title: Treatment of ovine oocytes with caffeine increases the accessibility of DNase I to the donor chromatin and reduces apoptosis in somatic cell nuclear transfer embryos.
abstract: Caffeine treatment of ovine oocytes increases the activity of maturation-promoting factor (MPF) and mitogen-activated protein kinases (MAPKs) and, in somatic cell nuclear transfer (SCNT) embryos, increases the frequency of nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC). At the blastocyst stage, caffeine-treated SCNT embryos have increased cell numbers. One explanation for this is that NEBD and PCC release chromatin-bound somatic factors, allowing greater access of oocyte factors involved in DNA synthesis and nuclear reprogramming to donor chromatin. This could advance DNA replication and cleavage in the first cell cycle, resulting in increased cell numbers. Alternatively, increased MAPK activity may affect localisation of heat shock proteins (HSPs) and reduce apoptosis. To investigate these possibilities, we investigated chromatin accessibility, the timing of DNA synthesis and first cleavage, the localisation of HSP27 during early development and the frequency of apoptotic nuclei at the blastocyst stage. Compared with control SCNT (non-caffeine treatment), caffeine treatment (10 mM caffeine for 6 h prior to activation) increased the accessibility of DNase I to donor chromatin (P < 0.05 at 1.5 h post activation (h.p.a.)), advanced DNA synthesis (43.5% v. 67.6%, respectively; P < 0.01 at 6 h.p.a.) and first cleavage (27.3% v. 40.5% at 20 h.p.a., respectively) and increased nuclear localisation of HSP27. Although development to the blastocyst stage was not affected, caffeine increased total cell numbers (98.5 v. 76.6; P < 0.05) and reduced the frequency of apoptotic nuclei (11.27% v. 20.3%; P < 0.05) compared with control SCNT group.
authors.affiliation: Animal Development and Biotechnology Group, Division of Animal Sciences, School of Biosciences, The University of Nottingham, Sutton-Bonington, Loughborough, Leicestershire, UK.
authors.forename: Inchul
authors.initials: I
authors.lastname: Choi
authors.forename: Keith H S
authors.initials: KH
authors.lastname: Campbell
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002110:Caffeine / Q000494:pharmacology*; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism*; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism*; D005307:Fertilization in Vitro; D020287:In Situ Nick-End Labeling; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000187:drug effects* / Q000378:metabolism; D010312:Parthenogenesis / Q000187:drug effects; D012756:Sheep
chemical_list: D002843:Chromatin; D002110:Caffeine; D003850:Deoxyribonuclease I