Effect of dibutyryl cyclic adenosine monophosphate on reactive oxygen species and glutathione of porcine oocytes, apoptosis of cumulus cells, and embryonic development

Abstract

SummaryThe present study was conducted to investigate the effect of dibutyryl cyclic adenosine monophosphate (dbcAMP) supplemented into porcine maturation medium on reactive oxygen species (ROS) and glutathione (GSH) levels of oocytes, and apoptosis of cumulus cells (CC). In addition, the effect of dbcAMP on embryonic development following in vitro fertilization (IVF) or parthenogenetic activation (PA) was determined. Cumulus–oocyte complexes (COCs) were cultured in 0 mM (control), 0.5 mM, 1 mM, 5 mM, or 10 mM dbcAMP-supplemented medium for 22 h, then for another 22 h without dbcAMP. GSH and ROS levels of oocytes were assessed at 44 h of culture by dichlorohydrofluorescein diacetate or 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin staining, respectively. Additionally, COCs were cultured in 0.5 mM or 1 mM dbcAMP and then fertilized in vitro or activated parthenogenetically. Embryonic development and blastocyst cell numbers and apoptosis levels on day 8 of culture were investigated. CC apoptosis at 44 h of culture and blastocyst apoptosis were assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. GSH levels in the 0.5 mM dbcAMP and control groups were increased (P < 0.05), while levels of oocyte ROS and CC apoptosis in the control, 0.5 mM, and 1 mM dbcAMP groups were significantly lower than the levels in other groups. Cleavage and blastocyst rates, cell numbers, and apoptosis levels were not significantly different in embryos derived by either IVF or PA among the groups, with the exception of significantly increased apoptotic levels in IVF blastocysts produced from oocytes treated with 1 mM dbcAMP. In conclusion, dbcAMP treatment during in vitro maturation (IVM) did not improve embryonic development under our study's parameters compared with control conditions, although 0.5 mM dbcAMP showed significantly higher GSH levels and lower blastocyst apoptotic levels compared with 1 mM dbcAMP.