Membrane and intracellular effects of ultraviolet irradiation with Hoechst 33342 on bovine secondary oocytes matured in vitro

Abstract

Ultraviolet (UV) irradiation in combination with the bisbenzimide stain Hoechst 33342 has been used to visualize the chromosomes before removal of oocytes to be used as cytoplasmic hosts for embryo nuclear transplantation. Short term effects of UV irradiation on the cytoplasmic viability of bovine oocytes matured in vitro were assessed by performing membrane and intracellular studies at 2 and 20 h after exposure to UV irradiation for 0, 30 or 60 s. At the membrane level, loss of integrity was shown by increased lysis and increased retention of the fluorescein diacetate dye in oocytes exposed to 60 s of UV irradiation and uptake of methionine was higher in both irradiated groups. At the intracellular level, methionine incorporation into protein was 5.8 times higher in controls than in oocytes exposed to UV irradiation for 60 s and there was a marked difference in the pattern of protein synthesis. Some changes in protein synthesis were also found in oocytes after 30 s exposure. Moreover, high levels of fluorescence with the dye rhodamine 123 at 20 h after exposure indicated large increases in mitochondrial membrane potential in both groups of UV-irradiated oocytes. Together, these findings indicate that exposure to UV irradiation for periods as short as 60 s causes alterations to both membrane and intracellular components of bovine oocytes matured in vitro. It is concluded that care must be taken when using this methodology to visualize or destroy metaphase chromosomes during enucleation in the embryo cloning protocol.