26 NUCLEAR REPROGRAMMING AND EMBRYONIC AND FETAL DEVELOPMENT OF CLONED OVINE EMBRYOS PRODUCED USING CAFFEINE-TREATED OOCYTES AS CYTOPLAST RECIPIENTS

Abstract

We have previously reported that treatment of ovine oocytes with 10 mM caffeine prior to their use as cytoplast recipients for somatic cell nuclear transfer (SCNT) increased the activities of both MPF and MAPK kinases, increased the occurrence of nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC), increased total cell numbers at the blastocyst stage, and altered the expression profiles of a number of developmentally regulated genes including Oct-4 and Oct-4-regulated genes and the stress response genes (HSP27 and HSP70). In addition, an increase in nuclear translocation of HSP27 was observed (Lee and Campbell 2006 Biol. Reprod. 74, 691–698; Choi et al. 2006 Reprod. Fertil. Dev. 18, 122; Choi et al. 2007 Reprod. Fertil. Dev. 19, 134). The objectives of the present study were to determine the potential mechanisms by which caffeine increased cell numbers and improved nuclear reprogramming. We previously hypothesized that caffeine treatment increased the degree of PCC and removed chromatin-bound somatic factors, allowing access of oocyte factors involved in reprogramming. With the use of a TUNEL assay, the accessibility of DNaseI (determined by fluorescence intensity) was increased in caffeine-treated embryos (P < 0.05), supporting the hypothesis. The timing of pronuclear formation, onset of DNA synthesis, and first cleavage were also studied. At 6 h post-activation (hpa), no difference in frequency of pronuclear formation was observed between groups but higher numbers of Brdu-labelled nuclei were detected in caffeine-treated embryos (67.6% v. 43.5%; P < 0.01). In caffeine-treated SCNT embryos, 40.5%, 54.3%, and 65% were cleaved at 20, 22, and 24 hpa as compared to 27.3%, 38.9%, and 48.4% in non-treated controls. On Day 7 there were no significant differences in the frequency of development to the blastocyst stage between treatment and control groups (26.06% v. 22.88%); however, treated embryos had increased total cell numbers (98.5 v. 76.6; P < 0.05) and lower percentages of apoptotic nuclei (11.27% v. 20.3%; P < 0.05). A total of 106 treated and 120 control blastocysts were transferred to the uteri of 43 and 49 recipient ewes, respectively. Five and six ewes established pregnancy, respectively. In the treatment group, three ewes maintained pregnancy for over 100 days and one live lamb was born; in contrast, all controls aborted within 100 days. Taken together, these results suggest that the increased cell numbers and improved reprogramming in SCNT embryos produced using caffeine-treated oocytes as cytoplast recipients may be mediated by increased NEBD and PCC facilitating chromatin access. In addition, increased MAP kinase activity may result in translocation of HSP27 to the nucleus, resulting in reduced apoptosis. Furthermore, caffeine-treated embryos maintained pregnancies for a longer period, supporting our previous observations (Lee et al. 2006 Reprod. Fertil. Dev. 18, 135–136) and suggesting that in sheep the use of caffeine to increase oocyte kinase activities results in embryos with a greater developmental competence.