Mitochondria are important determinants of developmental competence for oocytes and embryos owing to their central role in cellular metabolism, yet mitochondrial activity and morphometry during early porcine development have not been quantified. In this study, we examined the membrane potential Δψ(m) and the surface density Sv(in,m) of the inner mitochondrial membrane in pig oocytes and pre-implantation embryos using fluorescent probes and confocal microscopy. Mitochondria and their cristae were also examined by transmission electron microscope. Δψ(m) was consistently low from immature oocytes up to morulae and increased significantly in the early blastocyst before decreasing at the expanded blastocyst stage. This stage-dependent pattern of Δψ(m) changes differs from that reported for other mammals. We also determined that Δψ(m) is lower in cultured when compared to non-cultured porcine early blastocysts. Sv(in,m) was higher in immature oocytes than mature oocytes and remained constant up to the 4- to 8-cell embryo stage. It increased significantly at morula and early blastocyst stages. No differences in Sv(in,m) were found between developmentally matched non-cultured and cultured embryos. These results indicate that the inner mitochondrial membrane potential and surface density change significantly during pre-implantation porcine development in relation to metabolic alterations of the embryo. It is possible that modification of Δψ(m) by manipulating culture conditions may improve the performance of embryos that develop in vitro.
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 INOA, IMtOR, and IMtDR 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.
In comparison to in vivo derived pig embryos, in vitro culture conditions produce embryos with altered metabolic rates of carbohydrates and fatty acids (Romek M et al. 2010 Theriogenology 74, 265–276), which may compromise embryo viability. Because various energy substrates are metabolized via several aerobic pathways leading to generation of the inner mitochondrial membrane potential (ΔΨm), value of ΔΨm is a key indicator of embryo metabolic activity, closely related to oxygen consumption and cellular energy needs. Therefore, the aim of this study was to compare ΔΨm between non-cultured and cultured pig embryos during early development. The non-cultured embryos were obtained from 6-month-old gilts, whereas those derived in vitro were cultured from zygotes to the appropriate stage in North Carolina State University 23 (NCSU-23) medium supplemented with 4 mg mL–1 of bovine serum albumin. The ΔΨm measurements were carried out on both non-cultured and cultured 4 to 8 cell embryos, morulae, blastocysts and late blastocysts. For this, embryos were labelled with 0.5 μM Mito Tracker Orange CMTMRos (MtOR) for 30 min at 39°C and then with 0.5 μM Mito Tracker Deep Red (MtDR) for 30 min at 10°C. Using a LSM 510 Meta Zeiss confocal microscope, we measured the amounts of fluorescence (IMtOR and IMtDR) emitted from embryos and values of ΔΨm were estimated as the IMtOR/IMtDR ratios. The results were analysed by ANOVA and Tukey's test. From the zygote to morula stages, ΔΨm remained unchanged and did not differ between developmentally matched non-cultured and cultured embryos (P < 0.001). The value of ΔΨm increased significantly (P < 0.05) from 0.90 ± 0.26 arbitrary units (a.u.) for morulae to 3.92 ± 0.63 and 2.06 ± 0.38 a.u. for non-cultured and cultured early blastocysts, respectively. Whereas the mean value of ΔΨm was almost 2 times higher in non-cultured than in cultured early blastocysts, the mitochondrial membrane potential was statistically similar (P < 0.05) in the in vivo derived (2.10 ± 0.37 a.u.) compared to cultured (1.87 ± 0.30 a.u.) blastocysts. The lower ΔΨm in cultured early blastocysts may be explained by several-fold higher glucose concentration in NCSU-23 medium than in the oviductal fluid. It was reported that high levels of glucose decreases the Krebs cycle metabolism of pyruvate, glutamine, and glucose, and reduces oxidation rates of fatty acids in cultured pig embryos in comparison with in vivo counterparts. Hence, this impaired metabolism reflected by decreased ΔΨm may be responsible for insufficient energy production and reduced developmental competence of cultured early blastocysts. Therefore, because embryo-cavitation is a critical event in pig development, further effort should be focused on proper blastocyst culture. Research was partially supported by Grant NR 12 0036 06 from NCBiR, Poland.
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