It has been shown that the nuclei of different murine somatic cells (thymocytes, erythroleukaemia cells (MEL), embryonic red blood cells) undergo structural remodelling after transfer into the cytoplasm of acti- vated mouse oocytes (Czol / owska et al., 1984; Szöllösi et al., 1986b, 1988, 1998; Borsuk et al., 1996). The character of changes in these nuclei depends on the fate of the nuclear envelope after transfer. The nuclei which enter the egg cytoplasm soon after activation (not later than 60 min) undergo nuclear envelope breakdown and subsequently form pronucleus-like nuclei (PLN). The nuclei which are introduced later, after transition of the activated egg to interphase, maintain the nuclear enve- lope. In this case their remodelling is impaired and the decondensation of chromatin is less pronounced. The structural remodelling of introduced nuclei affects their transcriptional activity. In mouse zygotes and parthenogenetic eggs the activation of RNA synthesis in the pronuclei takes place no earlier than the late 1- cell stage, at the end of S-phase (Bouniol et al., 1995; Aoki et al., 1997; Bouniol-Baly et al., 1997). The trans- formation of a foreign nucleus into a pronucleus-like nucleus also leads to the termination of RNA synthesis, even if this nucleus was transcriptionally active before transfer. This activity is not restored during 3–6 h of culture of hybrid cells. In contrast, the nuclei which do not transform into pronuclei, continue (MEL) or restart Introduction Formation of cell hybrids has been widely used as a method to study the ability of different types of nuclei (originating from embryonal cells or from fully differ- entiated somatic cells) to undergo reprogramming under the influence of the cytoplasm of an activated egg. Successful cloning experiments carried out in farm animals such as sheep (Wilmut et al., 1997) or cat- tle (Vignon et al., 1998), and in laboratory mammals (mouse: Wakayama et al., 1998; Wakayama & Yanagimachi, 1999), were a consequence of those stud- ies. Although they proved that the reprogramming of fully differentiated nuclei is possible, they have not resolved all problems concerning the technique of cloning. The efficiency of normal development of embryos obtained as a result of nuclear transfer is still very low (approximately 1%) and the reasons for such poor development remain unknown. Improvement of the technique and an increase in the efficiency of cloning depend on better understanding of changes that foreign nuclei undergo in the egg cytoplasm. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 All correspondence to: Dr Ewa Borsuk, Department of Embryology, Institute of Zoology, Warsaw University, Miecznikowa 1, 02–096 Warsaw, Poland. Fax: +48 22 5541203. e-mail: borsuk@biol.uw.edu.pl DNA replication and RNA synthesis in thymocyte nuclei microinjected into the cytoplasm of artificially activated mouse eggs Ewa Borsuk and Marek Maleszewski Department of Embryology, Institute of Zoology, Warsaw University, Poland Date submitted: 14.1.02. Date accepted: 3.4.02 Summary Thymocyte nuclei were microinjected into the cytoplasm of parthenogenetic mouse eggs within 60 min or 3 h after egg activation and DNA replication and RNA synthesis were analysed in remodelled thy- mocyte nuclei and female pronuclei. We show that thymocyte nuclei which transform into pronucleus- like nuclei (thymocytes injected not later than 60 min after activation) enter S-phase 1 h earlier than the female pronuclei. At the beginning of the first cell cycle they remain transcriptionally silent, but in G2 undertake transcription earlier than the female pronuclei. Partly remodelled thymocyte nuclei (injected 3 h after activation) start to replicate DNA at the same time as the female pronuclei. They reinitiate RNA synthesis within 2 h after transfer and continue to transcribe irrespective of the transcriptional activity of the female pronucleus. We show that the observed transcription is only nuclear, i.e. RNA polymerase II- dependent. Keywords: DNA replication, Female pronucleus, RNA polymerase II, Thymocyte nucleus, Transcription Zygote 10 (August), pp 229–238. © 2002 Cambridge University Press DOI:10.1017/S0967199402002307 Printed in the United Kingdom