163 DEVELOPMENT OUTCOME AND MITOCHONDRIAL MEMBRANE POTENTIAL IN BOVINE PARTHENOTES SUBMITTED TO MITOCHONDRIAL SUPPLEMENTATION AND DEPLETION AT THE 1-CELL STAGE

Abstract

Apart from supplying ATP for cellular function by establishing a proton gradient across the inner membrane (Ψmm), mitochondria also play a central role in death pathways of most cells in the organism. Since mitochondria undergo major transformations during early embryogenesis, we hypothesize that the amount of these organelles at the 1-cell stage is correlated to development. Herein we evaluated the impact of mitochondrial supplementation and depletion on embryonic development and Ψmm. Immature oocytes were collected from ovaries of slaughtered cows and submitted to in vitro maturation for 26 h and parthenogenetically activated by 5 min exposure to 5 μM ionomycin followed by 3 h culture in 2 mM 6-DMAP (control, C). In order to deplete mitochondria (depletion, D), zygotes were centrifuged (15 min 10,000g in HEPES-SOF with cytochalasin B) and the mitochondria, at the mitochondria-enriched cytoplast fraction (MECF), were partially removed by micro-manipulation. Mitochondria supplementation was performed by introducing the MECF from the depleted zygote into another zygote (supplemented, S). Centrifugation and cytoplasm removal effects (7.1% of volume) were tested using 2 other control groups: centrifuged zygotes (centrifuged control, CC) and aspirated zygotes (aspirated control, AC). Embryos were submitted to Mitotracker CMXRos (500 ηM) staining to measure Ψmm levels. Zygotes were cultured in SOF medium for 3, 72, and 168 h in 5% O₂, 5% CO₂, and 90% N₂. Blastocysts were fixed and stained with Hoechst 33342 to count nuclei. At 72 h, development to the 8-cell stage was reduced in CC (41.6%), D (37.0%), and S (33.2%) groups when compared to C (52.5%) and AC (49.3%) control groups (P < 0.05, chi-square). Similar findings were observed at 168 h where the CC (31.9%), D (21.0%), and S (19.1%) group showed reduced development to the blastocyst stage when compared to the C (40.5%) and AC (41.8) control groups (P > 0.05, chi-square). Moreover, nuclear number was significantly reduced in the D (101.6 ± 12.4) and S (102.2 ± 7.8) groups when compared to the C (137.9 ± 6.7) and CC (126.6 ± 7.4) controls. The AC group (112.2 ± 7.8) is similar to CC, D, and S. These results indicate a sensitivity of embryos to changes (i.e. supplementation and depletion) in the number of mitochondria present at the 1-cell stage on further development to the 8-cell and blastocyst stages. With the exception of Ψmm of AC blastocysts at 168 (62.8 ± 6.8, P < 0.05; the Tuckey test), all other controls and treated blastocysts showed similar Ψmm levels (100.0 ± 6.2; 84.1 ± 8.3; 85.9 ± 6.6; 97.1 ± 19.1, respectively for C, CC, D, and S groups). Although, these results indicate that the Ψmm is not affected by the removal or addition of mitochondria. A reduction in cytoplasm volume (only the AC group) seems to adversely affect the ability of mitochondria to sustain normal Φmm levels at the blastocyst stage without affecting development or nuclear number. Together, supplementation and depletion of MECF are detrimental to development through mechanisms independent of mitochondrial Ψmm.

Financial support: FAPESP-Brazil.