Karyoplast-Cytoplast Volume Ratio in Bovine Nuclear Transfer Embryos: Effect on Developmental Potential VALERI ZAKHARTCHENKO, 1 MIODRAG STOJKOVIC, 2 GOTTFRIED BREM, 1 AND ECKHARD WOLF 2 * 1 Bayerisches Forschungszentrum fu ¨ r Fortpﬂanzungsbiologie, Oberschleissheim, Germany 2 Lehrstuhl fu ¨ r Molekulare Tierzucht und Haustiergenetik, Ludwig-Maximilians-Universita ¨ t, Mu ¨ nchen, Germany ABSTRACT To evaluate the effect of karyo- plast-cytoplast ratio on the development of nuclear transfer embryos, karyoplasts from day 4, day 5, and day 6 embryos were transferred to oocytes enucleated with different volumes of cytoplasm: Type 1, removal of a small volume of cytoplasm equivalent to the ﬁrst polar body, Type 2, removal of a volume of cytoplasm approximately equal to the volume of the respective karyoplast, and Type 3, removal of half of the oocyte volume. In addition, the effect of experimental reduction of karyoplast cytoplasm was investigated in day 4 and day 5 karyoplasts. Intact day 4 karyoplasts fused to Type 3 cytoplasts did not support development to blastocysts, whereas these karyoplasts yielded blasto- cysts in combination with Type 1 (7%) and Type 2 cytoplasts (12%). After experimental reduction of cyto- plasmic volume in day 4 karyoplasts, blastocysts (10%) were also obtained after fusion with Type 3 cytoplasts, probably due to reduction of cytoplasmic chimerism. With day 5 karyoplasts, blastocyst rate was higher in combination with Type 2 (34%) than with Type 1 (19%) and Type 3 cytoplasts (16%; P  0.05). The use of day 6 intact karyoplasts resulted in a signiﬁcantly ( P  0.05) higher proportion of blastocysts when fused with Type 2 (38%) or Type 1 cytoplasts (34%) than with Type 3 cytoplasts (16%). These results suggest that enucle- ation of oocytes with a volume similar to that of the respective karyoplast creates better conditions for cell cycle interactions with all types of karyoplasts than enucleation with minimal or large volume of cytoplasm. Mol. Reprod. Dev. 48:332–338, 1997.  1997 Wiley-Liss, Inc. Key words: embryo cloning; karyoplast; nucleus; cytoplast; ratio INTRODUCTION After successful transfer and fusion of the donor nucleus to the recipient cytoplasm, a whole cascade of interactions occurs between nucleus and cytoplasm. Cytoplasm of mature oocytes promotes nuclear enve- lope breakdown (Szo ¨llo ¨ si et al., 1988), chromatin conden- sation, and, if activated, chromatin decondensation, nuclear reformation (Newport and Kirschner, 1984; Fisher, 1987), nuclear swelling (Gurdon, 1964; Prather et al., 1990), translocation of cytoplasmic proteins into the nucleus (Di Berardino and Hoffner, 1975; Prather et al., 1989), and reprogramming of the genome (Wake- ﬁeld and Gurdon, 1983; Gurdon et al., 1984). In addi- tion, there is a release of proteins from the transferred nucleus into the recipient cytoplasm (Di Berardino and Hoffner, 1975; Leonard et al., 1982). The developmental potential of nuclear transfer em- bryos will depend primarily on the compatibility be- tween the donor nucleus and recipient cytoplasm. The compatibility between these two compartments of the nuclear transfer embryo is inﬂuenced by the ability of the oocyte cytoplasm to support the development of the transferred nucleus. Characteristics of the recipient cytoplast, mainly its stage of development and its position in the cell cycle (Robl et al., 1986; Prather et al., 1987; Collas and Robl, 1990; Modlinski and Smorag, 1991; Yang et al., 1993), have been proven to be important for the development of nuclear transfer embryos. Chromosome removal by physical enucleation prob- ably also removes important cytoplasmic components that may reduce cytoplast viability. Moreover, the re- moval of more cytoplasm during enucleation will result in nuclear transfer embryos with decreased cytoplas- mic volume, which may lead to problems later in development, since embryos with reduced cytoplasm often posses fewer cells at the morula or blastocyst stage (Evsikov et al., 1990; Westhusin et al., 1996). Donor nuclei also inﬂuence the developmental poten- tial of nuclear transfer embryos by their differentiation state (Gurdon, 1960; McGrath and Solter, 1984; Howlett et al., 1987; Prather et al., 1987), by their position in the cell cycle (Collas et al., 1992; Kono et al., 1992; Cheong et al., 1993; Otaegui et al., 1994) and also by treatment before transplantation (Bondioli et al., 1990; Yang et al., 1993). The method of nuclear transplantation in- volves transferring the nucleus as well as the cytoplasm Contract grant sponsor: Bayerische Forschungsstiftung; Contract grant number: 76/93. *Correspondence to Prof. Dr. Eckhard Wolf, Lehrstuhl fu ¨ r Molekulare Tierzucht und Haustiergenetik, Ludwig-Maximilians-Universita ¨t, Hackerstr. 27, 85764 Oberschleissheim, Germany. Received 4 February 1997; accepted 9 June 1997. MOLECULAR REPRODUCTION AND DEVELOPMENT 48:332–338 (1997)  1997 WILEY-LISS, INC.