PARENT SESSION

(T317) RELATIONSHIP BETWEEN MITOCHONDRIAL ACTIVITY, ENDOGEN HYDROGEN PEROXIDE AND APOPTOSIS IN OOCYTES, COMPETENT AND NON-COMPETENT EARLY BOVINE IN VITRO PRODUCED EMBRYOS.

Tarazona, Ariel¹, Jimenez, Marlene¹, Velez, Carlos¹, Olivera-Angel, Martha¹, ¹ Universidad de Antioquia, Medellín, Antioquia, Colombia

ABSTRACT- Immature oocytes (IO), mature oocytes (MO) incubated for 24 hours and in vitro bovine embryos (2,8,16-cell, morulae and blastocyst) were examinated to determine a possible relationship between mitochondrial activity (by staining with Jc-1 dye), hydrogen peroxide accumulation (by staining with di-hydrorodamine 123) and embryo death by apoptosis (by staining with acrydine orange and ethydium bromide). Embryos, IO, MO, were evaluated immediately after dying under conventional epifluorescence microscopy. The embryos cleaving synchronously were considered competent while asynchrony were a sign of non-competence. J-aggregates from MO were used as an internal positive control (100%). Six categories of activity were then estimated: 1) 0%, 2) 1-15%, 3) 16-30%, 4) 31-50%, 5) 51-75% and 6) 75-100%. For hydrogen peroxide and apoptosis the number of fluorescent /positive cells were counted in the embryo. Non-stained embryos were used as negative control. In this study, a total of 350 IO, 350 MO, 1.100 competent embryos and 1.300 non-competent embryos were used. The mitochondrial activity varied during the early cleavage, presenting the lowest level in IO (0%), the highest level in MO (75-100%) and an average activity during the cleavage (31-50%) in the competent group. Morulae and blastocyst presented lower activity than in early stages; the non-competent group showed a very low activity throughout all the cleavage period (0-15%). There were no positive cells to endogen hydrogen peroxide in IO and 3% in MO. The hydrogen peroxide accumulated in the blastomeres of competent embryos and reached maximum level at 8 cell stage (3.3 %) and decreased until 16 cells stage; morulae and blastocyst showed no peroxide accumulation. The non-competent group had positive cells during all the cleavages and they were blocked at 8-16 stages. There were less than 15% of apoptosis in oocytes and in competent group, but it was very high in the non-competent group (12.5% and 70% in two cells and 8-16 cells respectively). We concluded that competent embryos are able to clean the hydrogen peroxide produced by mitochondria and do not undergo apoptosis so much. In non competent embryos, however, this hydrogen would accumulate and trigger apoptosis, possibly by the caspase-c pathway.

KEY WORDS: Embryo, ROS, Apoptosis, Oocyte