133 MITOCHONDRIAL ACTIVITY OF IN VIVO DEVELOPING PIG EMBRYOS

Abstract

Preimplantation pig embryos undergo modifications in their metabolism. Glucose is metabolized via aerobic and anaerobic pathways at early cleavage stages, whereas glycolysis becomes the dominant pathway as development progresses. Moreover, fatty acid oxidation rate increases significantly at the early blastocyst stage. It is possible that mitochondria undergo structural and functional changes in response to altering embryo metabolism. Therefore, the aim of this study was to estimate changes in surface density of inner mitochondrial membranes (Sv) and mitochondrial activity (Av) per unit volume of mitochondria during the maturation of oocyte and early development of porcine embryos derived in vivo. The measurements were carried out on immature and matured oocytes, zygotes, 4 to 8 cell embryos, morula, blastocysts, and late and hatched blastocysts (10 to 39 embryos per stage). To estimate Sv, material was stained with mixture of 1.05 μM 10-N-nonyl Acridine Orange (NOA) and 0.5 μM Mito Tracker Deep Red (MtDR) for 30 min at 20°C. To measure Av, oocytes and embryos were labeled sequentially with 0.5μM Mito Tracker Orange CMTMRos (MtOR) for 30 min at 39°C and 0.5 μM MtDr for 30 min at 20°C. Embryos were then fixed in 3.8% formaldehyde and analyzed by confocal microscopy with a LSM 510 Meta Zeiss. The amounts of fluorescence emitted from embryos labeled as I_NOA, I_MtOR, and I_MtDR were measured because these ratios were directly proportional to Sv and Av, respectively. Values Sv and Av calculated for embryos at different stages of development were compared by ANOVA and Tukey’s intervals. During oocyte maturation, Av was low and remained unchanged, whereas Sv significantly decreased (P < 0.05) from 0.26 ± 0.021 f.a.u. (fluorescence arbitrary units) at immature oocytes to 0.11 ± 0.012 f.a.u. at matured oocytes. From the zygote to morula stage, Av was constant and then increased almost 5 times to the value of 0.95 ± 0.036 f.a.u. at the blastocyst stage, whereas late and hatched blastocyst characterized by significantly lower (P < 0.001) Av: 0.51 ± 0.023 and 0.34 ± 0.019 f.a.u., respectively. Surface density of the inner mitochondrial membrane in pig embryo during cleavage significantly (P < 0.05) increased from 0.16 ± 0.013 f.a.u. at zygote to 0.35 ± 0.020 and 0.75 ± 0.059 f.a.u. at morula and blastocyst, respectively. In conclusion, during the early stage of pig embryo development, mitochondria undergo structural and functional changes. Results demonstrate that the area density of inner mitochondrial membranes increases earlier (at 4 to 8 cell stages) than mitochondrial activity, which increase not before blastocyst stage. Furthermore, after blastocoel formation, when energy requirement is significantly lower, Av decrease but Sv remain constant.