zygotes cleaved to haploid embryos (89%). Blocking DNA synthesis with APC reduced cleavage (18%), while treatment with caffeine alone had no effect on cleavage when compared to controls (90% vs 91%). Electrofusion was similar between treatment and control fusion groups (94% vs 91%). Group 1 developed to the blastocyst stage more than the APC group (56% vs 19%), resulting from caffeine overcoming the cleavage block caused by APC preventing DNA synthesis. Caffeine, electrofusion and culture controls had similar development (73%, 76%, 79%). Transfer of two cell embryos resulted in resorbed fetuses after drug treatment which requires further research to obtain viable progeny. Caffeine overrides the mitotic check point when the cell cycle does not normally proceed until DNA synthesis has been completed. Caffeine increases cAMP by inhibiting phosphodiesterase activity and binding adenosine receptors, modulating adenylate cyclase activity. Cyclic AMP dependant protein kinase activates protein kinases and phosphatases ultimately involved in progression of the cell cycle. Murine embryos produced as described are considered an F1 generation. When extrapolated to domestic animal species, and coupled with IVF technology, hybrid embryos produced via disaggregation are an F2 generation. Removing an entire generation interval results in rapid advances in genetic analysis and progress.

Copyright © 1994 Butterworth-Heinernann

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