31 EFFECT OF TREATMENT OF OVINE CYTOPLASTS WITH CAFFEINE ON THE TRANSLOCALIZATION OF HSP27 IN SOMATIC CELL NUCLEAR TRANSFER RECONSTRUCTED EMBRYOS

Abstract

We have previously reported that treatment of enucleated ovine oocytes with 10 mM caffeine for 6 h increases the activities of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) (Lee and Campbell 2006 Biol. Reprod. 74, 691–698). In addition, when used as cytoplast recipients for somatic cell nuclear transfer (SCNT), the resultant blastocyst stage embryos show an increase in total cell numbers. Furthermore, we have also found that gene expression profiles observed following caffeine treatment more closely resemble those of IVF embryos than non-treated SCNT embryos. In particular, heat shock protein 27 (HSP-27, a molecular chaperon was down-regulated in non-treated SCNT embryos. In contrast, in caffeine-treated SNCT embryos, levels of transcripts were similar to those of IVF embryos (Choi et al. 2006 Reprod. Fertil. Dev. 18, 122). Several studies on heat shock protein 25 (HSP-25)/HSP-27 demonstrated that HSP-27 was underexpressed in SCNT bovine embryos (Pfister-Genskow et al. 2005 Biol. Reprod. 72, 546–555), and phosphorylated HSP-25 becomes sub-localized in nucleus following heat shock condition in mouse embryos (Kim et al. 2002 Mol. Reprod. Dev. 61, 3–13). In cultured cells, HSP-25 phosphorylation was mediated by mitogen-activated protein kinase (MAPK), suggesting that nuclear-translocated HSP-25 might function to protect nuclear structure, thereby preventing apoptosis (Geum et al. 2002 J. Biol. Chem. 277, 19 913–19 921). In this study, we investigated the effects of caffeine treatment on the localization of HSP-27 by fluorescene immunocytochemistry in SCNT and control embryos. HSP-27 was detected primarily in the cytoplasm of non-treated SCNT embryos from the 1-cell stage onwards; however, some nulear staining was observed in some embryos. In contrast, in caffeine-treated SCNT embryos, a more intense immunostaining of HSP-27 was observed in the nucleus than in the cytoplasm. This result indicated that HSP-27 was phosphorylated and translocated into nucleus in SCNT embryos treated with caffeine. In addition, we also examined the levels of transcripts of upstream genes of HSP-27 such as p38MAPKs, and MAPK-activated protein kinase 5 (Mapkapk5) by semi-quantitative RT-PCR. The levels of Mapkapk5 and p38MAPKs transcriptional expression were constant among groups. These observations suggest that the effects mediated by caffeine on HSP-27 occur before the onset of zygotic transcription, are independent of gene expression, but are maintained until the blastocyst stage. Taken together, our results suggest that the down-regulated expression of HSP-27 in SCNT embryos may be linked with the increased incidence of programmed cell death. In contrast, the elevated expression of HSP-27 and increased nuclear translocation observed in caffeine-treated SCNT embryos may contribute to the increase in total cell number we have previously reported at the blastocyst stage.